Vo Thu

An Nguyen

© Vo

Thu An Nguyen

O Ch Ch

C H Ch

Ch

Magnetic Polyion Complex Micelles as Therapy and Diagnostic Agents by

ii AUTHOR'S DECLARATION I hereby declare that I am the sole author of this thesis. This is a true copy of the thesis, including any required final revisions, as accepted by my examiners.

I understand that my thesis may be made electronically available to the public.

iii

Résumé / Abstract

Ce manuscrit de thèse présente la synthèse de nanoparticules d'oxyde de fer superparamagnétiques couramment appelées SPIONs servant d'agents de contraste pour l'imagerie par résonance magnétique (IRM) et la génération de chaleur pour la thérapie cellulaire par hyperthermie induite par champ magnétique radiofréquence (HMRF). Le contrôle des tailles et de la distribution en tailles des SPIONs et donc de leurs propriétés magnétiques a été obtenu en utilisant un copolymère arborescent G1 (substrat de polystyrène branché en peigne noté G0, greffé avec des groupements pendants poly(2-vinyle pyridine) ) comme milieu « gabarit », tandis que la stabilité colloïdale et la biocompatibilité des SPIONs ont été apportées par un procédé de poly-complexation ionique grâce à un copolymère double-hydrophile acide polyacrylique-bloc-poly(acrylate de 2-hydroxyéthyle) PAA-b-PHEA.

La complexation des segments de PAA-b-PHEA avec des nanostructures préformées contenant de la poly(2-vinyle pyridine) (P2VP) a été conduite dans l'eau afin de produire des micelles dynamiques unimoléculaires par poly-complexation ionique (PIC), par ajustement du pH dans une gamme étroite. Une fois formées, ces micelles PIC sont stables dans des tampons à pH neutre tels que des milieux de culture cellulaire. Le contrôle de la taille et de la structure des micelles PIC, allant d'espèces larges floculées à des entités stables unimoléculaires avec des diamètres hydrodynamiques entre 42 et 67 nm, a été accompli par l'ajustement de la densité de la couche polymère stabilisante autour du coeur G1. La preuve de l'unimolécularité par rapport à la formation de structures multimoléculaires a été apportée par des techniques de diffusion dynamique et statique de la lumière, tandis que la nature coeur-écorce des micelles PIC a été révélée par des images du signal de phase en microscopie de force atomique (AFM) ainsi que par la variation de la déflexion et de l'amplitude de vibration de la pointe AFM.

Notre revue de la littérature a rapporté les tentatives et les succès dans le contrôle des tailles et des formes dans la synthèse de nanoparticules magnétiques grâce à la variation de la iv taille et de la géométrie des gabarits polymères, dont il a été présagé qu'ils puissent servir de moules à l'échelle nanométrique. Bien que ces micelles PIC se soient révélées d'usage limité pour la synthèse directement in situ de nanoparticules magnétiques, le copolymère arborescent G1 (G0PS-g-P2VP) a été utilisé pour la première fois comme gabarit polymère pour contrôler les tailles et améliorer la distribution en tailles de nanoparticules d'oxyde de fer, comme prouvé par microscopie électronique en transmission (MET). La stabilité colloïdale à pH 7 des nanoparticles magnétiques, notées G1@Fe 3 O 4 , a aussi été améliorée par poly-complexation ionique avec le PAA-b-PHEA, produisant des micelles poly-complexes ioniques magnétiques (MPIC) de diamètre hydrodynamique D h  130 nm et d'indice de polydispersité PDI  0.136.

Le superparamagnétisme des nanoparticules de Fe 3 O 4 a été révélé par magnétométrie par vibration de l'échantillon, une technique aussi employée pour étudier l'influence de paramètres variés : stoechiométrie de complexation fer/azote, température, et nature du matériau de la couronne sur les propriétés magnétiques et relaxométriques des nanoparticules de Fe 3 O 4 , confirmant l'idée de pouvoir moduler les propriétés magnétiques et relaxométriques via les conditions de synthèse. On a effectué des tentatives pour comparer les résultats à des ajustements théoriques, pour discuter des différences entre échantillons (micelles nues ou recouvertes de copolymère dibloc), entre les échantillons et d'autres issus de la littérature, et entre les différentes mesures (taille relaxométrique, taille magnétique et taille issue d'images MET). Au final on a réussi à synthétiser des SPIONs présentant de fortes valeurs de relaxivité transverse r 2 = 335 s -1 mM -1 et de rapport de relaxivité transverse sur relaxivité longitudinale r 2 /r 1 = 31.4 (sous 1.47 T et à 37 °C ), comparables ou même supérieurs aux produits de contraste commerciaux, suggérant leur efficacité comme agents de contraste négatifs pour les séquences IRM pondérées en T 2 . La fraction volumique intraagrégat de Fe 3 O 4 à l'intérieur des micelles polymères a été estimée, et a mené à un nombre de 12 cristallites magnétiques par micelle, compatible avec les observations par AFM et MET.

Par ailleurs, une efficacité de chauffage magnéto-induite (SAR) jusqu'à 55. 6 Wg -1 a été mesurée par calorimétrie sous champ alternatif à la radiofréquence f = 755 kHz et l'amplitude v de champ H max = 10.2 kAm -1 . La dépendance des valeurs de SAR avec H max et f a été examinée dans une vaste gamme de ces deux paramètres. L'internalisation cellulaire et la cytotoxicité des micelles PIC et MPIC ont été évaluées par des expériences in vitro. L'internalisation cellulaire a été visualisée par microscopie par balayage laser confocale et par une étude histologique en MET, et quantifiée par tri par cytométrie de flux et mesure de fluorescence. L'utilité des micelles MPIC pour le chauffage par champ magnétique radiofréquence a aussi été confirmée, comme l'a révélé l'effet dose-dépendant des micelles MPIC sur la viabilité cellulaire. C'est bien à la dose d'incubation maximale (1250 µg/mL d'oxyde de fer) que la viabilité cellulaire sous champ magnétique alternatif radiofréquence la plus faible a été observée : environ 46-57% après une heure-et-demi de traitement, et 30-35 % après trois heures pour la lignée cellulaire murine de fibroblastes L929. Nous avons vérifié l'hypothèse que l'excitation magnétique RF des nanoparticules internalisées dans les cellules était bien le facteur principal conduisant à la mort programmée (apoptose), même en l'absence de chauffage macroscopique.

Mots clés : SPION, copolymère arborescent, poly-complexation ionique, agents de contraste IRM, hyperthermie magnétique, relaxométrie des protons de l'eau Laboratoire de Chimie des Polymères Organiques (LCPO) UMR5629 ENSCBP, 16 avenue Pey Berland 33607 Pessac Cedex, France vi This Ph.D. dissertation describes the synthesis of superparamagnetic iron oxide nanoparticles (SPIONs) designed to serve as magnetic resonance imaging (MRI) contrast agents and for heat generation in cellular radiofrequency magnetic field hyperthermia (MFH) treatment. Control over the size and size distribution of the iron oxide nanoparticles (NPs), and thus over their magnetic properties, was achieved using a G1 arborescent copolymer (comb-branched (G0) polystyrene substrate grafted with poly(2-vinylpyridine) side chains, or G0PS-g-P2VP) as a template. Good colloidal stability and biocompatibility of the SPIONs were achieved via the formation of polyion complex (PIC) micelles with a poly(acrylic acid)block-poly(2-hydroxyethyl acrylate) (PAA-b-PHEA) double-hydrophilic block copolymer.

The formation of SPIONs was first attempted using preformed PIC micelles as templates. Complexation of the PAA segment of PAA-b-PHEA with G0PS-g-P2VP was achieved in water over a narrow pH range to produce dynamic, unimolecular PIC micelles stable in neutral pH buffers such as cell growth media. Control over the size and structure of the PIC micelles, from large flocculated species to stable unimolecular entities with hydrodynamic diameters ranging from 42 to 67 nm, was accomplished by tuning the density of the polymer stabilizing layer surrounding the G1 core. Evidence for the formation of univs. multimolecular structures was provided by dynamic and static light scattering measurements, while the core-shell morphology of the micelles was confirmed by atomic force microscopy (AFM) phase images. Unfortunately, the preformed PIC micelles did not perform well as templates for the in situ synthesis of SPIONs. An alternate procedure was developed using the G0PS-g-P2VP copolymer as a template to control the size and size distribution of the iron oxide NPs, as evidenced by transmission electron microscopy (TEM) imaging. The colloidal stability of the G1@Fe 3 O 4 magnetic nanoparticles at pH 7 was improved by subsequent complexation with PAA-b-PHEA, to produce magnetic polyion complex (MPIC) micelles with a hydrodynamic diameter D h  130 nm and a polydispersity index PDI  0.136.

Vibrating sample magnetometry was employed to reveal the superparamagnetic character of the Fe 3 O 4 NPs, but also to investigate the influence of the Fe/N templating ratio, vii the temperature, and of the PAA-b-PHEA coating on their magnetic and relaxometric properties, and to demonstrate the possibility of tuning these properties via the synthetic conditions used. The results obtained were compared for samples in their bare state and after coating with the block copolymer, and with litterature values for relaxometric vs. magnetic and TEM measurements. The SPIONs synthesized in this work had values of transverse relaxivity of up to r 2 = 335 s -1 mM -1 , and a transverse-over-longitudinal relaxivity ratio r 2 /r 1 = 31.4 (1.47 T, 37 °C), comparable with or even larger than for commercial products, suggesting their efficiency as negative contrast agents for T 2 -weighted imaging. The estimation of the volume fraction of Fe 3 O 4 inside the polymer micelles yielded a number of ca. 12 magnetite crystallites per micelle, comparable with the AFM and TEM observations. Moreover, a maximum SAR value of 55.6 Wg -1 was measured by alternating magnetic field (AMF) calorimetry at f = 755 kHz, H max = 10.2 kAm -1 . The dependence of the SAR values on the magnetic field amplitude H and the frequency f was also examined.

The cytotoxicity and cell internalization of the PIC and MPIC micelles were evaluated in vitro. Cell internalization was visualized by confocal laser scanning microscopy and TEM, and quantified by fluorescence-activated cell sorting. The usefulness of MPIC micelles for cellular radiofrequency magnetic field hyperthermia was also confirmed, as the MPIC micelles had a dose-dependent effect on cell viability. At the maximum incubation dose (1250 µg/mL iron oxide), the lowest cell viabilities were observed with an applied AMF: about 46-57% after 1.5 h of treatment, and 30-35 % after 3 h for the L929 cell line.

We verified the hypothesis that AMF excitation of the intracellular MNPs was the main factor leading to programmed cell death (apoptosis), even in the absence of macroscopic heating.

Keywords: SPION, arborescent copolymer, poly-ionic complexation, MRI contrast agents, magnetic hyperthermia, water proton relaxometry viii

Acknowledgements

I would like to express my sincere gratitude to many individuals who have offered their precious support and encouragements throughout my Ph.D. journey. I am immensely grateful to Prof. Mario Gauthier, who introduced me to the world of polymer chemistry and to academic writing. He has always impressed me with his profound wisdom, interdisciplinary knowledge, massive support and endless patience. My heartfelt appreciation also goes to Dr. Olivier Sandre, who has patiently guided and unwaveringly supported me in my journey through the field of magnetic nanoparticles. I truly admire his interdisciplinary knowledge and his passionate enthusiasm for science. I am sincerely thankful to Prof. Marie-Claire De Pauw-Gillet, who introduced me to the world of cell culture; without her great encouragement and her generous support, I would not have had the chance to complete many of the valuable parts of my work. Yves Gossuin and Dr. Lam Quoc Vuong (Biomedical Physics Unit, UMONS, Belgium) for your precious support with magnetometry. I would like to thank Dr. Franck Couillaud and Coralie Genevois, at the Centre de Résonance Magnétique des Systèmes Biologiques, for allowing me to perform cellular hyperthermia in their lab and for being so helpful.

x I sincerely thank Erin for helping me with my academic writing, Uyxing, Wiljan and Mylène for being my squash partners, Marie for allowing me to stay at her house when I was in Liège, Vusala, Mathilde Champeau, Mathield Weiss-Maurin, and Tuyen for your support. Last but not least, I want to send my biggest and warmest thank you to my dearest Mother, whose love, passion, strength and bravery have enlightened everyday of my life. I am indebted to my Father, who sacrificed his life to love, to care and to ensure that we have the best education. I thank my brother who made me proud of him, and my Grandmothers and my Aunt Hang for always loving and trusting us.

I thank you all, a lot! Vo Thu An Nguyen xi Dedication I dedicate this work to my dearest Mother, whose love, passion, strength and bravery have enlightened every day of my life and to my Father, who sacrificed his life to love, to care and to ensure that we have the best education.

Con kính tặng luận văn này cho Mẹ yêu dấu. Tình yêu thương, sức mạnh và sự can đảm của Mẹ thắp sáng cuộc đời con mỗi ngày. Con kính tặng luận văn này cho Bố, người đã dành cả cuộc đời để yêu thương, chăm sóc và đảm bảo cho chúng con nhận được sự giáo dục tốt nhất.

Some people dream of success while others wake up and work hard at it.

Winston Churchill

Logic will get you from A to B. Imagination will take you everywhere.

Albert Einstein xii

Table of Contents

List of Figures

List of Tables

Chapter 1 Introduction

Chapter 1 2

Overview

Due to their small size and large surface-to-volume ratio as compared to their bulk counterparts, nanoparticles (NPs) display distinct physical and chemical properties making them attractive for applications in various fields including electronics, energy and optics, chemistry, and biology. [1][2] Among the broad spectrum of nanomaterials being investigated for biomedical uses, magnetic nanoparticles (MNPs) have received significant attention for applications in targeted drug delivery, as magnetic resonance imaging (MRI) contrast agents, and as heat mediators in the magnetic hyperthermia treatment of tumors. [3][4][5] Besides metallic and bimetallic MNPs, superparamagnetic iron oxide nanoparticles (SPIONs) have been favored because of their low cytotoxicity profile [6][7] and the possibility of surface modification with biocompatible coatings. 3,[8][9] SPIONs serve as contrast agents for MRI, a modern three-dimensional non-invasive diagnostic method based on the magnetic properties of body tissues, by effectively changing the relaxivity of water, thus enhancing the image contrast and improving its diagnostic value.

Since the first time they were used for liver imaging 20 years ago, SPIONs (60 nm < D h < 150 nm) serve as negative contrast agents, 10 most commonly by intravenous injection, while others designed for gastrointestinal imaging are administered orally. 11 Beside commercial SPIONs such as AMI-25 Ferumoxide (Endorem) 12 , IOs (Resovist), 13 etc., numerous other types of particles were also suggested as potential MRI contrast agents such as silica-coated Fe 2 O 3 , [14][15][16] as well as iron oxide encapsulated in a hydrogel, 17 in electrostatic complexes, 18 coated with amphiphilic block copolymers, 13 or enclosed in polymeric vesicles. 19 Controlling the size, shape and dispersity of the iron oxide crystals via the synthetic protocol, to allow tailoring of their properties, has been an ongoing research interest.

The therapeutic potential of SPIONs was further emphasized when hyperthermia was recognized as a promising form of cancer therapy. The heat generated by SPIONs when subjected to an alternating magnetic field (AMF) increases the local temperature and destroys temperature-sensitive cancer cells. Intracellular magnetic hyperthermia, [20][21] where heat is exclusively generated by internalized SPIONs during AMF exposure, must be differentiated Chapter 1 3 from magnetic fluid hyperthermia, where cells are dispersed in a magnetic medium. [22][23] In contrast to the belief that cancer cells can only be killed when the temperature is raised over 43 °C (defining the cumulative effective thermal dose at 43°C or CEM43), the induction of significant cell death by internalized SPIONs without a macroscopic temperature increase during AMF exposure (called "intra-lysosomal" or "cold" hyperthermia) has also been described. [24][25] Numerous studies have focused on tuning the magnetic properties of the materials used for that purpose by controlling the size, shape and dispersity of the iron oxide crystals.

Research Objectives and Thesis Outline

The main goal of this Ph.D. dissertation was to achieve the synthesis of SPIONs in situ within arborescent copolymers bearing a stabilizing hydrophilic shell. Control over the iron oxide nanoparticle synthesis and the stabilization process were demonstrated to provide a product with good potential as magnetic resonance imaging (MRI) contrast agent and for heat generation in cellular radiofrequency magnetic field hyperthermia (MFH) treatment. It is well-known that the magnetic properties of iron oxide NPs are strongly dependent on their average size and size distribution, as stated in many reviews 4,9,[26][27][28][29][30][31][32][33][34][35][36] on the optimal design of magnetic NPs for MRI and MFH. This is particularly true for the specific magnetization M s , defined as the weight concentration of magnetic moments at saturation (in a strong magnetic field), which is expressed in Am 2 kg -1 (SI) or emug -1 (CGS). An arborescent copolymer of overall generation G1, namely a comb-branched (G0) polystyrene substrate grafted with poly(2-vinylpyridine) side chains, was used as a templating medium to encapsulate iron salt precursors and disperse them in discrete compartments, enabling tuning of the nucleation process and improved control over the average size and size distribution of the particles.

These SPIONs adopt the shape and size distribution of the polymer template, which allows regulation of their hydrodynamic size and polydispersity. To enable the clinical applications ultimately targeted for these NPs, the SPIONs are shielded with a hydrophilic shell of poly(2hydroxyethyl acrylate) to provide good colloidal stability, enhance their biocompatibility, and achieve a sufficiently long blood circulation time to allow their accumulation in tumor tissues.

This dissertation is organized into seven main chapters. Following this Foreword, a detailed review on size and morphology control of MNPs using organic templates is presented in Chapter 2. This literature review highlights, among others, the impact of welldefined polymer structures on size and shape control of MNPs as compared to linear homopolymers and small molecules, differentiating studies with efficient templating effects from in situ syntheses whose results are often not signicantly improved over bulk syntheses.

Another point showcased in Chapter 2 is the extensive use of vinylpyridine-based polymers as templates for iron oxide MNPs, the evidence for templating effects being more pronounced as the complexity of the polymeric structure increases. Furthermore, polymer templates are usually preferred that also combine stabilizing, biocompatible materials enhancing cellular uptake, reducing cytotoxicity and increasing biocompatibility, once the MNPs are exposed to physiological conditions. The superparamagnetic and magnetic properties of the uncoated MNPs (G1@Fe 3 O 4 )

and the MPIC micelles were investigated in Chapter 5 using vibrating sample magnetometry.

Measurements of the r 1 and r 2 relaxivities and the ratio r 2 /r 1 for MPIC micelles demonstrated their potential as negative contrast agents for T 2 -weighted magnetic resonance imaging. Their use in inducing cell hyperthermia was also evaluated with specific absorption rate (SAR) measurements for radiofrequencies. The dependence of the SAR values on the magnetic field amplitude H and the frequency f was examined.

To be useful for biomedical applications NPs need to have good colloidal stability, but also good biocompatibility to achieve a sufficiently long blood circulation time and to allow their accumulation in tumor tissues. [37][38] In Chapter 6, the cytotoxicity and cell internalization of the PIC and MPIC micelles were evaluated in vitro. In agreement with University of Waterloo Thesis Regulations, the format of each chapter is organized as a manuscript for publication in scientific journals. Each chapter therefore includes a summary, an introductory section providing relevant background information on the subject matter, experimental methods, results and discussion, and conclusions. Additionally, an overall extended abstract for the whole Thesis is provided in the preliminary pages of this dissertation together with its French translation which is a requirement of the Université de Bordeaux, and a single list of references at the very end of the document is organized and numbered according to each individual chapter.

Chapter 2 Templated Synthesis of

Magnetic Nanoparticles Through the Self-Assembly of Polymers and Surfactants

Overview

The synthesis of superparamagnetic nanoparticles (NPs) for various technological applications continues to be an interesting research topic. The successful application of superparamagnetic NPs to each specific area typically depends on the achievement of a high magnetization for the nanocrystals obtained, which is determined by their average size and size distribution. The size dispersity of magnetic NPs (MNPs) is markedly improved when, during the synthesis, the nucleation and growth steps of the reaction are well-separated.

Tuning the nucleation process with the assistance of a hosting medium that encapsulates the precursors (such as self-assembled micelles), dispersing them in discrete compartments, improves control over particle formation. These inorganic-organic hybrids inherit properties from both the organic and the inorganic materials, while the organic component can also bring a specific functionality to the particles or prevent their aggregation in water. The general concept of interest in this review is that the shape and size of the synthesized MNPs can be controlled to some extent by the geometry and the size of the organic templates used, which thus can be considered as molds at the nanometer scale, for both porous continuous matrices and suspensions.

Introduction

The synthesis of superparamagnetic iron oxide nanoparticles (SPIONs) and other transition metal (e.g., cobalt) ferrite nanoparticles (NPs) targeting various technological applications such as magnetic storage media, magnetic actuators or biosensors is a topic of ongoing interest for many researchers. In the biological and medical fields, magnetic NPs (MNPs) with a large specific surface area provide innovative potential in targeted drug delivery, in magnetic resonance imaging (MRI) as both positive and negative contrast agents, and in the magnetic hyperthermia treatment of tumors. The successful application of SPIONs in each specific area depends on the attainment of high magnetization values for the nanocrystals obtained, which is necessarily determined by their average size, size distribution, and crystallinity (absence of defects in the magnetic order). Additionally, to facilitate their biomedical applications, the nontoxicity, biocompatibility and stability of the coated magnetic particles in biological media have also been the topics of numerous studies.

MNPs can be synthesized by numerous chemical techniques such as the alkaline coprecipitation of aqueous salts, hydrothermal reactions, microemulsion and sol-gel syntheses, sonochemical reactions, the hydrolysis and thermolysis of precursors, flow injection and electrospray syntheses, etc. Irrespectively of the method used, the formation of superparamagnetic NPs always involves nucleation and growth steps. Their separation is extremely difficult in the aqueous coprecipitation route, involving complicated pathways through oxo-hydroxide phases and eventually to magnetic iron oxide nanocrystals. 1 The size dispersity of the NPs prepared is markedly improved when these two steps are wellseparated, which is unfortunately hard to achieve without relying upon kinetic parameters, for example the temperature or mixing conditions. 2 These barriers have been investigated and might be overcome by tuning the nucleation process with the assistance of a hosting medium, that encapsulates the precursors and disperses them in discrete compartments (such as small molecule or polymeric micelles), thereby providing control over particle formation. These inorganic-organic hybrids are particularly interesting since they inherit the properties of both the polymer and the inorganic material. In addition, coating by polymer chains can efficiently hinder aggregation of the particles. 3 The focus of this review is on methods providing some degree of control over the shape and the size of the synthesized NPs through variations in the geometry and size of the organic templates. Two classes of molds at the nanometer scale can be considered, consisting of either pores contained within a continuous matrix, or of individual objects suspended in a reaction medium. The organic component can also bring other advantages in addition to colloidal stability in aqueous media, such as the presence of specific functionalities.

In Situ Synthesis in Non-Polymeric Templates

Complexing groups such as the carboxylate, phosphate, sulfonate, and sulfate functionalities are known to bind strongly to the surface of magnetite. Furthermore, surfactants containing such head-groups can be tailored for stabilization in oil or hydrocarbon carrier fluids, as well as with charged ligands (citrate, oxalate, etc.) for aqueous media. The same type of moieties can be used to complex the divalent or trivalent iron precursors selected for the reactions leading to the MNPs, for example by the coprecipitation of Fe 2+ and Fe 3+ salts in water or in a polyol medium, or the oxidation, reduction, or thermal decomposition of Fe 2+/3+ -ligand complexes in a high boiling point solvent.

Carboxylates

Oleic Acid Coating in Combination with Reduction 2.3.1.1

The templating ability of organic compounds bearing carboxylic functional groups, for example oleic acid and L-glutamic acid, was investigated in the aqueous phase reduction/hydrolysis of iron(III) acetylacetonate (acac) using NaBH 4. 4 The ratio of reducing precursors and the methylamine base added. 6 Interestingly, the influences of NP size and temperature on the magnetic properties were systematically studied. For example, a decline in the remnant magnetization of 8.5 nm CoFe 2 O 4 NPs with increasing temperature, and its disappearance leading to the demagnetization of the NPs at 290 K were noted. Using field cooled (FC)/zero field cooled (ZFC) superconducting quantum interference device (SQUID) magnetometry, the magnetic susceptibility of NPs of different sizes was shown to exhibit the usual temperature dependence: initially increasing as the temperature was lowered, it reached a maximum at the point referred to as the blocking temperature (T B ), and eventually decreased.

A correlation between higher T B and increasing NPs diameters was convincingly pointed out.

The superparamagnetism of the NPs above their T B was confirmed by neutron diffraction measurements yielding the magnetic mesh size from the magnetic reflections (as opposed to atomic reflections, giving the crystallographic mesh size by more common X-ray diffraction), while the increases in the T B , coercivity (H c ) and saturation magnetization (M s )

with the size of the particles were in agreement with the Stoner-Wohlfarth theory on single domain particles. Such a study could not have been completed without the exceptionally good control achieved over the size and size dispersity by the microemulsion templating process.

Sodium Dodecylbenzenesulfonate (NaDBS) Microemulsions 2.3.2.2

The same technique to control the size of MNPs by varying the relative concentrations of iron salts, surfactant, and solvent in a water-in-oil microemulsion system was demonstrated by Lee et al. 7 This efficient approach, using NaDBS microemulsions as nanoreactors and hydrazine as precipitating agent, allowed the size of the NPs to be tuned in the range of 2-10 nm with narrow size distributions and high levels of crystallinity. The lattice fringes appearing in HRTEM imaging again hinted at the highly crystalline structure of the MNPs obtained. The matching XRD patterns obtained for the MNPS and for a magnetite standard further confirmed the structure of the Fe 3 O 4 phase, while the peak widths calculated from the Debye-Scherrer equation were consistent with the observed TEM sizes. The magnetization vs. temperature curves followed a pattern similar to the one previously described, in which the T B and M s for larger particles had higher values. These results were in good agreement with the Stoner-Wohlfarth theory in terms of the increase in activation energy for larger particles, although determining the origin of the smaller H c values observed as compared to related studies would require further investigation. More importantly, the procedure was taken a step further by producing several mixed metal ferrites in a size range of 4-8 nm, with uniform particle size distributions and an inverse spinel structure.

In Situ Synthesis with Polymers in Solution or in the Bulk State

The preparation of aqueous magnetic fluids was been achieved more recently using polymers as steric stabilizers in solution and polymer templates in the bulk state. These studies will be presented according to the different types of polymers used for that purpose.

Dextran (DEX) and Polysaccharide Derivatives

The term dextran refers to a class of branched polysaccharides composed from a majority of α-1,6-and a minority of α-1,3-or α-1,4-glycosidic linked glucose units, which are non-toxic, biocompatible and biodegradable hydrophilic polymers extensively used in biomedical applications and in NP preparation procedures. The general role of dextran in enhancing the biocompatibility of metal oxides, using green coprecipitation and gel template strategies, was previously discussed in a review by Patrinoiu et al. 8 For the purpose of this discussion, we are primarily interested in the efficiency of dextran in templating and controlling the shape and size of MNPs.

Influence of Dextran End-Group Reduction 2.4.1.1

In an attempt to optimize the nucleation of ultra-small superparamagnetic iron oxide (USPIO) particles in the presence of dextran, the effects of chemical functionalities and the molecular weight (MW) on the formation, stability, size, and magnetic properties of the hybrid particles obtained were investigated by Paul et al. 9 Reduced dextran, generated by sodium borohydride treatment of native dextrans with different MW, has many hydroxyl groups existing in the native structure but only one additional hydroxyl group converted from the aldehyde (hemi-acetal) terminus. Despite this minor change in structure, reduced dextran was reported to successfully produce USPIOs with a volume-average diameter of less than 30 nm, while the size of native DEX-coated particles synthesized in the same conditions was larger than 700 nm in most cases and even reached 2.7 µm for 1 kDa dextran. Samples prepared with low MW reduced DEX (1 kDa and 5 kDa) possessed a magnetic susceptibility 5-25 times larger than their native counterparts. However, the influence of the reduced terminal unit on the size and the magnetic susceptibility of the USPIOs was diminished for dextrans of higher MW.

The minor change at the terminal sugar unit of 10 kDa MW dextran resulting from the reduction reaction also led to more efficient utilization of the templating material, as more stable autoclaved USPIOs with much smaller diameters (23 nm vs. 587 nm) were formed, with a higher percentage of metal-bound dextran (0.96 g DEX/g Fe vs. 0.59 g DEX/g Fe).

The size effect of reduced 10 kDa dextran was emphasized through similar experiments performed with 2.5-50 wt% addition of the simple sugar glucose vs. the amount of dextran used. The samples derived from reduced dextran with added glucose had radii similar to those prepared without sugar added, thus convincingly demonstrating binding enhancement arising from the additional OH groups in the linear polyol termini as compared to the aldehyde functionality present in the monosaccharide. It is worth mentioning that the size of the USPIO particles increased with the MW of dextran (20 nm for 1 kDa vs. 41 nm for 70 kDa dextran). However, stable colloids could only be created using reduced polysaccharides having a MW larger than 10 kDa as compared to 70 kDa for native dextran. This shows the dominant role of the MW of the chains in the growth of larger colloids from an initially unstable magnetic sol.

Dextran (DEX) Sulfate 2.4.1.2

Beyond the encapsulation enhancement brought by reduced DEX as compared to the native material, further modifications of this type of material may provide additional options leading to improved physicochemical properties. In addition to being considered a ligand for macrophage scavenger receptors, DEX sulfate was also proposed as an advanced coating material as compared to reduced DEX: A mixture of only 5 wt% DEX sulfate with reduced DEX yielded particles with a hydrodynamic diameter of over 30 nm and insignificantly larger core sizes (7.9 ± 2.0 nm vs. 7.0 ± 1.9 nm for reduced DEX). 10 Experimental parameters including the reaction time and the pH were varied to create DEX sulfate-coated NPs with good magnetic resonance properties. The delayed addition of Fe 2+ was mentioned as a technique to avoid overgrowth and cross-linking of the particles caused by interactions between excess Fe 3+ and the polysaccharide coating. While an increase in the polysaccharide/Fe 3+ ratio decreased the core size and thus the relaxivity of the particles, failure of particle formation was also reported at very low polysaccharide concentrations.

DEX-b-Poly(methacrylic acid)(Fe 3 O 4 )-b-Poly(N-isopropylacrylamide) with 2.4.1.3 Thiol End Groups

The synthesis of quadruple-responsive nanocomposites was achieved in solutions of the copolymer DEX-b-poly(methacrylic acid)-b-poly(N-isopropylacrylamide) (DEX-PMAA-PNIPAM) where complexation was achieved by the poly(methacrylic acid) (PMAA) block, the PNIPAM units provided thermosensitivity, the iron oxide NPs (IONPs) offered the magnetic response, and nucleated gold nanorods (AuNR) induced near infrared radiation (NIR) response. 11 The diameter of the SPIONs before the AuNR encapsulation was 10 ± 3.5 nm (based on TEM analysis) with a narrow size distribution, while the aqueous hydrodynamic radius (R h ) measured by dynamic light scattering (DLS) was 66-69 nm, suggesting that a thick copolymer shell covered the SPIONs. The dependence of the lower critical solution temperature (LCST) on pH (29 °C at pH 4.5 vs. 35 °C at pH 12) was explained by changes in the hydrophobic/hydrophilic balance caused by protonation of the PMMA block. Vibrating sample magnetometer (VSM) measurements at room temperature revealed superparamagnetic properties with neither remanence nor H c . At room temperature, SPIONs 10 ± 3.5 nm in size had limited stability in a 300 mT magnetic field, displaying slow aggregation under these conditions. The magnetic interactions were strongly enhanced above the LCST, however, (e.g., at 50 °C), due to the phase transition of the hybrids that induced aggregation via hydrophobic as well as magnetic dipolar interactions. Owing to the presence of the PNIPAM block the phase transition was reversible, providing water solubility to the recovered SPIONs when the temperature was brought back below the LCST, due to the hydrophilic DEX units.

Alginate (Alg) Beads 2.4.1.4

As a naturally occurring, biocompatible and inexpensive polysaccharide widely used in encapsulation processes, alginate is constituted of 1,4-linked α-L-guluronic acid (G) and 1,4-linked β-D-mannuronic acid (M) units (Figure 2-2), either segregated into homopolymer blocks or combined in copolymer structures. Commercially available alginates extracted from the cell wall of either brown algae or bacteria have different M/G compositions, MW, sequences, and physicochemical properties which strongly affect their efficiency in encapsulation applications. 12 The loading capacity of alginates with different M/G ratios was investigated in the in situ alkaline oxidation of ferrous ions to produce maghemite MNPs. 13 More precisely, this synthesis relied upon the alkaline coprecipitation of Fe 2+ and Fe 3+ , but with the particularity of the in situ production of Fe 3+ ions by the oxidation of Fe 2+ in air. After a first oxidation cycle, a higher iron content (ca. 31%) was recorded in a sample for which the M/G ratio was 0.6, as compared to ca. 14% for the alginate with M/G = 1.6. This result highlights the superior binding ability of guluronic vs. mannuronic acid units resulting from the "egg box" conformation of the guluronic acid segments that trap the Fe 2+ ions more efficiently. Larger M s values were recorded for the M/G = 0.6 samples, obviously due to their higher total iron content. As the total iron content in two samples reached the same maximum value after three oxidation cycles (approximately 47.1% vs. 48.3%), these two samples displayed similar magnetization curves. The superparamagnetic character of these fully oxidized samples at room temperature was confirmed by their large values of M s , zero hysteresis, and from the paramagnetic Mössbauer spectra obtained. Although no further data were provided about the size and the morphology of the maghemite NPs obtained, the hosting ability of alginate compounds was clearly demonstrated. Recently, questions related to the size and morphology of MNPs encapsulated in alginate networks were convincingly answered by the team of Srivastava et al. 12 who took the above approach one step further with the use of sodium alginate from Laminaria hyperborea (MW = 60,000-80,000 g•mol -1 , M/G = 32/68) to synthesize Fe 3 O 4 /Alg nanocomposites with a core-shell structure in a controlled low-temperature oxidation/precipitation process at pH 4, 10 and 14. The Fe loading, the size and, more interestingly, the magnetic properties of the core-shell composites were efficiently controlled through the final pH used in the precipitation process. It was indeed reported that higher Fe loading could be achieved at low pH (4-5) than at high pH (14). The Fe 3 O 4 content of the alginate composites synthesized at pH 4-5 was confirmed by XRD analysis. Calculation of the crystallite size from the XRD patterns gave a mean value of ca. 14 nm for Fe 3 O 4 /alginate, while the values determined for bare Fe 3 O 4 using the Scherrer equation and from TEM micrographs were ca. 10 and 15 nm, respectively. As compared to bare Fe 3 O 4 NPs, the alginate composites exhibited better dispersibility due to the alginate shell, essentially reducing the interactions between the MNPs. While neither H c nor remnant magnetization were detected in the M-H curves for the magnetite alginate composites, confirming their superparamagnetic behavior, the saturated magnetization values exhibited by the hybrids were much lower (below 6 emu/g) than for pure Fe 3 O 4 NPs prepared by the same process (above 31 emu/g).

This was assigned to differences in morphology among these two sample series: The Fe 3 O 4 synthesized at pH 4 was better dispersed than the sample produced at pH 10, which was agglomerated to some extent. The M s values for the composites were larger for the NPs synthesized at a higher pH, which is obviously in agreement with the Fe loading efficiency reported above.

Synthetic Linear Homopolymers

To maximize the in situ templating effect as well as to enhance the stability of the ferrofluids obtained, numerous attempts have been made in terms of: (i) changing the chemical nature of the template material; and (ii) varying the method employed to produce the NPs. The first approach is more convenient experimentally for synthetic linear homopolymer templates than for natural polysaccharide derivatives, due to the fact that the former materials can possess controlled chemical structures, MWs, and MW distributions, which facilitates magnetic property improvement and particle surface tailoring in terms of enhanced colloidal stability and biocompatibility.

Thermal Decomposition of Poly(vinyl alcohol)-Fe(OH) 3 2.4.2.1

One of the first synthetic homopolymers investigated as template for the preparation of MNPs was poly(vinyl alcohol) (PVA), containing a large number of hydroxyl functional groups available to interact with Fe 3+ ions and leading to water-soluble complexes. In their pioneering work, Yokoi and Kantoh 14 successfully combined different amounts of PVA to Fe(OH) 3 and obtained Fe 3 O 4 precipitates by thermal decomposition. Although agglomerates of sintered magnetite particles were obtained instead of uniform products, the diameter of the Fe 3 O 4 crystals in the composites evaluated from the line width of the (311) diffraction peak linearly increased with the heating temperature, indicating that the growth of Fe 3 O 4 crystals at higher temperatures, under the reducing effect of PVA, converted one third of the Fe 3+ into Fe 2+ , as required by the stoichiometry of the coprecipitation reaction. In continuation of the earlier work using PVA solutions as in situ templates, Sairam et al. 15 performed the alkaline coprecipitation of various amounts of Fe 2+ and Fe 3+ previously impregnated in PVA membranes. The free-standing composite membranes prepared were expected to be useful as pervaporation separation systems to dehydrate organic solvents. The encapsulation of IONPs in PVA increased the mechanical strength of the membranes but also enhanced their water affinity, as shown by the improvement in solvent drying performance with increasing iron oxide content.

Conversion of Fe 3+ in PVA into Hematite in Supercritical H 2 O 2.4.2.3

Another study dealing with antiferromagnetic iron oxide (α-Fe 2 O 3 or hematite)-PVA composites was conducted by Xu and Teja 16 using continuous hydrothermal processing (CHP) in supercritical H 2 O, a process allegedly providing size and morphology control for metal oxide NPs. Although the influence of the experimental variables on the particle characteristics was not convincingly determined, a higher ferric nitrate concentration, longer residence time, or elevated temperature did result in increased average particle radii and crystallinity, as confirmed by narrowing of the peaks in XRD patterns. A larger average particle size (27.4 nm vs. 15.6 nm) and morphological transformations from spherical to rhombic (elongated) shapes were also reported as a consequence of a higher precursor concentration (0.06 vs. 0.03 M). More importantly, the particle size and size distribution (standard deviation) both decreased as the PVA concentration increased, as demonstrated in three experiments with a reaction time of 2.6 s (controlled by the residence time in the high pressure conducts). As the molar ratio of vinyl alcohol units to Fe 3+ in the starting mixture was increased from 0:1 to 1:1 and 2:1, the average particle size of the samples decreased from 15.6 to 10.1 and 7.2 nm, respectively. This led the authors to conclude that a minimum amount of PVA was needed to coat all the particles, resulting in a polymeric layer with a thickness of approximately 10 nm that hindered further growth of the particles.

Fe(acac) 3 Decomposition/Diol Reduction in Octyl Ether (OE) with 2.4.2.4 Polyvinylpyrrolidone (PVP)

It is widely accepted that the main limitation of the thermal decomposition of Fe(CO) 5 to prepare monodisperse Fe 3 O 4 NPs is the high toxicity of the carbonyl precursor. In an attempt to solve this issue, Fe 3+ acac was used in an one-pot polyol reduction process in OE and in the presence of a water-soluble polymer, PVP, playing the role of polymeric stabilizer. 17 The PVP-coated, monodisperse Fe 3 O 4 NPs obtained had diameters < 5 nm and displayed well-defined superparamagnetic behavior at room temperature with zero H c .

Controlled Precipitation in the Presence of Poly(4-vinylpyridine) (P4VP) 2.4.2.5

The role of polymers containing nitrogen-based functional groups in the synthesis of MNPs was furthered investigated in the in situ controlled precipitation of Fe 3+ ions in Fe 3+ -P4VP coordination complexes prepared beforehand in water/acetone mixtures and dried at 60 °C. 18 In this approach, the iron-P4VP solid matrix played the role of a growth-restricted medium, where ion diffusion was efficiently slowed down and the growing iron NPs were effectively "trapped" due to Fe-N interactions with the pyridine groups. More importantly, the hydrophilicity of protonated P4VP before precipitation, that facilitated N-Fe coordination, was described as an important factor controlling particle growth. At the onset of precipitation the protonated pyridine groups became hydrophobic, inducing the collapse of the homogeneous iron-polymer gel and the creation of a non-uniform microstructure consisting of hydrophilic and hydrophobic regions. The subsequent growth process was restricted by the amount of Fe ions available only in the hydrophilic regions, which led to small particles. The hybrid polymer nanocomposites obtained after washing and heating to 150 °C yielded rod-like NPs in TEM images. These nanorods, with lengths ranging from 15 to 75 nm and antiferromagnetic properties affected by the Cl/Fe atomic ratio, consisted of the antiferromagnetic chlorinated iron oxy-hydroxide "akaganeite" (Fe 8 O 8 OH 8 Cl 1.35 , also noted β-FeOOH). It was speculated that incomplete filling of the Cl -sites affected the perfect compensation of antiferromagnetic sublattices, resulting in the appearance of a magnetic moment. Perfect sublattice alignment is indeed only achieved in a Cl --saturated crystal lattice (i.e., in a highly acidic P4VP environment). In that particular case, the templating effect of the polymer on the NPs can be partially ascribed to the chlorine counterions of this weak polyelectrolyte.

Poly(ethylene oxide) PEO in the "One-Pot" Synthesis of Pegylated Oxide NPs 2.4.2.6

Being known for its excellent solvating characteristics, good complexing ability for several transition metal cations, exceptional resistance to oxidation, reduction, and decomposition (by acids and bases at moderately high temperatures, hydrogen peroxide, and sodium borohydride), and the possibility of end-functionalization, poly(ethylene glycol) (PEG) or PEO has naturally become a good candidate to orient the synthesis of magnetic particles. Its stealth characteristics, that is, the ability to avoid non-specific interactions with proteins and uptake by the reticulo-endothelial system, provides to NPs pegylated on their surface a simple route for further medical applications. 19

PEO in Alkaline Hydrothermal Treatment 2.4.2.7

While conventional templating methods often result in the formation of polycrystalline nanowires or nanorods, monocrystalline 25 nm × 1.5 µm magnetite nanowires (Figure 2-3) were successfully produced in a PEG-assisted hydrothermal process. 20 There are numerous challenges in controlling the size, morphology, and crystallinity of Fe 3 O 4 nanowires due to their complex inverse spinel structure. Full control over the iron oxide nanostructure phase through careful variations in the PEG and H 2 O concentrations and the reaction temperature was claimed (see Table 2-1 andTable 2-2). A maximum M s value of ~91 emu/g, as obtained from vibrating magnetometer measurements, was achieved under the optimal conditions, confirming the crystalline perfection and extremely high purity of the sample (without γ-Fe 2 O 3 , having an M s value in the bulk of 80 emu/g). The nanowire formation mechanism proposed involved PEG in a critical role as a stabilizer, mild reducing agent, and growth-orienting agent for the newborn Fe 3 O 4 NPs into nanowires. Up to this point, the majority of synthetic polymeric stabilizers discussed did not contain functional groups able not only to bind efficiently to the surface of Fe 3 O 4 particles, but also to add strong enough repulsions (electrostatic and/or steric) between the MNPs and lead to enhanced colloidal stability desirable for true "ferrofluids". In a search for different homopolymers with suitable functional groups, Lin et al. 21 investigated PAA with MW~2000 g•mol -1 , whose carboxylic acid groups can theoretically form strong ionic bonds with Fe 3 O 4 , and thus act as template for iron oxide nucleation. Furthermore, the presence of excess COOH groups at the surface of the coated particles should provide both electrostatic and electrosteric stabilization enhancing the stability of aqueous magnetite suspensions.

The negative zeta potential obtained from laser light scattering velocimetry measurements demonstrated that the polymer molecules could bind to the surface of Fe 3 O 4 NPs (particularly at alkaline pH), providing both electrostatic and steric stabilization against particle aggregation. This repulsion (especially at high concentrations of stabilizing polymer) sterically hindered the growth of the particles, producing much smaller products with narrow size distributions as compared with traditional methods.

Pseudo-aggregates of NPs, with a size of about 150-450 nm, were also observed several weeks after the ferrofluids were synthesized. The explanations suggested for this were hydrogen bonding between the COOH groups, or the entanglement of the PAA chains when the polymer was present at a high concentration in the synthesis. The fact that these large, broadly distributed aggregates were destroyed by sonication before light scattering measurements was confirmed by the appearance of 10-40 nm ferrofluid species with a much lower size dispersity. The M s for the PAA-coated ferrofluid was about 35 emu/g, as compared with 50 emu/g for pure Fe 3 O 4 NPs. The reduced value of M s was explained by the presence in the particles of γ-Fe 2 O 3 (which is ~20% less magnetized than pure Fe 3 O 4 ), and by the significant reduction in M s expected for particle sizes below 10 nm. In another study, PEG with carboxylic acid functionalities at both chain ends (PEG diacid, M n ~ 600) was employed in a one-pot synthesis in a polar organic solvent, triethylene glycol (triEG), to yield surface-modified ultra-small SPIONs (USPIOs) with an average particle diameter (from TEM) of 1.7 nm. 22 These particles were nearly monodisperse and were highly water-dispersible, yielding a hydrodynamic diameter of about 5.4 nm, significantly larger than the diameter determined by TEM analysis (1.7 nm), due to the thickness of the hydrated PEG coating. The composition of USPIOs in the PEG-modified USPIOs, estimated from thermogravimetric analysis (TGA), was 30.7% and was used to correct the net magnetization values to yield 23.5 emu/g and 6.6 emu/g at T = 5 K and 300 K, respectively. Additional data obtained from the M-H curve indicated that the USPIOs were ferromagnetic at 5 K and superparamagnetic at 300 K. Fourier transform-infrared spectroscopy (FT-IR) spectra for PEG and the PEG-modified USPIOs exhibited a C-H stretch at 2890 cm -1 , and a C-O stretch at 1110 cm -1 in both cases, confirming the presence of PEG in the composites. More importantly, in the spectrum for the PEG-USPIO composites, the C=O stretch at 1745 cm -1 in PEG was not prominent and was shifted to 1645 cm -1 region, which suggests bonding between COO -(rather than -Oof the polyether) and Fe ions on the surface of the particles, as shown on Figure 234. Relaxivity measurements and magnetic resonance images obtained highlighted the potential usefulness of these particles as both T 1 and T 2 MRI contrast agents, due to their ultra-small size, and the possibility of achieving target-specificity and a long blood half-life. Another homopolymer containing multiple carboxylic acid groups is PMAA. A dualfunctionality PMAA with trithiol chain ends (PMAA-PTTM) was used in a single-step aqueous coprecipitation procedure leading to narrowly distributed Fe 3 O 4 nanocrystals. 23 Interestingly, an increase in the molar ratio of COOH to the Fe 2+ and Fe 3+ ions (from 0.7 to 3.4) clearly led to a decrease in size (from 6.1 to 4.5 nm), as well as narrowing of the size distribution of the particles (standard deviation decreasing from 1.1 to 0.4 nm), thus indicating the dominant role of the COOH groups vs. trithiol termini of the polymer in controlling the nucleating process. The less influential role of the trithiol chain ends in the impregnation step offers a facile post-synthesis functionalization path to the introduction of more reactive groups with further conjugation capacity, without causing large changes in size, size distribution or water-solubility of the NPs. The coated Fe 3 O 4 nanocrystals had a size of 4.5 nm, clearly exhibited superparamagnetic characteristics on the M-H isotherms measured at 300 K, and possessed a calculated M s of 48 emu/g Fe 3 O 4 , which is surprisingly high as compared with conventional magnetite particles of the same size (M s < 15 emu/g). This low magnetization is usually attributed to several factors such as crystallinity defects including non-stoichiometry of the spinel structure, surface spin-canting, surface disorder caused by interactions with surface ligands, cation site distribution, etc. In a study by another team, 24 PMAA was also used to produce coated agglomerates by an aqueous coprecipitation method, having magnetic core sizes around 8 nm by TEM analysis and hydrodynamic mean sizes ranging from 1.5 to 3 µm. The PMAA-encapsulated magnetite particles did not exhibit superparamagnetic behavior however, but rather a small remnant magnetization and H c , probably due to the aggregation of the magnetite NPs, although the individual 8 nm magnetite NPs displayed superparamagnetic properties. A low magnetization of 40 emu/g determined at 7 T, as compared to the magnetization values for bulk magnetic iron oxides (92 emu/g for Fe 3 O 4 , magnetite, and 80 emu/g for γ-Fe 2 O 3 , maghemite), was assigned to the particle surface spin disorder effects listed above 24 .

Coprecipitation in the

Poly(methyl glutarimide) (PMGI) Templated Iron NPs by Spin-Coating 2.4.2.11

While investigating a novel method to generate high-density and uniformly distributed carbon nanotube (CNT) mats, Lu et al. 25 employed spin-coating to prepare a film of PMGI loaded with various iron precursors (Fe 3+ chloride, nitrate or acac, and the organometallic complex ferrocene) for the templated formation of IONPs, subsequently exploited as catalysts for CNT growth. Following the trend of using polymeric templates with specific characteristics, PMGI, a polymer widely used in the bilayer lift-off process, was expected to form complexes with Fe 3+ ions by multidentate coordination.

The particle size, distribution, and overall yield in the spin-coating process were strongly influenced by the complexation tendency of the iron precursors used with the PMGI templates. The particles derived from strongly coordinated compounds like ferrocene and iron acac had a low density of iron oxide and broad size distributions, since the Fe 3+ ions in these materials were unable to complex with the PMGI chains due to steric hindrance caused by the bulky counterions. Iron nitrate and iron chloride produced the highest and second highest density of IONPs, respectively, with small mean diameters, since both monodentate anions were less bulky and more loosely bound to iron.

Synthetic Linear Copolymers

Block copolymers exhibiting microphase separation into ordered morphologies such as lamellar, cylindrical and spherical microdomains provide a convenient self-assembled template for the synthesis of nanocomposites. These microdomains represent nanoreactors within which a variety of NP clusters can be synthesized.

Aqueous Coprecipitation with Double-Hydrophilic Block Copolymers 2.4.3.1 (DHBCs)

To investigate the templating effects of DHBCs as well as tailored functional groups on the surfaces, Wan et al. 26 prepared stable aqueous ferrofluids using copolymers with poly(glycerol monoacrylate) (PGA) or poly(glycerol methacrylate) (PGMA) as one block, and either PAA, poly[(N,N-dimethylamino)ethyl methacrylate] (PDMAEMA) or poly(ethylene glycol) monomethyl (mPEG) ether as the other block.

When coated with PAA-b-PGA the surface of SPIONs carries negative charges, while the surface of particles obtained from PDMAEMA-b-PGMA, of which the PDMAEMA chains are protonated at neutral pH, possesses positive charges.

Multidentate coordination arising from the formation of five-membered rings between the 1,2-diol groups of the PGA or PGMA blocks and Fe 2+ or Fe 3+ cations at the surface of the magnetic particles was proposed to be more stable than the four-membered rings formed by the COOH groups in PAA. The disappearance of the COO -adsorption band at 1592 cm -1 in the FT-IR spectra of PAA-b-PGA-coated IONPs indicated that the carboxylic groups of PAA-b-PGA were not coordinated with the Fe 2+/3+ cations. Additionally, magnetic fluids obtained with PGA or mPEG-b-PGA were stable over a wider pH range as compared to those prepared from PAA or mPEG-b-PAA. PGA was considered to be a better stabilizer than PAA for the dispersions, such that the PGA block of the copolymer was coordinated to the surface of the magnetite NPs, while the PAA block was extended into water.

The same conclusion was drawn in the cases of PDMAEMA-b-PGMA and mPEG-b-PGA block copolymers, of which the PGMA or PGA blocks were chemisorbed onto the surface of the IONPs, thus playing a decisive role in orienting particle synthesis while the PDMAEMA or mPEG blocks, with a swollen conformation in water, contributed to postsynthesis stability.

Aqueous Coprecipitation with Triple-Hydrophilic Block Copolymers (THBCs) 2.4.3.2

Following this research trend, triblock copolymers constituted from the monomers listed above, but particularly mPEG-b-PMAA-b-PGMA, were used in the alkaline coprecipitation of Fe 2+/3+ . 27 PGMA influenced the nucleation step by tightly binding to the magnetite cores, while PMMA created an intermediate layer with a double function of bearing the mPEG corona and encapsulating the antibiotic and antitumor drug adriamycin (ADR), also called doxorubicin. The drug encapsulation was driven by both electrostatic interactions at pH 7.4 between anionic COO -of PMMA and the protonated glycosidic amine of ADR (pK a = 8.2), and by hydrophobic interactions between the PMAA main chain and the hydrophobic anthracycline ring of ADR (Figure 2345). The size of the Fe 3 O 4 core was estimated to be about 7-8 nm from TEM micrographs, whereas a narrow size distribution and an average R h of 23 nm were determined for the coated particles by DLS analysis. Superparamagnetic behavior at 300 K without magnetic hysteresis was observed for the MNPs obtained. The effectiveness of an amphiphilic random block copolymer of 3-(trimethoxysilyl)propyl methacrylate and ethylene glycol methyl ether methacrylate, denoted as poly(TMSMA-r-PEGMA), as stabilizer for MNPs was assessed by Lee et al. 28 In particular, the in situ synthesis of Fe 3 O 4 was compared to the stepwise addition of the same copolymer to preformed iron oxide colloids. In both cases, covalent binding was achieved between the magnetite surface and the siloxane functionalities of the polymer hydrolyzing to silanol groups, while PEG more likely remained on the outside of the SPIONs. The cross-linking reaction of the silanol groups by self-condensation, promoted by heating, resulted in more stable coating layers. The hydrodynamic radii of SPIONs prepared with and without copolymer in the NP synthesis step were 16.0 ± 2.2 nm and 12.3 ± 1.2 nm, respectively, with narrow size distributions and core sizes in the range of 4-8 nm in both cases, consistent with a thicker layer of coating by the in situ preparation as compared to sequential addition. However, the SPIONs synthesized in situ had an M s value of 65 emu/g Fe (equivalent to 45.5 emu/g Fe 3 O 4 ), significantly lower than the 80 emu/g Fe (56 emu/g Fe 3 O 4 ) value obtained for the post-synthesis coated SPIONs. This effect can be interpreted as reflecting the influence on crystallinity or magnetic order of the NPs of surface complexation by the silanol groups of the copolymer during the synthesis. However, unlike ligands such as carboxylic acids and polyethers, that can enter the "inner coordination sphere" of Fe 2+ and Fe 3+ cations, silanols mainly interact with the Fe-OH hydroxyls at the surface of the iron oxide nuclei already formed in the sol-gel process, i.e., the "outer-sphere" of the metal centers. The risk of magnetic properties decreases trough surface ligand or spincanting effects is therefore lower than for strong ligands introduced during the synthesis. Furthermore, the SPIONs synthesized were found to allow the detection of tumors in T 2weighted MR images within 1 h, as a result of the accumulation of the nanomagnets at the tumor site. Protein uptake by the surface of these coated SPIONs was reported to be significantly lower than by Feridex™ I.V., a popular contrast agent, thereby confirming the stealth effect in blood plasma of the PEG component of the copolymer. In an attempt to confirm the roles of a PEO-b-PMAA double-hydrophilic diblock copolymer (DHBC) in directing the nucleation, controlling the growth, and sterically stabilizing the dispersions, a comparison was made among three cases: precipitation without polymer present, in the presence of "non-interacting" PEO, and in the presence of PEO-b-PMAA. 29 TEM images (Figure 2-6c) revealed that the particles coated with PEO-b-PMAA were much smaller (5 ± 4 nm) and more uniformly dispersed than those created using either PEO homopolymer or without polymer, highlighting the decisive role of the carboxylic acid groups in the PMAA block in promoting nucleation and inhibiting growth of the magnetic iron oxide particles. Although crystallinity was confirmed by powder XRD (PXRD) analysis, the magnetite and maghemite phases could not be distinguished by that technique because these two phases have the same spinel structure and their XRD peaks positions are quite close to each other. The PEO-b-PMAA-coated oxide exhibited substantial magnetism, as seen on Figure 2-6d, with a magnetization of 50 emu/g at a field strength H = 275 kA/m or B = 0.34 T), from which we can infer a saturation level around 53 emu/g (so the value of 60 emu/g given by the authors appears a little overestimated). The hydrophilic layer covering the particles facilitated their redispersion in hydrophilic mixtures of hydroxyethyl methacrylate and methacrylic acid monomers, before evaporation of the water. The waxy solid obtained was redispersed in an oil phase (decane) via sonication with poly(ethylene-cobutylene)-block-poly(ethylene oxide) (PBO-b-PEO) as emulsifier. Inverse emulsion polymerization of this sol yielded magnetic poly(hydroxyethyl methacrylate-co-methacrylic acid) P(HEMA-co-MMA) latexes with sizes broadly distributed from 35 nm to 250 nm, with the same superparamagnetic properties as the initial magnetic core-shell NPs, but also with a lower overall magnetization (10 emu/g latex) corresponding to 18 wt% of iron oxide in the polymer matrix.

Coprecipitation in

Akaganeite Rods

In an effort to vary the chemical nature of the anchoring block as a polypeptide, while retaining PEG as a stabilizing moiety, Kumagai 30 prepared coordination complexes between FeCl 3 and PEG-b-PAsp in distilled water. Incubation of the mixture at 50 °C resulted in substantially homogeneous needle-shaped alkaganeite β-FeOOH particles, approximately 60 nm in length and 10 nm in width. Energy filtering transmission electron microscopy (EFTEM) zero-loss images revealed that the thickness of the PEG layer on the IONPs was 10-15 nm approximately (Figure 234567). The anchoring interactions of the carboxylic groups with the β-FeOOH particles were confirmed using FT-IR spectroscopy, with the successful resolution of simultaneous hydrogen and coordination bonding (complexation) interactions.

However, a control experiment without polymer also led to needle-like oxo-hydroxides. The formation of the needles may thus be more likely ascribed to the physical conditions used (e.g., pH, salts, etc.) rather than to a templating effect of the polymer. In a study by Hatton et al., 31 the use of PEO-b-PAA in an aqueous coprecipitation route followed a size sorting step using a high-gradient magnetic separator (HGMS) yielded MNP clusters with sizes larger than 100 nm, containing several particles with average core sizes of 9.5 nm. Both the bleed-off and the captured clusters in the magnetic separator were superparamagnetic (M s = 62.9 emu/g for the latter sample), thus indicating a single domain structure for the particles. The iron-to-polymer loading ratio (expressed as the molar ratio of the Fe 3+ /Fe 2+ cations to the polymer carboxylates) was varied, thus changing the polymer coverage of the MNPs. These results suggested that the formation of large clusters was induced by the limited amount of polymer added. Indeed, when a large excess of copolymer was used, the MNP clusters obtained became small enough to pass though the magnetic separator without being arrested. This HGMS method is thus very interesting to separate constituents such as free DHBC and individually dispersed MNPs from clusters with a relatively well-controlled spherical morphology and a finite size, as seen on Figure 2345678. Poly(norbornene) (PNOR) is a rubbery material with interesting properties, such as the ability to absorb up to 10 times its own weight of hydrocarbons while still retaining its high tear strength and vibration damping ability. Amphiphilic block copolymers of this material, suitable as templates for the loading and coprecipitating iron salts, were obtained by the sequential ring-opening metathesis polymerization (ROMP) of norbornene and deuterated norbornene dicarboxylic acid (to provide better contrast between the two blocks in smallangle neutron scattering (SANS) experiments). The sodium salt form of the NOR-b-NORCOOH copolymer was dissolved in a THF solution, to achieve ion exchange between Fe 3+ and the Na + ions of the carboxylate block. Thin films prepared from this solution were then soaked in NaOH solutions to produce γ-Fe 2 O 3 particles. 32 Variations in the constituent block volume fraction ratio (Ф NOR/NORCOOH ), while ensuring a constant 1:1 ratio of COOH/Fe 3+ , led to changes in the morphology (Figure 23456789) and characteristic distances for the microphase-separated block copolymer that were correlated with the size and the mean interparticle distance between the MNPs (as assessed by both SANS and the T B measured by magnetometry). Electron micrographs (with iodine staining) revealed that 6 nm average diameter γ-Fe 2 O 3 particles decorated the lamellar structure of the copolymer in thin film samples, appearing in the images as thin lines of NPs, the spacing between the lines being 80 ± 10 nm.

Poly(styrene sulfonate-alt-maleic acid) (PSS-alt-MA) Shell with Subsequent 2.4.3.8

Cross-Linking

The nucleating effects for the in situ coprecipitation of amphiphilic PSS-alt-MA and of hydrophilic PAA were nicely demonstrated by Yoon et al. 34 The coprecipitation was followed by cross-linking of the polymers with 1,6-hexanediamine, to prevent subsequent desorption of the polymer shell from the surface of the as-synthesized IONPs and to reach a good colloidal state through electrosteric stabilization. The zeta potential remained highly negative after cross-linking (Table 2-3), even at high salt concentrations (e.g., 8%) added to reduce the thickness of the electric double layer, thus weakening the electrostatic repulsions. The sub-100-nm IONP clusters obtained were therefore stabilized by a balance of van der Waals and electrostatic interactions. The primary, highly crystalline IONPs exhibited superparamagnetism without hysteresis and an excellent M s of ca. 90 emu/g Fe , approaching the theoretical magnetization value for bulk magnetite (92-100 emu/g Fe 3 O 4 , 35 equivalent to 127-138 emu/g Fe ), suggesting that the stabilizer did not lower the magnetization. These results confirmed good stabilizing effects for PAA and PSS-alt-MA, while not demonstrating a true template effect since the MNPs were still polydisperse.

Brush Linear Poly(oligo(ethylene glycol) methacrylate-co-methacrylic acid) 2.4.3.9

P(OEGMA-co-MAA) as Nucleating and Stabilizing Ligand

Rather than relying upon the same copolymer block for both nucleation control and stabilization of the MNPs, Lutz et al. 36 developed in 2006 the idea of using a doublehydrophilic brush-linear block copolymer in which each block played a different, specific task. The coordinating ability of methacrylic acid units and the stabilizing capability of oligo(ethylene glycol) segments were examined in a coprecipitation process of iron ions using hydrophilic P(OEGMA-co-MAA). The hydrodynamic diameter of the MNPs obtained was readily tuned in the range of 10-24 nm (z-average values, polydispersity indices around 0.2) by variation of the initial polymer amount used as compared to the iron salts (from 1:1 to 0.42:1 wt:wt), thus also tuning their zeta potential from -14 mV to -7 mV. The 10 nm hydrodynamic diameter NPs were revealed as nearly monodisperse 7 nm particles in the TEM micrographs. As evidenced by TEM, the NPs had a quasi-spherical topology, in contrast to the "rock-like" shapes often obtained in coprecipitation methods. The stabilizing chain density was estimated as being relatively constantly in the range of 0.2-0.3 chains/nm 2 in all cases, which could explain the correlation found between the size of the NPs and the polymer amount used in the reaction. Moreover, the colloidal stability of the ferrofluids in physiological media, enhanced by the POEGMA block, allowed their in vivo use as MRI contrast agents.

Coprecipitation with DHBCs of Poly(oligoethylene glycol acrylate) (POEGA) 2.4.3.10 and Different Binding Blocks

The groups of Boyer and Davis described the synthesis by the xanthate reversible addition-fragmentation chain-transfer (RAFT/MADIX) technique of DHBCs composed of the same repelling POEGA block and anchoring blocks with either phosphonic acid ethyl Chapter 2 38 acrylate (PAEA), carboxylic acid (PAA), or glycerol acrylate moieties in different copolymers. 37 These DHBCs were used for the in situ synthesis of IONPs by alkaline coprecipitation. The expected binding strength PAEA > PAA > PGA correlated nicely with the iron oxide loading, that also increased linearly with the feed-weight ratio of the iron salts (Figure 2-11b). The size of the MNPs measured both by TEM and XRD followed the reverse order (PAEA < PAA < PGA) for a given polymer-iron feed ratio, i.e., the stronger the affinity for iron of the ligand, the smaller the magnetic core size. The size also decreased for increasing polymer contents (Table 2-4). All these observations confirmed a high degree of control over the nucleation and growth of the IONPs achieved with the copolymer template.

The final products consisted in hybrid magnetic multi-core clusters (as seen on the TEM images) exhibiting good colloidal stability in biological buffers and high T 2 relaxivity, which had the authors envision their use for in vivo MRI.

a) (b) (c) (d) (f) (e)
As an intermediate case between (fully hydrophilic) DHBCs and amphiphilic block copolymers, graft polymers of composition PAA-g-PEO/PPO (PPO: poly(propylene oxide))

were studied by the group of Hatton. 39 In analogy with the commercial Pluronics ® systems (PEO-b-PPO-b-PEO), the idea was to vary the hydration level of the micelles by tuning their PEO/PPO content while using the PAA backbone to complex with Fe 2+ /Fe 3+ ions. After alkaline coprecipitation, the water-based magnetic fluids obtained consisted of ca. 7.3 nm (TEM) magnetite NPs decorating the amphiphilic graft copolymer micelles. The magnetic core radii obtained by TEM and neutron scattering analyses had broad size distributions and irregular particle shapes however. Therefore the self-assembly of such copolymers with iron oxide precursors does not appear to provide a sufficient template effect for the synthesis of well-calibrated MNPs, and better controlled nanostructures need to be explored as reactors.

Synthesis Templated by Preformed Structures

Rather than using individual polymer chains and inducing their self-assembly in the presence of metallic salts, loading can be achieved by exposing preformed self-assembled structures to these precursors. This strategy is expected to minimize changes in morphology or size dispersity, but the ability of the metallic precursors to reach the anchoring moieties might be affected. Consequently, several studies aimed at optimizing the conditions for the in situ synthesis of MNPs in individual nanosized, well-defined objects.

Microemulsions in an Organic Solvent

The size, shape, morphology and physical properties of in situ synthesized MNPs are not only strongly influenced by the nature of the templates, but also by the methods used to load the precursors and create the MNPs. Among the emulsion methods, the nanosized droplets of water-in-oil microemulsions appear ideal as reactors inside which NPs can be synthesized, with a monodisperse distribution of diameters below 20 nm. The results obtained in this approach depend on the type of amphiphilic species used to stabilize the microemulsion.

Chitosan Shells in Fe 2+ Microemulsions with Triton ® -X 2.5.1.1

Due to the presence of numerous amine and hydroxyl groups, chitosan, a derivative of chitin with an acetylation level below 60%, has been considered as a template for loading metallic ions. Combined with other useful properties such as being non-toxic, hydrophilic, biocompatible, biodegradable and anti-bacterial, chitosan can be useful to shorten the gap between the synthesis of the MNPs and their biomedical applications. In research combining the advantages of microemulsion systems with the benefits of chitosan templates, Zhi et al. 40 Quaternary microemulsions of cetyltrimethylammonium bromide (CTAB), water, cyclohexane and pentanol were used as microreactors for the coprecipitation of Co 2+ , Fe 2+ and oxalate anions, followed by the decomposition at high temperature of CoFe 2 (C 2 O 4 ) 3 (as "sacrificial template") and oxidation of Fe 2+ into Fe 3+ to produce spinel CoFe 2 O 4 nanorods. 41 The calcination treatment was indispensable to convert the amorphous nanorods into crystalline nanorods while maintaining control over their dimensions (50-100 nm in diameter, several micrometers in length), by stimulating internal nucleation and crystallization, in analogy to the natural petrification process which requires centuries to complete. Scanning electron microscope (SEM) images highlighted the overall rod-like shape of the particles (Figure 2 Regarding their formation mechanism, the authors noted that the direct calcination of a coprecipitated CoFe 2 (C 2 O 4 ) 3 suspension in the absence of the microemulsion template did not lead to long crystalline anisotropic magnetic structures. Therefore an "oriented attachment" mechanism between the inorganic nuclei growing through the organic template was proposed. The ionic nature of the CTAB surfactant may also have played a role at the beginning of the calcination process, by forming a molten salt also favoring oriented nucleation.

Spherical Micelles in Water

Templates with predetermined sizes and structures are good candidates to orient the nucleation process, as they can be designed with an appropriate anchoring block, carry highly stabilizing units, and optionally a cross-linkable segment providing additional versatility.

Triblock

Polyisoprene-block-Poly(2-cinnamoylethyl methacrylate)-block-2.5.

PEG-b-poly(4-vinylbenzylphosphonate) (PEG-b-PVBP) Micelles 2.5.2.2

Among the papers collected for this review, a small number concerned the surface coating density of pegylated IONPs (PIONs) for in vivo applications such as MRI. A recent study by Ujiie et al. 43 utilized PEG-b-PVBP block copolymers for in situ alkaline coprecipitation (Figure 2 -18). A correlation between the (PEG-b-PVBP)/(iron salt) feed ratio and the surface density of PEG chains in the PEG-PIONs was revealed, suggesting an optimal ratio to obtain sufficiently small particles with a high PEG chain density.

Interestingly, while the core sizes measured by TEM (≈ 7.7 nm) were typical for the aqueous Chapter 2 48 coprecipitation route, they were unrelated to the feed ratios used. Thus although a "template effect with micelle-like aggregates" was invoked, the main interest of this method is for the preparation of individually dispersed MNPs stable in biological buffers rather than achieving precise control over the size of the magnetic cores. It is worth noting that the phosphonate groups significantly contributed to the incorporation efficiency of the block copolymer, due to its well-known binding ability for various metal oxides. The R h ≈ 30 nm observed for the micelle-like aggregates before the ammonia addition was considered as reflecting a "templating effect" for metallic nucleation. This is the main reason why this study is reported in the preformed structures section instead of the block-copolymer section.

Multiarm Star-Like Amphiphilic or Double-hydrophilic Block Copolymers 2.5.2.3 (DHBCs)

For linear block copolymer templates the size, shape, and the colloidal stability of the nanocrystals are greatly influenced by the experimental conditions used (e.g., concentration, solvent, temperature, and pH). Multi-arm star-like unimolecular micelles derived from either amphiphilic or double-hydrophilic block copolymers were successfully employed to achieve better control over the size and shape of the MNPs. Starting from a multi-arm ATRP initiator (suitably functionalized β-cyclodextrin (CD)), the polymeric blocks (PtBA or P4VP) serving to bind the inorganic precursor were synthesized first. These star-like homopolymers were then coupled by "click" chemistry with either hydrophobic (PS) or hydrophilic (PEO) polymeric segments, which imparted solubility to the micelles in either organic or aqueous environments. 44 The FeCl 2 and FeCl 3 precursors were loaded in the hosts in appropriate solvents. After ammonium hydroxide addition, Fe 3 O 4 NPs were produced with different sizes (6, 10 and 16 nm, e.g., Figure 2 Copyright 2013 Macmillan Publishers Limited.

More accurately, the magnetite NPs were encapsulated within the P4VP core, and the PbTiO 3 or Au layers were grown within the PAA shell (derived from the PtBA block after the first encapsulation step), thus retaining the magnetic properties of the NPs. TEM imaging revealed a uniform size and narrow size distribution for the NPs, while the crystalline lattices were characterized by HRTEM, XRD and energy-dispersive X-ray spectroscopy (EDS) measurements. Hollow gold NPs were also reported, aside from the magnetic-gold core-shell systems. This work undoubtedly evidences a strong templating effect in the in situ synthesis of inorganic NPs, in particular magnetic ones, using unimolecular star-like PAA-PEO micelles.

Cylindrical Multimolecular Micelles

Different morphologies were obtained by the self-assembly of commercial Pluronic ® F127 PEO-b-PPO-b-PEO triblock copolymers in water/alcohol mixtures, to serve as templates for iron ion loading by coprecipitation. 45 Surprisingly, as the alcohol content in This shows that Pluronics ® can serve as structure-directing agents that control both the metal oxide mesoscale size and the crystalline structure. The decrease in M s of the samples observed was in agreement with this phase transformation from the superparamagnetic (maghemite) to the anti-ferromagnetic state (goethite).

Fe 3+ Loading in Poly(acrylic acid)-graft-Poly(n-butyl acrylate) Brush 2.5.

3.1

In an attempt to prepare hybrid magnetic unimolecular comb-like polymer micelles, Zhang and Müller reported a procedure employing amphiphilic core-shell cylindrical polymer brushes with poly(acrylic acid)-block-poly(n-butyl acrylate) (PAA-b-PnBA) side chains to impregnate Fe 3+ . 46 It is worth noting that in order to increase the rate and extent of iron ion uptake, the COOH groups in the PAA blocks were deprotonated with NaOH before the encapsulating step was performed with an excess amount of FeCl 3 . The polychelate effect was confirmed by FT-IR and ultraviolet visible spectroscopy (UV-VIS) measurements, while the TEM and atomic force microscopy images revealed the core of the unimolecular wormlike cylinder hybrids that had been mineralized by the ferric cations (Figure 2-22).

However the authors did not go further than precursor loading, i.e., they did not describe the in situ alkaline coprecipitation.

Poly(glycerol monoacrylate)-graft-Poly(PEG methyl ether acrylate) (PGA-g-2.5.3.3 PEG) Copolymers

In a detailed study 47 aimed at investigating the influence of the graft density and the MW of the graft copolymer poly(glycerol monoacrylate)-g-poly(PEG methyl ether acrylate) (PGA-g-PEG) on the average size and the magnetic properties of MNPs, the possibility of changing the diameter of particles in the 4-18 nm range by employing polymers with different grafting densities was suggested. It is understandable that since the hydroxyl groups in the PGA block were the anchoring functionalities, the decreased feed ratio of the monomeric precursors of glycerol monoacrylate to the PEG methyl ether acrylate side chains in graft copolymerization resulted in the increased graft density g, reducing the coordinating capability of each individual graft polymer micelle and thus leading to ascending particle sizes. This observation was confirmed by the data obtained from XRD measurement were the Bragg peak widths were analyzed by the Scherrer equation: The average particle sizes were 4, 7, 9 and 18 nm, respectively, for g = 0.14, 0.23, 0.30 and 0.42. Interestingly, graft copolymers with different MW (from 4.2 kD to 7.6 kD and 15 kD) having the same graft density g = 0.24 produced particles of similar size. The particles were superparamagnetic at room temperature, with normalized M s decreasing within the range of 30-42 emu/g for decreasing particle sizes, due to the smaller crystalline domains.

Poly(ethylene oxide)-graft-Poly(acrylic acid) (PEO-g-PAA) Graft Copolymer 2.5.3.4

The four-arm graft copolymers PEO-co-ethoxyethyl glycidyl ether-graft-PAA [PEO x -co-Gly y -g-PAA z ] 4 were employed in the same coprecipitation process reported above, leading to composite particles with narrow size distributions encapsulating 10-20 nm iron oxide single crystals inside the polymer capsules. 48 The TEM results were in good agreement with those from XRD analysis, with the exception that peak broadening was assigned to matrix constraints for the nanosized particles. The superparamagnetic properties were confirmed, and the calculated M s was 56 emu/g Fe 3 O 4 . The size and the size distribution of the hybrid NPs could be tuned by changing the mass ratio of polymer to Fe 3 O 4 and by varying the ratio of PAA/PEO segments in the graft copolymer (better template effects, with smaller particles and narrower size distributions, were reported for larger proportions of PEO segments). More importantly, star-shaped polymers with a more confined structure exhibited a clearer template effect as compared to graft copolymers with similar components.

From the three preceding examples, which originate from different teams, one can conclude that despite changes in the chemical nature of the polymer segments, the graft polymer structures always induce more reproducible trends in the variation of the average size of the magnetite NPs synthesized by in situ coprecipitation. However these studies may have overestimated the template effect, in the sense that the size distributions observed on TEM images were always broad and did not correspond to true uniform samples.

Lamellar Films

Polystyrene-block-Poly(2-vinylpyridine) (PS-b-P2VP) Lamellae Hosting 2.5.4.1 Pristine Iron Nanoparticles

The complexation ability of vinylpyridine moieties was investigated by the groups of Cohen and Ross 49 not only in the form of linear homo-and copolymers as discussed above, but also in bulk films of PS-b-P2VP. Nucleation was induced by the thermal decomposition of Fe(CO) 5 or Co 2 (CO) 8 . The impregnation of the organometallic complexes Fe(CO) 5 and nickelocene in vacuum-dried films produced monodispersed iron, iron-cobalt, and cobalt-nickel metallic NPs residing within the P2VP domains, that were superparamagnetic at room temperature. The lamellar structure of these composites was visible in TEM micrographs as a "cluster of grapes" morphology of pristine metal particles that were uniform in size when the metallic feed ratio of Fe to Co was 100:0 (Figure 2

Copolymer Micelles

To create monolayer films where the particles were arranged in a hexagonal array, PS-b-P4VP micelles (formed by self-assembly in toluene) encapsulating FeCl 3 within their P4VP core were spread onto cleaned silicon wafers by spin coating. 50 Oxygen plasma treatment was needed to convert the Fe 3+ precursor into iron oxide and to remove the polymer template. The γ-Fe 2 O 3 crystals formed at 1.0, 0.5, and 0.2 FeCl 3 /P4VP molar ratios appeared on the TEM images (Figure 2

Copolymers

Also targeting the preparation of hexagonal arrays of NPs, the in situ synthesis of metal oxide NPs through a gas phase oxidation pathway in oriented cylinder structures was investigated by Ghoshal et al. 51 The ordered thin films were prepared using a commercial diblock copolymer of polystyrene (PS), with PEO as the metal-anchoring block. The block weight ratio was selected to yield cylindrical PEO domains 20 nm in diameter and 28 nm in height, regularly distributed within the PS matrix. One of the strategic details differentiating this research from the methods reported earlier was the microphase separation induced by a solvent mixture after spin coating of a toluene solution of the PS-b-PEO block copolymer onto cleaned silicon wafers. Annealing with a water/toluene mixture efficiently weakened the preferential interactions between PEO and the substrate, thus allowing vertical alignment of the PEO cylinders (Figure 2 Control over the diameter and the height of the oxide nanodots was attained by varying the precursor concentration, which changed the height and the diameter of the nanodots without affecting their spacing. Precursor solution concentrations exceeding 1% led to saturation of the PEO cylinder template, however. This method nevertheless offers great potential to easily tune the NP size for iron oxide and several other inorganic materials. In addition, the nanodots could be sintered without losing their structural order, as seen on

Holey Membranes

Poly(vinyl alcohol) (PVA)-Fe 3+ /Citric Acid (CA)/Ethylene Glycol (EG) 2.5.6.1

Calcination

Poly(vinyl alcohol) (PVA), containing a high density of hydroxyl functional groups available to complex ferric ions, was employed in a so-called "polymer precursor" (PP) method to produce porous iron oxide films. 52 The first step in the PVA-PP method involved the preparation of a viscous solution of Fe 3+ ions complexed with PVA and the polyether produced in situ by the reaction of CA and EG. The solution was then spin-coated on a Pyrex ® glass substrate before repeated calcination to decompose the PVA and yield a uniform porous iron oxide film consisting of sintered 100 nm diameter ellipsoidal particles, with a microporosity volume (assessed by the N 2 adsorption isotherm method) increasing with the PVA/CA weight ratio. A change in crystal structure from α-Fe 2 O 3 to γ-Fe 2 O 3 was detected by PXRD and X-ray photoelectron spectroscopy (XPS) measurements when the PVA/CA ratio exceeded 1, which was interpreted as being due to the surface reduction of α-Fe 2 O 3 into Fe 3 O 4 crystals. The kinetic constant for this transformation was enhanced by a decrease in Gibbs energy for the reduction due to improved elimination of O 2 from the higher porosity films.

Loading of the Pores of a Nafion ® Membrane Followed by Reduction 2.5.6.2

In comparison with commonly used methods like coprecipitation or thermal decomposition, fewer studies relied upon a reduction reaction. This route was selected by Yoon et al. 53 using a polymeric ion-exchange membrane to produce nanosized magnetic pristine iron particles. The method basically consisted in the diffusion of Fe 3+ ions into a commercial porous perfluorinated sulfonate ionomer membrane, and the in situ reduction with NaBH 4 into magnetic Fe NPs. This led to the disappearance of the hysteresis loop at 120 K, corresponding to the T B for very tiny Fe NPs, as well as ferromagnetism at 10 K and superparamagnetism at 200 K. The observation of 1.5-2.5 nm diameter spherical particles dispersed in a clear polymer background in the TEM images, combined with crystalline lattices revealed by EDS measurements, confirmed the single-domain structure of the iron NPs obtained.

Tridimensional Scaffolds (Macroscopic Samples)

In an encapsulation process, the strength of interactions between the anchoring moieties in a polymer matrix and the NPs is considered the most important criterion preventing the diffusion of the NPs out of the template and leading to successful directed nucleation and growth. One approach suggested to realize this condition was to rely on the large surface area and the complicated inner structure of polymer gel networks to perform the Chapter 2 62 in situ synthesis of MNPs. Tridimensional polymeric scaffolds containing further structural hierarchical microstructures offer great potential for size, shape, crystalline structure, and orientation control, and have consequently motivated a large number of studies.

Porous Preformed Carbon Foams (CF) 2.5.7.1

In an innovative study by Yoon et al., 54 mesoporous CF produced by the resorcinolformaldehyde method, with a very large pore volume (0.87 cm 3 •g -1 ) and specific surface area HRTEM images and XRD patterns confirmed the Fe 3 O 4 composition of the crystals, which had a non-uniform size and shape distribution in the 5-50 nm range, i.e., larger and broader than the 7-15 mm particles obtained by the conventional (non-templated) polyol synthesis (same precursor but at 180 °C in EG). The width of the XRD peaks also yielded a significantly larger average crystallite size of 21.7 nm (as compared to 10.1 nm for the polyol pathway). Although physical adsorption was invoked rather than chemical interactions, due to the absence of coordinating functional groups, the roles of the carbon foam in orientating the growth of the iron oxide nanocrystals and preventing their agglomeration were clearly shown. When used as anode material for lithium-ion batteries, the as-prepared CF/Fe 3 O 4 composites exhibited superior charge/discharge capacities as compared to the unsupported materials.

Sponge-Like Polystyrene/Polyacrylate Copolymer Gel 2.5.7.2

In their early work, the teams of Antonietti and Mann 55 successfully developed magnetic iron oxide nanocolloids using elastic sponge-like polystyrene-polyacrylate copolymer gels providing a mesoporous open-cell structure with carboxylic groups readily coordinating the metallic ions. The hierarchical structures of the microspherical pores, whose size could be further adjusted during the synthesis, offered enormous surface areas that effectively facilitated the templating and nucleation processes (allowing up to 20 wt% iron loading). It is worth noting that the gel structure was not changed after the loading step (Figure 2-29a). There were no crystalline structures in the air-dried composites, but rather amorphous hydrated Fe 3+ oxide which gradually converted to 24 nm magnetic Fe 3 O 4 crystals (based on wide-angle X-ray scattering (WAXS) line width analysis). These results were in agreement with the TEM analysis of gel sections showing homogeneous spherical and cubic particles with a mean size of 16.3 ± 9.4 nm trapped in the wall of the gels (Figure 2-29b). No hysteresis was reported in room temperature measurements, indicating superparamagnetism of the particles. This pioneering research using an easily tailored material was followed by many other studies on gel templates. anchoring moiety. This copolymer was successfully prepared and used for the in situ synthesis of IONPs by deposition of Fe 2+ and multi-step NaOH treatment. 56 The data obtained from XRD analysis after two particle deposition cycles revealed the initial existence of maghemite (γ-Fe 2 O 3 ) NPs, which were gradually transformed to goethite (α-FeOOH). This was likely not due to oxidation as ascribed by the authors, but rather a result of the unavoidable pH increase in such a multi-step NaOH treatment. The constant value of mean particle size (14 nm) calculated from the Scherrer equation and XRD diffractograms after several deposition cycles indicates that growth of the particles was inhibited by the constrained architecture of the polymer network. The maghemite content increased after each loading cycle, but the fourth cycle led to agglomeration. The magnetization of the hybrids also increased after each loading cycle but was maximized after the third one (8.04 emu/g). There

was no subsequent change in M s due to the appearance of the goethite crystals, that are known to be antiferromagnetic. The non-zero H c values observed for the hybrids, even at room temperature, were assigned to the same reason, i.e., mixing of the two phases giving rise to spontaneous ferrimagnetism.

Semi-Interpenetrating (Semi-IPN) Polymer Networks of Alginate (Alg) and 2.5.7.4 Poly(N-isopropylacrylamide) (PNIPAM)

In an attempt to synthesize IONPs within networks of mixed polymer hydrogels, Hernández et al. 57 initially prepared semi-IPN polymer networks from alginate bringing chelating carboxylate groups, and PNIPAM providing thermosensitivity to these materials.

Cylindrical samples cut from the semi-IPN were submerged into a 1:2 solution of Fe 2+ and Fe 3+ ions, and then into a NH 3 solution for the in situ preparation of semi-IPN ferrogels. For samples with different numbers of alkaline bath treatments, the phase type of the IONPs within the gels was determined to be a combination of magnetite and maghemite, based on the diffraction patterns in wide angle X-ray analysis and characteristic peaks in Raman spectroscopy. The diameters calculated from the Debye-Scherrer equation for the (311) Bragg peak suggested larger sizes for the NPs seeded in the polymer networks (respectively D 311 = 10.1 ± 2.5 nm and D 311 = 11.2 ± 2.8 nm for one and two alkaline treatments) as compared to D 311 = 6.7 ± 1.6 nm for NPs created in an alginate solution.

Cross-Linked Polyacrylamide (PAAm) Hydrogels 2.5.7.5

To prepare water-dispersible magnetite NPs, Xiong and Sun applied an in situ synthesis approach involving cross-linked PAAm hydrogels and NaOH solution treatments.

As shown in Figure 2-30a, the PAAm hydrogel was photopolymerized with pentaerythritol triacrylate (PE-3A) as a hydrolysable cross-linker, and BDMBP as photoinitiator. 58 The polymer network was expected to act as a template possessing both sufficient capacity for ion impregnation and a restrained mesh structure limiting the growth of MNPs and preventing their aggregation.

Poly(2-Acrylamido-2-methyl-1-propansulfonic Acid) (PAMPS) and P4VP-2.5.7.6 Based Hydrogels

Hydrogels prepared by radical copolymerization, either with a photoinitiator for PAMPS, 59 or a redox (persulfate) initiator for poly(4-vinyl pyridine) (P4VP) and poly(4vinyl pyridine-co-2-hydroxyethyl metacrylate) (P4VP-co-HEMA), 60 were impregnated with FeSO 4 and FeCl 3 and employed in a coprecipitation reaction to create magnetic composite hydrogels. These hydrogels, designed for water remediation, absorbed toxic heavy metal ions efficiently in aqueous media. Unfortunately, no information was provided on the size and the morphology of the magnetic particles obtained.

Polyacrylamide (PAAm) Hydrogels 2.5.7.7

Polyacrylamide (PAAm) hydrogels were employed as templates for the coprecipitation of Fe 2+ and Fe 3+ to create pH-responsive magnetic hydrogels. 61 The magnetite particles obtained were 3-5 nm in size and homogeneously distributed inside the hydrogel network (Figure 2 The density of Fe 3 O 4 particles increased for higher iron ion feeds (from 25 wt% to 100 wt% relatively to PAAm), indicating the good loading capacity of the template. Despite a low M s value of 4 emu/g, ascribed to the small size of the MNPs and the large weight fraction of polymer matrix, the composite exhibited superparamagnetism with a small H c of 14.8 Oe.

Dispersed Colloids (Microscopic)

Poly(N-isopropylacrylamide-co-acrylic acid-co-2-hydroxyethyl acrylate) 2.5.8.1

(Poly(NIPAM-co-AA-co-HEA))

Microgels Cross-Linked with N,N′-

Methylenebis(acrylamide) (BIS)

Sub-micrometer gels of poly(NIPAM-co-AA-co-HEA), prepared by suspension polymerization and cross-linked with BIS, were loaded with ferrous iron ions in an ion exchange process (with the ions in the interior of microgels). 62 A post-loading oxidation step with H 2 O 2 was used to produce the IONPs (Figure 2-33a). The size of these microgels was successfully controlled by the polymerization conditions: A rise in pH from 2.3 to 9.2 transformed the PAA blocks from a highly compact and weakly hydrophobic conformation to a highly hydrophilic and expanded coil, and greatly increased the repulsive forces between COO -groups, leading to larger particle sizes (from 200 to 600 nm). Not surprisingly, the concentration of COOH functionalities in the copolymer was the dominant parameter affecting the size and the loading capacity of the microgels. However the Fe 2+ precursor concentration and the pH value range useable for the reactions were limited by the solubility product of Fe(OH) 2 (K s = 4.87 × 10 -17 ): For instance, the maximum allowable pH value was 7.1 for [Fe 2+ ] = 1.5 mM. Interestingly, the size distribution of the NPs obtained was relatively narrow (e.g., 8.5 ± 1.0 nm diameter by TEM). These Fe 3 O 4 NPs trapped in the 500 nm diameter microgels exhibited superparamagnetic properties at 300 K (both the remnant field and the H c were zero) with a specific M s of 32.4 emu/g (Figure 2-33b).

Poly(N-vinylcaprolactam-co-acetoacetoxyethyl methacrylate) P(VCL-co-2.5.8.2 AAEM) Microgels

An interesting characteristic of this system is that the chemical composition of the microgels could be adjusted to provide additional properties to the MNPs. One example of this is the work of Pich et al., 63 who successfully employed microgels obtained by the copolymerization of AAEM and VCL to synthesize thermosensitive magnetic hybrids (Figure 2-34). It is common knowledge that thermoresponsive polymers such as PVCL can extensively and reversibly swell in appropriate solvents at temperatures below the LCST, but shrink rapidly above the LCST. The PVCL-rich shell of the microgels, exhibiting an LCST, offers the possibility to control their size through temperature changes, while the AAEM-rich portions provide a high iron-loading capacity via bidendate coordination. The observed non-monotonic increase in R h of the microgels for increasing magnetite loadings was explained in terms of the chemical interactions formed. The slight decrease in R h observed when increasing the Fe 3 O 4 loading from 0 to 7 wt% was attributed to multivalent complexation between the β-diketone groups of AAEM and the Fe 2+/3+ ions linking simultaneously several AAEM units, thus leading to additional cross-linking and shrinkage of the microgels. When the loading level surpassed 7 wt%, repulsive forces among the crowded Fe 3 O 4 particles led to swelling of the microgels (increase in hydrodynamic size). An increase in magnetite content (e.g., from 4 to 9.4%), leading to larger magnetite-filled microgel cores, also affected the colloidal stability of the microgels in suspension as measured by their sedimentation velocity. Superparamagnetism was reported, with plateau values of the magnetization curve corresponding to 75 emu/g Fe 3 O 4 . The variation in the magnetic core size vs. magnetite-to-polymer content in the hybrid microgels was not investigated in this study.

Sulfonated Copolymer Beads 2.5.8.3

Polymer beads produced by the suspension copolymerization of styrene and a small amount of divinylbenzene were sulfonated to add complexing moieties to the polymer chains and facilitate the encapsulation of Fe 3+ and Co 2+ cations for the synthesis of MNPs in situ. 64 The presence of a CoFe 2 O 4 spinel phase was confirmed from its characteristic XRD peaks. TEM images obtained from ground powder displayed 7 nm, nearly spherical particles welldistributed within the polymer matrix. The absence of hysteresis, remanence and H c demonstrated the superparamagnetic character of these hybrids, the saturated magnetization being also low (0.818 emu/g) due to the low content of magnetic material in the beads.

Poly[poly(ethylene glycol diacrylate-co-acrylic acid) (PEGDA-co-PAA) 2.5.8.4 Copolymer Hydrogels Prepared by Microfluidics

Janus particles, combining two distinct materials on the opposite sides of the NP, were designed in the groups of Doyle and Hatton to work as selective nanoreactors for the in situ synthesis of magnetic species. 65 Figure 2-35 shows the Janus-like and other nonspherical hydrogel magnetic particles prepared by stop-flow lithography (SFL), with one part of PEG diacrylate (PEGDA) non-interacting with iron salts, and another part constituted of a copolymer of PEGDA and acrylic acid (AA) to coordinate the iron ions. Since the polymerization was initiated by a photosensitive radical initiator, the shape of the hydrogels could be varied at will (e.g., disks, triangles, etc.) by an appropriate mask shaping the UV beam illuminating the reactants in a Y-shaped laminar flow chamber (Figure 2-36). Then the transfer of the gels into an alkaline solution converted the COOH groups into COO -and induced swelling due to electrostatic repulsion between the anionic carboxylate groups.

Interestingly, the Fe 3+ /Fe 2+ feed ratio used (1:75) was much lower than the required 2:1 stoichiometric ratio, due to the different adsorption affinities of these two ions toward COO -.

The iron content of the particles was increased by repetition of the loading process, which allowed the adsorption of more ions at unoccupied nucleation sites and/or the surface of the magnetic particles, leading to larger, visibly darker and more magnetic particles. The hydrogel particles obtained after eight loading cycles reached a M s value of 42 emu/g. It is worth noting that the maximum magnetic domain diameter reported was 5.7 nm (but with a high σ of 0.3-0.4), which was considered to be limited by the swollen mesh size of the polymer network. This suggests partial size control for the MNPs synthesized in situ in such a hydrogel scaffold. The poly(VCL-co-AAEM-co-VIm) hybrid microgels were expected to exhibit thermosensitivity brought by the VCL units, pH responsiveness ascribed to the AAEM-rich core, and magnetic switchable properties owning to the encapsulated iron oxide particles.

Unfortunately, the concentration of VIm units in the copolymer was found to strongly influence the swelling behavior and the transition temperature of the hydrogels. Shrinkage of the hybrid particles was also observed at higher magnetite contents (which led to partial collapse of the network in some cases), in agreement with a previous study. 63 The magnetite particles had a diameter of ca. 10 nm determined from the TEM images (Figure 2-37). The highest M s measured was 30 emu/g for the highest loading obtained of 15.4 wt% magnetite.

Evidence for pH responsiveness and temperature-sensitive properties was shown for the hybrids, indicating the success of monomer blending to obtain copolymer microgels with combined characteristics and the creation of multifunctional materials.

Preformed Microspheres in Organic Solvents

Polystyrene (PS) Microspheres Swollen in CHCl 3 2.5.9.1 Interestingly, even water-insoluble cross-linked polystyrene (PS) microspheres were found to be capable of serving as hosts for the thermal decomposition of encapsulated ferric trioleate, after swelling of the microspheres in an organic solvent (CHCl 3 ) to facilitate the impregnation. 67 The magnetic character of the NPs thus obtained strongly depended on the decomposition temperature used, since the dissociation of the Fe(oleate) 3 complex only started around 200-240 °C but was completed at 300 °C, while crystal growth only took place above 310 °C and led to strong magnetism. TGA results showed that decomposition of the polymer matrix occurred between 380 and 450 °C, yielding an inorganic content of 15.9 wt% for the composite spheres. SEM and TEM images (Figure 2

Conclusions

In recent years, multiple routes have been explored for the synthesis of IONPs. This includes the alkaline coprecipitation of ferric and ferrous salts, the oxidation of FeSO 4 , the thermal decomposition of iron complexes, the polyol route, and hydrothermal processes in various organic templates, as summarized in Table 2-5. One feature common to all the systems explored so far is the ability of one of the components used to interact with the iron oxide precursor, most often through coordination with the iron ions. A number of the in situ syntheses did not give results significantly different from bulk synthesis methods, as these were realized under similar physicochemical conditions (pH, temperature, etc.). For instance, syntheses by alkaline coprecipitation leading to IONPs with rock-like shapes and a broad diameter distribution around 7 nm do not make a strong case for a true templating effect by the organic matrix. In contrast, several investigation listed in this review (in particular those using well-defined polymer structures) highlighted a significant influence of the polymer/precursor ratio on the outcome of the synthesis. This is the case, for instance, for tridimensional networks such as hydrogels, for which the importance of mesh size was recognized as a parameter limiting the diameter of the MNPs grown in situ. It was also demonstrated on several occasions that strong ligands (such as phosphate groups) directed the synthesis towards smaller particle sizes, which seems to be a general well-known trend in coordination chemistry that can be ascribed to hampering of ion diffusion through the ligand shell during the growth of nanocrystals. On the contrary, other studies reported MNP diameters that increased from 10 to 20 nm with the template. This is the case for chemical reactions performed at high temperatures, were an important property required from the organic template is to prevent the aggregation of the growing nanocrystals. This protecting effect was observed in particular in the last study reported, where a polystyrene matrix could withstand temperatures as high as 310 °C necessary for the thermal decomposition of iron oleate. hexane/Triton ® -X/Chitosan, CTAB Regarding shape control, several studies claimed success of the organic templating method to orient the synthesis towards rod-like NPs. However, caution should be used in that the physicochemical conditions used may themselves favor the precipitation of antiferromagnetic ferrihydrite phases such as goethite or akaganeite. A more convincing case of needle-like cobalt ferrite microparticle formation was reported for a microemulsion process, based on the oriented aggregation of nanocrystals. One interesting aspect that can be brought by polymer-templated synthesis is the ability to prepare NPs with highly magnetic properties (useful for MRI contrast or magnetic hyperthermia) directly covered by a polymer shell, providing colloidal stability and stealth properties in biological media. This was achieved in several investigations by employing either double-hydrophilic or amphiphilic copolymers, in particular those forming micelles with a dense core strongly binding to the precursor species and a shell with multiple hydrophilic arms providing steric repulsion.

In all these studies, both with macroscopic matrices and individually dispersed objects, loading of the inorganic precursor into the adsorption sites was critical and needed to be optimized (by an iterative process or by the careful removal of excess Fe 2+/3+ ions) to achieve a true templating effect.

Overview

This Chapter focuses on the preparation of water-dispersible polyion complex micelles from a G1 arborescent copolymer, G0 polystyrene-graft-poly(2-vinylpyridine) or G0PS-g-P2VP. Poly(acrylic acid)-block-poly(2-hydroxyethyl acrylate) (PAA-b-PHEA) double-hydrophilic block copolymers (DHBCs) were employed as polymeric stabilizers in this process, whereby electrostatic interactions between the PAA and P2VP segments were used to couple the block copolymer chains with the arborescent substrate while the PHEA tails provided a stabilizing shell. Four PAA-b-PHEA copolymers with tailored chain lengths were prepared by atom transfer radical polymerization (ATRP), by producing successively a poly(tert-butyl acrylate) macroinitiator and the corresponding poly(2-trimethylsilyloxyethyl acrylate) block copolymer, followed by deprotection of both blocks. The hydrodynamic radius, size distribution and zeta potential of the micelles obtained by the polyion complex (PIC) process were monitored by the dynamic light scattering (DLS) technique, whilst the absolute molar mass, radius of gyration and second virial coefficient of the micelles were extracted from multi-angle laser light scattering (MALLS) data. Tuning the length of the copolymer blocks and varying the amount used with respect to the arborescent substrate provided control over the size and the structure of the PIC micelles obtained, from large flocculated species to stable unimolecular PIC micelles with diameters ranging from 42 to 67 nm. Higher stability was observed when decreasing the length of the PAA block from 27 to 13 units at a constant PHEA block length of ca. 50 units, presumably due to increased packing density of the block copolymers at the surface of the PIC micelles. However stabilization was most effective when the PHEA chain length reached 150 and 260 units, even when using lesser amounts of stabilizer in comparison to the copolymers with shorter PHEA blocks. Changes in morphology of the arborescent copolymer before and after complexation were observed by atomic force microscopy (AFM), especially when varying the force applied with the AFM tip on the samples.

Introduction

Colloidal dispersions are ubiquitous in our everyday lives, from polymer latexes in paints and plastics, through agricultural emulsions and dispersions, to blood and other biological fluids. 1 In biomedical applications, colloidal stability is of critical importance to ensure the safety and efficacy of nanoscale products, 2 since particles larger than 5 μm can cause capillary blockage and embolism. 3 Modification of the surface of nanoparticles by selfassembly or complexation with a copolymer is a technique commonly applied to the delivery of DNA (gene therapy) [4][5][6][7] and oligonucleotides (anti-sense therapy), 4,6 dendrimer porphyrins (photodynamic therapy), 8-11 drugs [12][13] and other types of hydrophobic nanomaterials, [14][15][16] by protecting (shielding) specific functions within the micellar core. Recently reported approaches to bind copolymers onto the surface of particles and improve the stability of colloidal dispersions will be first considered. This will be followed by a discussion of the polyion complexation process, particularly for materials incorporating polyvinylpyridine (PVP), in both organic solvents and aqueous dispersions. Finally, the types of interactions imparting stability in the complexation process, and control over the complexation parameters in relation to the structure of the PIC micelles obtained will be addressed.

Colloidal Stability

Van der Waals (VDW) interactions are considered the main driving force inducing instability in particle dispersions. 1,[17][18] The magnitude of the attraction force (V A ) between two core-shell particles approaching each other (Figure 3-1) is given by 1,19 where A* is the effective Hamaker constant,  the radius of the core, and H  is the minimum distance of approach between the surface of the cores. Taking two sterically stabilized polystyrene (PS) latex particles (A* = 5×10 -21 J or 1.2 kT) as an example, the magnitude of the VDW forces calculated when the particles just touch (H  = 2) reflects the stability of the dispersion. If V A is larger than kT flocculation occurs despite the existence of the polymer coating, as seen for large cores stabilized by a low w polymer (Table 3-1). Increasing the w of the stabilizing polymer efficiently reduces the magnitude of the VDW forces relatively to kT and thus imparts stability to the dispersed phase. In our work, the particle core is an arborescent PS substrate grafted with P2VP chains (likewise mostly hydrophobic in their neutral state). Given the small diameter of the G1 substrate (below 50 nm), this calculation suggests that even a polymer with w = 2500 g/mol may suffice to stabilize this system (V A is just 0.5 kT and the layer thickness is slightly larger than the VDW effective radius of 5 nm). However a minimum value of w for stabilizers of 10,000 g/mol was suggested to ensure good colloidal stability. 1 There are limited options available to stabilize colloidal dispersions, which are mainly electrostatic stabilization and polymer (steric) stabilization. Electrostatic stabilization tends to be problematic since the thickness of the electrical double layers is very sensitive to the ionic strength and the dielectric constant of the dispersion medium. The thickness of the double layer typically exceeds the effective range of VDW attraction (5-10 nm) in water at low ionic strengths (I < 1 mM), but it shrinks to less than 1 nm in a 0.1 M ionic strength environment. 1 Electrostatic repulsions thus become insufficient to outweigh VDW attractions under these conditions. For that reason, polymeric stabilization (also referred to as steric stabilization), or a combination of steric and electrostatic (electrosteric) stabilization, have been more frequently employed to achieve the colloidal stability of dispersions in the literature and will be thus preferred in this work. Different strategies have been suggested to attach polymeric stabilizers to the surface of particles, namely physical adsorption, ionic bonding and covalent grafting. 14 Considering the composition of the arborescent PS-g-P2VP copolymer used in this investigation, whose 2VP units can be easily protonated, a polymer stabilizing shell bound through ionic interactions appears promising, as it offers stronger interactions than physical adsorption and is much more versatile than covalent bonding.

Polymeric Stabilization by the Polyion Complexation Process

Electrostatic interactions between two linear and oppositely charged polymers to form polyion complex micelles, first reported in the mid-1990s by Harada and Kataoka, 20 Kabanov et al., 21 and Cohen Stuart et al., 22 immediately drew considerable attention. The main driving force for polyion complexation comprises two contributions: Coulombic attractions, and an entropy gain through the release of counterions. [20][21][22][23][24][25][26] More precisely, complex formation is ascribed to a decrease in total electrostatic free energy, due to a change in translational entropy for the microions and the total electrostatic field energy. 25 Hydrophobic interactions and hydrogen bonding are sometimes considered as additional contributions to the PIC formation process. Steric constraints oppose complexation of the polyelectrolytes however, resulting in a lower degree of effective charge neutralization. 25 The complexation process has been investigated for different polymeric materials including PVP homopolymers and PVP-containing block copolymers.

PVP Homopolymers 3.2.2.1

Poly(2-vinylpyridine) (P2VP) and poly(4-vinylpyridine) (P4VP) homopolymers were complexed with poly(acrylic acid) (PAA) and poly(methacrylic acid) (PMAA) in ethanol/water (1:1) solutions. 27 The presence of hydrogen bonds between the PMAA and P2VP or P4VP chains was confirmed by Fourier transform-infrared (FT-IR) spectroscopy, based on the absence of pyridinium ions (protonated form) in the spectrum and the shift in the pyridine ring band. The acidic PAA formed complexes with both P2VP and P4VP through ionic interactions, as evidenced by the appearance of a ring stretching band for pyridinium ions at 1632 cm -1 in FT-IR spectroscopy, and also confirmed in X-ray photoelectron spectroscopy (XPS). When the PAA content in the complex was increased, a higher intensity of the pyridinium ion band and a shift in the pyridine ring band at 1595 cm -1 toward higher wavenumbers were reported. Since the complexes were dried and ground to fine powders, no information was reported on their hydrodynamic size. Hydrogen bonding was also cited as the driving force for the self-assembly of P4VP homopolymer with polystyrene-block-poly(acrylic acid) (PS-b-PAA) to micelles in ethanol. 28 It is interesting to note the influence of the complexation solvent on the nature of the driving force for the complexation between PAA and P4VP segments: Weak hydrogen bonding was observed in ethanol, while strong polyionic interactions were reported in ethanol/water (1:1) solutions.

Pergushov et al. 29 a corona of excess PMAA fragments not involved in polyion complexation was concluded on the basis of i) fluorescence spectroscopy measurements using pyrene as a polarity probe, and ii) small-angle neutron scattering (SANS) data showing an increase in size for the micellar nucleus, attributed to the penetration P4VP•EtBr into the core (R core increased from 3.4 nm to 6.3 nm, yielding a much higher scattering intensity and a larger slope at intermediate qvalues). The penetration of P4VP•EtBr into the core was explained by their preference to be localized in the vicinity of the PIB cores. "Dynamic" micelles were formed under these conditions rather than "frozen" micelles, the latter being usually obtained by micellization in organic solvents, followed by transfer to aqueous media either by heating or stepwise dialysis.

Polyion complexation in an aqueous environment was not as straightforward for the weak base P2VP as for quaternized P4VP•EtBr as described above, due to their difference in base strengths. Warnant et al. 30 thus investigated the physicochemical properties of pHsensitive PIC micelles derived from a P2VP homopolymer and a double-hydrophilic block copolymer (DHBC) of poly(acrylic acid)-b-poly(acrylate methoxy poly(ethylene oxide))

PAA-b-PAMPEO. It was suggested that the pH used for complexation was a crucial parameter influencing the micellization process, as it was necessary to ionize both weak polyelectrolytes before complexation. The selection of a hydrophilic block (PAMPEO) rather than a hydrophobic block (PIB) as described above 29 eliminated the complication of concurrent self-assembly of the amphiphilic copolymer before complexation. A core-corona morphology for the PIC micelles was suggested on the basis of i) fluorescence spectroscopy measurements using Auramine O as probe, and ii) variable density contrast experiments between the core and the corona in small-angle X-ray scattering experiments (SAXS). The hydrodynamic diameter of the PIC micelle was reported to be 36 nm, with a narrow size distribution (PDI = 0.28). The PIC micelles were unstable above pH 5 as the P2VP became insoluble, collapsed and dehydrated, leading to macroscopic precipitation.

PVP-Containing Block Copolymers 3.2.2.2

Unusual rod-like and buckle-like micelles were produced by the complexation of poly(2-vinylpyridine)-b-poly(ethylene oxide) (P2VP-b-PEO) with sodium poly(4styrenesulfonate) (PSS) homopolymer in water acidified to pH 5. 31 The formation of these different morphologies was assigned to stretching of the core-forming blocks (the P2VP and PSS segments in this case), the surface tension between the micelle core and the solvent outside the core, and to the corona-chain segment interactions. 32 The dissociation of the PIC micelles observed at elevated pH and their reassembly upon decreasing the pH was highlighted as an interesting feature of this system, for biomedical applications requiring pH responsiveness over a narrow range, e.g. from pH 5 to 7-8.

Attempts at using PAA homopolymers to complex with amphiphilic block copolymers including PS-b-P4VP in DMF 33 and poly(3-caprolactone)-block-poly(2-vinyl pyridine) (PCL-b-P2VP) in THF/ethanol 34 were also reported. Reasonably, in both cases micelle formation was ascribed to intermolecular hydrogen bonding between the PAA units and the P2VP segment of the block copolymers, as shown by FT-IR analysis. The size of the PIC micelles formed in DMF depended more on the length of the P4VP segment than the PAA segment. Small variations in hydrodynamic diameter were observed for the PIC micelles produced from PCL-b-P2VP in organic solutions when the amount of PAA was varied, suggesting that the complexes were saturated. 34 By changing the solvent, tuning the chain length and adjusting the stoichiometry ratio of the complex, the extent of hydrogen bonding could be controlled. Likewise using DMF as the complexing medium, Jérôme and 35 Hydrogen bonding between PAA and P2VP stabilized the micellar core, while the outer corona contained both PS and PEO chains. Since the hydrophilic PEO chains were relatively insoluble in DMF, they tended to cluster to avoid interactions with the hydrophobic PS blocks, which resulted in nanophase separation within the corona.

Layered core-shell-corona micellar complexes were formed by the complexation of preformed polystyrene-b-poly(acrylic acid) (PS-b-PAA) micelles and poly(ethylene glycol)block-poly(4-vinyl pyridine) (PEG-b-P4VP) in ethanol. 36 The complexation of PEG-b-P4VP preformed micelles having a P4VP corona resulted in shrinking of the micellar structures from 75.8 nm to 57.6 nm for a weight mixing ratio of 0.25. Assuming that there was no change in the size of the PS core (R c = 15.1 nm), which was kinetically frozen in ethanol at 25 °C, the shell thickness of the PIC micelles obtained ranged from 10.5 to 14.5 nm, depending on the weight ratio of the two complexing components.

Efforts were also made to control the PIC process in water by changing the complexation parameters. The formation of PIC micelles by mixing protonated poly(2vinylpyridine)-block-poly(ethylene oxide) (P2VP-b-PEO) with neutralized poly(methacrylic Chapter 3 90 acid)-block-poly(ethylene oxide) (PMAA-b-PEO) was thus achieved within a limited pH range (2 < pH < 6.1). This pH range was employed to avoid both the self-assembly of PMAA-b-PEO at acidic pH and of P2VP-b-PEO at pH > 6. 37 The fact that the PIC micelles dissociated at NaCl concentrations above 0.35 M due to electrostatic screening highlights the sensitivity of PIC micelles to ionic strength and, more importantly, it confirms the role of electrostatic interactions in the micellar structures.

Interesting phase behavior and micellization phenomena were also observed at various pH in the self-complexation of a poly(2-vinylpyridine)-b-poly(methyl methacrylate)b-poly(acrylic acid) block terpolymer (P2VP-b-PMMA-b-PAA) in aqueous dilute solutions. 38 At low pH (1-2) heteroarm star-like micelles were formed via interactions of the P2VP and PAA arms in the corona, wrapping the hydrophobic PMMA core. As the pH was increased from 2 to 3, electrostatic intermicellar attraction forces led to the formation of irregular aggregates. An insoluble two-phase region was observed between pH 3.2 and 5.8, i.e., around the isoelectric point of the ampholytic chains. Finally, under alkaline conditions, aggregated amphiphilic heteroarm star-like micelles were observed after deposition on mica substrates.

The penetration of poly(N-isopropylacrylamide-co-4-vinylpyridine) statistical copolymers (PNIPAM-co-P4VP) into rigid preformed core-shell microspheres of poly(styrene-co-methacrylic acid) (PS-co-PMAA), synthesized by a one-step surfactant-free emulsion polymerization technique, was also reported by Wang et al. 39 The complexation was performed in water, with PNIPAM-co-P4VP dissolved in an HCl solution, thus allowing electrostatic interactions to dominate as the driving force for self-assembly. The diameter of the resulting composite microspheres formed was slightly larger than that of the core-shell PS-co-PMAA microspheres, indicating the insertion of the copolymer of PNIPAM-co-P4VP into the PMAA shell layer of the core-shell microspheres.

A similar approach was used by Xiong et al. 40 to obtain a core-shell structure starting with the self-assembly of a PS-b-PAA block copolymer in ethanol, a good solvent for the PAA block but a non-solvent for the PS segment. Complexation of the preformed micelles with a poly(4-vinyl pyridine)-b-poly(N-isopropylacrylamide) (P4VP-b-PNIPAM) block copolymer was then achieved in DMF/ethanol. Dialysis of the PIC micelles against water at pH 4 and heating to 45 °C led to the collapse of the thermosensitive PNIPAM corona onto the interacting PAA/P4VP sites, thus leaving an excess of P4VP segments as the corona. It was argued that the reversible response of PNIPAM upon temperature and pH variations provided the flexibility needed for applications in drug delivery, as catalyst carriers, templates, and so on.

Summary Remarks and Approach for the Current Work 3.2.2.3

Most of the studies discussed above were performed with PVP homopolymers or block copolymers, using PAA or PMAA as the block interacting with the PVP segments.

Until now, to the best of our knowledge, no studies have been performed on the complexation of complex polymer architectures containing PVP segments. Organic solvents would allow the two components to be combined by hydrogen bonding, while aqueous environments at a suitable pH should give rise to electrostatic interactions. It is noteworthy that micelles formed in organic solvents remain frozen when transferred to aqueous environments, while direct complexation in aqueous solution tends to yield dynamic micelle structures, with additional electrostatic repulsions from excess ionic groups increasing the stability of the system. 28,38 Using an arborescent copolymer G0PS-g-P2VP as a platform for the complexation reaction, we expect the PAA block of the complexing copolymer to penetrate into the arborescent structure and interact with the 2VP units. Water was used as the solvent for complexation, since we wanted to create dynamic PIC micelle systems and to characterize them in their dispersed form before examining their structure in the dry state, as it was done in most investigations mentioned above. The purpose of the PIC process was to stabilize the G1 hydrophobic arborescent copolymer with the double-hydrophilic copolymer PAA-b-PHEA, to obtain micellar structures colloidally stable in water at pH 7, and most importantly biocompatible due to PHEA, a highly biocompatible material. [41][42][43][44][45] The PIC formation process relies upon the presence of electrostatic interactions between the PAA and P2VP segments in Chapter 3 92 water after suitable adjustment of the pH (Figure 3-2). These interactions are assumed to anchor the block copolymer chains onto the surface of the arborescent copolymer, while the PHEA tails provide a swollen shell increasing the separation distance between the particles to the extent that van der Waals attractions become negligible (> 5 nm, as observed for many colloidal systems). 1 Moreover, electrostatic stabilization due to repulsion between the negatively charged PIC micelles at neutral pH would also be expected to impart colloidal stability to the dispersion, at least at low ionic strengths. Changes in morphology of the arborescent polymer before and after complexation were furthermore revealed by atomic force microscopy (AFM), by varying the force applied with the AFM tip on the samples.

Experimental Procedures

Materials

Acetone

Hydrolysis of PtBA-b-PHEATMS

The hydrolysis of the protecting groups on the four PtBA-b-PHEATMS block copolymer samples was performed to produce the corresponding PAA-b-PHEA derivatives.

The procedure reported here was applied to P(tBA) 27

Polyion Complexation Process

Complexation of the G1 arborescent copolymer with various amounts of different PAA-b-PHEA block copolymers was carried out in aqueous solutions. Hereafter the parameter f = CO 2 H/N will be used to quantify the molar ratio between the CO 2 H groups (from PAA chains of the block copolymer) and N moieties (2VP units of the arborescent copolymer):

The following procedure describes the complexation of the G1 copolymer with

Characterization

Nuclear Magnetic Resonance Spectroscopy 3.3.7.1

The number-average degree of polymerization, the silylation of HEA, the composition of the polymers, and the deprotection reaction were monitored by nuclear magnetic resonance 1 H, 13 C and heteronuclear single quantum coherence (HSQC) spectroscopy analysis on a 400 MHz (100 MHz for the 13 The measurements were performed on a Zetasizer Nano ZS90 (Malvern Instruments) equipped with a 4 mW He-Ne laser operating at 633 nm and 25.0 °C, at a scattering angle of 90°. Each sample was measured in triplicate using disposable PS cuvettes or quartz cuvettes.

The samples were prepared at concentrations of 3-5 mg/mL in Milli-Q water (at 25 °C, refractive index 1.330, viscosity 0.8872 cP), and filtered through a nylon membrane with 0.45 μm nominal pore size. The light scattering data were analyzed with the Zetasizer 7.11 Software (Malvern Instruments). The intensity-weighted autocorrelation function g (2) () produced by the digital correlator was used to extract the particle size distributions. The second order cumulants analysis algorithm was applied for relatively monodisperse particles, while the non-negatively constrained least-squares method (NNLS) or CONTIN analysis were employed for polydisperse samples. The hydrodynamic radius of the particles was determined from the Stokes-Einstein equation with the diffusion coefficient (D T ) obtained from the decay rate (Γ) and the instrumental component .

Static Light Scattering 3.3.7.4

The static light scattering (SLS) measurements to determine the absolute weightaverage molar mass w , the second virial coefficient (A 2 ) and radius of gyration (R g ) were performed using an ALV/CGS-3 platform-based goniometer system equipped with a He-Ne For selected PIC systems, the refractive index increment (dn/dc) needed for the calculations was determined using a differential refractometer (Wyatt Technology) at 658 nm, by injecting a series of 5 micellar solutions at 0.2-1 mg/mL. The data were processed with the ASTRA 6 software. The linear correlation found between dn/dc vs. composition was used to interpolate the dn/dc values for the other PIC systems.

Zeta Potential Measurements 3.3.7.5

The electrical double layer surrounding a particle is composed of an inner region (called the Stern layer, where the ions are strongly bound) and an outer, diffuse region (where the ions are less firmly attached). A slipping plane is a notional boundary within the diffuse layer, inside which the ions move with the particle as it moves. The potential existing at this boundary is known as the Zeta potential. 48 Zeta potential (ZP) measurements were performed on a Zetasizer Nano ZS90 instrument (Malvern) equipped with a 4 mW He-Ne laser (633 nm) and at 25.0 °C. In this instrument the laser beam is split into a reference beam and an incident beam, before passing through the sample placed in a capillary cell. The scattered light detected at 17° is then combined with the reference beam to provide the rate of fluctuation, which is proportional to the velocity of the particles (electrophoretic mobility U E ). The zeta potential () is calculated from the electrophoretic mobility U E using the Henry equation 49 where  is the dielectric constant,  the viscosity, and ƒ(Ka) is Henry's function. This function was set at 1.5, as the system was fitted to the Smoluchowski model. 50 Atomic Force Microscopy 3.3.7.6

Freshly cleaved mica was used as sample substrate. The polymer solutions were prepared in Milli-Q water at concentrations ranging from 0.05 to 0.1 mg/mL. A 20 μL aliquot of PIC dispersion was deposited on the mica substrate and spin-coated at 3000 rpm for 180 s under ambient conditions. Atomic force microscopy (AFM) images were recorded in the tapping mode in air on a Veeco Dimension Icon System equipped with a Nanoscope V controller belonging to the NsySA group at ENSCBP headed by Pr Neso Sojic. The probes used were Olympus ® AC160TS-R3 micro cantilevers with a spring constant of 26 -56 N/m, a resonance frequency of 300 -399 kHz and a curvature radius of 8 -10 nm. The scan rate was typically between 0.7 and 1.2 Hz, at a scan angle of 0°, acquiring 512 samples/line. Image analysis was done using the Nanoscope Analysis V1.20 software from Bruker.

Results and Discussion

Synthesis of the Double-Hydrophilic Block Copolymers

The synthesis of the double-hydrophilic block copolymers (DHBC) PAA-b-PHEA was carried out in a multistep process involving monomer protection, atom transfer radical polymerization (ATRP), and deprotection steps. ATRP is a well-known controlled radical polymerization (CRP) technique allowing the synthesis of homopolymers and copolymers with narrow size distributions, in particular for polyacrylates. 47,[51][52][53] Low molar mass PtBA samples were first prepared in acetone at 60 °C, using methyl 2-bromopropropionate as initiator, CuBr as catalyst, and PMDETA as ligand as shown in Scheme 3-1. 47 The 1 H NMR spectrum of PtBA (Figure 3-3a) is dominated by the tert-butyl protons at δ 1.14 -1.65 ppm (d). The n value of the two homopolymers P(tBA) 13 and P(tBA) 27 was determined by integrating the methine protons in the repeat units (δ 2.01-2.34 ppm, a2) and Chapter 3 103 in the terminal unit (next to the bromine atom) at 4.00-4.19 ppm (a1). The peak area ratios for the methoxy proton (b) to (a1), and for the methyl group (e) to (a1) are both in a 3:1 ratio, which confirms the presence of a single ω-bromine group on every polymer chain and allows this material to work as macroinitiator in the next ATRP reaction to synthesize the desired block copolymers. In our work, the protected form of HEA was used as the monomer to increase the monomer conversion (otherwise limited to < 50 %) 54 and to achieve fast kinetics, while obtaining well-defined block copolymers with a low dispersity. 41,54 The protection of HEA with a trimethylsilyl group is depicted in Scheme 3-2, whilst the structure of the protected monomer was confirmed by 1 H NMR analysis (Section 3.3.3).

PIC Micelles in Water

The arborescent G0PS-g-P2VP copolymer is insoluble in aqueous media at neutral pH, while PAA-b-PHEA is soluble, but no micelles or aggregates are formed despite the fact that PAA is a weak polyelectrolyte. 55 At pH values below the pK a of PAA, more than 50% of the carboxyl functionalities of PAA exist in their hydrophobic form (COOH), 56 which may turn the double-hydrophilic block copolymer into amphiphilic species but also reduce the complexation efficiency. The pK a of PAA-based polyelectrolytes in solution in the absence of added salts was reported to fall within a narrow range from 5.7 to 6.5. [56][57][58][59] Based on the similarities in the polymer structures tested and our double-hydrophilic block copolymer, pK a = 6.5 56,59 was used in the theoretical calculations for this work. Such a decrease in acidity is usually noted for polyelectrolytes as compared to small molecule carboxylic acids. Water is a non-solvent for the PS segments, while P2VP is also a weak polyelectrolyte (pK a = 5) 30 with a lower basicity than its monomer (pK a = 5.6). 60 As previously mentioned, the presence of electrostatic interactions between the PAA and P2VP segments is expected to be vital to couple the BC chains with the G1 substrate. To maximize polyelectrolyte interactions, it is crucial to ionize of these components before the complexation, 30 by protonating the amine groups of P2VP (pK a = 5) to NH + in an acidic environment, and ionizing the COOH moieties of PAA (pK a = 6.5) to CO 2 -. Ideally, a pH range where both components can be mostly present in their ionic form is required to form stable electrostatic interactions between polycations and polyanions, 30,61 but this was not possible for the system investigated here (Figure 345). Complexation is unfavorable at extreme pH values, but pH 4.7 was selected such that ca. 67% of the 2VP moieties remained protonated, and 1.6% of the carboxylic groups stayed in their anionic form. To compensate for the low level of COOH ionization, a larger amount of COOH (from PAA-b-PHEA) was used as compared to N, i.e., a higher f ratio. The effective f* = CO 2 -/NH + ratios for various f at pH 4.7 are listed as supporting information (Table S3-1). It should be mentioned that intermolecular hydrogen bonding between neutral COOH groups in the PAA segments and neutral N groups in P2VP, as illustrated in Figure 3456, has been suggested by many authors. 34,40 This interaction is claimed to be the main driving force for complexations performed in organic solvents. [33][34][35]40,62 In aqueous environments at pH 4.7, the presence of multiple intermolecular hydrogen bonding interactions may also have assisted electrostatic interactions in the complexation process. However at pH 7 these hydrogen bonds should be perturbed due to increased ionization of the COOH groups, allowing electrostatic interactions to play a dominant role in the polyion complexation process. 61 It is noteworthy that complexation at pH 4.7 was carried out for 1 h before the addition of NaOH to increase the pH to 7, at which point ca. 76% of the COOH functionalities should exist in their ionic form while much less (< 1%) of the N moieties stay protonated, which results in the negatively charged PIC micelles obtained at every f Nanoparticles with a diameter below 200 nm are preferable for in vivo biomedical applications in terms of systemic circulation. [63][64][65][66][67] The PIC process was therefore optimized in terms of its ability to yield exclusively unimolecular micellar structures, with only one G1 arborescent copolymer forming the core. The intensity-weighted hydrodynamic diameter of the G1 copolymer at pH 4 was measured to be 29  4 nm (PDI = 0.04), much larger than the hypothetical hard-core diameter (D G1 = 14.6 nm calculated from the density of PS and P2VP and the molar mass of the G1 polymer) for a collapsed PS-P2VP hydrophobic sphere as expected at pH 7. This D G1 = 14.6 nm value is however in good agreement with i) a diameter of 15 nm derived from a similar calculation by Sheiko et al. 68 for an arborescent grafted polystyrene G0PS-g-PS with the same w ; ii) R g = 7.5 nm obtained from small-angle neutron scattering (SANS) measurements on G0PS-g-PS in d-cyclohexane and d-toluene, 69 and iii) R g = 7.3 nm estimated by fitting a core-shell model to the SANS data for an arborescent G0PS-g-P2VP copolymer in d-methanol. 70 The coiled dimensions of the PHEA segment in the different block copolymers, ranging from 7.1 to 12.2 nm, suggests that the absolute maximum diameter of unimolecular PIC micelles (derived from a single G1 molecule) D max should be in a range of ca. 29-40 nm. These should also have a narrow, unimodal size distribution, in analogy to the G1 copolymer substrate.

G1@PAA 27 -b-PHEA 56

The first polymeric stabilizer investigated had n = 8,090 g/mol, close to n  10,000 g/mol suggested to achieve good colloidal stability (Section 3.2.1). That polymer may thus impart sufficient steric stabilization to the system even without the additional contribution from electrostatic repulsion. The coiled dimensions of the PHEA block l PHEA was calculated to be 7.3 nm, which suggests a maximum diameter for unimolecular PIC micelles of ca. 29 nm. As shown by the charge equivalence calculation provided in Table S3-1, this DHBC may stabilize the G1 copolymer, but only for f > 1. It should be mentioned that for f = 1, the G1 and BC molecules agglomerated to form 1.6 µm diameter particles, confirming the inability of the BC chains to form a dense-enough polymeric shell to stabilize the G1 molecules. The very negative zeta potential of the aggregates (-29 mV) also confirms the excess of CO 2 -at pH 7 in the aggregates, as predicted previously. At this ratio, even a combination of electrostatic and steric stabilization was insufficient to prevent flocculation of the G1 substrate. The arborescent polymers assembled into larger particles to reduce their surface area, until the protecting shell composed of the limited amount of BC chains and the negative charge from CO 2 -groups sufficed to stabilize the system.

For f = 3, 6 and 9, stable PIC micelle dispersions were obtained with values of 80, 61 and 44 nm, respectively, closer to the size of the collapsed G1 core. Increasing the f ratio provided more BC chains to the stabilizing shell and increased the CO 2 -density surrounding the G1 molecules, leading to larger electrosteric repulsions, as well as smaller and more stable particles due to their enhanced colloidal stability. However even for the highest ratio (f = 9), the micelles do not seem to be unimolecular since the = 44 nm is much larger than the maximum calculated diameter D max  29 nm. This multimolecular hypothesis is confirmed by the larger thickness  15 nm as compared to l PHEA  7.3 nm. Due to its lowest diameter observed among the PIC micelles tested, f = 9 was selected as the optimal ratio for this system.

G1@PAA 13 -b-PHEA 50

This DHBC, designed with a PAA chain length half as large as the previous one whilst maintaining a comparable length for the PHEA block, would be expected to provide better stabilization for the G1 substrate. Indeed when PAA 27 -b-PHEA 56 and PAA 13 -b-PHEA 50 are used at the same f ratio, the latter corresponds to twice as many BC chains for the same number of COOH functionalities. Although this polymer has n = 5,580 g/mol, lower than PAA 27 -b-PHEA 56 and the 10,000 g/mol limit (Section 3.2.1), it could perform comparably to the previous polymer. The coiled dimensions of the PHEA 50 block was calculated to be l PHEA  7.1 nm, which suggests that the maximum diameter of unimolecular PIC micelles would be ca. 29 nm.

For f = 3 the of the PIC micelles was 75 nm, i.e., as large as for PIC micelles with PAA 27 -b-PHEA 56 at the same f, whilst the size distribution was much narrower (PDI = 0.07).

A decrease in number-weighted PIC micelle diameter (to 45 and 36 nm) was also observed for f = 6 and f = 9, respectively, lower than for PIC micelles derived from PAA 27 -b-PHEA 56 .

This indicates that the particles are better shielded by a higher density of stabilizing segments. The contribution from electrostatic repulsion to PIC micelle stability may also be more important in this case, due to reduced steric repulsion caused by shortening of the BC chains.

The optimal composition of this system was determined to be for f = 9, at which point 36 nm diameter PIC micelles with a very narrow size distribution (PDI = 0.07) were obtained. This is still larger than the maximum diameter D max  29 nm and the thickness > l PHEA , which suggests that these micelles are not unimolecular. In spite of the lower molar mass of PAA 13 -b-PHEA 50 as compared to PAA 27 -b-PHEA 56 , the DHBC with a shorter PAA segment produced smaller but still stable PIC micelles. The optimal f = 9 noted is still relatively high however. To provide better protection, a DHBC with the same PAA chain length but a longer PHEA block, PAA 13 -b-PHEA 150 , was designed and tested as stabilizer.

G1@PAA 13 -b-PHEA 150

This block copolymer, with n = 18,300 g/mol and a PHEA chain three times larger than PAA 13 Upon reducing the f ratio further to 0.5, 0.25 and 0.125, the decreased density of BC chains in the PIC assemblies led to flocculation. The fact that the optimal f for this DHBC is f = 1 stills confirms the benefits of using longer PHEA chains as stabilizer. The influence of steric stabilization imparted by longer BC chains is also evident for f = 0.5. Indeed, the PIC micelles G1@PAA 27 -b-PHEA 260 f = 0.5 had = 26 nm, fairly close to the size of the collapsed G1 core (D G1  14.6 nm,  6 nm), and a narrow size distribution (PDI = 0.08) confirms their unimolecular structure. The G1@PAA 27 -b-PHEA 260 f = 0.5 PIC micelles are the smallest colloidally stable micelles obtained. The ca. 6 nm shell thickness gives a closest distance of approach between two cores of 12 nm, still exceeding the 5 nm minimum effective distance 1 and further confirming the superior stabilization ability of this BC. Further reductions in the f ratio led to fewer BC chains per micelle. For f = 0.25 and 0.125, the BC content was insufficient for stabilization of the G1 copolymer in its unimolecular form, which led to the formation of larger, multimolecular species.

G1@PAA

The most efficient BC tested for the preparation of unimolecular micelles was therefore PAA 27 -b-PHEA 260 , as it maintained a sufficient thickness for the protecting shell while allowing a sufficient density of BC chains to cover the G1 substrate. For these polymers, the optimal f was about 0.5-1.

Determination of Refractive Index Increment 3.4.2.2

Further investigations using static light scattering measurements were performed to confirm the unimolecular character of the PIC structures. To determine the molar mass of polymers accurately by light scattering, the refractive index increment (dn/dc) of the samples must be determined. The refractive index increment of three of the PIC micelle dispersions was thus determined at 25 °C and  = 658 nm, i.e., the same conditions used in the static light scattering measurements. Among the PIC micelles obtained, the ones incorporating the long chain BC were of particular interest due to their good stability and uniform size. We also selected these three systems due to their smaller size, to minimize intraparticle scattering effects and to avoid sedimentation of the particles. Moreover, the concentration of the dispersions was maintained in a low range (0.2-1.0 mg/mL) for the analysis. The refractive index increment of the three selected PIC micelle dispersions was determined by plotting the refractive index of each dispersion vs. concentration. The dn/dc values, corresponding to the slope of the graphs, are listed in Table 3- The dn/dc values for the PIC micelles display consistent trends, with a magnitude increasing with the weight fraction of the higher refractive index components (PS and P2VP) as expected. The correlation coefficients were higher than 0.9999 in all cases. The linear correlation found between dn/dc vs. composition (Figure 3-10) was used to interpolate the dn/dc values for the other PIC systems (Table 3-3. bottom).

Uni-vs. Multimolecular Micelles

The unimolecular character of PIC micelles prepared from the BC with long PHEA segments, G1@PAA 13 -b-PHEA 150 with f = 3 and 1, and G1@PAA 27 -b-PHEA 260 with f = 0.5 was previously concluded from the DLS measurements. This is also confirmed by the SLS data since w(Exp) < 2× w(G1) with w(G1) = 1.1×10 6 g/mol. This is probably also true for G1@PAA 27 -b-PHEA 260 f = 3 and f = 1, for which w(Exp) < 3× w(G1) and unimolecular character was likewise concluded from the DLS measurements when comparing the of the arborescent substrate and the PIC micelles. This assumption allows the calculation of the The w(Exp) reported for PIC micelles G1@PAA 13 -b-PHEA 150 with f = 0.5 and 0.25, and G1@PAA 27 -b-PHEA 260 with f = 0.25 being larger than w(Theor.) , the G1 cores clearly flocculated due to the lack of stabilizer within the PIC shell. Contrary to our expectations, the G1@PAA 13 -b-PHEA 50 and G1@PAA 27 -b-PHEA 56 systems with f = 9, thought to be multimolecular on the basis of the DLS measurements, were found to have w(Exp) < 2× w(G1) . The expected structure of these two assemblies will be further discussed on the basis of their -parameter below.

-Parameter

The ratio R g /R h , also known as the -parameter, is structure-sensitive and may provide information on the morphology of PIC micelles. For example,  = 0.775 is expected for spheres of uniform density 71 (also called rigid spheres), while  = 1.7-2.0 is typically observed for randomly coiled polymer chains. [72][73] The situation can be more complicated when the density of the molecules or particles is highly heterogeneous however, e.g., for particles combining a dense core and partly coiled, less dense chain segments forming a shell. Due to the small radius of gyration of these systems resulting from concentration of the mass within their core,  values below the rigid sphere "limit" are often observed.

Furthermore, values close to 0.775 do not necessarily reveal a rigid sphere morphology for inhomogeneous systems (as is customarily assumed for homogenous materials), but it more Chapter 3 126 likely reflects a core-shell structure with non-homogenous density. The -parameter remains a useful tool to obtain information on particle morphology, but it must be used with caution and one must also consider the composition of the material and its behavior under the conditions used for its characterization, as illustrated in the following examples.

1.  Below the Rigid Sphere Limit Spherical PS microgels synthesized by microemulsion polymerization with crosslinking within the restricted micellar volume were reported to have 0.58 <  < 0.68. 74 The solvent used for the measurements was toluene, a good solvent for linear and lightly crosslinked PS, but a bad solvent for a rigid, highly cross-linked PS. The low -parameter values found for both lightly (0.58 <  < 0.61) and moderately cross-linked microgels (0.61 <  < 0.68) were attributed to the different swelling behaviors of the outer shell and the core. This phenomenon decreased the mass concentration of material in the shell relatively to the core.

The result  = 0.741 was also obtained for highly cross-linked microgels, that acted essentially like spheres of homogenous density (R g = 58, R h = 78 nm).

Micelles formed by the self-assembly of polystyrene-block-poly(methacrylic acid)

PS-b-PMAA and of triblock PMAA-b-PS-b-PMAA copolymers were reported to have  values ranging from 0.37 to 1.23, when characterized in a dioxane : water 80:20 v/v solvent mixture. 75 Values between 0.37 and 0.6 were assigned for structures with polystyrene segregated within the micellar core, surrounded by less dense PMAA segments. More interestingly,  ~ 0.9 was explained by the presence of micellar clusters corresponding to two micelles touching each other to form a dumbbell. Similar values were obtained for other small clusters, while longer linear (pearl necklace-like) clusters yielded much larger values. 75

 Above the Rigid Sphere Limit

It is also possible for a core-shell structure to have  > 0.775, as reported for CdS@PS-b-PMMA nanoparticles with  = 1.1-1.3. 76 In toluene, the quantum dots were covered by a shell of both swollen PMMA and PS chain segments (R g = 41 nm, R h = 31 nm,  = 1.3). Collapse of the PS chains onto the particles was observed when replacing toluene with acetone however, which gave rise to a dense core and partly coiled PMMA chain segments yielding a smaller -value (R g = 24 nm, R h = 21 nm,  = 1.1). The shift in mass concentration to the core was concluded to be the main factor responsible for the smaller parameter.

-Parameter for the PIC Micelles

A collapsed core-loose BC stabilizing shell morphology was postulated earlier for the arborescent PIC micelles, so these assemblies may fall in the second category above, depending on the relative masses of the G1 core and the BC shell components. For that reason, the trends in  observed among the different samples will be discussed separately below.

For the PIC micelles G1@PAA 27 -b-PHEA 56 with f = 9, the value  = 0.80 obtained could suggest a structure behaving similarly to a rigid sphere. However the unexpectedly large average hydrodynamic diameter ( = 44 nm) as compared to the calculated maximum diameter for unimolecular PIC micelles (D max = D G1 + 2l PHEA  29 nm) suggests an aggregation pattern corresponding to a mixture of unimolecular and multimolecular species. The average w(Exp) of these species being larger than w(G1) but less than 2× w(G1) , this most likely results from a dominant population of unimolecular species in comparison to the other species. The situation could be similar for G1@PAA 13 -b-PHEA 50 with f = 9, with  = 1.89 and a slightly larger w(Exp) = 1.4×10 6 g/mol (as compared to w(Exp) = 1.3×10 6 g/mol in the previous case) corresponding to more extensive aggregation.

It was previously concluded that PIC micelles G1@PAA 13 -b-PHEA 150 with f = 3 and 1 were unimolecular. These micelles have  = 1.43 and 1.20, respectively, consistent with a core-shell structure (below the 1.7-2.0 range expected for randomly coiled chains). These values are still well above the rigid sphere value however, which evidences the large contribution from the mass of swollen BC chains in the shell surrounding the collapsed arborescent core. Among these two samples, the protecting shell was likely less dense for the Chapter 3 128 G1@PAA 13 -b-PHEA 150 micelles with f = 1 than for f = 3, as evidenced by their somewhat lower w(Exp) . This also led to smaller R g (27.7 vs. 35.8 nm) and  (1.20 vs. 1.43) for f = 1, in agreement with our interpretation of the results.

The aggregates produced by PIC micelles G1@PAA 13 -b-PHEA 150 with f = 0.5 had R g = 49.6 nm and  = 0.95, suggesting the formation of a dense core from multiple G1 molecules, as indicated by the large w(Exp) = 3.9×10 7 g/mol obtained, surrounded by a loose shell of BC chains. This seems a normal consequence of decreasing the "feed" amount of BC in the self-assembly process. However this lower -parameter value could also be due to the presence of small clusters composed of many PIC micelles, as shown in the work of Qin et al. 75 On the other hand, the  = 1.47 value obtained for the large aggregates of G1@PAA 13 -b-PHEA 150 micelles with f = 0.25, apart from incorporating an even larger number of G1 molecules, more likely correspond to linear cluster structures, given that  is significantly larger than in the previous case.

Among the R g values obtained for the G1@PAA 27 -b-PHEA 260 system, only the values for f = 3 and 0.25 are considered reliable since the other two samples had R g < /20  15 nm, i.e., dimensions in a range leading to large errors. On that basis,  = 0.76 obtained for the PIC micelles with f = 3 is considered consistent with a core-shell morphology. It is tempting to come to the same conclusion for f = 1 and 0.5, in spite of the large errors expected on the R g values. Furthermore, the unimolecular PIC micelles in the G1@PAA 27 -b-PHEA 260 series with f = 3, 1 and 0.5 exhibit a larger -value for f = 3. This could be due to a more extended conformation for the BC chains in the shell of the f = 3 sample, resulting from increased steric crowding in this sample which has the highest molar mass among all the unimolecular samples ( w(Exp) = 3.2×10 6 g/mol).

The aggregates produced by the PIC micelles G1@PAA 27 -b-PHEA 260 with f = 0.25 were characterized by  = 0.40, indicating the formation of a dense collapsed core with a relatively small number of G1 molecules ( w(Exp) = 9.7×10 6 g/mol). This is presumably due to the low colloidal stability achieved for f = 0.25. Chapter 3 129

Second Virial Coefficient A 2

The second virial coefficient (A 2 ) of polymers in solution obtained from SLS data is a measure of solvent quality. Its magnitude depends on the solvent-polymer interactions, as well as the molar mass of the polymer and its architecture through scaling relationships of the type A 2  w a . 72 The second virial coefficient is thus a weakly decreasing function of the polymer molar mass as A 2  w -0.2 for linear polymers, 77 but this molar mass dependence is more pronounced for star-branched structures. 72 For highly branched polymers displaying rigid sphere behavior such as the arborescent polymers, the scaling relationship reaches A 2  w -1 . 78 Since structural variations are fairly small among the different PIC micelles obtained, the A 2 values should reflect mainly solvent-polymer interactions. All the measured A 2 values are positive, reflecting favorable interactions between the -OH groups on the PHEA segments of the PIC micelles with the solvent (water). However, the range of values observed (from 0.2×10 -5 to 19.3×10 -5 cm 3 molg -2 ) is indicative of variations in the quality of these solvent-polymer interactions as follows:

1. Values in the high end were observed mostly for systems with f > 1, regardless of the specific BC used, thus reflecting the importance of interactions between the -OH groups on the PHEA chains and water.

2. A decrease in A 2 values was observed for lower f ratios among the series of PIC micelles derived from the same BC type. This is also consistent with the first statement.

3. Comparisons among the PIC micelles G1@PAA 13 -b-PHEA 150 and G1@PAA 27 -b-PHEA 260 at the same f ratio produce the same result: The A 2 values for the former are smaller than for the latter. This is again related to differences in the number of -OH groups per PIC micelle. Taking f = 3 as an example, the G1@PAA 13 -b-PHEA 150 micelle had ca. 5,000 -OH groups provided by 33 BC side chains; G1@PAA 27 -b-PHEA 260 carried ca. 17,500 -OH groups on 68 BC side chains.

The lowest A 2 values are observed for the aggregated systems as expected: The largest PIC micelle G1@PAA 13 -b-PHEA 150 with f = 0.25 ( w = 170×10 6 g/mol) had A 2 = 0.2×10 -5 Chapter 3 130 and so on. There is no obvious correlation between A 2 and the w of the PIC micelles for the aggregated systems however, possibly because the PIC micelles have a heterogeneous chemical composition: the hydrophobic G1 and the hydrophilic PAA-PHEA components.

Considering the dominating contribution from -OH/water interactions to A 2 , a correlation was found between the number of -OH groups in the molecules and the A 2 values for nonaggregated systems with f > 1 (Figure 3-14).

Atomic Force Microscopy 3.4.2.5

The size, size distribution, morphology and morphology of the G1 substrate and the PIC micelles were investigated by atomic force microscopy (AFM) imaging in the tapping mode. The samples were prepared by spin coating deposition of the materials onto freshly It is noteworthy that the PIC micelles obtained after complexation appeared as uniformly distributed, almost spherical particles with a narrow size distribution on the mica surface. The spherical morphology of the PIC micelles, shown in the 3D images and in section analysis of the images (Figure 3-17, column 1), are attributed in part to electrostatic repulsions between the PIC micelles and mica, both negatively charged. In this case the particle diameter of the solid PIC micelles determined from section analysis was 27 nm, which was smaller than the = 42 nm measured in the aqueous dispersions, and the height of particles was ca. 2.3 nm. Phase imaging in the tapping mode can reveal nanometer-scale information on surface structure and properties often not detected by other microscopy techniques. [79][80][81] The phase lag (or phase shift) of the cantilever oscillations with respect to the applied force is sensitive to variations in composition, adhesion, friction, viscoelasticity, as well as other factors. [82][83][84][85] The dark domains of phase images have been assigned to "soft" materials in several reports, 83,[86][87] whilst the bright regions were ascribed to stiffer phases. The surface of PIC micelles changed dramatically in comparison to the G1 substrate, from a "stiff" G0PS-g-P2VP matrix appearing in bright yellow color to a "soft" coating layer. This could be due to plasticization by water absorbed in the hydrophilic PHEA phase (indicated by black particles on the phase images). The size, size distribution and uniformity of the PIC particles provides strong evidence for the success of the polyion complexation process using DHBC PAA 27 -b-PHEA 260 , providing PIC micelles with good hydrophilicity and colloidal stability.

In the tapping mode, the cantilever is excited by the piezoelectric driver to lightly tap the surface. Feedback from the oscillation amplitude is used to both measure topographic variations in the sample, and to monitor and minimize amplitude variations. 80 The tapping force applied during scanning is related to the ratio of the set-point amplitude to the freeoscillation amplitude, usually defined as the set-point ratio. 80 For set-point ratios close to but less than 1, a low force is applied on the surface and the tip just lightly taps the surface whilst at smaller set-point ratios, the AFM tip interacts more strongly with the sample. 83 By tuning the amplitude set-point and thus varying the interaction forces between the AFM tip and the particles, the morphology of PIC micelle can be probed.

The G1@PAA 27 -b-PHEA 260 PIC micelles (Figure 3-17) appear as spherical particles surrounded by a layer of "soft" PHEA (in dark color) at a set-point ratio 0.96 (column 1). A stronger force applied at a set-point ratio of 0.92 (column 2) reveals the "hard" PS core (bright color) inside each PIC micelle, with a surrounding softer ring (dark color), thus confirming the core-shell morphology of the micelles. This structure was further confirmed by the "mountain-like" morphology of PIC micelles (shown in the 3D models and in the section analyses) when the set-point ratio was further decreased to 0.86 (column 3), at which point the PIC micelles appeared in a "sunny side up" egg-like morphology on the height AFM images. Only the rigid PS component could be detected in these phase images. The morphology of aggregates of G1@PAA 27 -b-PHEA 260 for f = 0.25, and of G1@PAA 13 -b-PHEA 150 for f = 0.5 and 0.25 is also shown in Figure S3-2 in the supporting information. It may thus be concluded beyond any doubt that AFM analysis confirmed the formation of a DHBC stabilizing layer on the G1 arborescent substrate, and of unimolecular micellar species as deduced from the DLS and MALLS measurements.

Conclusions

The synthesis of four PAA-b-PHEA block copolymers was achieved by ATRP, followed by hydrolysis of the protecting groups on both blocks. These copolymers were employed as polymeric stabilizers for a G1 (G0PS-g-P2VP) arborescent copolymer through the formation of polyion complexes in water. This is the first report on the complexation of PAA-b-PHEA segments with preformed P2VP-containing nanostructures in water to produce unimolecular PIC micelle species. The electrostatic interactions between PAA and P2VP segments enabled coupling of the block copolymer chains on the surface of the arborescent polymer, while the PHEA tails provided a stabilizing shell. Control over the size and structure of the PIC micelles, from large flocculated species to stable unimolecular entities with sizes ranging from 42 to 67 nm, was accomplished by controlling the density of the polymer stabilizing layer around the G1 core, either by varying the BC stoichiometry or the length of the PAA anchoring block, and most notably by varying the length of the nonionic PHEA block. Short PHEA tails were insufficiently bulky to impart good colloidal stability to the PIC micelles, and stable dispersions with low degrees of aggregation were only achieved at high BC contents (f ratios) in the complexation process. Slightly better stability was achieved by decreasing the PAA block length from 27 to 13 units at constant length of the PHEA block, which led to increased coverage by the block copolymers on the surface of the micelles. However most effective stabilization was achieved when the length of the nonionic segments was increased to 260 units, imparting good colloidal stability to the PIC micelles Chapter 3 136 even when a complexation ratio f = 0.5 was used. The hydrodynamic diameter of the micelles, when compared with the dimensions calculated for the collapsed core, provided evidence for uni-vs. multimolecular structure formation.

The MALLS measurements provided additional information on the structure of PIC micelles: The w and -parameter values hinted at uni-vs. multimolecular structures formation, while the second virial coefficient A 2 highlighted the dominating role of -OH/water interactions. The soft shell of PHEA surrounding the stiff G1 core was observed in AFM images, confirming the success of the complexation process in producing unimolecular micelles. Most notably, the core-shell morphology of PIC micelles was confirmed through variations in the AFM amplitude set-point. The PIC process using double-hydrophilic block copolymer to stabilize hydrophobic G1 was thus optimized, to be applied to the templated preparation of iron oxide nanoparticles, which will be discussed in detail in Chapter 4.

Chapter 4 Templating and Stabilizing

Magnetic Nanoparticles

Overview

Vinylpyridine-containing polymers have been employed extensively as templates in the preparation of iron oxide magnetic nanoparticles (MNPs). Polymer templates may also be combined with stabilizing and biocompatible materials to enhance cellular uptake, while reducing cytotoxicity, once the MNPs are exposed to physiological conditions. We prepared a substrate combining templating and stabilizing effects for the MNP synthesis, which is a polyion complex (PIC) micelle derived from a G1 arborescent copolymer, G0 polystyrenegraft-poly(2-vinylpyridine), and a poly(acrylic acid)-block-poly(2-hydroxyethyl acrylate) copolymer, PAA-b-PHEA, forming a hydrophilic shell. As it turns out, the direct in situ application of these PIC micelles as templates in MNP synthesis was of limited usefulness since the MNPs formed had weak magnetization and large sizes unsuitable for biomedical

applications. An alternative approach to this was to first use the G1 copolymer as a template for the synthesis of Fe

Introduction

Due to their small size and large surface-to-volume ratio, nanoparticles (NPs) display different physical and chemical properties as compared to their bulk counterparts since a significant fraction of their atoms are located at the surface. 1-2 Some NPs combine optical, catalytic, electronic and magnetic properties making them attractive for applications in various fields from optics, energy and electronics, to chemistry and biology. [1][2] Among the broad spectrum of nanomaterials being investigated for biomedical uses, magnetic nanoparticles (MNPs) have received significant attention for uses in targeted drug delivery, as magnetic resonance imaging (MRI) contrast agents, and as heat mediators in the magnetic hyperthermia treatment of tumors. [3][4][5] Besides metallic and bimetallic MNPs, superparamagnetic iron oxide nanoparticles (SPIONs) have been favored because of their low cytotoxicity profile [6][7] and the possibility of surface modification with biocompatible coatings. 3,8-9 Among numerous chemical techniques by which SPIONs can be synthesized, the ferric/ferrous ions alkaline coprecipitation method has been preferred as the simplest and most efficient route. 9

Coprecipitation Method

Introduced in 1981 by Massart 10 in Europe and by Molday and Mackenzie 11 in the USA, the coprecipitation method is probably the most efficient and reproducible, yet simplest route to prepare magnetic particles, and is still the method of choice in the industry to produce large quantities of superparamagnetic MRI contrast agents. 12 A stoichiometric mixture of ferric and ferrous salts (Fe 3+ /Fe 2+ 2:1) in aqueous media precipitates upon addition of a strong base (NH 4 OH or NaOH) in a non-oxidizing environment to yield a finely dispersed magnetic iron oxide solid phase 13 (Scheme 4-1a). Complete precipitation is expected for a molar base-to-total-iron ratio greater than the stoichiometric value R = 8/3  2.67, leading to a final pH between 9 and 14 depending on the excess of base present.

Magnetite NPs produced by the coprecipitation route typically have magnetic saturation

Chapter 4 140 values in the range of 30-60 emug -1 , lower than the value for bulk magnetite (92 emug -1 ).

Oxidation of the air-sensitive Fe 3 O 4 to -Fe 2 O 3 occurs easily in the presence of oxygen under acidic conditions, through desorption of Fe 2+ ions from the surface into solution as

[Fe(H 2 O) 6 ] 2+ (Scheme 4-1b), or by direct oxidation of superficial ferrous ions under basic condition. 9 In the magnetite crystal structure (called "inverse spinel"), the iron ions are distributed within the octahedral (Oh) and tetrahedral (Td) sites of a face-centered cubic (fcc) stacked oxygen mesh according to ([Fe 3+ ] Td [Fe 3+ Fe 2+ ] Oh O 4 ) 14 (Figure 4-1). Fast electron hopping between Fe 2+ and Fe 3+ in the octahedral sublattice, evidenced by Mossbauer spectroscopy, plays an important role in the crystallization of the spinel structure and facilitates the transformation of this inverse spinel structure to a non-stoichiometric spinel -

Fe 2 O 3 , [Fe 3+ ] Td [Fe 3+ 5/3 V 1/3 ] Oh O 4
, where V stands for a cationic vacancy 14 (Figure 4-1).

Scheme 4-1. Alkaline coprecipitation and oxidation reactions.

The main disadvantage of alkaline coprecipitation is the lack of control over the particle size distribution since the size of the crystals is mainly determined by nucleation, followed by rapid crystal growth. In the nucleation step, the collision of iron ions or molecules in solution leads to the formation of stable "embryonic" nuclei. These nuclei are involved in a breakup and reforming process until their size exceeds a critical limit at which the particles start to grow. 15 Crystal growth occurs either through the diffusion and adsorption of the solute on the surface of the crystals, or by the oriented aggregation of nuclei. [16][17][18][19] The growth kinetics is determined by the lowest rate between mass transport and surface equilibration. The driving force for dissolution increases as the size decreases, thus facilitating the dissolution of small particles and their re-deposition onto larger particles (Ostwald ripening). 19 Chapter 4

141 Since nucleation and crystal growth occur simultaneously, particles obtained by the coprecipitation route are polydisperse in sizes. Decoupling of the nucleation process from crystal growth is mandatory for the formation of iron oxide nanoparticles of uniform size. 20 Ideally, if the nucleation process can be controlled in a way such that all the nuclei are created at the same time, subsequent slow crystal growth from these nuclei is expected to produce particles with a very narrow size distribution. Jolivet et al. 21 is a single Fe 3 O 4 phase produced in the coprecipitation process. 21 The first superparamagnetic iron oxide particles (with an average diameter of 8 nm, as measured by XRD) were produced by the alkaline coprecipitation of FeCl 3 and FeCl 2 by Massart. 13 Tailoring the size, size distribution, and thus the magnetic characteristics of the iron oxide NPs has been attempted in multiple investigations by adjusting the pH, temperature and ionic strength of the reaction medium, or by changing the Fe 2+ /Fe 3+ ratio, the medium composition, the order of addition of the reactants, the rate of mixing, or the nature of the base used. Higher pH values (facilitating the dissolution-crystallization process) and low ionic strengths (corresponding to low Cl -concentrations) resulted in smaller NPs with narrower size distributions. 14,22 Elevated temperatures led to the formation of more ordered crystals but a decrease in magnetite content. 23 The ferrous-to-ferric ratio had a strong impact on the mean size, and thus on the properties of iron oxide NPs: higher Fe 2+ /Fe 3+ ratios yielded larger NPs, [23][24] but only particles formed in the ratios range from 0.4 to 0.6 were Chapter 4 142 capable of enhancing the contrast in magnetic resonance imaging (MRI). 23,25 Interestingly, maghemite (-Fe 2 O 3 ) obtained for a Fe 2+ /Fe 3+ ratio of 0.118 in the preparation step (with dextran coating) exhibited higher genotoxicity than magnetite (Fe 3 O 4 ) prepared for a ratio of 0.435 (also dextran-coated), indicating that Fe 3+ ions are more potent at inducing DNA damage. 26 However, adjusting the reaction conditions as discussed above is not likely to provide significant improvements in size dispersity as compared to the unmodified coprecipitation method. A solution to this issue must be searched elsewhere. The addition of organic complexing agents during coprecipitation can assist in the synthesis of more uniform NPs. The chelation of metal ions by organic agents can hinder nucleation, thus limiting the number of nuclei formed and leading to fewer but larger particles since the system is dominated by crystal growth. However if the chelation of metal Chapter 4 143 ions by organic agents inhibits crystal growth, the formation of small NPs is favored. For example, the presence of citrate anions in various molar ratios resulted in 2-8 nm diameter -Fe 2 O 3 NPs. 27 Among numerous methods developed to improve the uniformity of iron oxide NPs, creating nanoreactors with surfactants to provide a constrained environment that prevents further nucleation or inhibits crystal growth has been reported. The water-in-oil microemulsion system reported by Pillai et al. 28 is an excellent example of this approach to produce iron oxide nanoparticles with a narrow size distribution. This was achieved by adding tetraethylammonium hydroxide to a water-in-oil microemulsion of ferrous sulfate in a CTAB/n-butanol/n-octane mixture to induce the hydrolysis of Fe 2+ ions. A second microemulsion with sodium nitrite as the dispersed aqueous phase was then added. Frequent collisions and coalescence of the droplets allowed the reaction of the Fe 2+ ions and sodium nitrite, resulting in the formation of Fe 3+ species, and then of iron oxide, within the aqueous core of the droplets. The surfactant concentration played an important role in controlling the droplet size, thus restricting the growth of the iron oxide NPs. The advantages of using microemulsions to control the size of iron oxide NPs were further enhanced by adding chitosan, a biocompatible hydrophilic stabilizing agent, into the system. 29 The size of the hybrid nanoparticles (10 to 80 nm) could be controlled with chitosan having different molar masses, while the weight ratio of chitosan to Fe 3 O 4 greatly impacted the magnetization properties of the composites.

The separation of nucleation and growth may be achieved by tuning the nucleation process through a hosting medium such as small amphiphilic molecules or polymeric micelles, able to encapsulate the iron precursors and disperse them in discrete compartments, thereby providing control over particle formation. These inorganic-organic hybrids inherit the properties of both the inorganic material and the organic component. In the case of polymers, compartmentalization provides confinement for the reactants and steric stabilization, thus improving the colloidal stability and in some cases, introducing new functional groups. Control over the size and the morphology of MNPs achieved through adjustment of the geometry and the size of the organic templates has been considered in our review, 30 whereby well-defined polymer structures exerted stronger impact on the outcome of size-and shape-controlled MNPs than simple homopolymer chains. Although the accessibility of metallic precursors complexed by anchoring moieties might be affected, welldefined polymer templates offer predetermined structures with minimal changes in morphology and size dispersity.

Surface Modification of Templated MNPs for Improved Biocompatibility

Uncoated iron oxide NPs can easily precipitate under the influence of gravitational forces (sedimentation), agglomerate under physiological (high salinity) conditions, or when submitted to a magnetic field (dipolar interactions) due to their insufficiently repulsive interactions. In a clinical setting for example, precipitation and agglomeration of the iron oxide NPs can lead to clotting in blood vessels. 31 There are theoretically four types of interactions contributing to the overall equilibrium state of a colloidal magnetic suspension:

-forces originating from electric dipoles in matter, either permanent, induced or dispersive componly refered to as "van der Waals forces", that create strong short-range isotropic attractions; -electrostatic repulsive forces, which are long-range, but according to the Debye-Hückel, theory, they can be partially or totally screened out as the ionic strength (defined as the sum of all the concentrations of ionic species, weighted by the square of their elementary charge) of the dispersion medium is increased;

-magnetic dipolar forces between the magnetic particles, which are basically anisotropic attractions that can be attractive or repulsive, depending on the field orientation; -steric repulsion forces observed for coated particles, that are related to the entropy loss when macromolecular chains highly swollen by a solvent are brought into contact.

Chapter 4 145

The stabilization of magnetic suspensions can be achieved most conveniently using electrostatic and/or steric repulsions. 32 Electrostatic repulsion appears when the NPs carry charges on their surface, which may originate from the adsorption of charged ions or molecules, or from the ionization of pre-existing functional groups on their surface. Charged particles have a double-layered structure: the inner (Stern) layer, consisting of highly organized solvated H 2 O molecules and chemically adsorbed ions, and a diffuse layer composed of counterions balancing the charge at the surface, called the Debye layer. The iron atoms at the surface of iron oxide NPs act as a

Lewis acid capable of coordination with electron-donating ligands. In aqueous solutions, the Fe 3+ and Fe 2+ cations form octahedral coordination complexes with water that dissociate into hydroxyl groups. These hydroxyl groups may react with acids or bases, or exchange with other functional groups such as carboxylates (citrate, [33][34] gluconic acid, 35 dimercaptosuccinic acid 36 ), sulfates, 37 or phosphates (alkyl phosphonates, 37 poly(vinyl alcohol phosphate) 38 ).

Phosphonate and phosphate ligands, due to their biocompatibility, 39 enable the use of magnetic nanoparticles for biomedical applications. The coordination complexes between iron cations and small molecule stabilizers are reversible, the stability of the magnetic suspension depending strongly upon the pH and the concentration of non-adsorbed molecules. [40][41][42][43][44] The sensitivity of electrostatic forces to ionic strength of the dispersion environment is well-documented. Increased ionic strength drastically decreases the thickness of the electrical double layer: An ionic strength above 100 mM typically reduces the thickness of a double layer to less than 1 nm, which is insufficient to prevent aggregation driven by van der Waals forces. 45 At physiological salt concentrations (around 150 mM), electrostatic repulsion is thus unable to impart sufficient stability to hinder the precipitation of NPs.

The limited usefulness of electrostatic repulsion leaves steric stabilization as the preferred option to enhance the colloidal stability of magnetic NP suspensions. Steric stabilization arises upon coating the NPs with a corona of macromolecular stabilizer chains, that effectively increase the distance between NPs and outweighs the van der Waals Chapter 4 146 attraction. A surfactant layer with a thickness of 2 nm yields steric repulsion sufficient to compensate for the attractive forces between magnetic particles, thereby preventing their agglomeration. 46 These steric stabilizers can be adsorbed or grafted onto the NP surface during or after their synthesis. If the macromolecules can interact strongly with the NP surface and efficiently prevent clustering, they have the potential to serve as steric stabilizers.

However, the chemical nature of the coating on NPs has strong if not vital impact on their cellular uptake efficiency, cytotoxicity, biocompatibility, and biodegradability once they are exposed to physiological conditions. [47][48] To enable any clinical application, the coating should consist of biocompatible materials or biological molecules such as antibodies, peptides, polysaccharides, etc. to enhance their biocompatibility 49 and to maintain a sufficiently long blood circulation half-life time that permits accumulation of the MNPs in tumor tissues through the so-called "enhanced permeation and retention effect" (EPR). 50 Biocompatible materials such as dextran, 51 citrate [33][34] and PEGylated starch 7 have been used to coat MNPs. Fe 3 O 4 NPs coated with poly(ethylene glycol) (PEG), 52 pullulan 53 or poly(maleic anhydride-alt-1-octadecene)-g-poly(ethylene glycol) PMAO-g-PEG 54 displayed no apparent cytotoxicity in in vitro assays when exposed to cells at various concentrations (1-2 mg/mL), owing to the biocompatible coating preventing the iron oxide core from interacting with cells. The anti-biofouling property (resistance to protein adsorption) of PEG 50,[55][56] makes this polymer a very useful coating material. Methoxypoly(ethylene glycol)-oligo(aspartic acid) MPEG-Asp 3 -NH 2 -coated iron oxide nanoparticles thus had no cytotoxicity on OCTY mouse cells at a concentration of 400 µg/mL, but methoxypoly(ethylene glycol)-block-poly(acrylic acid) MPEG-b-PAA-coated and PAAcoated nanoparticles killed 84% of the cells. 57 The relation between cellular uptake and the repulsive properties of the polymer shell on MNPs was recently studied by Berret et al. 58 in the case of PEO chains with phosphonic acid side chains, that are much more efficient than a PAA coating. Apart from these polymer coatings, silica-coated iron oxide nanoparticles, 49,59- 63 Ferumoxsil (AMI-121), has also been proposed and tested in clinical trials by oral administration. [64][65] Other inorganic materials such as gold [66][67][68][69][70] or gadolinium(III) [71][72] have Chapter 4 147 also been used not only to stabilize the nanoparticles in solution, but also to help in binding various biological ligands to the nanoparticle surface.

The stability of the coatings and their biodegradation characteristics also affect the biocompatibility of MNPs. Commercially available contrast agents such as Ferridex ® , Resovist ® , Supravist ® and Sinerem ® are coated with dextran or carboxy-dextran. 7,73 These materials are not ideal for coating due to their weak binding to iron oxide NPs however, which makes them more prone to detachment and aggregation. [73][74] Dextran-magnetite NPs thus caused cell death and reduced cell proliferation as strongly as uncoated iron oxide particles, 51 because the dextran coating can collapse and expose iron oxide NP aggregates to the cellular components, leading to lipid membrane disruption by often invoked intracellular production of reactive oxygen species (ROS). However, only limited information is known on the effects of these coatings on DNA damage and oxidative stress. 31 The molar mass ( w ) of the stabilizer is another criterion determining its usefulness for in vivo applications. Magnetic nanoparticles (MNPs) with hydrodynamic diameters < 50 nm can be distributed to both lymph nodes and the reticuloendothelial system (RES, comprising in particular the spleen and liver), while MNPs with diameters > 50 nm are located only in the reticuloendothelial system. 48 Large-sized magnetic NPs are also cleared by the reticuloendothelial system from the bloodstream faster than smaller-sized particles. 75 Very high w stabilizers, while imparting good colloidal stabilization to the coated NPs, also yield hybrid NPs with a large hydrodynamic size, leading to different biodistribution and clearance pathways and thereby limiting the potential applications of these NPs. with PAA-b-PHEA was designed to produce magnetic polyion complex (MPIC) micelles with improved colloidal stability at pH 7.

Two approaches for the preparation of stable

Advantages of Using Arborescent Copolymers as Templates for MNPs

The preparation, structure and properties of dendritic graftalso called arborescent or dendrigraftcopolymers will be briefly reviewed to highlight the expected advantages of this kind of polymer as a template for MNP synthesis. The linear PS substrate, with n = 5200 gmol -1 and w / n = 1.06, was acetylated and grafted with n = 5500 gmol -1 living polystyryllithium chains to obtain a G0PS substrate with n = 9.7×10 4 gmol -1 , w / n = 1.03, and 17 PS side chains. The G0PS product, after Chapter 4 149 purification by precipitation fractionation (to remove the linear PS contaminant) and acetylation, was grafted with living P2VP having n = 5500 gmol -1 and w / n = 1.15. The resulting G0PS-g-P2VP or G1 arborescent copolymer, purified by fractionation, had n = 1.1×10 6 gmol -1 , w / n = 1.08, and 182 P2VP side chains (corresponding to 9500 2VP units). Arborescent graft copolymers PS-g-P2VP (G0) and G1PS-g-P2VP (G2), also briefly examined as templates, were prepared by the same procedure. The P2VP arborescent copolymers are soluble in polar solvents (DMF, methanol), and in water upon protonation by a strong acid (e.g., HCl or trifluoroacetic acid). The apparent pK a of such a polymer depends on the degree of protonation of the chains, the electrolyte concentration and particularly on steric hindrance, which is difficult to define precisely. 78 The hydrodynamic diameter of G0-G2 arborescent copolymers measured in HClacidified aqueous solution are shown in Figure 4-2. Clustering was observed when dissolving the G0 copolymer, probably due to its smaller number of P2VP chains (f n = 13) leading to insufficient electrostatic repulsion between the molecules. Based on this observation, the G0 copolymer was not explored as a magnetic nanoparticle template. Both the G1 and G2 copolymers exhibited narrow size distributions and no agglomeration (PDI = 0.04 and 0.05, Chapter 4 150 respectively) when dissolved in HCl solutions (pH 4). The bulkier and more rigid structure of the G1 and G2 molecules could be useful in templating MNPs, but potentially also induce colloidal instability. The smaller G0 hydrophobic PS core of the G1 molecules may lessen these disadvantages. Last but not least, our goal was to generate hybrid magnetic polymeric nanoparticle structures ideally in a range of 10-100 nm, considered optimal for intravenous administration, since particles > 200 nm are sequestered by the spleen and those < 10 nm are removed by renal clearance. 79 The smaller size of the G1 copolymer (D h = 29 nm) as compared to G2 (D h = 58 nm) is thus considered another advantage. The high content of the copolymers in 2VP, a ligand that coordinates with iron ions, combined with the very compact structure of the G1 arborescent copolymer, should make it a very good platform for the complexation of iron and templating of the MNPs. Iron(III) chloride (FeCl 3 , Sigma-Aldrich, anhydrous, powder, ≥ 99.99% trace metals basis), iron(II) sulfate heptahydrate (FeSO 4 7H 2 O, Sigma-Aldrich, ACS reagent, ≥ 99.0%), hydrochloric acid (HCl, Sigma-Aldrich, ACS reagent, 37%), ammonia (NH 4 OH Sigma-Aldrich, 28-30% solution), and sodium hydroxide (NaOH, Sigma-Aldrich, pellets, semiconductor grade, 99.99% based on trace metals basis) were used as received. Milli-Q water (18.2 MΩ•cm) was obtained from a Milli-Q water purification system (Purelab Classic, Elga Lab Water).

The G1 arborescent graft copolymer used in this study was synthesized by anionic polymerization and grafting. 58 The graft copolymer G0PS-g-P2VP ( n = 1.1×10 6 gmol -1 ; M w /M n = 1.08; branching functionality f n = 182) consisted of 9500 2VP units, corresponding to 91 mol% of 2VP (Table 4-1). 77 Polyion Complex Micelle Preparation The polyion complex (PIC) micelles used 4.3.1.2 as templates for MNP synthesis in this work were prepared from a G1 arborescent graft copolymer (G0PS-g-P2VP) and a DHBC according to the procedures described in Section 3.3.6. The complexation of G1 with the PAA 27 -b-PHEA 260 block copolymer was carried out in aqueous solution. The parameter f = CO 2 H/N was used to quantify the molar ratio of CO 2 H groups (from the PAA segment of the block copolymer) and the amine moieties (from the 2VP units of the arborescent copolymer):

The following procedure describes the complexation of the G1 copolymer with Sedimentation of the magnetic nanoparticles (MNPs) in the magnetic field of an NdFeB permanent magnet allowed complete removal of the excess reagents. The MNPs were rinsed with water (2 × 5 mL), acetone (2 × 5 mL), and diethyl ether (2 × 5 mL) before being redispersed in 2 mL of dilute HNO 3 (pH 1.4) to produce a black homogenous ferrofluid that was a relatively stable colloidal dispersion of MNPs.

Stabilization

The procedures and the characterization techniques for the synthesis of the doublehydrophilic block copolymers (DHBC) poly(acrylic acid)-b-poly(2-hydroxyethyl acrylate) by atom-transfer radical polymerization (ATRP) were previously described (Section 3.3).

Among the four block copolymers obtained, PAA 13 The measurements were performed on a Zetasizer Nano ZS90 (Malvern Instruments) equipped with a 4 mW He-Ne laser operating at 633 nm and 25.0 °C, at a scattering angle  = 90°. Each sample was measured in triplicate using disposable PS cuvettes or quartz cuvettes.

The samples were prepared at concentrations of 3-5 mg/mL in Milli-Q water (at 25 °C, refractive index n o = 1.330, viscosity = 0.8872 cP), and filtered through a nylon membrane with 0.45 μm nominal pore size. The light scattering data were analyzed with the Zetasizer™ 7.11 Software (Malvern Instruments). The intensity-weighted autocorrelation function g (2) () produced by the digital correlator was used to extract the particle size distributions. The second order cumulants analysis algorithm was applied for relatively monodisperse particles, while the non-negatively constrained least-squares method (NNLS) or CONTIN analysis were employed for polydispersed samples. The hydrodynamic radius R h of the particles was determined from the Stokes-Einstein equation with the diffusion coefficient (D T ) obtained from the decay rate (Γ D T  q 2 ) of the amplitude autocorrelation function g (1) ()

and the instrumental scattering wave vector defined as ⁄ .

In this Chapter, for simplicity, we will use D h (rather than ) for the intensityweighted hydrodynamic diameter, and for the number-weighted hydrodynamic diameter.

Transmission Electron Microscopy 4.3.3.2

Transmission Electron Microscopy (TEM) images were obtained at the Bordeaux Imaging center (BIC) on a Hitachi H7650 microscope working at 80 kV. Samples were prepared by depositing 0.7 μL of the MNPs aqueous solution onto a copper grid (200-mesh coated with holey carbon film) and removing the excess after 5 min. The grids were dried overnight in a fume hood. The metal-free samples were stained by exposing the grids to iodine vapors for 60 min.

Thermogravimetric analysis 4.3.3.3

Thermogravimetric analysis (TGA) was carried out on a Q500 (Q Series™ TA Instrument) using the following program: Freshly cleaved mica was used as sample substrate. The polymer solutions were prepared in Milli-Q water at concentrations ranging from 0.05 to 0.1 mg/mL. A 20 μL aliquot of MPIC micelle dispersion was deposited on the mica substrate and spin-coated at 3000 rpm for 180 s under ambient conditions. Atomic force microscopy (AFM) images were recorded in the tapping mode in air on a Veeco Dimension Icon System equipped with a Nanoscope V controller. The probes used were Olympus ® AC160TS-R3 micro cantilevers with a spring constant of 26-56 N/m, a resonance frequency of 300-399 kHz, and a curvature radius of 8-10 nm. The scan rate was typically set between 0.7 and 1.2 Hz, at a scan angle of 0°, acquiring 512 samples/line. Image analysis was done with the Nanoscope Analysis V1.20 software from Bruker.

Results and Discussion

In this Chapter the term magnetic nanoparticles (MNPs) will be used for G1@Fe 3 O 4 NPs, while the magnetic polyion complex (MPIC) micelles will be identified as G1@Fe 3 O 4 @PAA-b-PHEA NPs. The superparamagnetic properties of the MNPs and MPIC micelles will be confirmed in Chapter 5, but the term superparamagnetic iron oxide nanoparticles (SPIONs) will not be used until then. The number-weighted hydrodynamic diameter, , will not be discussed frequently, thus for simplicity we will use D h (rather than ) to represent the intensity-weighted hydrodynamic diameter.

Using PIC Micelles as Templates for MNPs

The PIC micelles have an arborescent polymer structure with 2-vinylpyridine units (2VP, a ligand that strongly coordinates with iron ions) and a hydrophilic shell of poly(2hydroxyethyl acrylate) PHEA. Unfortunately, the direct in situ application of PIC micelles to the Fe 3 O 4 alkaline coprecipitation reaction resulted in weak magnetization for the products, causing difficulty in MNP sedimentation with a permanent magnet (Figure 4-3). The orangebrown color and the weak magnetization of the products were probably due to the formation of different phases of iron oxyhydroxides rather than magnetite (Figure 4-1). We hypothesize that mixing of the PIC micelle solution at pH 7 with an aliquot of Fe 2+ and Fe 3+ salts at pH 1.4 yielded a solution with pH 2.6, insufficiently low to prevent the precipitation of ferric hydroxide (with a very low solubility product K s = [Fe 3+ ][OH] 3 = 10 -44 ) 80 and ferrous hydroxide (K s = [Fe 2+ ][OH] 2 = 10 -34 ). 80 Another reason for the failure of templating may be the presence of the PAA-b-PHEA block copolymer in the shell, designed to improve the colloidal stability and biocompatibility of the end product, interfering with the accessibility of the iron precursors to the 2-vinylpyridine moieties, since PAA is also a complexing ligand for ferrous and ferric cations.

As shown on

Templating -In Situ Coprecipitation

Due to its high content (91 mol%) of 2VP units (a strong iron-coordinating ligand) grafted in a very compact structure with small dimension (D h = 29  4 nm, PDI = 0.04, The size and size distribution of the MNPs strongly influence their magnetization properties. Among iron oxide NPs with a size of 5-14 nm, the 14 nm NPs were indeed found to have the highest Specific Absorption Rate (SAR) value of 447 Wg -1 when submitted to a 400 kHz, 24.5 kA/m alternating magnetic field. 82 De la Presa et al. 83 reported a four-fold increase in the SAR value (from 10 to 40 Wg -1 ) when increasing the size of maghemite NPs from 8 to 11 nm, and 1.3-fold (from 40 to 55 Wg -1 ) from 11 to 13 nm. In general, higher SAR values are also obtained for samples with lower polydispersity values, regardless of the NP size. 84 To emphasize the advantages of the G1 arborescent copolymer as a template, coprecipitation in the presence of linear poly(4-vinylpyridine) P4VP ( n = 65,000 gmol -1 )

was also carried out, and Transmission Electron Microscopy (TEM) served to determine the crystallite size of the Fe 3 O 4 NPs (composed of both crystalline and amorphous components).

The size measured by TEM is a number-weighted value, and we performed size analysis based on greater than 100 particles to obtain meaningful statistical results. The crystallite size of P4VP@Fe 3 O 4 determined by TEM analysis was 7.0  1.4 nm, which is typical for samples prepared by non-templated coprecipitation. 13 The crystallite size of the Fe 3 O 4 NPs increased to 9.1  1.7 nm in the presence of G1 template (Figure 456). The encapsulation of Fe 2+/3+ ions within the G1 template presumably distributed the ions into a smaller volume, which allowed a short burst in the nucleation rate as compared to homogeneous nucleation.

In comparison to homogenous nucleation, heterogeneous nucleation within the P2VP phase of the G1 micelles forces the Fe 2+ and Fe 3+ cations closer to each other, since complexation partly overcomes electrostatic repulsion. In so doing, the nucleation and growth steps are more likely to be separated.

G1-80

Growth of the Fe 3 O 4 crystallites to 12.1  2.0 nm was achieved when the G1templated coprecipitation was performed at 80 °C (Figure 456). The elevated temperature probably accelerated both the nucleation and growth steps, resulting in a larger crystallite size. An attempt at 100 °C led to a higher oxidation level: the ferrofluid had a brownish color. We therefore decided to produce subsequent G1@Fe 3 O 4 samples at 80 °C. The coprecipitation method can produce MNPs in a size range of 2-25 nm, but with a broad size distribution (usually greater than  25% from the mean). 85 The microemulsion technique can narrow the size distribution to within  5% from the mean, at the expense of a more challenging purification procedure and a much smaller quantity of product. 85 In our work a slight improvement in size distribution from  20% to  16.5% from the mean was observed, and the crystallites appeared separated by an organic layer (bright stripe around the dark cores), which is consistent with their better dispersibility (bare inorganic grains in close contact experience strong van der Waals attraction and usually cannot be separated).

Selected area electron diffraction (SAED) was employed to reveal and characterize the phases present in the oxide. The Bragg diffraction pattern confirmed a spinel structure of either magnetite Fe 3 O 4 or maghemite Fe 2 O 3 (Figure 456). Unfortunately, the SAED patterns cannot distinguish magnetite from maghemite due to their isostructural character:

both are cubic, with similar unit cell parameters. A few peaks are different for these two phases, but they usually appear with a low intensity and are easily blurred by background noise or crystallite size effects. 86 Thermogravimetric analysis (TGA) served to determine the relative amounts of iron oxide and polymer template in the nanocomposites. Decomposition of the G1 polymer started at ca. 350 °C and ended at ca. 450 °C (dotted curve, Figure 4567). The thermogravimetric trace for a physically blended mixture of dry G1 powder and Fe 3 O 4 paste was similar to that of the pure G1 sample except for a weight loss after 140 °C, much sooner than for G1 alone. This is attributed to the loss of physically adsorbed water (on the surface of the nanoparticles) and surface hydroxyl groups (forming H 2 O molecules by elimination from adjacent hydroxyl groups). [87][88][89][90][91] This step differs from the evaporation of unbound water ending earlier, at ca. 130 °C. -A small weight loss at 140 °C, attributed to the elimination of physically adsorbed water and surface hydroxyl groups, [87][88][89][90][91] as observed for the mixture of dry G1 powder and -A weight loss that accelerated rapidly with the introduction of O 2 (at 470 °C), indicating burning of the P2VP phase coordinated with the Fe cations.

The stepwise thermal degradation of polymers in magnetic hybrids has been previously reported by different groups. [87][88][89]92 Zhang et al. 92 overestimation is attributed to incomplete degradation/combustion of the organic material in TGA analysis, leaving some carbonaceous material in the residue. This phenomenon has been observed before, for example by Huang et al. 93 who used TGA to determine the iron content in cross-linked poly(methacrylic acid)/iron oxide magnetic microspheres, and reported 25.8 % residual weight (measured by TGA), higher than the Fe 3 O 4 content of 19.8 wt% determined by spectrophotometry analysis, indicating the presence of organic components in the residue even after a temperature of 750 °C was reached. The thermogravimetric analysis of paramagnetic microparticles functionalized with poly(ethylene glycol) (PEG) and carboxyl groups also resulted in a slightly larger mass fraction of residue at the end of the TGA experiment as compared to the Fe 3 O 4 content measured by atomic absorption spectroscopy. 91 Wang et al. 94 likewise expected a theoretical value for the Fe 3 O 4 content of 62.4 wt% for nanoparticles capped with oleic acid, but obtained a residual mass fraction of 68 wt% after TGA. The small discrepancy was attributed to the surface oxidation of Fe 3 O 4 to Fe 2 O 3 in the presence of oxygen in the TGA experiment in that case, which slightly increased the inorganic component weight. 94 The actual mass of Fe 2 O 3 in the residue was determined by UV-VIS analysis in 5 M

HCl. The residue (ca. 1.73 mg) was not completely soluble in 5 M HCl, leaving some black particles clearly evidencing the presence of organic material. The Fe content was determined from a linear calibration curve relating the iron concentration to the optical density at  = 350 nm (OD 350 ) as suggested by Arosio et al., 95 and inspired from the literature 96 (Figure 45678).

The absorbance peak at this wavelength is ascribed to the hexachloride complex [FeCl 6 ] 3-in 5 M HCl medium. An OD 350 = 0.3695 was obtained for sample, corresponding to 1.29 mg

Stabilization of Magnetic Nanoparticles by the Polyion Complexation Technique

Block copolymers (BC) with a long PHEA segment, PAA 13 -b-PHEA 150 and PAA 27 -b-PHEA 260 , proved to be the most efficient stabilizers among the BC series tested as they could form stable unimolecular micelles with the G1 arborescent copolymer, even at low f = CO 2 H/N ratios. These BCs were therefore tested to form micelles with G1@Fe 3 O 4 by the

Conclusions

We observed poor performance for preformed PIC micelles as templates for the synthesis of MNPs: The product formed displayed weak magnetization and a large size.

Neither templating nor stabilization were efficient by this approach, presumably because: i) chains. A slight improvement in size distribution from  20% to  16.5% from the mean was observed, i.e. lower than the  25% variation usually reported for non-templated NPs. 85 When using thermogravimetric analysis (TGA) to determine the amount of iron oxide in the nanocomposites, we observed a three-stage weight loss profile for the thermal decomposition/combustion of the templated NPs G1@Fe 3 O 4 . The overestimation of the Fe 2 O 3 content by that method was ascribed to the incomplete degradation (in N 2 ) or combustion (in O 2 ) of the organic phase in the TGA experiment, which is in good agreement with literature reports. 91,[93][94] The presence of carbonaceous material in the residue was confirmed by UV-VIS analysis in 5 M HCl. UV-VIS analysis therefore proved to be more reliable to determine the Fe (and corresponding Fe 3 O 4 ) content of the MNPs, so UV-VIS analysis was preferred to TGA.

the
The G1@Fe 3 O 4 MNPs were then complexed with a PAA-b-PHEA block copolymer to produce stable magnetic micelles whose core-shell morphology was revealed by tapping mode AFM height and phase imaging. We observed the stabilization of G1@Fe 3 O 4 to yield MPIC micelles even for smaller amounts of block copolymer that were insufficient to prevent aggregation of the corresponding metal-free PIC micelles. The presence of Fe 3 O 4 in G1@Fe 3 O 4 likely increased the volume of the G1 template, exposing more coordination sites and resulting in more efficient complexation even at lower f ratios. The MPIC micelles obtained, with suitable hydrodynamic diameters D h  130 nm and good colloidal stability, not only under physiological conditions (pH 7) but also over a wide pH range, hold great promise for the biomedical applications targeted in this work.

Chapter 5 Magnetic, Relaxometric and

Hyperthermic Properties Measurements

Overview

The properties of magnetic nanoparticles (MNPs) obtained using a G1 arborescent copolymer as template, both in their bare and block copolymer-coated forms, were investigated by vibrating sample magnetometry, relaxometry, and alternating magnetic field ). The dependence of the SAR values on the magnetic field amplitude H and frequency f was also investigated.

Introduction

Superparamagnetic iron oxide nanoparticles (SPIONs) can be used for numerous biomedical applications including magnetic resonance imaging contrast agents and heat mediators in magnetic field hyperthermia (MFH). To begin with, single domain theory and superparamagnetism will be briefly reviewed and followed by an introduction on vibrating sample magnetometry, a method commonly used to measure the magnetization curve vs.

applied magnetic field strength. The contrast enhancement effect by iron oxide nanoparticles in magnetic resonance imaging will also be presented. Finally, heat dissipation mechanisms will be discussed and followed by a literature review on optimizing the specific absorption rate (SAR) values of SPIONs.

Single Domain Theory and Superparamagnetism

The internal structure of a bulk ferromagnet (iron, cobalt, nickel…) is split into magnetic domains to minimize the internal energy. Inside a magnetic domain all the magnetic moments are coupled in the same direction, thus allowing the representation of that domain by a single magnetization vector accounting for all of the magnetic moments per volume unit. Magnetic domains are separated from each other by domain walls, a transition region between two domains where a gradual reorientation of the magnetic moments (usually by 90° or 180°) occurs. The domain wall thickness depends on several energetic, crystallographic and geometric factors, but usually spans across ca. 100-150 atoms. 1 Domain walls can be moved across the sample by a small magnetic field. A larger ferromagnet has more possibilities of forming domain walls, due to its higher need for energy minimization and greater ease to anneal structure defects. Decreasing the sample size leads to lower energy minimization, fewer structural defects to anneal, and thus reduced wall formation. The sample size or volume may be decreased until a critical value is reached, at which point the system prefers one single domain configuration called a "Weiss domain"; below this threshold size, multidomain configurations are energetically unfavorable since the energy cost for domain wall formation becomes greater than the energy reduction obtained by splitting domains. The critical radius R C is estimated by the following formula given in the texbook on solid state physics by Kittle: 2

√

where M s is the spontaneous magnetization, K is the anisotropy constant (in J/m 3 ), and A is the exchange energy density or constant (energy per unit length). One can define also the lengthscale below which the attractive exchange energy dominates the repulsive dipolar energy between neighboring magnetic moments:

√

A sample with dimensions below R C acquires the single domain configuration; Its magnetization can be represented by one single vector. In the absence of an external field, the magnetization of the sample is dictated to spontaneously point in a particular crystallographic direction, called the easy magnetization axis. The energy that prevents the magnetization from changing freely between all possible orientations is called the anisotropy energy, which depends upon the angle between the magnetization vector and the crystallographic direction.

The direction minimizing the anisotropy energy is called the anisotropy direction or easy axis. The anisotropy energy increases very rapidly as the crystal radius increases, because it is proportional to the crystal volume (the proportionality constant being K). The assumption that the anisotropy has uniaxial symmetry is usually applied to systems of higher symmetry based on cubic models, since the calculations for more complicated symmetries are timeconsuming without producing a complete solution. 3 Stoner-Wohlfarth 4-5 proposed the magnetic anisotropy energy of a noninteracting single-domain spherical particle with uniaxial anisotropy in zero magnetic field as where K is the magnetocrystalline anisotropy constant, V is the sample volume, and  is the angle formed between the magnetic moment and the easy axis.

The dependence of the anisotropy energy on θ is depicted schematically in Figure 5-1. Both orientation states with minimum energy (θ = 0 or π) are equally probable and are separated by an anisotropy energy barrier E a =KV. An external magnetic field H applied along the easy axis reduces the magnetic anisotropy energy barrier, facilitating switching of the magnetization from an energy-unfavorable state (opposite to the field) to an energy-favorable state (along the field and oriented in the same direction), thus the interaction energy between the moment and the external field appears as a negative correction to the previous equation, where M nr is the nonrelaxing magnetization. and K S =39 µJ/m 2 for Fe 3 O 4 NPs. 6 Nevertheless, reductions in size can decrease the magnetic anisotropy energy barrier E a = KV down to a critical level where it becomes comparable to or Chapter 5 178 even lower than the thermal activation energy (k B T, k B being Boltzmann constant). The energy barrier for magnetization reversal is thermally overcome; the magnetic moments rapidly fluctuate from one orientation state to another, a behavior also found in paramagnetism, but with magnetic moments several orders of magnitude higher. The sample then enters the superparamagnetic regime. A system is commonly described as superparamagnetic when i) the M/M s vs. H/T curves for a range of temperatures above the socalled "blocking temperature" T B superimpose on each other; ii) neither coercive field H c , remanence magnetization M r , nor hysteresis exist (Section 5.2.2).

Another important magnetic parameter for single domain particles is the Néel relaxation time. This is the time that a magnetic moment needs to return to thermal equilibrium after a perturbation. When the anisotropy energy is sufficiently large, it keeps the magnetization locked along an easy axis. Once the Stoner-Wohlfarth energy of magnetization exceeds the anisotropy barrier, the Néel relaxation time is defined as the fluctuation time for a magnetic moment to flip between easy axes. The Néel relaxation time is defined as:

where E a is the anisotropy energy, is the Boltzman constant, T is the absolute temperature, and is the pre-exponential factor of the Néel relaxation time expression also called "attempt time" because it is the repetition time between attempts for the magnetic moment to cross the barrier: 7

√ [ ( ) ] √ ( )

where V is the crystal volume, M s (0) is the specific magnetization of the crystal extrapolated at 0 K, M s (T) is the specific magnetization at the experimental temperature, γ e is the gyromagnetic ratio of the electron, and η f is a dimensionless constant.

The parameter  0 depends on several factors: the temperature, magnetic field strength, magnetization, particle size, etc. but it is usually assumed to be a constant within a range of 10 -9 -10 -13 s. The Néel relaxation time varies with the temperature and the size of the magnetic nanoparticles. For small size particles with a small anisotropy energy at high temperatures, the anisotropy energy is easily exceeded by the thermal energy (E a << k B T), For well-dispersed magnetic NPs in a magnetic fluid, the effective relaxation time of the magnetic moments is determined not only by the Néel relaxation time but also by the Brownian relaxation time , reflecting the rotation of the particles in a viscous medium:

and
Large particles have short and long values, since is proportional to the crystal volume V but has V in its exponential function. The relaxation time is therefore determined by the shorter relaxation time, in this case , which explains why the relaxation time for a magnetic fluid is faster than when the solvent is frozen or for a dry powder, and why the magnetization curve is reversible: Even if their moment is blocked along an easy axis, the particles in a superparamagnetic regime can physically rotate when the magnetic field changes its polarity; thus the overall magnetization of the suspension can follow the field (some authors refer to this phenomenon as "extrinsinc superparamagnetism" as the magnetic suspension in a fluid carrier still exhibits superparamagnetism even in the case where the moments are blocked, corresponding to ferro-or ferrimagnetism behavior in the solid state).

Vibrating Sample Magnetometry -Magnetization Curve 9-11

Vibrating sample magnetometry (VSM) is a method to measure the magnetic moment of a sample by its continuous vibrational motion parallel to a uniform magnetizing field, through the synchronous detection of the magnetic flux induced in a "pickup coil" at the same frequency as the mechanical oscillation of the sample. 10 The first instruments invented by Foner had a sensitivity of up to 10 -5 -10 -6 emu (the detectable range for a modern VSM instrument is as low as 10 -6 -10 -8 emu) 9 and a stability of 0.01%. VSM is simple, less expensive than a magnetometer with a superconducting quantum device (SQUID), and versatile. VSM indeed enables the minimization of many sources of error found in other methods and permits precision magnetic moment measurements in a uniform magnetizing field as a function of temperature, magnetizing field strength, and crystallographic orientation. 10 In a VSM experiment, the sample is placed inside the uniform magnetic field created by an electromagnet to magnetize the sample before it is oscillated at fixed frequency (about proportional to the magnetic moment of the sample, but independent of the strength of the applied magnetic field (that is homogeneous and constant, thus also its flux through the coil):

; where C is a coupling constant, A is the oscillation amplitude, f is the oscillation frequency, and m is the DC magnetic moment of the sample. The induced voltage measured by the detection coil can be written, according to the Lenz law:

( ) ( )
where  is the magnetic flux of the magnetic induction B lines enclosed by the pickup coil, z is the vertical position of the sample with respect to the coil, and t is time.

When applying cyclic positive and negative magnetic field values to a sample, a hysteresis loop indicating irreversible changes in magnetization M with respect to the applied field strength H applied can be obtained (Figure 5-2a). Since the sample is initially unmagnetized, the initial path indicating the increase in M as H is increased is called a "minor loop". The most useful parameters extracted from the hysteresis loop are the saturation magnetization M sat , the remanence magnetization M r , and the coercive field H c .

The saturation magnetization M sat is the limiting value that the curve can reach within the high-magnetization-field regionwhen all the magnetic moments are aligned with the magnetic field. When the field is decreased the magnetization of the sample decreases, but it is unable to recover its unmagnetized state. The amount of magnetization retained in the sample at zero-field is called the the remanent magnetization M r or simply remanence. The coercive field H c is the field needed to demagnetize a sample exhibiting (irreversible) magnetic hysteresis. A system is commonly described as superparamagnetic when i) the M/M s vs. H/T curves for a set of temperatures above T B superimpose on each other; ii) neither coercivity H c , remanence M r , nor hysteresis exist (Figure 5-2b). It is possible to extract parameters of interest from the room temperature magnetization curve of a ferrofluid if a log-normal particle size distribution is assumed to describe the size dispersity: the saturation magnetization M sat ; the specific magnetization M s ;

the width of the size distribution expressed as Ln(D) (); and the median particle diameter of the distribution (D 0 ), which is defined as a value separating the particles into 50% of largerthan-D 0 particles and 50 % of smaller-than-D 0 particles. In work published in 1978, Chantrell et al. 12 reported discrepancies between the magnetic-size-distribution parameters (measured by VSM) and the physical-size-distribution parameters (measured by microscopy): D 0 < D 0phy and  >  phy , which was later confirmed by Popplewell et al. 13 Later on, Bacri et al. 14 suggested the treatment of superparamagnetic colloidal solutions (or suspensions) as an ensemble of polydisperse magnetic moments using the Langevin's equation of superparamagnetism convolved with a size distribution P(D). Each ferromagnetic single domain particle carries a permanent magnetic moment  that progressively aligns along the magnetic field H, starting from a random orientation at zero-field. The magnetization M (D) of a monodisperse population of particles of diameter D in a magnetic field of strength H was given by Langevin:

0 M b) a) [ ( ) ]
where M sat is the magnetic moment of the sample under saturation conditions, μ 0 is the vacuum magnetic permeability, M s is the specific magnetization of the magnetic material in the suspension of nanometric grains (always smaller than in the bulk); is the solid volume fraction, related to the molar content through the molar volume (15.77 cm 3 /mol for Fe 2 O 3 );

T is the temperature, and k B is the Boltzmann constant. One salient feature of Langevin's law is its linear variation at low field, whose slope is called magnetic susceptibility and writes:

showing the 1/T dependence characteristic of (super)paramagnetism (Curie's law).

In reality, ferrofluid samples are never monodisperse; a log-normal distribution P(D) is usually assumed as probability law for quasi-spherical magnetic particles of diameter D:

√ ( )
where D 0 is the median particle diameter of the distribution and  is the size distribution width, defined as the standard deviation for the Ln(D/D 0 ) distribution.

The mean-average and volume-average diameters may then be calculated from the equations:

〈 〉 ( ) 〈 〉 〈 〉 〈 〉 ( )
and the diameter-dispersity can be defined by analogy to the molar mass dispersity of polymers: .

Size dispersity introduces a distortion in the magnetization curve ∫ since M/M sat becomes a function of and . From the shape of the magnetization M versus field intensity H curve, a computational fit of the experimental data to a log-normal probability law of median diameter D 0 yields M s ,  and D 0 . In this work, we used the fitting technique developed by Bacri et al. using the least mean-square solver of MS Excel. 14 The susceptibility is also shifted towards larger values by size dispersity according to ( )

Magnetic Resonance Imaging -How Iron Oxides Affect Proton Relaxivity Magnetic Resonance Imaging 5.2.3.1

Among the nuclei commonly found in high abundance in the body hydrogen 1 H (but not 12 C nor 16 O), with an odd mass number (number of nucleons) is selected for magnetic resonance imaging (MRI) because it has a non-zero spina quantum mechanical property reflecting the rotation of the nucleus around its own axis. This rotation can be considered as a circulation of charge or a tiny current loop producing its own magnetic moment. Each magnetic moment undergoes a precession motion, and a population of hydrogen atoms has its magnetic moments pointing in random directions. When subjected to an external field B 0 , the magnetic moments precess, i.e. they are animated by a rotary motion with a tilt angle around the direction of B 0 . In thermal equilibrium, according to the Boltzmann distribution, there are slightly more nuclei oriented along B 0 (zero tilt angle) than against it (tilt angle is ), inducing a small net longitudinal magnetization, M 0 (Figure 5-3). However all the magnetic moments precess with random phases; therefore they produce no net transverse magnetization. The frequency at which the nuclei precess around the magnetic field is called the Larmor frequency, ω L , which is proportional to the strength of the magnetic field B 0 :

where is the gyromagnetic ratio of the nucleus ( 2.6710 8 rad/s/T for the proton 1 H). Applying a radio frequency (RF) magnetic pulse with pulsation in the direction perpendicular (B 1 ) to the population of oriented nuclei induces two events:

1. The nuclear magnetizations are tipped away from the longitudinal axis with a flip angle, increasing the net energy of the nuclei above their equilibrium value. The nuclear magnetization continues to precess, but now around the B 1 axis.

2. The nuclei that had been oriented by B 0 now precess in phase/in synchrony, producing a transverse magnetization in the transverse plane, called M xy .

These induced magnetizations can be detected with an RF coil, also called a "detection antenna". When the RF pulse is turned off, there are two relaxation regimes occuring:

1. The nuclei gradually lose the additional energy through interactions with neighboring nuclei and the environment, bringing the system back to its equilibrium state and producing an oscillating magnetic field that can be detected with a radio frequency coil.

The recovery of the longitudinal (M z ) magnetization toward its equilibrium value M 0 occurs during a time interval T 1 , termed longitudinal or spin-lattice relaxation. The value of T 1 varies according to the host molecule (e.g. in body tissues, it differs for water and lipids in fat, and in brain for grey matter, i.e., the cellular body of neurons vs.

white matter) and its immediate surroundings. The T 1 for water in body fluids (blood and cerebrospinal fluid) is longer than 1 s at 1.5 T, much longer than in fat (250 ms at 1.5 T) and much longer than in solid tissues, where motions are more restricted. The T 1 variations among tissues produce signal contrast on the MR images: Fat and solid tissues relax quickly, producing a signal with a high intensity appearing bright on a T 1 -weighted image while water, with much slower longitudinal relaxation, induces a lower signal and appears dark. T 1weighted sequences are used to create T 1 -weighted images, demonstrating differences in T 1 relaxation times of the tissues. The production of T 1 -weighted images with good contrast requires the repetition of excitation and signal acquisition. The repetition time between successive excitations TR needs to be sufficiently long to allow the longitudinal magnetization of tissues of interest to fully recover their equilibrium state before the next excitation, but it also needs to be sufficiently short to create tissue contrast within a reasonable time frame.

Transverse relaxation happens more rapidly than longitudinal relaxation, because it involves both microscopic and mesoscopic processes. The transverse relaxation caused by microscopic processes (due to intrinsic factors, namely the molecular size and tissue type) is denoted as T 2 . Mesoscopic factors such as the magnetic field inhomogeneity and magnetic susceptibility differences among the tissues in fact decrease the transverse magnetization relaxation time to a T 2 * value. Free water in body fluids, due to its constant rotation, relaxes relatively slowly in ca. 3-4 s, more slowly than immobilized fat tissues (T 2  100 ms), which explains why water appears bright while fat tissues appear dark when sufficiently long repeat time TR and echo time TE are used. T 2 and T 2 * differences between the tissues can be used to produce contrast on T 2 -and T 2 *-weighted images.

The dephasing caused by mesoscopic factors follows a free induction decay (FID) regime and can be reversed by applying a 180° RF pulse of B 1 (a refocusing pulse). This pulse creates inverse field inhomogeneities that gradually attenuate the phase differences among faster and slower spins, until they precess in phase again, producing a brief signal called "spin echo". The time for the spins to rephase equals the time for which they were allowed to dephase, and the total is known as the echo time, TE. Microscopic interactions, varying as the molecules under thermal agitation rotate and diffuse, cannot be compensated by the refocusing procedure. Therefore FID acquisitions provide T 2 * weighting contrast, whereas spin-echo acquisitions offer T 2 weighting contrast.

A longer delay between the RF excitation and the signal acquisition allows more time for transverse relaxation, so that tissues with short T 2 or T 2 * will appear darker than those with longer relaxation times.

To obtain good tissue contrast for T 1 -weighted images, short TR and TE are usually selected to record the signal differences between the tissues. Setting TR to a long value allows the T 1 effect on tissue contrast to disappear and for a sufficient long TE, the differences in signals induced by the transverse relaxation will emphasize the tissue T 2contrast, defining the sequence parameters for T 2 -weighted images (long TR and long TE).

Individual nuclei obey the laws of quantum mechanics. It is therefore possible to apply the rules of quantization and the quantum mechanical description of RF excitation and signal emission to describe MR physics. 15 However since the number of nuclei in a macroscopic sample is extremely large, a discussion of MR in terms of classical physics is more adequate to explain many aspects of their collective behavior and to understand the imaging techniques. 15 Magnetic Resonance Contrast Agent -How SPIONs Affect Proton Relaxation 5.

2.3.2

Magnetic resonance contrast agents (MR CAs) reduce the T 1 and T 2 relaxation times of proton nuclei in the target tissue. Depending on whether the relative reduction in relaxation times is greater for T 1 or T 2 , the MR CAs are categorized as either 'T 1 agents' or 'T 2 agents'. The efficiency of SPION as MRI contrast agents is determined by their relaxivities r 1 and r 2 : the enhancement of the longitudinal or transverse relaxation rates induced by an aqueous solution containing 1 mmol of iron per L:

where R i(obs) is the total relaxation rate of the aqueous system (s -1 ), T i(diam) is the relaxation time of the system without addition of the contrast agent, C is the concentration of the paramagnetic center (mmolL -1 ), and r i is the relaxivity (s -1 mmol -1 L).

The origin of contrast enhancement lies in the different properties of healthy and diseased tissues. Diseased tissues are metabolically different from healthy tissues: Due to an anarchic and leaky vasculature, they have a much higher CA uptake, resulting in enhanced contrast in the MRI images. T 1 agents produce positive image contrast due to the increased image signal intensity at the tissue site where the T 1 shortening effect dominates, through the contribution ( ⁄ ) that makes the signal larger than without the CA. The negative image contrast induced by T 2 agents is ascribed to the predominant effect of T 2 shortening, lowering the term ⁄ . 16 Iron oxide NPs could thus be designed either as positive CAs to detect metastatic lymph nodes, or negative CAs for liver tumor imaging. 17 The influence of MR CAs on the proton relaxation of a system stems from two contributions: the inner-and outer-sphere relaxations. Inner-sphere relaxation is assigned to the direct exchange of energy between protons and electrons located in the first hydration sphere of the paramagnetic metallic cation or superparamagnetic particle surface. For SPIONs, the inner-sphere relaxation is minor because most of the Fe 3+ cations are located inside the crystals, and thus their coordination sphere is already full. It is thus negligible as compared to the outer-sphere relaxation, which is related to the behavior of water protons in the magnetic field gradients created by the SPIONs in their vicinity (Figure 5-5). A SPION behaves differently from a paramagnetic complex: While each individual atom of a paramagnetic molecule is influenced independently by an external magnetic field, the magnetic moments of the entire SPION particle are all aligned together and re-orient cooperatively with the magnetic field. The magnetic moments of the individual ions do not cancel out but are collectively aligned, providing the SPION with a permanent magnetic moment that is much larger than that of a single molecule of paramagnetic Gd chelate (typically by a factor of 1000) in the presence of a magnetic field. The fluctuations in dipolar magnetic coupling between the water proton spins and the magnetic moments of the SPIONs are influenced by the translational diffusion of water protons and by the Néel relaxation (flip of the particle magnetic moment between easy axes).

The relaxation rate increases with the fluctuation correlation time and with the magnitude of this fluctuation. The translational diffusion of water protons varies with the minimal approach distance between the surface of a SPION and the water molecules. The translation correlation time , defined as the time for a water molecule to diffuse within a radius r, is given by ⁄ , where D is the water diffusion coefficient and r is the outer-sphere radius, that is either the crystal radius for a bare SPIO particle, or the total radius including the shell thickness in the case of a SPIO particle or multi-core cluster coated by a nonmagnetic layer impermeable to water. The correlation time of the Néel relaxation is called the Néel relaxation time ( ), which depends on crystal anisotropy. The combination of these two relaxation processes results in a global relaxation time with ⁄ ⁄ ⁄ .

If is longer than the precession period of the spin within the external magnetic field B 0 , i.e. ⁄ (where is the angular frequency of the proton precession), the fluctuation is averaged by the precession and thus inefficient to relax the proton spins. Rapid fluctuations in the interactions between the magnetic moment of the particle and the proton spins of water is expected to efficiently relax the proton spin magnetization, i.e. when is shorter than the precession period of the proton spin:

⁄

The correlation between proton relaxation and the applied magnetic field B 0 or frequency is discussed in details in Section 5.4.2

In small crystals on the contrary, the anisotropy energy is comparable to the thermal energy and the particle magnetization is no longer locked onto the anisotropy directions, allowing some electron spin precession around the direction of the B 0 field. Under these conditions the assumption of rigorous locking of the magnetization along the easy axes becomes less valid, which leads to the requirement of a new theory that introduces anisotropy energy as a quantitative parameter in the problem. However this model does not provide an analytical solution for the NMRD profile, and the fitting of experimental NMRD curves by a heuristic method, which in theory can lead to the determination of the anisotropy energy E a , is in practice time-consuming in numerical computing. 3,19 SPIONs are therefore mostly used with T 2 or T 2 *-weighted MR pulse sequences, introducing a "negative contrast" on images, i.e. they produce darker regions where they accumulate in comparison to pure buffer or tissues. The classification of MR CAs as either 'T 1 agents' or 'T 2 agents' is not always accurate, since any CA that shortens T 1 also shortens T 2 . However, any agent that reduces T 2 does not necessarily reduce T 1 , at least at the high magnetic field strengths used in clinical MRI scanners. Whether the CA functions as a 'T 1 agent' or 'T 2 agent' depends on the imaging sequence used, the magnetic field strength, the size of the CA, and how the CA is compartmentalized in the tissue. 16 Nevertheless, a good performance criterion for these materials is the ratio of relaxivities r 2 /r 1 at a given field/frequency value: One can expect positive contrast enhavement only for r 2 /r 1 < 4 at most (since the signal is measured by the antenna in the M xy plane, it is cancelled by any too large T 2 effect).

Magnetic Field Hyperthermia

Heat Dissipation Mechanism 5.

2.4.1

Both ferri/ferromagnetic materials and superparamagnetic NPs dissipate heat when subjected to an alternating magnetic field (AMF) due to several mechanisms. While ferri/ferromagnetic materials produce heat as a result of hysteresis losses, which can be measured by integrating the area of the static hysteresis loop of magnetization vs. DC field strength, superparamagnetic NPs dissipate heat through relaxation losses, categorized as Néel relaxation and Brownian relaxation. 20 The equations for the Néel and Brownian relaxation times were given earlier. The size of the superparamagnetic NPs determines the predominance of one relaxation type over the otherlosses by Néel relaxation are prevalent for small NPs, while Brownian relaxation dominates for larger NPs. [21][22][23] The anisotropy energy of a magnetic material also assigns the primary mode of relaxation since the Néel relaxation time varies exponentially with the anisotropy energy, while the Brownian relaxation time depends on both the suspension viscosity and the hydrodynamic volume of the particles. 24 For hyperthermia applications, relaxation through the Néel mechanism is preferable since a change in the medium viscosity is unavoidable once the particles are internalized in the cells, which hampers the free rotation of particles.

The combination of the two magnetic relaxation rates gives the overall effective relaxation rate of the particles: ⁄ ⁄ ⁄ . The reorientation of the magnetic moment between easy axes in a Néel relaxation regime with each oscillation of the external magnetic field creates an internal 'friction', while the rotation of the entire particle in the Brownian relaxation regime induces solvent velocity gradients and an external viscous friction. 23 Under exposure to an AMF oscillating faster than the relaxation time of the MNPs, both internal and external frictions lead to a phase lag between the magnetic moment of the particles and the external magnetic field. The delay in the relaxation of the magnetization response is described by a complex magnetic susceptibility ̃ , the thermal losses being related to the imaginary part of the susceptibility. 18,20 The dissipated heat power (P) is given by 25 :

where P is the heat dissipation value, is the permeability of free space, is the AC magnetic susceptibility (imaginary part), f is the frequency of the applied AC magnetic field, and H max is the amplitude of the applied AC magnetic field.

The heat dissipation ability of magnetic nanoparticles in an AMF is usually quantified through the specific absorption rate (SAR) expressed in Wg -1 (also referred to as the specific loss power -SLP): 21 Chapter 5

194
where C is the specific heat capacity of the sample (usually assimilated to the value of the solvent, C=4.18 J/°C/g for water), m is the mass concentration of magnetic material in suspension (g of Fe 3 O 4 / g of water) and ⁄ is the initial slope of the time-dependent heating curve (°C/s).

Tuning the SAR Values 5.2.4.2

A suspension of MNPs with a high SAR is highly desirable for clinical magnetic field hyperthermia (MFH), because a smaller amount of MNPs would then be required for in vivo injection. The SAR value of a magnetic fluid depends on the frequency and the amplitude of the alternating magnetic field, [26][27] and on instrinsic properties of the particles: their chemical composition, [27][28] size, 26,[29][30] size distribution, 30 shape, 29 saturation magnetization, and on their surface modification. 31 Maximizing the hysteresis loop area of ferri-or ferromagnetic materials requires a high magnetic field to overcome the coercive field H c , which is not clinically practical due to technical and physiological restrictions. The frequency for biomedical purpose has to be higher than 50 kHz to avoid neuromuscular electrostimulation, and lower than 10 MHz to achieve an appropriate penetration depth for the RF field. 32 In general, to avoid any detrimental effects on healthy tissues due to electromagnetic radiation exposure, the product of H max by f should not exceed an experimentally estimated threshold of 510 9 Am -1 s -1 , which is already less strict than the usually called "Brezovich criterion" H max f < 4.8510 8 Am -1 s -1 . 26,33 Multi-domain hysteretic NPs exhibit high SAR values at lower radiofrequencies (e.g. 100 kHz) but can require magnetic field strengths outside the allowed range (e.g., H max > 50 kA/m). On the contrary, superparamagnetic NPs can produce much more heat at lower field amplitudes (e.g., H max = 10 kA/m), characterized by high SAR values (up to hundreds of W/g),but they require radiofrequencies of few hundreds of kHz. 30 The chemical composition of particles is the most commonly cited parameter affecting magnetization, and thus the magnetic energy loss power and heating rate. Although they have the highest saturation magnetization values, pure transition metals are not ideal for biomedical applications as they are prone to oxidation, liberating toxic reactive oxygen species, and exhibit poor chemical stability. 20 The good magnetic properties and chemical stability of various pure and mixed metal ferrites have drawn the attention of many research groups. For example, mixed Co-Zn ferrite Co 1-x Zn x Fe 2 O 4 nanoparticles exhibit higher saturation magnetization than the corresponding mixed Mn-Zn ferrite Mn 1-x Zn x Fe 2 O 4 nanoparticles, and thus exhibit higher SAR values. 34 The magnetization and SAR values of mixed Fe-Mn ferrite nanoparticles Fe 1-x Mn x Fe 2 O 4 was found to vary with the Mn concentration: The values initially increased with x up to x = 0.4 before decreasing. 28 The dependence of the SAR on H max varied from linear to quadratic relations, which was attributed by the authors to "changes in ferrofluid composition", which may mean a transition from a superparamagnetic to a ferromagnetic state depending on the value of x. 28 The presence of both Zn and Mn in the ferrite structure (Zn 0.4 Mn 0.6 )Fe 2 O 4 increased the SAR value to 432 Wg -1 , 4 times higher than 115 Wg -1 reported for commercial Ferridex ® iron oxide nanoparticles under identical magnetic field conditions (500 kHz, 3.7 kAm -1 ). 35 Changing the composition is also a good method to modulate the Curie temperature of the particles (T C : the temperature above which MNPs exhibit zero magnetization, in the range of 500-600 °C for iron oxides), which allows the preparation of low Curie temperature MNPs that can perform "self-controlled" hyperthermia and prevent overheating. [36][37] The T C value of Y 3 Fe 5-x Al x O 12 ( 0 ≤ x ≤ 2 ) garnet sub-micron MNPs ranged from 280 to -40 °C, and their saturation magnetization decreased for increasing x, avoiding overheating at a desired temperature close to T C . 38 Replacing partially magnetic Fe 3+ ions by half of the same number of non-magnetic Ti 4+ ions in Mn 1+x Ti x Fe 2-2x O 4 structure decreased T C from 300 °C at x = 0 to 46 °C at x = 0.55, restricting heating to below 46 °C. 39 Similar substituted ferrite structures ZnGd x Fe 2-x O 4 x = 0.02 (T C = 45 °C) 40 and Mg 1+x Fe 2-2x Ti x O 4 x = 0.37 (T C = 46 °C) 41 were reported as potential heat mediators for self-controlled hyperthermia avoiding overheating of the surrounding healthy tissues. However due to their ease of synthesis and surface modification, combined with low toxicity and good biocompatibility, iron oxide NPs are still the most intensely studied materials for hyperthermia applications. 20 Since magnetic behavior is strongly size-dependent, varying the size can readily tune the magnetization, coercivity, blocking temperature, and thus the SAR values. The particle size d is a determining parameter of the SAR value, since it dictates the heating mechanisms that arise from either hysteresis losses or Néel and Brownian relaxation processes when the particles are placed in an alternating magnetic field. Hysteresis losses are responsible for the heating of large ferro-or ferrimagnetic nanoparticles while Néel relaxation dominates for NPs with d < 20 nm, and Brownian relaxation for NPs with d > 20 nm. 42 A threshold size of ca. 9.8 nm was established by Jeun et al. below which the SAR of particles was insufficient for hyperthermia applications, while particles in a size range of 11.8 to 22.5 nm had SAR values ca. one order of magnitude larger. 43 According to Lartigue et al. the threshold should be 10 nm, since no significant heating was generated below this particle size, while 35 nm rhamnose-coated Fe 3 O 4 MNPs had a SAR ~ 76 Wg -1 at 168 kHz and 21 kAm -1 . 44 Some 14 nm diameter Fe 3 O 4 MNPs yielded a SAR of 447 Wg -1 at 400 kHz and 24.5 kAm -1 , larger than for 5, 10, and 12.8 nm diameter particles. 45 A 4-fold increase in SAR values (from ∼10 to 40 Wg -1 ) was reported when the particle size increased from 8 to 11 nm, whereas only 40% increase in SAR values (from ∼ 40 to 55 Wg -1 ) was observed for an increase from 11 to 13 nm. 46 Additional values of SAR vs. particle size can be found in a recent review by Kolhatkar et al., 37 although comparisons among literature values are not straightforward since many authors quote different magnetic field intensities and frequencies (most of the time without stating how they were measured), and the experiments suffer from different sources of uncertainties and the lack of standardization of the setups and methods.

In an attempt to rationalize the design of MNPs for magnetic hyperthermia, the optimal volume of MNPs with random orientation of the moments was suggested by Carrey et al. to achieve the highest SAR value as: 47

( ) ⁄ Chapter 5
197 where is the Boltzmann constant, T is the absolute temperature, f is the applied field frequency, is the attempt time, K eff is the anisotropy constant, is the field amplitude, is the magnetic permability of vacuum, and M s is the saturation magnetization. Along the same line, according to Krishnan et al., for an AMF at f = 373 kHz with = 14 kAm -1 , depending on the anisotropy constant K eff of magnetite (which varies from below 10 to above 20 kJ/m 3 , depending on the samples and authors), the optimal diameter can range from 13 to 17 nm. 48 One parameter that is often not taken into account in the literature is the direct effect of temperature on the SAR, even for iron oxide NPs far below T C . For example, Garaio et al. 49 observed a 40% decrease in SAR between 10 and 60 °C in a multi-parametric study of dynamic hysteresis losses with size-graded maghemite NPs by AC magnetometry.

Size control alone may be insufficient to ensure the desired results since the size, shape and structure of particles have a complex influence on the SAR. 29 The optimal size to achieve the highest possible SAR value was suggested to be near the transition from superparamagnetic to ferromagnetic behavior. 30 Hergt et al. used magnetic separation to investigate the influence of the size distribution on the SAR values, and found that the imaginary magnetic susceptibility spectra "(f) and the SAR both varied remarkably with the mean particle core diameter. 29,50 A narrow size distribution and an optimal mean core size (near the transition from superparamagnetic to ferromagnetic behavior) resulted in high SAR values. [29][30] Later on Garaio et al. 49 showed that passing the theoretical size maximizing the SAR also resulted in a transition from a negative thermal behavior (SAR decreasing with temperature) to a positive one (SAR increasing with temperature), as predicted by the socalled "linear relaxation theory" (LRT) introduced by Rosensweig. 26 Negative influence of the size dispersity of magnetite particles on the SAR values is also expected from the LRT model, indicating the advantages of more uniform samples. 50 Fractionation of a ferrofluid to improve the size distribution of the particles thus increased the SAR values from 60 to 332 Wg -1 (at f = 400 kHz, H max = 10 kAm -1 ). 51 Pegylated Fe 3 O 4 @PEG 6000 with a smaller magnetically active core with diameter d = 10 nm, a broader size distribution (standard deviation 3 nm), and a lower anisotropy constant yielded a significantly lower SAR of 60 Chapter 5 198 Wg -1 in comparison to Fe 3 O 4 @PEG200 reaching 195 Wg -1 for d = 12 nm, a standard deviation of 2 nm, and a higher anisotropy constant. 52 Even though this has been the scope of intensive research, there are still no clear conclusions on the effects of particle shape on the SAR, based on the limited studies currently available. A maximum SAR of 2450 Wg -1 was measured for cubic Fe 3 O 4 with an edge length of 19 ± 3 nm (at f = 520 kHz, H max = 29 kAm -1 ), 53 while larger-sized Zn 0.4 Fe 2.6 O 4 nanocubes (edge length 40 nm) exhibited a maximum SAR of 4060 Wg -1 , but at even higher field strength (at f = 500 kHz, H max = 37.4 kAm -1 ). 54 Highly uniform quasicubical (9.6 nm) MNPs synthesized by thermal decomposition exhibited a higher SAR value than spherical ones with an equivalent size. 55 As another significant example, a SAR of 1990

Wg -1 under an AMF at f = 700 kHz of moderate intensity, H max = 21.5 kAm -1 , was obtained for nanoparticles qualified as 24 nm flower-like assemblies composed of multiple smaller grains of maghemite (11 nm as determined by TEM and X-rays diffraction). Such a high value was ascribed to the cooperative combination of moments (these nanoflowers behaving like large magnetic monodomains). 56 Another possibility to obtain high SAR values is to play on an exchange-bias mechanism between a superparamagnetic shell built around an antiferromagnetic core. This was achieved for FeO@Fe 3 O 4 nanospheres of 19 nm ± 15% diameter, exibiting SAR values at 700 kHz (484 W/g for 2 mM [Fe]) analogous to Fe 3-x O 4 nanocubes with an edge of 16 nm ± 12% (450 W/g for 2 mM [Fe]. 57 According to the heat dissipation equations from the linear rexation theory (LRT), the SAR values increase with an increase in frequency f or applied field amplitude H max , thereby reducing the therapeutically needed concentration of MNPs. 30 However, this strategy also unavoidably increases the power dissipated by the electric field via Joule effect in healthy tissues surrounding the tumor ("eddy currents" heating) and reduces the selectivity of the treatment. 58 To minimize unwanted and non-selective heating generated in healthy tissues, the product H max f should not exceed an experimentally estimated threshold of 510 9 Am -1 s - 1 . 26 Various studies focused on selecting an optimal set of H max f and d to be used in hyperthermia. 30,47 The SAR heating characteristics of MNPs are often studied at various applied fields, to reveal its dependence on magnetic field amplitude and frequency.

In order to be useful for biomedical applications, MNPs are usually coated with a shell that prevents them from aggregating (which can also change their magnetic properties) and increases their biocompatibility by excluding them from reticulo-endothelial clearance, thus extending their circulation time in the bloodstream. The nature of the protecting material determines how it interacts with the crystal core and modifies the magnetic properties of the whole system. Silica coating was thus found to increase the surface spin disorder of Fe 3 O 4 @SiO 2 nanoparticles, inhibiting a fraction of Fe 2+/3+ cations from being magnetically active at room temperature and resulting in low SAR values. 59 Negative effects for surface coating by phosphorylated methoxy poly(ethylene glycol) (mPEG) on the SAR values of magnetite NPs were also reported. 60 Short mPEG chains ( w = 2,000 g/mol) provided Fe 3 O 4 cores with d(Fe 3 O 4 ) = 9 and 19 nm with good colloidal stability, and maintained good magnetization properties. Lower SAR values were observed when the w of mPEG was increased to 5,000 and 20,000 g/mol, but larger cores d(Fe 3 O 4 ) = 31 nm needed mPEG w = 5,000 g/mol to be stable since shorter chains (2,000 g/mol) were insufficient to prevent aggregation, while longer chains (20,000 g/mol) formed a layer too thick for efficient heating. 60 A surface coating with optimal thickness can maintain and sometimes improve the magnetization properties by hindering agglomeration of the MNPs, which otherwise leads to strong dipolar intearactions hampering Néel relaxation. Chitosan coating also improved the SAR value of Fe 3 O 4 @chitosan to 119 Wg -1 , as compared to 79 Wg -1 for bare NPs (f = 265 kHz, H max = 26.7 kAm -1 ). 61 A similar influence on the heating efficiency of ferromagnetic La 0.7 Sr 0.3 MnO 3 particles was observed with a dextran coating. 62 Similarly, a bilayer of oleic acid improved the hyperthermia properties of La 0.7 Sr 0.3 MnO 3 NPs from 25 to 40 Wg -1 ( f = 267 kHz, H max = 26.7 kAm -1 ) by enhancing the colloidal stability of the suspension. 63 The physicochemical properties of a magnetic suspension such as the ionic strength, medium viscosity, and temperature also influence its heating efficiency. A decrease in the chitosan coating thickness of La 0.7 Sr 0.3 MnO 3 @chitosan, induced by increased ionic strength, Chapter 5 200 also resulted in agglomeration and a decreased SAR value. 63 A viscous medium slows down particle rotation, therefore limiting heat generation by Brownian relaxation. 20 A systematic drop in heating efficiency for various MNPs was reported when Brownian relaxation was inhibited, either in the cellular environment or when the nanoparticles were attached to the cell membrane. 64 The SAR values of NPs with several volume-average sizes (12, 14, and 15 nm) decreased with the temperature of the measurements, whereas the opposite trend was observed for the largest size sample (D v = 16 nm). 49 The extent of SAR variations with temperature was also reported as being frequency-dependent for maghemite NPs, as interepreted within the frame of the LRT model for these size-graded samples. 49 In order to compare the SAR values obtained by different research groups, measured at different frequencies and applied field amplitudes, the intrinsic loss power 65 parameter defined as ⁄ was introduced. Since the ILP is obtained by normalization of the SAR values through the frequency and the square of the field amplitude, it is theoretically independent of the magnetic field parameters. However the linear dependency of the SAR on frequency and the quadratic dependency on the field strength can be questioned in practice, so the usefulness of the ILP is not universally recognized among researchers.

Experimental Procedures

Magnetic Polyion Complex Micelle Preparation

The uncoated (UC) magnetic nanoparticles G1@Fe 3 O 4 used for the magnetization measurements were prepared by alkaline coprecipitation in the presence of the G1 arborescent copolymer template (Section 3.3.6). The Fe 2+ and Fe 3+ iron oxide precursors were loaded in the template by mixing the iron salts stock solution with G1 in HCl solution at pH 1.4. Concentrated NH 4 OH was then added to trigger the formation of colloidal Fe 3 O 4 .

Chapter 5

201

The magnetic nanoparticles (MNPs) were washed and redispersed in HNO 3 solution at pH 1.4 to produce a black homogeneous ferrofluid.

The magnetic polyion complexation (MPIC) micelles used for the magnetization measurements were prepared from the G1 templated magnetic nanoparticles G1@Fe 3 O 4 and the PAA 27 -b-PHEA 260 block copolymer with f = CO 2 H/N = 0.5. Briefly, the G1@Fe 3 O 4 ferrofluid prepared in HNO 3 solution at pH 1.4 (0.99 mg Fe 3 O 4 , 1.9 mg of G1, 16.7 µmol of N functional groups) was loaded in a 10 mL vial. An aqueous solution of PAA 27 -b-PHEA 260 at pH 7 (9.6 mg, 5 mg/mL, 8.3 µmol of CO 2 H functional groups) was quickly added, the mixture was left to react for 1 h with stirring before adjusting the pH to 4.7, and stirring for 1 h with a magnetic stirring bar at 400 rpm. The pH was adjusted further to 7 and the solution was stirred for 30 min. The solution was dialyzed against Milli-Q water (5 L) for 48 h before it was collected and stored at 10 °C.

Characterization

Vibrating Sample Magnetometry 5.

3.2.1

Magnetometry curves for the Fe 3 O 4 NPs (for field strengths up to 5 T) were recorded using a Quantum Design™ Physical Property Measurement System (PPMS ® ) with the vibrating sample magnetometer (VSM) device. At room temperature, a sufficiently concentrated (ca. 1 mg/mL) aqueous suspension of Fe 3 O 4 NPs was moved periodically (at fixed frequency) in a magnetic field varying in strength from 0 to 5 T. In this experiment the voltage induced in the detection coils, which is proportional to the magnetic moment, is recorded vs. field strength or temperature. The diamagnetic contribution from water and the container are subtracted from the data obtained.

Relaxometry

5.3.2.2

These measurements were performed in the NMR and Molecular Imaging Laboratory of Professors Sophie Laurent and Luce Vander Elst, and the Biomedical Physics Unit of Professor Yves Gossuin at UMONS, Belgium.

The longitudinal (R 1 ) and transverse (R 2 ) relaxation rates were measured at 0.47 and 1.41 T on Minispec mq20 and mq60 relaxometers (Bruker, Karlsruhe, Germany). Nuclear magnetic relaxation dispersion (NMRD) profiles were measured using a Stelar Spinmaster fast-field-cycling (FFC) NMR relaxometer (Stelar, Mede PV, Italy) over a magnetic field strength range of 0.24 mT to 0.7 T. A Bruker AMX 300 (7 T) spectrometer was also used for the high-field measurements. The measurements were performed on 0.6 mL samples contained in 10 mm (outside diameter) Pyrex tubes at 37 °C.

The saturation magnetization and the size of the superparamagnetic crystals, , were determined by fitting the NMRD curves with the MINUIT minimization program using the outer-sphere model and a standard phenomenological approximation. [66][67] In most of the plots shown in this work, the magnetic field is expressed in terms of the proton Larmor frequency: A field of 1 T corresponds to a Larmor frequency of 42.6 MHz. The results are represented in terms of longitudinal and transverse relaxivities r 1 and r 2 , respectively.

The iron concentration was determined from the longitudinal relaxation rate (R 1 ) measurements at 0.47 T and 37 °C after microwave digestion (MLS-1200 MEGA, Milestone) into ferric ions with a mixture of nitric acid and hydrogen peroxide. 68 The iron content of the sample was also verified by UV-VIS analysis. 69

Specific Absorption Rate Measurements 5.3.2.3

To minimize the effects of magnetic field inhomogeneities, a small NMR plastic tube was totally filled with the sample. The diameter (5 mm external) was not so small as to cause large thermal losses, and the volume (0.35-0.4 mL) was as reported in the literature for MFH experiments on cells. 24 The well-defined sample geometry (cylindrical shape and large aspect Chapter 5 203 ratio), and the sample was oriented parallel to the coil axis to allow minimization of the socalled "demagnetization effect", similarly to a VSM experiment. 14 The setup for hyperthermia measurements in Bordeaux (Figure 56) was used to create a high frequency alternating magnetic field (f = 755 kHz, H max = 10.2 kAm -1 ). The tubes were placed in a double-walled jacketed vessel, maintained at a temperature of 20 °C during the treatment by a thermostated circulating bath (Huber Polystat CC). The whole system was inserted in an induction coil (4 turns with 1 cm spacing, 55 mm diameter, cooled by internal water circulation at 18 °C). The coil was fed with a current of 234 A at full power of the generator (3 kW MOSFET solid state resonant circuit, Seit Electronica unior™, Treviso, Italy) producing a high frequency alternating magnetic field (f = 755 kHz, H max = 2.8 kAm -1 at minimum power, H max = 10.2 kAm -1 at full power according to a calibration by Dr Eneko Garaio with RF current measurement techniques). The temperature of the sample was monitored with a fiber optic thermometer (OTG-M420, Opsens, Québec, QC, Canada). designed for in vivo hyperthermia measurements with quad frequency capability. The simulation shows that the coil was specially designed with an observation window.

Results and Discussion

The terminology G1@Fe 3 O 4 used to refer to the magnetic nanoparticles (MNPs), and G1@Fe 3 O 4 @PAA-b-PHEA for the magnetic polyion complex (MPIC) micelles in Chapter 4, does not fit well within the scope of this Chapter, since we deal with MNPs and MPIC micelles synthesized under different conditions. Consequently, we will hereafter use a three- Therefore a sample labeled as 0.7-80-BC corresponds to a Fe/N ratio of 0.7, a reaction temperature of 80 °C, and particles coated with the block copolymer.

Since the superparamagnetic character of the MNPs and MPIC micelles will be confirmed by vibrating sample magnetometry in Section 5.4.1, the term superparamagnetic iron oxide nanoparticles (SPIONs) will also be used to refer to them.

Vibrating Sample Magnetometry

The magnetic properties of the Fe3O4 nanoparticles were characterized with a vibrating sample magnetometer (VSM) at 300 K. The magnetization curves of M versus the applied field strength H (between -4000 kAm -1 and +4000 kAm -1 ) obtained for the various samples are shown in Figure 5 As the applied field was increased from 0 to 1000 kAm -1 , the magnetization M rose sharply and gradually reached saturation around 2000 kAm -1 . Since only 1 or 2 experiments were used to obtain these results, their statistical significance is unfortunately limited and they should be viewed with caution. However, the Fe/N ratio in the template, the temperature, and the coating material all influenced the magnetization properties of the NPs to some extent (Table 5-1).

At both temperatures tested (80 and 100 °C), the G1@Fe 3 O 4 @BC samples prepared with a molar ratio Fe/N = 0.7 had a higher magnetization than those produced with Fe/N = 1, due to the formation of magnetite NPs with a larger median diameter and a narrower size distribution. Figure 5-8a depicts the higher saturation value of magnetization for 0.7-80-BC as compared to 1.0-80-BC. The former sample, with less Fe 2+/3+ ions involved in the coprecipitation reaction, also had a larger median diameter D 0 (6.26 vs. 6.08 nm), a more These results can be explained as follows:

1. An increased number of nuclei formed in the nucleation step of the akaline coprecipitation reaction when the Fe 2+ /Fe 3+ ions were present at a higher concentration (for Fe/N = 1.0 as compared to 0.7), leading to a decrease in the radius of the MNPs. As stated in the LaMer Chapter 5 207 model of nucleation-growth, 70 a minimum concentration of monomer (in our case: Fe 3 O 4 ) is needed to overcome the energy barrier of the nucleation process: burst nucleation occurs at that point, and stable nuclei are formed. A higher monomer concentration results in a larger number of nuclei and thus of final NPs, but with a smaller median diameter (e.g. D 0 = 6.08 nm for 1.0-80-BC), while a lower monomer concentration leads to a smaller number of nuclei and NPs, but with a larger median diameter (e.g. D 0 = 6.26 nm for 0.7-80-BC). templating effects achieved with a proper Fe/N ratio. The templating effect for a material with limited accessibility such as the G1 copolymer may be complex, especially since the influence of simple ligands like oleic acid and citric acid on NP size still is unclear. [71][72][73][74][75][76] The ratio of metal cations to organic agents can either lead to the hindrance of nucleation (resulting in larger particles) or the inhibition of crystal growth (resulting in smaller particles).

3. Uneven/non-homogeneous crystal growth of the embryonic nuclei due to limited accessibility of the G1 template. The diffusion and adsorption of the solute on the surface of the crystals were presumably facilitated for particles located near the G1 template surface as compared to those buried inside, leading to the formation of both small and large particles. Samples coated with block copolymer (BC) PAA 27 -b-PHEA 260 had a higher magnetization, a larger median diameter and a narrower size distribution than the UC (uncoated) samples (Table 5-1). The magnetization of the "optimal" G1@Fe 3 O 4 @PAA 27 -b-PHEA 260 nanoparticles (denoted as 0.7-80-BC) was 59.6 emu/g at 300K, which is 65% of the magnetization of bulk magnetite (∼92 emu/g). The good magnetic properties are attributed to the morphology of these G1@Fe Attempts to oxidize G1@Fe 3 O 4 to G1@Fe 2 O 3 with Fe(NO 3 ) 3 , followed by a size grading step using a concentrated HNO 3 solution, led to destruction of the templating effect:

The G1@Fe 2 O 3 @PAA 27 -b-PHEA 260 NPs had a large  300 nm, which is less useful for in vitro applications.

For samples 0.7-80-UC and 0.7-80-BC, we observed a discrepancy between the diameters calculated from the VSM analysis (D n = 6.8-7.2 nm) and measured by TEM (D TEM = 12.1  2.00 nm), which has often been ascribed to spin canting occurring at the surface or within the particles. 71,77 The spin canting effect at the surface of nanoparticles has been described as misaligned spins or non-colinear arrangement, and related to local changes in magnetic anisotropy induced by breaking of the quantic exchange bonds between adjacent metal centers. [77][78][79] Spin canting within the volume of the particles occurs because of Fe 3+ vacancies, which are more frequent in maghemite. 77,79 spin canting as a result of a disordered structure induced by the perturbed oxidation state of these NPs. Larger NPs displayed a core-shell morphology of magnetite and maghemite, which explained why their magnetization depended on the oxidized shell thickness, defects and surface spin canting.

Relaxation Properties

Relaxivity Measurements at 0.41 and 1.47 T

5.4.2.1

Relaxivity measurements were performed for all the uncoated (UC) and block copolymer-coated (BC) magnetic nanoparticles at 0.41 T (20 MHz) and 1.47 T (60 MHz) field strengths, that closely match the magnetic field strengths of most clinical MRI systems (0.5 T and 1.5 T, although human 3 T MRI is also frequent in hospitals). The longitudinal r 1 and transverse r 2 relaxivities acquired at 0.41 and 1.47 T displayed similar trends (Table 5-2).

Since only 1 or 2 experiments were used to obtain these results, their statistical significance is unfortunately limited and they should be viewed with caution.

A good T 2 contrast agent has a high r 2 and r 2 /r 1 ratio. The transverse relaxivity r 2, the most important parameter for negative CAs, expresses the increase in the T 2 -relaxation per millimolar concentration of magnetic centers. The ratio r 2 /r 1 provides a precise picture of the availability of nuclear longitudinal magnetization after each subsequent acquisition for signal averaging. 80 This ratio increases with the particle volume, and the threshold value for an effective negative contrast is above 3-4. 80 Ultrasmall superparamagnetic iron oxide particles (USPIOs, D h < 40 nm) composed of a monocrystalline core can produce a low r 2 /r 1 < 3-4 (at 20 MHz) allowing them to perform as "positive contrast agents" (producing brighter regions where they are delivered) as long as they remain individually dispersed in the suspension. [81][82][83] Superparamagnetic iron oxide particles (SPIOs, 60 nm < D h < 150 nm) containing polycrystalline clusters are often reported to have high r 2 /r 1 ratios, making them "negative contrast agents", i.e. producing dark zones associated with a high local accumulation of Chapter 5 211 CA. 80 Values of r 2 /r 1 reaching several hundreds can be obtained, depending on the magnetic field strength of the MRI scanner. Block copolymer-coated SPIONs display higher r 2 and r 2 /r 1 values than uncoated SPIONs. The transverse relaxivity of SPIONs is significantly greater than their longitudinal relaxivity, which explains their effectiveness as negative contrast agents for T 2 -weighted imaging. The lack of block copolymer coating favors the agglomeration of uncoated samples, leading to decreased relaxivities due to exchange between the water molecules inside the agglomerates (which relax rapidly) and the bulk water (relaxing more slowly), as previously reported by Roch et al. 84 By preventing agglomeration, a block copolymer coating is thus essential to maintain good MRI contrast properties.

Sample 0.7-80-BC (with Fe/N = 0.7) displayed larger r 2 and r 2 /r 1 values than 1.0-80-BC, suggesting that 0.7-80-BC would be a better contrast agent. Increasing the coprecipitation temperature to 100 °C led to some agglomeration, as can be interpreted from the decreases in both r 1 and r 2 observed at both frequencies. The most promising sample, 0.7-80-BC, has r 2 reaching 269 s -1 mM -1 with r 2 /r 1 = 9.9 at 20 MHz, and r 2 = 336 s -1 mM -1 with r 2 /r 1 = 31.4 at 60 MHz.

In comparison with different commercial materials, sample 0.7-80-BC can be viewed as an efficient negative contrast agent characterized by comparably high relaxivities, which allows smaller injection amounts for the patients. Sample 0.7-80-BC is more efficient than AMI-25 Ferumoxide (Endorem) 85 and IOs (Resovist) 86 , but less effective than IO-loaded PLGA-mPEG. 86 The relaxivities of commercial USPIOs, used as positive contrast agents, are also listed in Table 5-3. The relaxivities and magnetization properties of superparamagnetic iron oxide, both commercialized and in the literature, were compiled in a paper published by Vuong et al. 19 These authors suggested a universal scaling law to compare the efficiency of magnetic Chapter 5 213 nanoparticles as MRI T 2 -contrast agents (Figure 5-9), using data from their own samples and from the literature, provided that these follow the outer-sphere theory or the so-called Redfield condition Δω < 1, where =  0 M v /3 is the angular frequency shift experienced by a proton at the equator of a particle, = 2.67513×10 8 rads -1 T -1 is the gyromagnetic factor of the proton, and µ 0 = 410 -7 TmA -1 is the magnetic permeability of vacuum. The square of the specific magnetization (denoted as M s in this work) was used as a normalization parameter. The intra-aggregate volume fraction of magnetic material  intra was used for clusters and hybrids to correct the relaxivity as r 2 ' = r 2 ×  intra .

A good correlation 88 was found, as shown in Figure 56789:

⁄

We applied this correlation to evaluate the intra-aggregate volume fraction for sample 0.7-80-BC, using r 2 = 335.8 s -1 mM -1 (measured at 1.41 T), , and . The specific mass-magnetization of the MPIC micelle extracted from the NMRD profile (Table 5-4)

was also converted, using the respective densities  1 gcm -3 for the polymer and  5 gcm -3 for iron oxide, into the specific volume-magnetization Am Finally, the transverse relaxivity r 2 = 335.8 s -1 mM -1 is located in the high end of the overall spectrum, which suggests good potential of this suspension as a T 2 MRI contrast agent.

NMRD Profiles 5.4.2.2

The relaxivities of the uncoated (UC) and block copolymer-coated (BC) magnetic nanoparticles prepared by alkaline coprecipitation at various Fe/N templating ratios and temperatures were studied. The nuclear magnetic relaxation properties of a compound can be further explored through its nuclear magnetic resonance dispersion (NMRD) profile, a curve of relaxivity change vs. the external magnetic field strength, providing a tool to estimate the spectral density . The NMRD profiles of longitudinal relaxivity r 1 vs. frequency (Figure 5-10) were obtained from measurements on a fast field cycling (FFC) relaxometer.

Since only 1 or 2 experiments were used to obtain these results, their statistical significance is unfortunately limited and they should be viewed with caution.

These curves (Figure 5-10) exhibit no low-field dispersion (the left part of curve is flat), which is typical of large superparamagnetic iron oxide nanoparticles for which the anisotropy energy E a is sufficiently large in comparison with the thermal energy for the direction of the crystal magnetic moment to be locked to the anisotropy axes, precession of the electronic magnetization being thus forbidden. 89 This plateau observed at low magnetic fields is ascribed to the magnetic fluctuations arising from jumps of the moment between different easy axes (Néel relaxation). The precession limitation is introduced in the "outersphere" model equations by setting the electron Larmor precession frequency to zero. The longitudidal relaxation dispersion profile r 1 () is generally governed by the so-called Freed equations and originates from the modulation of dipolar interaction by the characteristic times of Néel relaxation ( N ) and water diffusion ( D ), respectively. 18,89 At high fields the magnetic moment is locked onto the magnetic field direction, following the Curie law of paramagnetism (SPIONs rather follow the equivalent Langevin law for superparamagnetism, but the "outer-sphere" model was originally designed for paramagnetic contrast agents like gadolinium complexes). Therefore Néel relaxation is restricted and the so-called Curie relaxation dominates. Curie relaxation refers to the relaxation of water protons in a locally fluctuating inhomogeneous dipolar magnetic field induced by motions of the water molecules relatively to the SPIONs. Here the translational diffusion correlation time is much shorter than the Néel relaxation time ), thus is dominated by water diffusion and the Freed equations are reduced to the Ayant equations. In particular, the drop of r 1 () to zero for high fields / frequencies is explained by the absence of a "secular term" in the right-hand part of one of these equations (too long to be reproduced here). At intermediate magnetic fields / frequencies, both the local magnetic field (Curie relaxation) and the fluctuating magnetic moments (Néel relaxation) contribute to the relaxation of water protons spins. 3,18 The degree of orientation of the magnetic moment depends on B 0 , and therefore the relaxation rate is dominated by the terms proportional to the squared Langevin function. 89 This gives a maximum in the curve of r 1 () proportional to the square of the saturation magnetization of the sample, which is caused by the increased magnetization and the successive alignment of the particle magnetic moments under the influence of the increased magnetic field. 66,91 Numerical fits were done using the MINUIT minimization program within the framework of the "outer-sphere" model and a standard phenomenological approximation [66][67] assuming that each profile corresponds to a log-normal size distribution. The divergence between the NMRD curves for the uncoated G1@Fe 3 O 4 magnetic micelles (UC samples) and the fits (Figure 5-11) is most likely due to the multimodal size distribution and the significant contribution from the hydrophobic polymeric G0PS-P2VP core to the NMRD profiles, that may not simply lead to reduced magnetization but also to other effects such as reduced water diffusivity in the vicinity of the micelles. Storage of the uncoated G1@Fe 3 O 4 magnetic micelles in dilute HNO 3 (pH 1.4) may also have caused partial oxidation and/or agglomeration, thus resulting in a multimodal size distribution. The interference of nonmagnetic materials partially impermeable to water (for example SiO 2 ) with the relaxivity of SPIONs was reported earlier: A good fits was observed for bare Fe 3 O 4 SPIONs, but not for the SiO 2 -coated NPs. 92 Subtraction of the diamagnetic contribution from silica was suggested to obtain better fits. 92 This solution is not applicable to our magnetic micelles however, due to their inhomogeneous morphology with Fe 3 O 4 NPs encapsulated within the porous G0PS-g-P2VP micelles, with their internal polar P2VP surface that i) disturbs dipolar coupling between neighboring crystals, decreasing the anisotropy energy, and ii) partially inhibits the exchange of water molecule in both directions (from the outside in, and from the inside out), thus interfering with the translational diffusion of water molecules.

The fits to the NMRD curves for the block copolymer-coated (BC) samples G1@Fe 3 O 4 @PAA-PHEA obtained by the same protocol suffers from less interference (Figure 5-12), presumably because the Fe 3 O 4 crystals are covered by a layer of doublehydrophilic copolymer, which prevents their aggregation. For every pair of analogous BC and UC samples, the BC sample exhibits a higher relaxivity (suggesting lower anisotropy Chapter 5

218 energy E a ) than the UC sample at very low magnetic fields. The presence of a hydrophilic layer such as PHEA should increase the inter-crystal distance, reducing the dipolar coupling energy between neighboring crystals. Large dipolar interactions are known to increase the anisotropy energy, slowing down Néel relaxation, and thus decreasing the proton relaxivity at very low magnetic fields (and also the SAR, which is likewise related to  N ). Therefore the repulsive effect of the PHEA shell is favorable. Similar phenomena were observed by Laurent et al. 93 for dextran-coated SPIONs, and by Sharifi et al. 94 for carboxyethylsilanetriolcoated SPIONs. 84 This effect was ascribed to exchange between the "rapidlyrelaxing-water-inside-the-agglomerates" and the "slowly-relaxing-water-in-the-bulk". The essential role of the block copolymer coating in preventing the agglomeration is thus demonstrated for the magnetic G1@Fe 3 O 4 arborescent copolymer micelles. (1) Average crystal radius ( ). At high magnetic fields, the relaxation rate only depends upon the translational correlation time , which can be extracted from the inflection point where . An equivalent crystal / outer-sphere radius can be calculated from the equation ⁄ , where D is the translational diffusion coefficient of water at the measurement temperature. 66,80,95 (2) Specific magnetization (M s ). At high fields, M s can be obtained from the equation

√ ⁄
, where C is a constant and R max is the maximum relaxation rate. 66,80,95 Significant divergence was observed when fitting the data for all the samples synthesized with Fe/N = 1.0, probabely due to clustering of non-templated Fe 94 which is analogous to our results ( ). Laurent et al. reported a larger increase from 40 to 55 nm, definitely related to the increase in the w of dextran coating the particles from 35,000 to 200,000 g/mol. 93 Hannecart et al. also reported an increase in outer-sphere radius between MNPs coated with small molecules (silanes) vs. NPs grafted with a thermosensitive polymer shell: The gap increased from 0.5 nm when the chains were swollen by water at low temperature, to 1.8 nm when the shell became impermeable to water above the lower critical solution temperature (LCST), a plausible value based on the radius of gyration estimated under poor solvency conditions for PPO-co-PEO (2,000 g/mol). 83 The UC samples stored in diluted HNO 94 On the other hand, the temperature of the coprecipitation reaction did not show a significant influence on the M s and values. The radius obtained by relaxometry is 33% larger than from TEM, and 43% larger than from magnetometry (assuming a log-normal distribution of sizes when calculating the volume-average diameters), which can be explained by the fact that fitting of the NMRD profiles leads to the distance of closest approach (also called the outer sphere diameter) of water molecules to the superparamagnetic surface, thus comprising the hydrophobic polymer part (excluding water penetration), whereas the TEM and VSM data give the radius of the crystallites and the magnetic core, respectively. The ratios of crystal radii found were in the range of 1.4-1.5, likely due to the presence of the hydrophobic PS-P2VP core.

Bare SPIONs and SPIONs with a thin dextran coating were reported to have 

1.1-1.4, [92][93]96 while this ratio rose to 1.5-3.9 for SiO 2 -coated SPIONs as the SiO 2 coating thickness was increased, even though it was partially permeable to water, since silica synthesized by a sol-gel process has a mesoporous struture. 92 The magnetization obtained from NMRD is lower than by magnetometry, as a result of a larger sample size probed by relaxometry. The magnetization value for individual Fe 3 O 4 crystals is not changed, but rather diluted in a larger volume: The particles behaves as though they have a larger size, but are less magnetized. 92 We found ratios  0.4-0.5, close to values reported by other groups (  0.5-0.8). 91,96 Increasing the thickness of the diamagnetic SiO 2 coating reduced this ratio to 0.1. 92 In conclusion, the transverse relaxivity r 2 and r 2 /r 1 ratio measured for magnetic micelles 0.7-80-BC are relatively large when compared to commercial products, indicating good potential of these materials for T 2 -contrast enhancement. The minimal proton approach size and relaxivity of this magnetic micelle are well-fitted by the "outer-sphere" (OS) model extensively described previously in the literature. The NMRD profiles for the uncoated and coated magnetic micelles revealed: i) the determining effect of size dispersity on the quality of the fits; ii) the contribution of the hydrophobic polymer template to the dipolar coupling energy between neighboring crystals, affecting the anisotropy energy through its shape and demagnetization-effect component; iii) the limited accessibility of water near the superparamagnetic surface, hampering the translational diffusion of water; and iv) the agglomeration of uncoated samples due to the lack of a protecting layer, flattening the curves and resulting in poor fits. The role of a hydrophilic stabilizing shell in preventing agglomeration is therefore essential. The NMRD curves for samples synthesized with the optimal ratio Fe/N = 0.7 can be fitted quite well with the OS model, suggesting that these samples contain a narrow size distribution. The presence of the double-hydrophilic diblock copolymer led to a larger equivalent OS radius and was found crucial to maintain a high magnetization for the SPIONs. The discrepancy observed when comparing parameters obtained by relaxometry and magnetometry was explained by the different nature of these methods: the equivalent OS radius derived from relaxometry is larger than from magnetometry, due to the diamagnetism and the partial impermeability of the polymer shell to water. The magnetization obtained from NMRD was lower than by magnetometry due to dilution of the magnetic moment in the micelle volume (since the magnetization M s is, by definition, the concentration of moments per unit micelle volume). The specific magnetization and Néel relaxation phenomenon will be of utmost importance when dealing with the last property for which the magnetic micelles were designed, namely magnetic field hyperthermia intended for anticancer therapy (cytotoxicity).

Hyperthermia

The specific absorption rate (SAR) of the magnetic micelles was determined by AMF calorimetry. The SAR values were calculated from ⁄ , the initial rate of temperature rise measured in water when the magnetic micelles suspension was placed in the AMF generated by a solenoid coil fed by the power generator. The SAR values were corrected for thermal losses of the calorimeter and parasitic heating of the coil by the Joule effect (in spite of being refrigerated by internal cold water circulation) using pure water as a reference blank, and were normalized by the iron oxide content. The specific absorption rate of the magnetic micelle suspensions increased with the magnetic field amplitude H max and frequency f, and was found to be independent of the concentration of magnetic material. 50 A maximum SAR value of 55.6 Wg -1 was measured at f = 755 kHz and H max = 10.2 kAm -1 . Recently, some authors 97 suggested subtracting from the positive slope of the T versus time curve when the AMF is ON, the negative slope when the AMF is OFF (for reach instantaneous temperature)

to correct for non-adiabatic conditions, a procedure which we have also used.

The heating characteristics of SPIONs are a complex function of the individual crystal properties, and of the structure and state of the crystals in the particles and in suspension. 98 Therefore, the SAR heating characteristics need to be evaluated at various field amplitudes and frequencies. According to the heat dissipation equations of the linear relaxation theory (LRT) introduced by several authors (Rosensweig, Hergt, Pankhurst…), the SAR values increase with the frequency f and the applied field amplitude H max . We determined the SAR values as a function of magnetic field amplitude H max for five fixed frequencies f available on the two experimental setups used (Figure 5-13). In the literature, it is described that the field amplitude dependency of SAR follows power laws of nearly second order for superparamagnetic samples, and higher order (e.g. three to four) for ferromagnetic samples above a threshold field strength identified as the anisotropy field H K , as observed for various ferrofluids. [30][31]50,53,[99][100][101] For the magnetic micelles studied here, the SAR vs. H max power trend lines at high and medium frequencies were below, but nevertheless close to second order (slopes on double-logarithmic plots of 1.73 at 755 kHz,

1.68 at 473 kHz, and 1.56 at 344.5 kHz), which is consistent with the superparamagnetic character of the magnetic micelles, and with Néel relaxation being the main contribution to the heating process. Moreover, the size dispersity of samples was also suggested as a factor responsible for the deviations between experimental results and LRT predictions. 50 However, a closer look at the fundamental equation of LRT giving the dissipated power shows that the SAR cannot be perfectly linear with frequency, since P is a linear function of f but also proportional to the imaginary part of magnetic susceptibility, , which reaches a maximum near the characteristic relaxation rate = + of the magnetic NPs. As a result of the near-to-square dependence of the SAR of magnetic micelles on the field magnitude and its near-to-linear dependence on the field frequency, the linear correlation of the SAR with the product H max 2 f is only valid at the highest frequency tested (755 kHz) but not for the lower ones, with much lower correlation coefficients R 2 (Figure 5-15). The parameter H max 2 f has actually been suggested as a normalization factor to yield the intrinsic loss power ⁄ parameter, 65 with the idea to allow a more or less direct y = 0.003x 1.203 y = 0.003x 1.295 y = 0.001x 1.598 y = 0.003x 1.487 y = 0.005x 1.421 y = 0.010x 1.331 y = 0.246x 0.760 In conclusion, double-hydrophilic BC-coated magnetic micelle suspensions exhibited good SAR values for different field conditions. Nearly square correlation between the SAR values and the magnetic field amplitude was observed for high-and medium-range frequencies, while a less pronounced dependence was obtained at lower frequencies. These variations in comparison to the LRT predictions are most likely related to i) the complex internal structure of our magnetic micelles leading to dipole-dipole interactions between neighboring crystals, moderated by complexation of the iron oxide surface with the doublehydrophilic block copolymer, and ii) restricted Brownian relaxation, particularly at lower radiofrequencies. Both of these characteristics conflict with the models routinely used to characterize the SAR as a function of H max and f. Nevertheless, the good SAR values Frequency f determined for these coated magnetic micelle suspensions justify the next step of the work, which is in vitro hyperthermia-induced cytotoxicity assays on cancer and control cells.

Conclusions

The magnetic properties of Fe3O4 nanoparticles (bare and coated with a doublehydrophilic block copolymer) were characterized by vibrating sample magnetometry. All the The relaxivities of all the uncoated (UC) and block copolymer-coated (BC) magnetic nanoparticles were measured. The coated SPION 0.7-80-BC had superior colloidal stability, which prevented their agglomeration and led to higher r 2 = 335 s -1 mM -1 and r 2 /r 1 = 31.4

values (1.47 T, 37 °C) than the uncoated SPIONs, suggesting that they could be efficient negative contrast agents for T 2 -weighted imaging. Samples produced with Fe/N = 0.7 also displayed better relaxivity (larger r 2 and r 2 /r 1 ratio) than for Fe/N = 1 ratio, and are thus considered better contrast agents. When compared with commercial products and from the literature, sample 0.7-80-BC fared well as an efficient negative contrast agent, characterized by comparably high relaxivities, allowing smaller injection doses for the patients.

The nuclear magnetic resonance dispersion (NMRD) curves for the MNPs exhibited no low-field dispersion. The divergence between the NMRD curves obtained for the uncoated magnetic micelles and fits using the equations from the outer-sphere theory was assigned to the broader size distribution and to significant interference from the hydrophobic PS core and P2VP surface on the water diffusivity and exchange rates probed in the NMRD profiles. Uncoated (UC) samples were characterized by low relaxation rates and flat NMRD profiles indicating agglomeration, thus confirming the essential role of the block copolymer coating. The radii obtained from NMRD analysis were larger than those from TEM (by ca.

33%) and from magnetometry (by ca. 43%), which can be explained by the different nature of these characterization methods, for which the polymer component of the micelles can be invisible (TEM), non-magnetic (VSM), and partially impermeable (NMRD). The magnetization values obtained from the fits to the NMRD profiles were smaller than those determined by magnetometry, as a result of a larger effective sample size for relaxometry and dilution of the iron oxide in the polymer (pushing further the limit of the outer-sphere).

The specific absorption rate (SAR) of the magnetic micelles was determined by AMF calorimetry. A maximum SAR value of 55.6 Wg -1 was measured at f = 755 kHz, H max =10.2 kAm -1 . The SAR values obtained had a nearly square dependence on the magnetic field amplitude H max at fixed frequency f in the high-and medium-frequency ranges, consistent with the superparamagnetic nature of the coated micelles and a main contribution from Néel relaxation to the heating process. Closer-to-one exponents of the SAR vs. H max were observed at lower frequencies and were explained by the contribution of Brownian relaxation of the MNPs to the heating process, and the enhancement of dipole-dipole inter-crystal interactions, despite the positive effect of the hydrophilic polymer coating separating multiple magnetic cores within the same G1 micelle.

Chapter 6 Biocompatibility Assessment

and In Vitro Cell Hyperthermia

Introduction

Nanomaterials have significantly different properties and reactivity as compared to their bulk material counterparts, in part due to their larger surface area which enables an exponential increase in the number of molecules exposed on the surface as the particle size decreases to < 100 nm. 1 These unique properties provide nanomaterials with immense potential for various applications. Nanomaterials offer innovations in biomedical imaging, sensing, diagnosis, and in designing novel approaches to the treatment of diseases.

Translating an application from the laboratory to clinical settings requires the evaluation of the impact of nanomaterials on cellular and animal models under acute or chronic exposures, and of the fate of nanoparticles in biological systems (i.e., their clearance mechanisms). 2 Biocompatible nanoparticles must resist premature enzymatic degradation and aggregation, be able to target specific tissues, cross biological barriers such as epithelia (e.g., the blood brain barrier), extracellular matrix, and eventually the cell plasma membrane, and to perform their desired function without inducing toxicity or an immune response. [3][4] Potential toxicity and the response of cells upon exposure of nanomaterials must therefore be investigated.

Toxicity is a complex event in vivo since it is related to direct cellular damage, as well as physiological, inflammatory, and other systemic effects. Even for in vitro assays it is currently difficult to monitor systemic and physiological effects, so most assays investigate effects at the cellular level, or cytotoxicity. 5 Although cytotoxicity assays simplify the events that might occur, they are still useful as a first screening step before further investigations are performed because they are more economical, easily quantified, reproducible, and more ethically acceptable than animal testing. The evaluation method for cytotoxicity used herein was adapted from ISO 10993-5 (2009) guidelines, using the mouse fibroblast L929 line. The MTS assay was used to determine whether the NPs have effects on cell proliferation or have direct cytotoxic effects leading to cell death. In this assay, cells in the exponential phase of growth are exposed to the nanoparticles for a set duration for maximal damage to occur. Cell proliferation is allowed to occur for two to three population-doubling times. All the viable cells, those capable of proliferation as well as those that remain viable but cannot proliferate, are distinguished from dead cells. 5 Cell viability in the cultures was monitored by the reduction of clear yellow tetrazolium salt (MTS) to its brown soluble formazan product upon exposure to NADH-dependent cellular oxidoreductase enzymes found in the mitochondria of metabolically active cells. [6][7] While negatively charged MTS does not readily penetrate cells and must be used with an intermediate electron acceptor to transfer electrons from the cytoplasm or plasma membrane and facilitate its reduction, the MTS assay is still preferable to MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), because the formazan products generated by the reduction of MTS are soluble in the cell culture medium rather than in organic solvents such as DMSO, thus making the protocols more convenient. 8 Understanding how nanoparticles interact with cells is a first step in assessing the biomedical functions and toxicity of nanomaterials, allowing the prediction of the toxicological consequences of nanoparticles in vivo. It is known that nanoparticles, due to their small size, readily penetrate through cell membranes and can translocate among targeted cells, tissues, and organs, as required for efficient diagnostic or therapeutic functions. 9 Study of the cell internalization mechanism of nanoparticles is thus necessary towards understanding their biological fate. It is noteworthy recalling the dynamic structure of the plasma membrane, composed of lipid bilayers and membrane proteins that segregate the cytoplasm from the external medium and regulate the transport of matter into the cells. Cell membranes are semi-permeable and allow only small molecules to pass through via diverse mechanisms that are either passive or active (consuming the biochemical fuel ATP molecules). The free diffusion of oxygen, carbon dioxide, water, and small hydrophobic molecules through the plasma membrane is driven by concentration gradients, while ions and amino acids penetrate the cell via the active transport system of integral membrane protein pumps or ion channels. Nanoscale hydrophilic biomacromolecules are usually transported by endocytosis. 8 Endocytosis is a form of active transport in which cells uptake objects by invagination of the plasma membrane, enclosing them into vesicles called "endosomes".

These processes occur mainly via phagocytosis, pinocytosis, and caveolae-dependent or Chapter 6 235 clathrin-mediated endocytosis. [10][11][12] Particles with large diameters (> 750 nm) are uptaken via the phagocytosis pathway, through the formation of an internal phagosome usually observed in specialized mammalian cells (like monocytes, macrophages, and neutrophils). 13 Pinocytosis or macropinocytosis occurs in almost all cell types to uptake smaller particles ranging from a few to several hundred nanometers. Clathrin-mediated endocytosis is an energy-dependent pathway that allows cargo deposited in small endocytic vesicles (usual diameter < 100 nm) to fuse with early endosomes. [14][15] The relationship between NP size, 9,16- 17 shape [17][18] or charge 14,17,19 and its uptake kinetics and mechanism have been extensively studied. It is well-known that nanoparticles of uniform size with diameters below 200 nm and homogeneous in composition are desirable for systemic circulation, i.e., long blood halflives, especially if they coated with a neutral polymer sheath. [3][4] Spherical NPs with a diameter of 50 nm are taken up by mammalian cells at a faster rate and at higher concentrations than other sizes and shapes. 9,16 Positively charged particles are expected to be more efficiently endocytosed than negatively charged ones due to their effective binding to the negatively charged cell surface. This expectation was proven by a 2-fold higher uptake of positively charged particles into HeLa cells when compared with negatively charged nanoparticles of equal size (80 nm). 14 However the work of Zhang et al. depicted higher uptake for negatively charged quantum dots into HEK cells, which was explained by a lipid raft-mediated mechanism, different from the clathrin-dependent pathway reported for positively charged NPs. 19 Further investigation is therefore needed to determine the relationship between NP size, surface charge, and internalization pathways.

To target biomedical applications for MPIC micelles as MRI contrast agent or in hyperthermia treatment, the cytotoxicity and cell internalization of the MPIC micelles need to be evaluated in vitro. The same protocols were performed with PIC micelles, to provide an in vitro biocompatibility platform for their future biomedical use (e.g., biosensor, drug delivery, gene delivery, cancer therapy). The study of the interactions of nanoparticles with the cells is the first step to predict the toxicological consequences of nanoparticles in vivo and to reveal the fate of the NPs once they enter the organism. The cellular uptake, location of the Chapter 6

236

NPs and any biological consequences, such as cytotoxicity of the MPIC micelles were thus investigated herein. An in vitro study of magnetic field hyperthermia was also performed. In this case a colloidal suspension of MNPs (the MPIC micelles) was incubated with the cells, allowing the MNPs to be internalized through the cell membrane. The application of an external alternating magnetic field (AMF) induced heat in the MNPs to kill the cells, presumably by an oxidative pathway (ROS production) as described in recent literature. [20][21][22] In our work, the incubation concentration of MPIC micelles and the AMF exposure time strongly influenced the viability of murine L929 and human glioblastoma U87 cells.

Experimental Procedures

Block Copolymer Synthesis

The chemicals, procedures and characterization techniques for the synthesis of the

Polyion Complex Micelle Preparation

The polyion complex (PIC) micelles used for the biocompatibility tests in this

Chapter were prepared from a G1 arborescent graft copolymer (G0PS-g-P2VP) and the DHBC by the procedure described in Section 3.

Magnetic Polyion Complex Micelle Preparation

The magnetic polyion complex (MPIC) micelles used for biocompatibility tests in this Chapter were prepared from the G1 templated magnetic nanoparticles G1@Fe 3 O 4 and the DHBC obtained by the procedure of Section 3.3.6. Briefly, the magnetic nanoparticles G1@Fe 3 O 4 were prepared by coprecipitation in the presence of the G1 copolymer as a template. The iron oxide precursors were loaded by mixing Fe 2+ sulfate and Fe 3+ chloride salts stock solutions (at 1:2 stoichiometric ratio) with the G1 template in HCl solution at pH

Cell Cultures 6.3.4.2

The fibroblast-like L929 cells derived from normal subcutaneous areolar and adipose tissue of a 100-day old male C3H/An mouse 23 was purchased from LifeTechnologies. This murine fibroblast cell line is recommeneded for cytotoxic assays of nanomaterial in the ISO10993-1:2009 procedure "Biological evaluation of medical devices". Both cell types (U87 and L929) were cultured in DMEM supplemented with 10 vol% of heat-inactivated fetal bovine serum (FBS), 1 vol% of antibiotics (10,000 units of penicillin and 10,000 units of streptomycin/mL), 1 vol% sodium pyruvate and 1 vol% GlutaMAX. The cells were maintained at 37 °C in a 5% CO2 humidified incubator. After rinsing with PBS (Ca 2+ /Mg 2+ free) solution to remove the serum, the cells were detached with trypsin solution 10× diluted 10-fold with PBS (Ca 2+ /Mg 2+ free) buffer solution.

Cytotoxicity Assessment 6.3.4.3

Cell viability was evaluated via the MTS assay. After detachment, the L929 or U87 cells were seeded in a 96-well plate at a density of 2×10 3 cells/well, and grown in DMEM complete medium in a humidified incubator (5 vol% CO 2 ) at 37 °C for 24 h prior to treatment. The PIC micelle solutions with different final concentrations in the culture medium (10, 2, 1, 0.4 and 0.2 mg of PIC micelles/mL) were prepared in a mixture of 10% PBS solution (pH 7.4, 154 mM NaCl, Ca 2+ /Mg 2+ free) and 90% DMEM complete medium before cell treatment. The solutions of MPIC micelles were also prepared in the same mixture consisting of 10 vol% PBS solution (pH 7.4, 154 mM NaCl, Ca 2+ /Mg 2+ free) and 90 vol% DMEM complete medium with 700, 140, 70, 28, and 14 µg of Fe 3 O 4 /mL final concentrations before use. The highest Fe 3 O 4 concentration of 1250 µg/mL was tested only with the G1@Fe 3 O 4 @PAA 27 -b-PHEA 260 f = 0.5 MPIC micelles. The cells were exposed to either the PIC or MPIC micelle solutions, incubated for 48 h, and rinsed with PBS (with Ca 2+ /Mg 2+ ) solution (100 μL/well). A 120 μL aliquot of MTS solution (containing 20 μL of MTS solution and 100 μL of PBS with Ca 2+ /Mg 2+ ) was then added into each well. The plates were incubated at 37 °C for 120 min. Cell viability in the cultures was monitored by the reduction of clear yellow trezolium salt (MTS) to the brown formazan product by NADHdependent cellular oxidoreductase enzymes found in metabolically active cells. [6][7] The absorbance at 490 nm was measured on a PowerWave X (BioTek Instrument Inc.) microplate UV-VIS spectrometer. The positive controls were cells incubated with the complete media alone. The results were expressed as the percentage of metabolic activity of treated cells relatively to untreated cells (control, 100% viability). Independent experiments were performed 3 times with 4 replicates per condition. According to ISO 10993-5 ( 2009) guidelines, the cytotoxicity potential was considered less than 70% of the blank control.

Cell internalization Studies 6.3.4.4

Confocal Laser Scanning Microscopy

The mouse fibroblast-like L929 cells were seeded on the surface of a sterile glass coverslip placed in the well of a 12-well plate at a density of 5×10 4 cells/2 mL/well and Chapter 6 241 allowed to attach and to grow at 37 °C in a 5% CO2 humidified incubator. The fluorescently labeled PIC micelle solution (2 mg of PIC micelles/mL) was prepared in a mixture of 10% PBS solution and 90% DMEM complete medium before cell treatment. The solutions of fluorescently labeled MPIC micelles were prepared in the same mixture consisting of 10 vol% PBS solution and 90 vol% DMEM complete medium at 140 µg of Fe 3 O 4 /mL. After 24 h of cell growth, the DMEM complete medium was replaced with 2 mL of either fluorescently labeled PIC or MPIC micelle suspensions. From this point the plate was protected from light exposure by wrapping in aluminum foil when it was not in use. After 24 h of exposure to the nanoparticles, the suspension was removed and the cells were rinsed twice with PBS (with Ca 2+ /Mg 2+ ) solution to completely remove non-uptaken nanoparticles.

The cells were then fixed with 2 mL of paraformaldehyde cross-linking agent (4 vol%) diluted in PBS (with Ca 2+ /Mg 2+ ) solution at 4 °C for 20 min. The fixative solution was then removed and the cells were washed twice with PBS (with Ca 2+ /Mg 2+ ) buffer solution.

Permeabilization of cell membranes to provide access to intracellular or intraorganellar antigens 24 was performed by treating the cells with 2 mL of 0.5 vol% Triton X-100 in PBS (with Ca 2+ /Mg 2+ ) solution for 20 min at 4 °C. The Triton X-100 surfactant solution was removed and the cells were rinsed twice with PBS (with Ca 2+ /Mg 2+ ) buffer solution. The protein-binding sites on the membranes were blocked by incubating with 1 vol% BSA in PBS (with Ca 2+ /Mg 2+ ) solution (2 mL) at 37 °C for 30 min. The blocking agents were removed and the cells were washed twice with PBS (with Ca 2+ /Mg 2+ ) solution before 2 mL of 1.67 vol% of Alexa Fluor® 594 phalloidin in PBS (with Ca 2+ /Mg 2+ ) solution was added to label the F-actin cytoskeleton of the fixated cells. The actin staining was allowed to occur for 1 h at 37 °C in the humidified incubator before the cells were washed twice with PBS (with Ca 2+ /Mg 2+ ) buffer solution. The cell nuclei were then stained with 2 mL of 0.025 vol% of DAPI in PBS (with Ca 2+ /Mg 2+ ) solution for 10 min at 4 °C. The staining solution was finally removed and the cells were rinsed twice and stored in 0.05 vol% of Tween-20 in PBS (with Ca 2+ /Mg 2+ ) buffer solution, to improve the assay sensitivity by reducing background interference. The coverslips were picked up, allowed to dry for 2 h, turned over and permanently mounted to the glass slide using one drop of VECTASHIELD HardSet Mounting Medium (refractive index 1.452).

Analysis of the treated L929 cells was performed using a Leica TCS SP5 confocal laser scanning microscope equipped with a 405 nm diode UV laser for blue fluorescent labeling (DAPI filter set, Ex: 405 nm, Em: 461 nm), an argon laser for green labeling (Green Fluorescent Protein filter set, Ex: 488 nm, Em: 525 nm) and a DPSS 561 laser for red fluorescent labeling (Alexa filter set, Ex: 581 nm, Em: 609 nm). The scan speed was 400 Hz.

The used objective was a Leica HCX PL APO 63x magnification /1.3 numerical aperture objective lens embedded in 100% glycerol (refractive index 1.46). Images were taken at 1024 pixel × 1024 pixel resolution. Photomultiplicator gain and offset configurations were set up on control cells so as to correct the images for green auto-fluorescence. A Z-stack of 50 frames covering a depth of 10 μm was recorded for assessment of the NP distribution across the section.

Transmission Electron Microscopy

The mouse fibroblast-like L929 cells were seeded in a 6-well plate at a density of 2.5×10 5 cells/2 mL/well and allowed to grow at 37 °C in a 5% CO2 humidified incubator. The MPIC micelle solution (140 µg of Fe 3 O 4 /mL) was prepared in a mixture of 10% PBS solution and 90% DMEM complete medium before cell treatment. After 24 h the DMEM complete medium was replaced with 2 mL of MPIC micelle suspension. After 24 h of exposure to the nanoparticles, the suspension was removed and the cells were rinsed twice with PBS (without Ca 2+ /Mg 2+ ) solution to completely remove non-uptaken nanoparticles.

After being detached with trypsin solution 10× diluted 10-fold in PBS (without Ca 2+ /Mg 2+ free) buffer solution, the cells were centrifuged at 200 rcf for 5 min. The trypsin solution was carefully removed and the cells were fixed with a 2.5% glutaraldehyde solution diluted in PBS solution at 20 °C for 30 min. The cells were then centrifuged at 200 rcf for 5 min, the fixative was removed, and the cells were washed and redispersed in H 2 O.

For observation by transmission electron microscopy, the cells were first placed in vials where they were post-fixed for 30 min in 1% OsO 4 , washed with water, and air-dried.

Epoxy resin (Epon 812, AGAR 100) was directly cast and polymerized in the vial. To facilitate sectioning, the cell resin was isolated and cut in two halves before re-embedding in resin prepared in flat silicone rubber molds. Sections with a thickness estimated at approximately 200 nm were cut with a glass knife. Observation was made without further staining on a Philips 301 electron microscope at an accelerating voltage of 80 kV.

Fluorescence-Activated Cell Sorting

The mouse fibroblast-like L929 cells were seeded in a 24-well plate at a density of 4×10 5 cells/well and allowed to grow at 37 °C in a 5% CO2 humidified incubator. For the kinetics experiments, the fluorescently labeled MPIC* micelle solution (140 µg of Fe 3 O 4 content/mL) was prepared in a mixture of 10% PBS solution and 90% DMEM complete medium before cell treatment. For concentration effect experiments, the MPIC* micelle solutions were also prepared in a mixture of 10% PBS solution and 90% DMEM complete medium at various concentrations (1.4, 14, 28, 70 and 140 µg of Fe 3 O 4 /mL). After 24 h the DMEM complete medium was replaced with 1 mL of MPIC micelle suspension. The magnetic nanoparticle exposure time periods for cells in the kinetics experiments were 1, 4, 8, 12 and 24 h, while all the cells used in the concentration tests were exposed to the magnetic nanoparticles for 24 h. After desired exposure to the nanoparticles, the suspension was removed and the cells were rinsed twice with PBS (without Ca 2+ /Mg 2+ ) solution to remove non-uptaken nanoparticles. After being detached with trypsin solution 10 diluted 10-fold in PBS (without Ca 2+ /Mg 2+ ) solution, the cells were centrifuged at 200 rcf for 5 min.

The trypsin solution was removed before the cells were fixed with a 4% solution of paraformaldehyde diluted in PBS solution at 20 °C for 30 min. The cells were then centrifuged at 200 rcf for 5 min, the fixative was removed, and the cells were washed and redispersed in PBS solution. The control cells were prepared using the same protocol but without being exposed to fluorescently labeled magnetic nanoparticles. Quantitative studies were performed using the fluorescence-activated cell sorting (FACS) technique on a FACScan system (FACS, Becton-Dickinson). The mean fluorescence intensity (MFI) of 10,000 treated L929 cells was compared with that of 10,000 untreated cells. The percentage of fluorescence-positive cells per 10,000 cells was also reported. The mean values are reported with the standard deviations from three independent experiments.

In Vitro Magnetic Field Hyperthermia

The mouse fibroblast-like L929 cells and human glioblastoma U87 cells were seeded in a 48-well plate at a density of 2×10 4 cells/0.5 mL/well and allowed to grow at 37 °C in a 5% CO2 humidified incubator. The MPIC micelles were prepared in DMEM complete medium at 1250, 700, and 140 µg of Fe 3 O 4 /mL before cell treatment. After 6 h the DMEM complete medium was replaced with 0.5 mL of magnetic PIC micelle suspension and the control cells were incubated in complete media alone. After overnight exposure (15 h) to the nanoparticles, the suspension was removed and the cells were rinsed twice with PBS (without Ca 2+ /Mg 2+ ) solution to remove non-uptaken nanoparticles. After being detached with trypsin solution 10 diluted 10-fold in PBS (without Ca 2+ /Mg 2+ free) buffer solution, both control and treated cells were centrifuged at 200 rcf for 5 min. To minimize the effects of magnetic field inhomogeneities a small sample volume (0.35-0.4 mL) is recommended, 25 so the cells were suspended in small NMR plastic tubes. This choice was also made to control the local magnetic field experienced by the cells, through well defined sample geometry (cylindrical shape with a large aspect ratio), location in the solenoid (6 NMR tubes can be placed simultaneously near the center of the coil) and orientation (parallel to the coil axis). This minimizes the so-called "demagnetization effect". On the other hand, the diameter was not so small as to increase the effect of thermal losses. Each magnetic field treatment was applied on 6 tubes (3 samples, 3 controls). The trypsin solution was removed before the cells were redispersed in 0.35 mL of DMEM complete medium (containing 10 mM HEPES, to maintain the pH in the absence of a controlled CO 2 pressure) and transferred to sterile vials (0.4 mL, 3 mm diameter). These vials, containing cells with or without internalized MPIC micelles Chapter 6 245 (controls), were placed in a small bath of de-ionized water inside a double-walled jacket maintained at a temperature of 37 °C during the treatment. The whole system was inserted in an induction coil (4 turns with 1 cm spacing and 55 mm diameter, cooled by internal water circulation at 18 °C, as shown on Figure 6-1. calibration by an electrical engineering method). Two field exposure times were tested: 1.5 and 3 h. The temperatures of the media in one control sample and in one treated sample were simultaneously monitored with two fibre optic thermometers (OTG-M420, Opsens, Québec, QC, Canada). After exposure to the RF magnetic field, the cells were reseeded in a 48-well plate and incubated at 37 °C in a 5% CO2 humidified incubator. After 15 h of incubation the complete medium was removed and the cells were rinsed with PBS (with Ca 2+ /Mg 2+ ) solution (0.5 μL/well) before the addition of 60 μL of MTS solution (prior dilution in 300 μL of PBS with Ca 2+ /Mg 2+ ). The plates were incubated at 37 °C for 120 min before measurement of the absorbance at 490 nm. The results were expressed as the percentage of intracellular reductase activity for the treated cells relatively to the untreated cells (control, 100% viability). Independent experiments were performed twice.

Results and Discussion

Fluorescent Labeling of Block Copolymer

As discussed in the previous Chapters, among the four PAA-b-PHEA block copolymers synthesized, PAA 27 -b-PHEA 260 exhibited the best stabilizing ability when complexed with the G1 arborescent copolymer and G1@Fe 3 O 4 magnetic nanoparticles, and thus was selected to be labeled with the fluoresceinamine dye. Fluorescenly labeled PAA 27 *b-PHEA 260 was obtained via amidation of a fraction of the carboxylic groups in the PAA block with fluoresceinamine in the presence of EDC 26 as illustrated in Scheme S6-1. EDC reacted with the carboxylic acid groups to form a highly reactive but short-lived Oacylisourea intermediate, which reacted with the primary amine group on fluoresceinamine (FA) to form an amide bond.

An emission band, absent for the non-labeled block copolymer, appeared when PAA 27 *-b-PHEA 260 was excited at 488 nm (Figure 6-2). A bathochromic shift in the emission peak from 530 nm (for free FA) to 543 nm (for FA conjugated with the block Chapter 6 247 copolymer) was observed, evidencing covalent linking of the dye to the block copolymer. This phenomenon has also been reported for fluorescein isothiocyanate (FITC) and is related to local changes in the environment of the dye (pH, temperature, ionic strength), [27][28][29] and in particular to polarity (dielectric constant) changes when conjugating FITC with bovine serum albumin (BSA). 30 Therefore the bathochromic shift in the emission peak observed for PIC* and MPIC* micelles (Figure 6-3) indicates the presence of conjugated FA molecules in the micellar structures. The presence of fluorescence emission for MPIC* also shows that the absorbance by iron oxide in the short wavelength region of the UV-VIS spectrum does not impede the fluorescence of the dye, at least within the iron oxide concenration range tested. of up to 40 mg/mL. 31 The cell toxicity of poly(2-vinylpyridine) has not been studied as much as for its quaternized derivatives, likely due to its hydrophobicity. For example, quaternized poly(4-vinylpyridine) is a microbicidal polymer that is effective to kill a wide range of bacteria; however this polymer is not biocompatible, with LD 50 = 2330 mg/kg obtained in acute toxicity experiments. [32][33][34] Due to the hydrophobicity of PS and P2VP at physiological pH, these polymers would be most likely isolated within the core of the micellar system, thus shifting the need for biocompatibility to the protecting shell, in our case, PAA and PHEA.

PAA has been used as an adhesive and surface-protective agent in dentistry [35][36] and for the slow release of drugs in eyedrops, [37][38] suppositories, capsules, wart creams, [39][40][41] and in ophthalmic gels. 42 PAA used as a coating polymer for superparamagnetic iron oxide NPs increased the colloidal stability and the biocompatibility of the magnetic particles. [43][44][45][46] PHEA has similar biocompatibility including low cytotoxicity and cell compatibility, [47][48][49][50][51] similarly to the more widespread poly(2-hydroxyethyl methacrylate) (PHEMA). PHEA-based materials have been used in controlled release drug delivery 52 and in cell culture media without significant cytotoxicity, indicating that PHEA-based materials are useful for in vitro applications. 50 Cell viability after exposure to the PIC micelles was assessed using the MTS cell metabolic activity test. The fibroblast-like L929 cells were incubated for 48 h with micelle concentrations ranging from 0.2 to 10 mg/mL, to reveal toxicity exerted by the polymeric nanoparticles. Within the concentration range tested, none of the PIC micelles displayed any cytotoxicity on the L929 cells (Figure 6

MPIC Micelles 6.4.2.2

The cytotoxicity of the MPIC micellesif significantcould be due to their hybrid polymeric and iron oxide composition. The cytotoxicity of bare Fe 3 O 4 nanoparticles has been reported to be dose-dependent based on MTT cell viability studies conducted with a short time exposure (24 h). [53][54] At the lowest concentration tested (50 µg of Fe 3 O 4 SPION/mL), 20% loss of cell viability was reported as compared with the control. NPs reported in the literature, indicating that the G1 template had no significant effect on L929 cell viability in itself. SPIONs. [57][58][59][60] Among various mechanisms for iron oxide toxicity, one suggestion was the highly localised accumulation of NPs within the targeted tissue (necessary to maximize the therapeutic or diagnostic benefits), in combination with the degradation of Fe 3 O 4 into dissolved Fe 2+ and Fe 3+ ions. 61 The iron overload can cause aberrant cellular responses including cytotoxicity, DNA damage, oxidative stress, epigenetic events and inflammatory processes. 57,[62][63][64] Several mechanisms for iron-induced carcinogenesis have been suggested

and associated to the damage of DNA and proteins, and to lipid peroxidation. [65][66][67][68] The generation of reactive oxygen species (ROS) was also suggested as a mechanism by which Fe 3 O 4 NPs induce (geno)toxicity. 60 Dissolved Fe 2+ ions can cross the mitochondrial membrane and react with hydrogen peroxide and oxygen (produced by the mitochondria), forming highly reactive  OH and radicals which causes damage in vivo to DNA, proteins, polysaccharides and lipids. 69 It is well-established that NPs without any coating designed to avoid this are rapidly covered by a layer of proteins and other biomolecules when they are in contact with a biological fluid such as serum. 10,[70][71][72][73] This biomolecule corona confers a new identity to the NPs and is sufficiently stable to be retained when the NPs are internalized into the lysosomes. The degradation of this biomolecule corona inside lysosomes exposes the bare surface of the NPs, resulting in lysosomal damage, cytosolic release of the lysosomal content and apoptosis (assuming that the NPs have cytotoxicity). [74][75][76] However in more complicated physiological environments such as the bloodstream, surface opsonisationinteraction of the NPs with opsonins, i.e., blood-circulating proteinsleads to conformational rearangements of the opsonins, inducing biorecognition by phagocytes that triggers a protein complement activation and results in a phagocytic removal process. 77 Surface coatings using flexible and hydrophilic polymers provides a solution to prevent opsonisation, thus extending the circulation time of the NPs. [78][79] Various natural (dextran, hyaluronic acid, chitosan, etc.) and synthetic polymers (poly(vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol) (PEG), PEG-based copolymers, etc.) have been used for that purpose. [77][78][79][80][81][82] Coated Fe 3 O 4 NPs were thus found to be relatively nontoxic, even at high concentrations. In one study, 99% of telomerase-immortalized primary human fibroblast cells remained viable relatively to the control after 24 h of exposure to PEG-coated nanoparticles at a concentration of 1 mg Fe 3 O 4 /mL. 53 Using the same cell type, only 8% cell loss was observed for incubation with pullulan-coated Fe 3 O 4 NPs (2 mg of Fe 3 O 4 /ml) for 24 h, preventing the iron oxide core from interacting with the cells. 54 Solutions of 100 nM of poly(maleic anhydride-alt-1-octadecene)-co-poly(ethylene glycol) PMAO-PEG-coated Fe 3 O 4 in water (0.023 g Fe 3 O 4 /mL) led to less than 5% of SK-BR-3 human breast cancer cell death after 24 h of incubation. 83 However cell cytotoxicity strongly depends on the type of cell tested, the density and the chemical nature of the coating. Thus after 24 h of incubation with 170 µg of Fe 3 O 4 /mL solution of amino-SPIONs (polymer/Fe coating ratio 20), less than Chapter 6 254 40% of human melanoma metastasis Me237 cells were viable; the viability reported for Me275 cells was even lower, below 20%. 84 SPIONs coated with less polymer agglomerated and were no longer internalized by cells, thus showing no cytotoxicity. 84 Wan et al. attested that for an iron concentration of 400 µg/mL (ca. 550 µg of Fe 3 O 4 /mL), MPEG-Asp 3 -NH 2coated iron oxide nanoparticles had no cytotoxicity on OCTY mouse cells, while MPEG-PAA-coated and PAA-coated iron oxide nanoparticles significantly reduced cell viability, resulting in 84 % cell loss. 85 In our work, we studied the cytotoxicity for four types of MPIC micelles. Two of them were the magnetic nanoparticles G1@Fe 3 O 4 coated with the PAA 13 -b-PHEA 150 block copolymer at two different ratios, f = 1 (optimal in term of colloidal stability) and f = 0.5, while the other two were for the same magnetic core complexed with Our work was performed with fibroblast-like L929 cells derived from normal subcutaneous areolar and adipose tissue of a 100-day old male C3H/An mouse. 23 Diseased tissues such as tumors have a metabolism different from healthy tissues and a much higher NP uptake, 93 suggesting that the effectiveness of the NPs in their targeted biomedical applications (MRI contrast enhancement and hyperthermia) would be even greater in diseased tissues.

Transmission Electron Microscopy 6.4.3.2

The internalization mechanism of MNPs into cells has been the topic of different studies. Bertorelle et al. reported that rhodamine-grafted iron oxide nanoparticles were internalized into HeLa cells through small 100 nm vesicles, followed by tranfer into larger Chapter 6 259 600 nm endosomes. 88 Lu et al. 87 relied upon various pharmacological inhibitors to investigate the uptake mechanism. The uptake of SiO 2 -coated superparamagnetic iron oxide nanoparticles labeled with FITC (SPIO@SiO 2 -FITC) was impeded by clathrin and actin inhibitors but not by caveola or microtubule inhibitors, which strongly suggested that the internalization pathway was via clathrin-and actin-dependent endocytosis, with subsequent intracellular localization in late endosomes/lysosomes. The colocalization of the NPs inside the lysosomes was further confirmed by overlapping of the green fluorescence from the SPIO@SiO 2 -FITC nanocomposites and the red fluorescence from the lysosome marker, which resulted in a merged yellow fluorescent signal. 87 Negatively charged SPIONs coated with poly(isobutylene-alt-maleic anhydride) were first found in endosomes and subsequently in lysosomes, while positively charged SPIONs coated with polyethylenimine were found exclusively inside lysosomes. 94 An energy-dependent clathrin-mediated endocytosis pathway was suggested by Soenen et al. 90 based on the localization of MNPs in lysosomes, and the fact that none of the MNPs tested were incorporated by endocytosis at 4 °C, the ATP hydrolysis-dependent mechanisms being impeded at this low temperarure.

In our work, TEM was used to confirm internalization of the MPIC micelles, and to determine the distribution of MNPs after the cellular uptake. Iron oxide crystallites appeared with high contrast inside large intracellular endosomes or lysosomes visible in a clearer area that could be differentiated from the grey background of the cytoplasm (Figure 6-10). This location of the iron oxide crystallites is in good agreement with the results reported in the literature, suggesting that MPIC micelles were also internalized through the cell membrane by clathrin-dependent endocytosis (Figure 6 Extending the incubation period further enhanced the cellular uptake. After 12 h of incubation, 45.6% of the cells fluoresced with an MFI = 444 units while after 24 h, these figures were 97.2% and 1067 MFI units. Linear regressions for the MFI and % positive cells vs. incubation time extracted from the data suggests a steady rate of MNP internalization (within the time interval tested), which could be used as a predictive tool for cellular uptake kinetics (Figure 6

In Vitro Cellular Radiofrequency Magnetic Field Hyperthermia

Hyperthermia is a medical treatment that brings body tissues to slightly higher temperatures than the highest tolerable physiological value (42 °C) for the purpose of making the cells more sensitive to radiotherapy or to specific drugs. [95][96] However this therapy has been used more widely to damage or kill cancer cells by provoking cell apoptosis [97][98] (also called programmed cell death 98 that occurs spontaneously in cells), thus retarding tumour growth. While the exact mechanism by which hyperthermia kills cells is still an open question, membrane alteration and cytoskeletal damage such as cell rounding and blebbing due to heat-induced protein denaturation are found to be the most apparent phenomena leading to cell death. [99][100] As compared to normal cells which can maintain physiological temperatures (i.e., up to 42 °C) during hyperthermia treatment, tumour cells were discovered to have a disorganized and abnormal vasculature architecture, resulting in poor heat dissipation capability and thus are more sensitive to heat-induced-apoptosis. 93,101 As compared to whole-body hyperthermia and regional hyperthermia, local hyperthermia is considered a safer and less invasive technique. 102 In hyperthermia treatments using MNPs as mediators of heat, a suspension of MNPs is injected directly into the tumor, allowing the MNPs to be internalized by the cancer cells or at least in the tumor microenvironment (extracellular matrix). 103 The heat produced by the oscillating magnetic moments of the MNPs exposed to an external alternating magnetic field (AMF), usually at a frequency of several hundreds of kHz, will lead to cell death. It is believed that cancer cells can only be killed when the temperature is raised over 43 °C, defining the cumulative effective thermal dose at 43°C or CEM43. However work by Creixell et al. 104 and Asín et al. 20 demonstrated the possibility of causing significant cell death with internalized MNPs without increasing a macroscopic temperature increase during AMF exposure, an effect sometimes called "intralysosomal" or "cold hyperthermia".

It is important to categorize published in vitro magnetic hyperthermia research into magnetic fluid hyperthermia, where cells are dispersed in a magnetic fluid during AMF exposure, and intracellular magnetic hyperthermia, where heat is exclusively generated by internalized MNPs. In the first category, Ota et al. 105 reported cell viability reduced to 50% by exposing a suspension of cells dispersed in a magnetic fluid to an AMF (210 kHz, 250 Oe, 19.9 kA/m, 1 h) that brought the temperature to 46.2 °C. Cell viability was further reduced to 30 % by performing the same treatment using CH11 antibody-complexed MNPs. Higher cell death rates were also observed for a 5-15 min exposure to an AMF of 114 kHz, 89.9 kA/m using a Mn 0.6 Zn 0.4 Fe 2 O 4 -based magnetic fluid, with exceptional magnetic and heating properties (SAR 1102.4 W/g). 106 In the second category, intracellular magnetic hyperthermia was carried out by Fortin et al. 25 MNPs into needles under the influence of the AMF was also observed.

In vivo magnetic hyperthermia following the direct injection of an iron oxide nanoparticle fluid has also proven successful in many animal models: rat glioma, [110][111] melanoma (in combination with immunotherapy), 112 prostate cancer [112][113] and liver tumors in rats (arterial embolization hyperthermia). 114 The promising results of the first clinical magnetic fluid hyperthermia conducted in 2003 on 14 patients suffering from glioblastoma multiform led to a phase II study involving 66 patients. 115 The magnetic hyperthermia method after intra-tumor injection of MNPs was authorized (in combination with conventional radiotherapy) to treat patients with this uncurable brain cancer in 2011, but this method only extended their life expectation by 2 months. 116 Since our experiments showed that MPIC micelles could be easily internalized by L929 cells, we studied their use in intracellular magnetic hyperthermia on both L929 and U87 cells. Because the amount of internalized MPIC micelles showed strong dependence on the incubation concentration, the cells were incubated with MPIC micelle solutions at Fe 3 O 4 concentrations of 1250, 700, and 140 µg/mL in complete DMEM culture medium for 15 h.

The cells were then extensively rinsed with PBS to ensure that all the MPIC micelles not Chapter 6 268 taken up (e.g., simply adsorbed on cell membranes) were removed and that the hyperthermia effect, if present, was only due to intracellular nanoparticles. The cells were suspended in culture medium during AMF exposure (755 kHz at 10.2 kA/m, for 1.5 or 3 h) before they were seeded for the cell viability assay. The control cells were incubated in complete medium and exposed to the same AMF. Exposure to the AMF alone did not affect cell viability nor the propotion of cells at any cell cycle stage. 117 The temperature of the control sample and the incubated sample was monitored during AMF exposure and, in both cases, no increase in temperature was observed (Figure 6-14), indicating that there was no macroscopic heating and that the amout of heat dissipated by the intracellular MNPs was insufficient to raise the temperature of the whole sample. This observation seems to confirm our hypothesis that AMF excitation of the intracellular MNPs was the main factor leading to programmed cell death (apotosis), without heating of the bulk medium. The search for a mechanism leading to cell death focused on answering two main questions:

1. What kind of and how is the energy generated? Asín et al. 20 proposed a mechanism leading to cell death involving sudden disruption of the lysosomes containing the MNPs releasing toxic agents into the supernatant triggering the death of neighbouring cells. This mechanism was supported by the fact that 0% cell viability was achieved even though only 50-60% of the cells internalized NPs. Replacing the medium after the AMF treatment with fresh medium resulted in about 15% cell viability.

Zero cell viability was also reported when treating cells with the toxic medium removed after AMF, even though these cells were neither treated with MNPs nor subjected to AMF.

In our study, lysosomal membrane permeabilization due to local heating is deemed to be the most likely mechanism leading to cell death because i) macroscopic heating was absent; and ii) MNPs were found in vesicles, probably lysosomes, after they were internalized through the cell membranes. Extending the AMF exposure time resulted in killing more cells, probably due to the variable number of MNPs internalized observed by FACS. The cells uptaking a sufficiently large number of MNPs should be more sensitive to AMF exposure, while cells with a lower number of MNPs may be killed only for longer AMF exposure times. In order to confirm apoptosis and/or necrosis, a fluorescent labeling method can be used with the assistance of fluorescent microscopy and flow cytometry.

Conclusions

The cytotoxicity of PIC micelles and MPIC micelles was investigated. None of the PIC micelles incorporating a G1 arborescent core and a shell of PAA-b-PHEA (tested at concentrations of up to 10 mg/mL) showed any significant cytotoxicity for L929 cells, in spite of their different coating densities and sizes. The good colloidal stability and noncytotoxicity of the PIC micelles are likely due to their high biocompatibility and hydrophilicity, and to the neutrality of the PHEA shell. Within the range of coating densities and polymeric stabilizer chain lengths tested, no significant differences in cell viability were observed. Similarly, the MPIC micelles were non-cytotoxic up to the highest concentration tested (1250 µg Fe 3 O 4 /mL) after 48 h of incubation, resulting in over 91% cell viability as compared to 53% for the bare MNPs. A slightly higher cell loss was reported for the MPIC micelles coated with a lower amount of polymeric shell material, but only at high concentrations. This confirms the efficiency of the PAA-b-PHEA copolymers as protecting agents preventing the interaction of the Fe 3 O 4 cores with the cells. accomplished by tuning the density of the stabilizing polymer layer around the G1 core, either by varying the block copolymer stoichiometry or the length of the PAA anchoring block, and most notably by varying the length of the nonionic PHEA block. Most effective stabilization was achieved when the length of the nonionic segments was increased to 260 units, producing stable unimolecular PIC micelles even when a complexation ratio f = CO 2 H/N = 0.5 was used. This approach was later on applied to improve the colloidal stability of magnetic nanoparticles G1@Fe 3 O 4 at pH 7, enabling their use in physiological environments. Evidence for uni-vs. multimolecular structure formation was provided by the difference between the hydrodynamic diameter of the PIC micelle to that of the collapsed core, as well as the w and -parameter values obtained from static light scattering measurements. We used atomic force microscopy (AFM) phase images to distinguish the soft shell of PHEA surrounding the stiff G1 core, and used different AFM amplitude set-points to reveal the core-shell morphology of the PIC micelles.

Control over Fe 3 O 4 Crystallite Size Using an Arborescent Copolymer

In a context where many reviews were published earlier with more general goals such as summarizing pathways for the synthesis of magnetic nanoparticles, [3][4][5][6][7][8] their surface modification, 3-4,6-7,9-10 their physicochemical characterization 3,5,8,[11][12] and biological applications, [3][4][5][6][8][9][10][11][12][13][14][15][16][17][18][19] our literature review aimed at a more specific aspect: the possibility of controlling the shape and size of the synthesized magnetic nanoparticles (MNPs) through the size and geometry of the polymer templates, as well as the idea of using polymer templates, in the forms of both porous continuous matrices and polymer self-assembled suspensions, as nanometer-scale molds. Investigations demonstrating significant influence of the polymer/precursor ratio on the outcome of the synthesis (in particular those using welldefined polymer structures) were highlighted in comparison to other in situ syntheses not making a strong case for a true templating effect, and those affected by a high reaction temperature. Micelles formed by double-hydrophilic or amphiphilic copolymers, having a dense core and a shell with multiple hydrophilic arms, emerged as efficient templates to prepare multi-functional NPs with highly desirable magnetic properties, and good colloidal stability and biocompatibility. The polymers used in this Ph.D. work not only represent a further development of this core-shell structure concept, but also take advantage of the good templating effect of a preformed arborescent structure (a well-defined structure that compacts a large number of vinylpyridine moieties into a small volume), allowing the targeting of lower hydrodynamic sizes and dispersities for the MNPs (in contrast to those synthesized without these self-assembled systems).

For the first time, a G1 arborescent copolymer (G0PS-g-P2VP) was used as a polymer template to control the size and improve the size distribution of iron oxide NPs. The templating effect of G1 was evidenced by: i) the formation of larger Fe 3 O 4 crystallites with D TEM  12 ± 2 nm, ii) enhanced colloidal stability due to electrostatic repulsion from protonated NH + groups of the P2VP chains, iii) a slight improvement in the size distribution as compared to non-templated magnetic NPs, and iv) separation of the crystallites appearing in the TEM images with an organic layer (bright stripe around the dark cores), lowering their dipolar interactions, which has many positive effects on the properties such as yielding a higher specific absorption rate (SAR) in magnetic hyperthermia, and a higher relaxivity, i.e. a higher MRI contrast, and better dispersability (inorganic grains at close contact usually experience strong van der Waals attraction and cannot be separated). The three-stage weight loss thermal decomposition and combustion profile observed for the templated MNPs G1@Fe 3 O 4 was comparable to previously published results, evidencing strong interactions between P2VP and Fe 3 O 4 , and thus successful templating. The slight overestimation of the Fe 2 O 3 content by thermogravimetric analysis was ascribed to incomplete degradation (in N 2 ) or combustion (in air) of the organic material, in agreement with literature reports. [20][21][22] The presence of residual organic material was supported by UV-VIS analysis in 5 M HCl, a method that proved to be reliable to determine the equivalent Fe (and corresponding Fe 3 O 4 ) content of MNPs.

Application of the Polyion Complexation Process to Produce Magnetic Micelles Stable Under Physiological Conditions

The experience gathered from the PIC process investigation reduced the time needed to optimize the complexation between G1@Fe 3 O 4 MNPs and the PAA-b-PHEA block copolymer to produce magnetic micelles stable at pH 7. The presence of Fe 3 O 4 in the G1@Fe 3 O 4 structure probably swelled the G1 template to a larger volume, exposing more binding sites and thus resulting in more efficient complexation even at smaller f = CO 2 H/N ratios. Tapping mode AFM height and phase imaging were again applied to reveal the coreshell morphology of the MNPs. The magnetic micelles obtained had D h  130 nm (PDI  0.136) and good colloidal stability not only under physiological conditions (at pH 7 with salts), but also over a wide pH range, which shows great promise in terms of the biocompatibility and biomedical applications being targeted.

Magnetic, Relaxometric and Hyperthermic Properties Measurements

The superparamagnetism of the Fe 3 O 4 nanoparticles (bare and coated with the double-hydrophilic block copolymer) was revealed by the absence of a hysteresis loop, coercivity and remanence in the magnetization (M vs. H) curves. The Fe/N templating ratio, temperature, and the presence of the coating material were found to influence the magnetic and relaxometric properties of Fe 3 O 4 NPs to different extents, confirming the possibility of tuning their magnetic and relaxometric properties via the synthetic procedure used.

Attempts to compare the results obtained by fitting the experimental data with literature, and among the different measurement techniques (relaxometric size vs. magnetic and TEM size) were made. On the basis of these results: 1) The divergence between the nuclear magnetic resonance dispersion (NMRD) curves for the uncoated/coated magnetic micelles and the outer-shell model fits was ascribed to the multimodal size distribution, and to significant interference from the hydrophobic PS-g-P2VP polymeric core.

2) The low relaxation rates and flat NMRD profiles observed for the bare G1@Fe 3 O 4 samples were indicative of agglomeration, thus confirming the essential role of the block copolymer coating.

3) The discrepancy between the magnetic domain diameters determined by VSM and by TEM was ascribed to spin canting occurring at the surface of or within the volume of the nanoparticles. 4) The radii obtained from NMRD analysis were larger than from TEM and magnetometry, which can be explained by the different nature of these characterization methods. The polymer component of the micelles is invisible in TEM, non-magnetic in VSM, and partially impermeable to water in NMRD.

3) The magnetization obtained from the fits to the NMRD profiles was lower than that determined by magnetometry, as a result of the larger relaxometry sample size and dilution of the iron oxide by the polymer (pushing further the limit of the Outer-Sphere). The scaling law introduced by Vuong et al. 23 to relate the transverse relaxivity of magnetic hybrid particles to their structure enabled the estimation of the intra-aggregate volume fraction of Fe 3 O 4 in the polymer micelles to  intra 1%. This estimated volume fraction corresponds to roughly twelve magnetite crystallites per micelle, a number compatible with the AFM and TEM observations.

We successfully synthesized SPIONs with high values of transverse relaxivity r 2 = 335 (s -1 mM -1 ) and transverse-over-longitudinal relaxivity ratios r 2 /r 1 = 31.4 (1.47 T, 37 °C), comparable with or even larger than commercial products, suggesting their efficiency as negative contrast agents for T 2 -weighted imaging. Moreover, a maximum SAR value of 55.6

Wg -1 was measured by AMF calorimetry at f = 755 kHz, H max =10. CLSM confirmed the successful internalization of the labeled, negatively charged PIC micelles through the L929 cell membrane, suggesting that this micellar system is a highly biocompatible platform with good potential for biomedical uses (e.g. as biosensorss, for drug delivery, gene delivery, or cancer therapy).

In Vitro MFH Assessment

An in vitro MFH assessment was conducted with fibroblast-like L929 cells and U87 human glioblastoma cells, using custom-built MFH equipment and a newly proposed cell treatment protocol, under prolonged treatment with an AMF at 37°C in a HEPES buffer to avoid the need for CO 2 pressure control. Cell viability strongly decreased as a function of both increased incubation concentration and increased exposure time to the AMF, evidencing a dual dose-effect. At the maximum incubation dose (1250 µg/mL iron oxide), the lowest cell viabilities with applied AFM were observed: about 46-57% after 1.5 h of treatment, and 30-35 % after 3 h for the L929 cell line. We verified the hypothesis that AMF excitation of the intracellular MNPs was the main factor leading to cell death, based on the fact that cell viability was decreased in a dose-dependent manner with the concentration of internalized MNPs in the cells, even in the absence of macroscopic heating.

including iron, which has already enabled the templated synthesis of gold nanoparticles by reduction of the HAuCl 4 precursor. 30

Next Generation MNPS

Due to their low cytotoxicity profile, [31][32] their ease of synthesis, [3][4][5][6][7][8] and the possibility of surface modification with biocompatible coatings, [3][4][6][7][9][10]33 superparamagnetic iron oxide nanoparticles (SPIONs) have been intensively studied, particularly for bioimaging applications such as magnetic resonance imaging (MRI) contrast agents, and for therapeutics by magnetic hyperthermia. 6,[34][35] However the potential use of SPIONs for magnetically targeted drug delivery is limited by their relatively low saturation magnetization (M S  300-400 emu•cm -3 ). [35][36][37] Thanh et al. 35 have published an interesting review on the 'Next Generation Magnetic Nanoparticles for Biomedical Applications', highlighting the importance of developing new superparamagnetic (metallic, bimetallic) materials that have a higher magnetocrystalline anisotropy energy and higher saturation magnetization (the M S of metallic Fe is ca. 1,700 emu•cm -3 , 38 and the M s of bimetallic FePt, CoPt, FeCo and SmCo are in the range of 800-1,900 emu•cm -3 ), 29,[32][33][34] much larger than SPIOs, to overcome this limit. The aqueous reduction of metallic salts by reducing agents is a common method to produce uniform MNPs, in which the presence of suitable surfactants can assist in separating the nucleation and growth steps, and prevent MNP aggregation. 35 Since alkyl thioether endfunctionalized poly(methacrylic acid) (PMAA-DDT) was reported as a capping ligand for Co MNP synthesis, 38 it would be interesting to test a similarly modified G0PS-g-P2VP arborescent copolymer for its ability to load metal precursors (although the hydrophobicity of this polymer in a basic environment could be a challenge in the reduction step). It is however noteworthy that most of these new materials are not biocompatible and, particularly, not suitable for in vivo applications, although their superior magnetic properties and enhanced hyperthermia performance allow their application at sufficiently low concentrations to reduce their toxicity. 35 Otherwise, a coating strategy is needed to mitigate their toxicity.

Cell Internalization

In our work, cell internalization of the NPs was visualized by CLSM and TEM, revealing their presence mainly at the periphery of the nuclear membrane and in intracytoplasmic vesicles. Possible work complementary to ours would be to study the internalization mechanism of the MNPs into cells. Specific pharmacological (clathrin/actin/caveolae/microtubule) inhibitors can be used to reveal the internalization pathway. 39 Another solution would be to label the lysosomes with a red fluorescence marker (e.g. LysoTraker™ Red), to visualize the colocalization of the NPs inside the lysosomes.

Overlapping of the red fluorescence with the green fluorescence from the NPs would result in a merged yellow fluorescent signal, as shown for another system. 39 The fate of the MNPs after they are internalized in the cells (isolated or aggregated) could be further investigated by TEM, by increasing the MNP incubation concentration to 1250 g/mL (rather than 100 g/mL). When the MNPs are internalized at a high incubation concentration, different phenomena such as transcytosis can be observed, 40 i.e. the transport of macromolecular cargo from one side of a cell to the other within a membrane-bound carrier, which causes leakage of the MNPs into the medium.

Active Targeting by the MNPs

A most significant development for our work would be to enable active targeting by attaching targeting molecules (e.g., antibodies that specifically bind to antigens overexpressed by tumor tissue, but have a low affinity for healthy tissue) to the surface of the MNPs. Antibody-conjugated MNPs were indeed shown to have higher accumulation at tumor sites, [41][42][43] and thus enhanced the effectiveness of magnetic hyperthermia. 44 However, monoclonal antibodies (mAbs) contain elements that may be recognized by the recipient as foreign and can activate immune and innate reactions. [45][46] Acute reactions following the infusion of mAbs were reportedly caused by various mechanisms: acute anaphylactic and anaphylactoid reactions against the mAb, serum sickness, the tumor lysis syndrome, and the cytokine release syndrome. 46 The use of antibodies has been associated with various antibody-induced adverse effects including immunosuppression, immunostimulation and hypersensitivity (immunogenicity), 47 which can manifest themselves clinically as local skin reactions at the injection site, pyrexia and an influenza-like syndrome, or even acute anaphylaxis and a systemic inflammatory response syndrome, which could be fatal. 46 The attachment of antibodies on the surface of MNPs can therefore trigger active targeting and enhance the accumulation at tumor sites, but at the cost of increased potential for immunotoxicity. Antibody-conjugated Fe 3 O 4 MNPs targeting the epithelial growth factor receptor (often overexpressed in tumoral vasculature) thus resulted in 40% of cell viability even in absence of magnetic field vs. results without targeting. 44 The unique ability of MNPs to be guided by an external magnetic field 48 can also be used to enhance their accumulation in specific organs. However this option depends more upon the design and control of the magnet system to ensure that the nanoparticles can efficiently accumulate at specific sites such as deeper tissue tumors. 49

Theranostics MNPs: Controlled Drug Release

There have been a number of studies demonstrating hyperthermia-based controlled drug delivery, in which the magnetic nanoparticles generate local heat triggering the release of drugs, 50 either by breaking bonds binding the drugs to the MNP or to a polymer, [51][52] or by enhancing permeability to release drugs encapsulated within a polymer matrix, or dispersed in magnetic polymer carriers. [53][54][55][56][57][58][59][60] This possible development for our magnetic polyion complex (MPIC) micelles is inspired by Carregal-Romero et al., 60 who designed layer-bylayer assembled polyelectrolyte microcapsules of poly(allylamine hydrochloride) and poly(styrene sulfonate), incorporating 18 nm iron oxide nanocubes within their walls. The microcapsules were loaded with an organic fluorescent polymer (Cascade Blue-labeled dextran), used as a model molecular cargo. The authors reported the successful use of AMF to destroy the microcapsule walls, leading to release of the embedded cargo to the surrounding solution. 60 Based on the structure of the MPIC micelles, a hydrophilic drug could be embedded into the arborescent P2VP matrix of the G1@Fe 3 O 4 NPs just before polyion complexation with the PAA-b-PHEA block copolymer. Encapsulation of the drug could be enhanced if it is negatively charged. A negatively charged drug would consume some of the 2VP moieties subsequently serving for the polyion complexation; however this interference may be minor, since there should be enough 2VP moieties left to form strong electrostatic interactions with a negative polyelectrolyte like PAA. The heat generated by Fe 3 O 4 MNPs sandwiched between the polyelectrolyte layers under AMF irradiation would be expected to weaken the electrostatic interactions and release the encapsulated drug, thus producing a new example of "magneto-chemotherapy".

Chapter 3 Supporting Information

I

  would also like to thank my defense committee members, Prof. Nguyen Thi Kim Thanh, Prof. Harald Stöver, Prof. Étienne Duguet, Prof. Xiaosong Wang, and Dr. Caroline Robic for accepting to read my work, for their fruitful discussion and their guidance provided at different stages of the work. I wish to express my warm and sincere thanks to Prof. Sébastien Lecommandoux and everyone in the TH2 team and at LCPO, Bordeaux, France who have made my experience in the lab so much more valuable. Thank you Annie and Camille for teaching me how to work professionally, brightening my Ph.D. student life, and fulfilling it with English lessons, running, and a heartfelt friendship. My gratitude also goes to you, our magnetic nanoparticles team: Kevin, Hugo, Eneko and Gauvin for your solid support, and Colin for your drawings. Thank you Edgar for always caring, and Lise for being my running best friend. I appreciate Elisabeth, Elizabeth, Julie, Silvia, Cony, Charlotte, Pauline, Ariane, Deniz, Paul, and Winnie who have been very supportive, and kept me strong with the LCPO spirit. I am grateful to Manu (the AFM expert), Nico (the SEC and ALV master), Anne-Laure (NMR lady), Sabrina (TEM) and Cédric (TGA). I thank Prof. Neso Sojic and Mr. Patrick Garrigue (NSysA, ix ENSCBP) for allowing me to use their AFM. My special appreciation goes to Madame Catherine Roulinat, who gave me her warm welcome and tremendous support. And also, thank you Ms. Bernadette, Ms. Corin, Ms. Nicol and Mr. Claude. I would further like to thank my colleagues and friends in the Polymer Chemistry Laboratory at the University of Waterloo, Canada, who have shared my lab life and made it more interesting: Deepak, Toufic, Firmin, Joanne, Mosa, Ala, Ryan, Mehdi, Priscilla, Xiaozhou, Liying, Yan, and Timothy. Thank you Olivier Nguon, my ATRP mentor and Aklilu, my friend with whom I can discuss many topics. I am grateful to Prof. Jean Duhamel, for his knowledge and the fruitful discussions that helped me to strengthen my work. Thank you also to all the members of Prof. Duhamel's group, who have been helpful and welcoming. I am grateful to Prof. Michael Tam for inspiring me with his Nano-courses. I wanted to thank Dr. Yi-Shiang, who mentored me to work with cells, Dr. Sandra Ormenese, who trained me to use confocal microscopy, Dr. Pierre Colson and Ms. Nicole Decloux , for their valuable help with TEM for cells, and Dr. Ji Liu, for sharing his experience and for his fruitful email discussions. I am grateful to Prof. Sophie Laurent, Prof. Luce Vander Elst, Coralie Thirifays, Corinne Piérart, Adeline Hannecart, and my colleagues in the NMR and Molecular Imaging Laboratory (UMONS, Belgium) for their treasured help with relaxometry. Thank you, Prof.

  Thank you Varsity Squash team for a good practice time. Thank you Van U, Vi Beo, Thuy Vy, Vi Vi, Mien, and Thao for always listening. Thank you, Chi Phuong for sharing my brightest and darkest days. I wish to thank the International Doctoral School in Functional Materials (IDS-FunMat), an Erasmus Mundus Program of the European Union, for financial support and for giving me the opportunity to work in six different labs, in three different countries, and allowing me to establish a solid network beneficial to my work. I also thank Prof. Mario Gauthier for funding my work during my 4 th year, and the University of Waterloo Graduate Office for their financial support. I am grateful to Dr. Olivier Sandre for sponsoring my cellular hyperthermia work, and for giving me the chance to develop my work at the University of Mons. I sincerely appreciate Ms. Audrey Sidobre, Prof. Stéphane Carlotti, Ms. Marianne Delmas, Ms. Elodie Goury, Mr. Christopher Niesen, Ms. Catherine Van Esch, Ms. Susanna Fiorelli, and Ms. Marta Kucharska for their wonderful job as administrative coordinators.

  AUTHOR'S DECLARATION .................................................................................................................. II RÉSUMÉ / ABSTRACT ........................................................................................................................ III xvi

Figure 2 - 1 . 11 Figure 2 - 2 .

 211122 Figure 2-1. TEM images clearly showing: (a) lattice fringes; and (b) facetted shapes for nanoparticles coated with oleic acid, using 10 equivalents of reducing agent (NaBH 4 ) at room temperature. Reprinted with permission from Reference 4. Copyright 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim...................................................................... 11

Figure 2 - 3 . 21 Figure 2 - 4 . 24 Figure 2 - 5 . 28 Figure 2 - 6 . 31 Figure 2 - 7 . 32 Figure 2 - 8 . 33 Figure 2 - 9 . 34 Figure 2 - 10 . 35 Figure 2 - 38 Figure 2 - 12 . 40 Figure 2 - 42 Figure 2 - 14 . 44 Figure 2 - 44 Figure 2 - 46 Figure 2 - 47 Figure 2 - 48 Figure 2 - 50 Figure 2 - 50 Figure 2 - 51 Figure 2 - 52 Figure 2 - 23 . 53 Figure 2 - 56 Figure 2 - 57 Figure 2 - 26 . 59 Figure 2 - 60 Figure 2 - 62 Figure 2 - 64 Figure 2 - 66 xxiiFigure 2 - 67 Figure 2 - 32 . 68 Figure 2 - 70 Figure 2 - 71 Figure 2 - 35 . 73 Figure 2 - 36 . 74 Figure 2 - 75 Figure 2 - 77 Figure 3 - 1 . 84 Figure 3 - 2 . 92 Figure 3 - 3 . 1 HFigure 3 - 4 .Figure 3 - 5 .Figure 3 - 6 .Figure 3 - 7 .Figure 3 - 8 .Figure 3 - 9 .Figure 3 - 12 .Figure 3 - 13 .Figure 3 - 14 .Figure 3 - 15 .Figure 3 -Figure 3 -Figure 4 - 1 .Figure 4 - 2 .Figure 4 - 3 .Figure 4 - 5 .Figure 4 - 6 .Figure 4 - 7 .Figure 4 - 8 .Figure 4 - 9 .

 2321242425282631273228332934210352382124024221444244246247248250250251252223532562572265926026226426626723268270271235732367427527731843292331343536373839312313314315334142434546474849 Figure 2-3. TEM image for magnetite nanowires synthesized with poly(ethylene glycol) (PEG)/H 2 O (1:2 by volume) at 150 °C for 24 h in an autoclave. The selected area electron diffraction (SAED) pattern is shown in the inset. Reprinted with permission from Reference 20. Copyright 2008 Elsevier B.V. ....................................................................... 21 Figure 2-4. Suggested bond formation between COO -and the Fe 3+ ions at the ultrasmall superparamagnetic iron oxide (USPIO) surface. Reprinted with permission from Reference 22. Copyright 2008 IOP Publishing Ltd. ............................................................. 24

Figure 4 - 10 .

 410 Figure 4-10. Size distributions for magnetic micelles G1@Fe 3 O 4 @ PAA 27 -b-PHEA at different f ratios obtained at 25 °C and pH 7. .................................................................

Figure 4 -

 4 Figure 4-11. TEM images for Fe 3 O 4 crystallites in G1@Fe 3 O 4 @PAA 27 -b-PHEA 260 f = 0.5. .................................................................................................................................

Figure 4 -

 4 Figure 4-12. AFM images for magnetic micelles G1@Fe 3 O 4 @PAA 27 -b-PHEA 260 f = 0.5. ....................................................................................................................................

Figure 4 -

 4 Figure 4-13. Magnetic micelles at various amplitude set points (the lower the value in mV, the higher the average deflection of the cantilever and thus the exerted force). ...........

Figure 4 - 14 .

 414 Figure 4-14. Zeta potential and intensity-weighted hydrodynamic diameter D h of MPIC micelles G1@Fe 3 O 4 @PAA 27 -b-PHEA 260 at f = 0.5 as a function of pH. ..................

Figure 5 - 1 .

 51 Figure 5-1. Switching of the magnetization in a particle across the anisotropy barrier under a) no external magnetic field; b) external magnetic field pointing downward; and c) external magnetic field pointing upward. ...........................................................................

Figure 5 -

 5 Figure 5-2. a) Main parameters of interest extracted from a generic hysteresis loop; b) Room temperature magnetization curve for superparamagnetic particles. ..........................

Figure 5 - 3 .

 53 Figure 5-3. Nuclei spin oriented along B 0 , with a slight excess in its direction producing a net longitudinal magnetization, M 0 . Reprinted with permission from Reference 15. Copyright 2006 Humana Press Inc. ..............................................................................

Figure 5 - 4 .

 54 Figure 5-4. 180 o Refocusing pulse and the formation of a spin echo. Reprinted with permission from Reference 15. Copyright 2006 Humana Press Inc. ...................................

Figure 5 - 5 .Figure 5 - 6 .Figure 5 - 7 .Figure 5 - 8 .Figure 5 - 9 .Figure 5 - 10 .Figure 5 - 11 .Figure 5 - 12 .Figure 5 - 13 .Figure 5 - 14 .Figure 5 - 15 .Figure 6 - 1 .Figure 6 - 3 .Figure 6 - 4 .Figure 6 - 5 .Figure 6 - 6 .Figure 6 - 7 .Figure 6 - 8 .Figure 6 - 9 .Figure 6 -Figure 6 - 11 .Figure 6 - 12 .Figure 6 - 13 .Figure 6 - 14 .Figure 6 - 15 .

 555657585951051151251351451561636465666768696611612613614615 Figure 5-5. Outer-sphere relaxation of water protons in the vicinity of a superparamagnetic CA. Reprinted with permission from Reference 19. Copyright 2009 John Wiley & Sons, Inc. ............................................................................................................



  effective relaxation time of magnetic moments pre-exponential factor of the Néel relaxation time expression xli

Chapter 3

 3 describes the preparation of a polymeric substrate combining both templating and stabilizing effects for the MNP synthesis, which is a polyion complex (PIC) micelle composed of a G1 arborescent copolymer, namely G0 polystyrene-graft-poly(2vinylpyridine) or G0PS-g-P2VP, and a hydrophilic shell of the block copolymer poly(acrylic acid)-b-poly(2-hydroxyethyl acrylate), PAA-b-PHEA. The size and the core-shell morphology of the PIC micelles were investigated by different techniques including atomic force microscopy (AFM), dynamic light scattering (DLS), and multiangle laser light scattering (MALLS). Varying the length of the PAA and PHEA chain segments and the stoichiometric ratio of the arborescent and block copolymer components provided a means to tune the size and the shell density of the PIC micelles. As it turns out, the direct in situ application of these PIC micelles as templates in MNP synthesis was of limited usefulness, since the PAA-b-PHEA added in the shell to improve the colloidal stability and the biocompatibility of the MNPs interfered with loading of the iron precursors. Chapter 4 therefore presents an alternative approach to this, which is first to use the G1 copolymer as a template for Fe 3 O 4 NP synthesis, and then bind the PAA-b-PHEA copolymer to enhance the colloidal stability and biocompatibility. The high density of 2-vinylpyridine units in the very compact structure of arborescent copolymers assisted the formation of G1@Fe 3 O 4 MNPs by increasing the Fe 3 O 4 crystallite size. The subsequent complexation of G1@Fe 3 O 4 MNPs with PAA-b-PHEA created magnetic polyion complex (MPIC) micelles with improved colloidal stability at pH 7. The Fe 3 O 4 crystallites of the MPIC micelles were characterized by transmission electron microscopy (TEM) and selected area electron diffraction (SAED), while the dimensions of the micelles were determined by DLS and AFM measurements.

  Cell internalization was visualized by confocal laser scanning microscopy (CLSM) and TEM, and quantified by fluorescence-activated cell sorting (FACS). Cell internalization by an endocytosis pathway was hypothesized. The usefulness of MPIC micelles for cellular radiofrequency magnetic field hyperthermia was also confirmed, as the MPIC micelles had a dual dose-dependent effect (concentration and duration of magnetic field exposure) on cell viability. The overarching conclusions drawn from the Thesis work are presented in Chapter 7, which provides an overall summary of the body of research, the original contributions to knowledge, and suggestions for future work. Chapter 1 6

  agent to iron precursor required to produce the largest diameter bare NPs (8.08 ± 0.93 nm by transmission electron microscopy (TEM) and 8.95 nm by X-ray diffraction (XRD) was first determined and then applied to the in situ coating method. Both the uncoated and the coated NPs displayed clear lattice fringes in high resolution transmission electron microscopy (HRTEM) images (Figure2-1a) with spacings of 2.9 Å and 2.5 Å, consistent with a cubic spinel iron oxide structure and confirming the highly crystalline character of the NPs. It is noteworthy that some of the NPs coated with hydrophobic oleic acid had a facetted particle shape on the HRTEM images (inset on Figure2-1b) rather than the spherical morphologies observed in most other cases, indicating that monolayer formation by the hydrophobic tail of oleic acid molecules brings a different templating effect as compared to several hydrophilic carboxylic compounds tested in the literature,5 most often leading to a mere reduction in the final size of the MNPs due to capping of the crystallites.

Figure 2 - 1 .

 21 Figure 2-1. TEM images clearly showing: (a) lattice fringes; and (b) facetted shapes for nanoparticles coated with oleic acid, using 10 equivalents of reducing agent (NaBH 4 ) at room temperature. Reprinted with permission from Reference 4. Copyright 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Figure 2 - 2 .

 22 Figure 2-2. Representation of two 1,4-linked α-L-guluronic acid (G)-blocks forming an "egg box" structure with ferrous ions. Reprinted with permission from Reference 13. Copyright 2000 Elsevier Science B.V.

Fe

Figure 2 - 3 .

 23 Figure 2-3. TEM image for magnetite nanowires synthesized with poly(ethylene glycol) (PEG)/H 2 O (1:2 by volume) at 150 °C for 24 h in an autoclave. The selected area electron diffraction (SAED) pattern is shown in the inset. Reprinted with permission from Reference 20. Copyright 2008 Elsevier B.V.

FeCl 3 Thermal

 3 Decomposition with PEG in Triethylene Glycol (Polyol) 2.4.2.9

Figure 2 - 4 .

 24 Figure 2-4. Suggested bond formation between COO -and the Fe 3+ ions at the ultra-small superparamagnetic iron oxide (USPIO) surface. Reprinted with permission from Reference 22. Copyright 2008 IOP Publishing Ltd.

Figure 2 - 5 .

 25 Figure 2-5. Schematic structure of mPEG-b-PMAA-b-PGMA-Fe 3 O 4 NPs loaded with adriamycin at neutral pH, designed to release the anticancer drug in the acidic environment of a tumor. mPEG: poly(ethylene glycol) monomethyl; PMAA: poly(methacrylic acid); PGMA: poly(glycerol methacrylate); and ADR: antitumor drug adriamycin. Reprinted with permission from Reference 27. Copyright 2008 The Royal Society of Chemistry.

  Water in the Presence of Poly(ethylene oxide)

Figure 2 - 6 .

 26 Figure 2-6. Transmission electron micrographs for magnetic iron oxide precipitated in: (a) water alone; (b) the presence of poly(ethylene oxide) (PEO) homopolymer; and (c) the presence of PEO-b-poly(methacrylic acid) (PMAA) block copolymer. Note that Figure 2-6c is at somewhat higher magnification than the others; (d) magnetization curves for the PEO-b-PMAA coated NPs and for P(HEMA-co-MMA) magnetic latexes obtained by inverse emulsion polymerization. Reprinted with permission from Reference 29. Copyright 2001 Elsevier.

Figure 2 - 7 .

 27 Figure 2-7. EFTEM zero-loss images of: (a) β-FeOOH NPs; and (b) poly(ethylene glycol) (PEG)-b-poly(aspartic acid)-coated β-FeOOH NPs. Reprinted with permission from Reference 30. Copyright Elsevier B.V.

Figure 2 - 8 .

 28 Figure 2-8. Size distribution by dynamic light scattering (DLS) (a, b, c, d) and from TEM images (a', b', c', d') of: (a, a') the initial suspension after coprecipitation in presence of PEO-b-PAA; (b, b') the non-captured particles; (c, c') the bleed-off samples; and (d, d') the captured clusters. Reprinted with permission from Reference 31. Copyright Elsevier B.V.

Figure 2 - 9 . 2 35

 292 Figure 2-9. Morphology of poly(norbornene-block-deuterated norbornene dicarboxylic acid) loaded with iron oxide NPs (IONPs) at: (a) Ф PNOR/PNORCOOH = 0.64/0.36 (disordered spheres); and (b) Ф PNOR/PNORCOOH = 0.40/0.60 (interconnected spheres). Reprinted with permission from Reference 32. Copyright 2005 Elsevier Ltd.

Figure 2 - 10 .

 210 Figure 2-10. Electron micrographs for poly(norbornene methanol)-block-poly(norbornene dicarboxylic acid) (PNORMEOH/PNORCOOH) diblock copolymer: (a) stained with I 2 vapor; and (b) doped with iron oxide by submerging a thin film in FeCl 3 solution. Reprinted with permission from Reference 33. Copyright 2006 Elsevier Ltd.

Figure 2 - 11 .

 211 Figure 2-11. (a) In situ coprecipitation of iron oxide NPs (IONPs) at different polymer-to-Fe ratios; (b) polymer content measured by thermogravimetric analysis (TGA) for IONPs coated with different anchoring groups at a 1:2 [Polymer]:[Fe] weight concentration ratio. Reprinted with permission from Reference 37. Copyright 2014 Royal Society of Chemistry.

(

  PAEMA), to promote the nucleation of iron oxide. Papaphilippou et al.38 investigated the use of this hydrophobic polymer in a copolymer with PEGMA as a hydrophilic and biocompatible second block, but also having a LCST near 60 °C. Highly stable hybrid micelles, visualized by atomic force microscopy (AFM) as spherical micelles loaded with IONPs (Figure 2-12), were obtained at various precursor salt concentrations. The maximum Chapter 2 40 amount of iron that could be encapsulated without precipitation was unfortunately not determined. A plateau magnetization M s = 300 A/m was reached for the PEGMA 70 -b-AEMA 16 -coated MNPs at a 3:1 [Fe 3+]/[AEMA] molar ratio, but the specific magnetization cannot be calculated for this system since the corresponding iron concentration used was not specified. In vitro biocompatibility and macrophage uptake tests were also conducted and were encouraging in terms of toxicity and the minimization of recognition and phagocytosis.

Figure 2 - 12 .

 212 Figure 2-12. Strategy for the preparation of stabilized magnetic IONPs in aqueous media (top): (a) micelle formation by PEGMA x -b-AEMA y diblock copolymer in water; (b) addition of the Fe 3+ /Fe 2+ mixture to the micellar solution, leading to complexation of the iron salts by the βketoester units inside the micellar core; and (c) transformation of the iron salt "precursor" into IONPs inside the micellar core upon addition of NH 4 OH solution; atomic force microscopy (AFM) images for the block copolymer micelles loaded with IONPs (bottom): (d) height image; (e) amplitude image; and (f) phase image. Reprinted with permission from Reference 38. Copyright 2009 American Chemical Society.

(

  proposed a route in which NaOH solution was quickly added to a microemulsion system of cyclohexane, n-hexanol, HCl, chitosan, and a ferrous salt stabilized by the Triton ® -X 100 surfactant, with controlled exposure to oxygen. The type of iron oxide obtained in the NPs was efficiently controlled by the amount of oxygen in the N 2 gas added during the synthesis, with 0.05% of oxygen yielding 60-80 nm spherical chitosan particles encapsulating cubic-shaped Fe 3 O 4 cores (Figure 2-13).

Figure 2 - 13 .

 213 Figure 2-13. TEM of magnetic NPs (MNPs) obtained with: (a) molecular weight (MW) = 5.0×10 5 g•mol -1 non cross-linked chitosan; (b) cross-linked; (c) w = 1.0×10 5 g•mol -1 non cross-linked chitosan; (d) cross-linked. Reprinted with permission from Reference 40. Copyright 2006 Elsevier B.V.

  -14), and TEM images revealed the morphological transformation and the ripening of the rod-like particles upon heating, leading to the final state with individual CoFe 2 O 4 crystals linearly arranged along the annealed nanorods (Figure 2-15a). The crystallographic alignment of the FeCo 2 O 4 nanocrystals became visible after calcination in the high resolution TEM images as atomic planes (Figure 2-15c) and grain boundaries between the interconnected nanocrystals (Figure 2-15b). After annealing, the nanorods also exhibited magnetic H c with a Curie temperature well above room temperature.

Figure 2 - 14 .

 214 Figure 2-14. Scanning electron microscope (SEM) images for: (a) as-synthesized CoFe 2 (C 2 O 4 ) 3 suspension after coprecipitation in a microemulsion; and (b) CoFe 2 O 4 rods annealed at 720 °C. Reprinted with permission from Reference 41. Copyright 2005 Wiley-VCH.

Figure 2 - 15 .

 215 Figure 2-15. (a) TEM image for an individual CoFe 2 O 4 nanorod annealed at 720 °C; the insets illustrate SAED patterns acquired from two individual nanocrystals of size about 80 nm. The inset (a) shows the indexed diffraction pattern for the fcc crystals in the [011] beam direction, and inset (b) in the [001] beam direction; (b) high resolution transmission electron microscopy (HRTEM) image showing a grain boundary between two CoFe 2 O 4 nanocrystals; and (c) HRTEM image for a CoFe 2 O 4 nanocrystal. Reprinted with permission from Reference 41. Copyright 2005 Wiley-VCH.

  spherical micelles with a structure locked by photo-cross-linking were prepared using a triblock copolymer PI-b-PCEMA-b-PtBA, in which PI forms the corona, PCEMA a solvent-insoluble shell, and PtBA a hydrolysable core.42 Hydroxylation of the PI corona and cleavage of the tert-butyl groups to convert PtBA to PAA enabled the coordination of Fe 2+ ions in the template. Finally, alkaline oxidation with NaOH and H 2 O 2 yielded watersoluble Fe 2 O 3 -impregnated nanospheres. This multi-step pathway is represented schematically on Figure 2-16.The diameter of the polymeric template (90 nm) did not change after the tert-butyl removal step (as seen by comparison of Figure2-17b with Figure2-17a), indicating the benefits of using templates with a predetermined size, shape and morphology. Inorganic cores with diameters ranging from 4 to 16 nm were visible on the TEM images (Figure2-17c). The Bragg peaks detected in selected area electron diffraction (SAED) experiments were matched with the Miller indices of the maghemite (γ-Fe 2 O 3 ) phase. Therefore such templates with a cross-linked polymer shell surrounding a porous polymer core coordinating ferrous precursors were efficient as templates for the preparation of IONPs, although further studies were suggested to control the size distribution of the magnetic cores.

Figure 2 - 16 .

 216 Figure 2-16. Polyisoprene-block-poly(2-cinnamoylethyl methacrylate)-block-poly(tert-butyl acrylate) (PI-b-PCEMA-b-PtBA) as template: Photolysis cross-links the PCEMA shell (gray to black); the PI corona chains are made water-soluble by hydroxylating the double bonds (wavy lines to free-hand lines); the core is made inorganic-compatible by removing the tertbutyl groups (light gray to gridded pattern). Soaking the nanospheres in aqueous FeCl 2 leads to proton exchange (slanted to vertical grids) and the Fe 2+ ions are precipitated (NaOH) and oxidized (H 2 O 2 ) to yield cubic γ-Fe 2 O 3 magnetic particles (last step). Adapted with permission from Reference 42. Copyright 2000 American Chemical Society.

Figure 2 - 17 .

 217 Figure 2-17. TEM images for PI-b-PCEMA-b-PtBA nanospheres at each step of the synthesis: (a) after PCEMA cross-linking and PI hydroxylation (stained with OsO 4 ); (b) after removal of the tert-butyl groups (stained with OsO 4 ); (c) after Fe 2 O 3 loading (no staining); and (d) attraction by a magnet. Adapted with permission from Reference 42. Copyright 2000 American Chemical Society.

Figure 2 - 18 .

 218 Figure 2-18. (a) Chemical structure of PEG-b-poly(4-vinylbenzylphosphonate) (PEG-b-PVBP) and schematic illustration of the proposed morphology of the pegylated IONPs (PIONs); and (b) TEM image for the PIONs and their size distribution as inset. Adapted with permission from Reference 43. Copyright 2011 Elsevier B.V.

  -19a and Figure 2-20a), depending on the MW of the PAA block derived from PtBA (4.5, 8.4 and 16.8 kDa). These exhibited superparamagnetic properties at 300 K without hysteresis, and M s increasing with the NP size. The advantages of triblock structures (β-CD-P4VP-PtBA-PS) and (β-CD-P4VP-PtBA-PEO) were also highlighted by producing NPs with Fe 3 O 4 cores and PbTiO 3 (Figure 2-19b and Figure 2-19c) or Au shells (Figure 2-20b).

Figure 2 - 19 .

 219 Figure 2-19. (a) Representative TEM images for Fe 3 O 4 NPs synthesized with star-like PAA-b-PS templates, D(Fe 3 O 4 ) = 10.1 ± 0.5 nm; (b) TEM; and (c) HRTEM images for Fe 3 O 4 -PbTiO 3 core-shell NPs formed with the star-like triblock copolymers nanoreactors. Reprinted with permission from Reference 44. Copyright 2013 Macmillan Publishers Limited.

Figure 2 - 20 .

 220 Figure 2-20. TEM, HRTEM and digital camera images for core/shell Fe 3 O 4 /Au NPs: (a) Fe 3 O 4 core (D = 6.1 ± 0.3 nm); and (b) Fe 3 O 4 /Au core/shell NPs at different magnifications (Au shell thickness is 2.9 ± 0.2 nm). Fe 3 O 4 appears dark in the center. The magnetic properties of Fe 3 O 4 were retained, as evidenced by the response of the NP dispersion in toluene to a magnet (right panel in (b)). Reprinted with permission from Reference 44.

  the solvent mixture increased, a gradual phase transformation from 15 nm Fe 3 O 4 NPs (Figure 2-21a) to uniform (ca. 20 nm diameter, 200-300 nm length) α-FeOOH (goethite) nanorods (Figure 2-21b) was observed.

Figure 2 - 21 .

 221 Figure 2-21. TEM images for samples prepared in alcohol/water mixtures with various volume ratios of alcohol to water: (a) 0:1; (b) 1:1; and (c) 5:1. Reprinted with permission from Reference 45. Copyright Springer Science Business Media, LLC 2008.

Figure 2 - 22 .

 222 Figure 2-22. (a) AFM height image for the polychelate of a poly(acrylic acid)-graft-poly(nbutyl acrylate) brush and FeCl 3 after dialysis; and (b) TEM image for the unimolecular polymer brush polychelate of FeCl 3 (after dialysis). Adapted with permission from Reference 46. Copyright 2004 Springer-Verlag.

Figure 2 - 23 .

 223 Figure 2-23. Synthesis of Fe 3 O 4 /polymer nanocomposite in the presence of poly(ethylene glycol) methyl ether acrylate (PPEGMEA)-g-PMAA densely grafted double-hydrophilic copolymer. Reprinted with permission from Reference 3. Copyright The Royal Society of Chemistry 2008.

  -24a) or 80:20 (Figure 2-24b), and bimodal when increasing the Co content to 50:50 (Figure 2-24c) or 20:80.

Figure 2 - 24 .

 224 Figure 2-24. (a) TEM micrograph for a lamellar PS-b-P2VP copolymer containing 1.2 meq of iron/g P2VP; sample heated to 195 °C for 24 h, scale bar = 50 nm; (b) TEM micrograph for the same copolymer containing a total of 1.2 meq of Fe and Co/g P2VP, with an atomic ratio of Fe to Co of 80:20; sample heated to 161 °C for 24 h, scale bar = 100 nm; and (c) scanning transmission electron microscopy micrograph for the copolymer containing a total of 1.2 meq of Fe and Co/g P2VP, atomic ratio of Fe to Co = 50:50; sample heated to 161 °C for 24 h; the atomic ratio of Fe:Co at the center of the large particles was 48.9:51.1 from energy dispersive X-ray spectroscopy (EDX); scale bar = 50 nm. Reprinted with permission from Reference 49. Copyright 2003 Elsevier B.V.

  -25a) as 25.0, 18.0, and 6.0 nm sized particles, respectively, arranged in a hexagonal self-assembled pattern. The wide range of IONP diameters obtained and the constant spacing of the IONP pattern in all three cases demonstrated that PS-b-P4VP is a powerful template that effectively controls the nucleation and the growth of the particles. AFM (Figure 2-25b) and field emission-scanning electron microscopy (FE-SEM) imaging (Figure 2-25c) revealed the hemispherical topology of the IONPs.

Figure 2 - 25 .

 225 Figure 2-25. (a) TEM image for a self-assembled pattern of IONPs with hexagonal packing. The inset is a SAED pattern typical for γ-Fe 2 O 3 ; (b) AFM image for the hexagonal pattern of IONPs on a silicon wafer; and (c) field emission-scanning electron microscopy (FE-SEM) image in tilt view. The molar ratio of FeCl 3 precursor to 4-vinylpyridine was 0.5 in all cases. Reprinted with permission from Reference 50. Copyright 2005 American Chemical Society.

59 CeO 2

 592 -26A). A subsequent treatment with an ethanol solution at 40 °C for 15 h (Figure 2-26B) appeared necessary as an "activation step" for the PEO cylinders to create a functional chemical pattern for nanodot development, which was interpreted by the authors as being due to the crystallization of the PEO blocks induced by ethanol. The affinity of Fe(NO 3 ) 3 for PEO as compared to PS allowed selective inclusion of the metallic salt by spin-coating of the 0.4 wt% precursor solution (Figure 2-26C). Finally, ultraviolet (UV)/ozone treatment (Figure 2-26D) produced highly ordered Fe 3 O 4 particles (or Chapter 2 or CuO, depending on the precursor). This treatment not only produced the oxides from the precursors but also removed the polymer template. The Fe 3 O 4 particles appeared in HRTEM images as nanodots 24 nm in diameter and 9 nm in height, with a regular spacing of 42 nm between their centers.

Figure 2 - 26 .

 226 Figure 2-26. Schematic illustration of the fabrication of oxide nanodots: (A) highly ordered PS-b-PEO thin film prepared by a solvent annealing process; (B) nanoporous template produced by activation of the PEO cylinders upon exposure to ethanol at 40 °C for 15 h; (C) the metal oxide precursor diffuses into the cylinders after spin coating of the metal nitrate solution; and (D) oxide dots remaining after the ultraviolet (UV)/ozone treatment. (a) TEM cross-sectional image for iron oxide nanodots; the inset shows a higher magnification image; (b) cross-sectional HRTEM image for a single nanodot; and (c) HRTEM image for the nanodots after the UV/ozone treatment; the inset shows crystalline fringes corresponding to Fe 3 O 4 . Reprinted with permission from Reference 51. Copyright 2012 The Royal Society of Chemistry.

Figure 2 - 27 .

 227 Figure 2-27. For example at 800 °C in air, the IONPs shrunk in diameter by 3 nm and in height by 2 nm, which was ascribed to oxide densification.

Figure 2 - 27 .

 227 Figure 2-27. (a) AFM; and (b) SEM images for hexagonally ordered iron oxide nanodots after UV/ozone treatment. The inset of (b) shows the iron oxide nanodots after annealing at 800 °C for 1 h. Reprinted with permission from Reference 51. Copyright 2012 The Royal Society of Chemistry.

(

  834 m 2 •g -1 ) were loaded with a Fe(NO 3 ) 3 solution by repeated impregnation. After in situ thermal decomposition of the precursor at 450 °C under argon, Fe 3 O 4 nanocrystals were obtained in the confined pores of the carbon template (Figure 2-28).

Figure 2 - 28 .

 228 Figure 2-28. TEM images for unsupported Fe 3 O 4 NPs synthesized by: (a, b) the hydrothermal method; (c, d) the in situ formation of Fe 3 O 4 NPs on the CF composite; and (e) schematic representation of the in situ formation of Fe 3 O 4 nanocrystals in the confined pores of carbon foam. Reprinted with permission from Reference 54. Copyright 2011 The Royal Society of Chemistry.

Figure 2 - 29 .

 229 Figure 2-29. TEM images for sample sections: (a) 3.5 wt% Fe-loaded polystyrene-polyacrylate gel prior to reaction with NaOH (scale bar = 500 nm); (b) after reaction with NaOH, showing the distribution and uniformity of the magnetite nanocrystallites (scale bar = 500 nm); and (c) synthesis of the magnetic sponge-like copolymer gel. Comparison between an unloaded polymer gel (c, left), after exposure to a 0.2 M Fe 2+ solution (c, middle), and after reaction with NaOH (c, right). The gels imaged with a digital camera in (c) were in the swollen state. Reprinted with permission from Reference 55. Copyright 1998 WILEY-VCH Verlag GmbH.

Figure 2 - 30 .Magnetite

 230 Figure 2-30. (a) Schematic representation and (b) appearance of a magnetic hybrid hydrogel obtained by photopolymerization and in situ coprecipitation; (c) in the second step, the ferrogel was exposed to NaOH to hydrolyze the cross-link points and induce the release of water-dispersible IONPs. Adapted with permission from Reference 58. Copyright 2011 Elsevier B.V.

Figure 2 - 31 .

 231 Figure 2-31. (a) TEM image and (b) SAED pattern for magnetic Fe 3 O 4 NPs synthesized by the coprecipitation of iron salts within a polymer disk; (c) typical HRTEM image for a single Fe 3 O 4 NP, and size distribution of the Fe 3 O 4 NPs for samples containing the cross-linker at: (d) 0.46 mol% and (e) 6.14 mol%. Reprinted with permission from Reference 58. Copyright 2011 Elsevier B.V.

  -32).

Figure 2 - 32 .

 232 Figure 2-32. Schematic views and pictures of polyacrylamide (PAAm) hydrogel networks at three steps of loading with magnetite NPs: (a) swollen hydrogel; (b) iron ion-loaded hydrogel; and (c) magnetite NPs in the hydrogel matrix, inset: TEM image showing well-dispersed MNPs of uniform size. Reprinted with permission from Reference 61. Copyright 2009 Elsevier B.V.

Figure 2 -

 2 Figure 2-33. (a) TEM image for hybrid poly(N-isopropylacrylamide-co-acrylic acid-co-2hydroxyethyl acrylate) (poly(NIPAM-co-AA-co-HEA)) microgels loaded with 0.618 g Fe 3 O 4 /g polymer; the scale bar is 150 nm; and (b) magnetization curve at 300 K. Reprinted with permission from Reference 62. Copyright 2004 American Chemical Society.

Figure 2 - 34 .

 234 Figure 2-34. (a) Chemical structure of a poly(N-vinylcaprolactam/acetoacetoxyethyl methacrylate) copolymer; and SEM images for: (b) empty cross-linked microgels; (c) magnetite NPs; (d) composite microgels with 4% magnetite; (e) SEM; and (f) AFM images for microgels with 9.4% magnetite (the height scale of the AFM image in the height mode is 0-100 nm.) Reprinted with permission from Reference 63. Copyright 2004 American Chemical Society.

Figure 2 - 35 .

 235 Figure 2-35. Optical images for various magnetic microparticles: (a) homogenous magnetic disks; (b) homogenous triangular particles; (c) Janus disks; and (d) gradient particles. Reprinted with permission from Reference 65. Copyright 2012 American Chemical Society.

Figure 2 - 36 . 5 (

 2365 Figure 2-36. Coprecipitation process in photopolymerized microgels with ~ 20 µm diameter prepared in a microfluidic channel. After deprotonation in 0.5 M NaOH and rinsing with 0.5% Tween 20 to reach pH 7, 0.2 M FeCl 3 and 1 M FeCl 2 solutions were mixed with the microgels at a Fe 3+ :Fe 2+ ratio of 1:75. After diffusion of the iron ions in the polymer particles, excess salts were removed. After the addition of NH 4 OH at 60 °C, the MNPs were nucleated and grown in situ before a final rinse with Tween 20. All these steps were repeated several times for successive growth cycles. Reprinted with permission from Reference 65.Copyright 2012 American Chemical Society.

Figure 2 - 37 .

 237 Figure 2-37. TEM images for microgels with different magnetite contents: (a, b) no magnetite; (c, d) 8.4 wt%; (e, f) 15.4 wt%. Inset: EDX iron-mapping image. Reprinted with permission from Reference 66. Copyright 2007 Wiley-VCH Verlag GmbH & Co., Weinheim.

  -38) revealed nearly spherical IONPs with a mean diameter of 11 nm and a low size dispersity (21% standard deviation), distributed uniformly within the network serving as template, by allowing nucleation but inhibiting the overgrowth of NPs. XRD analysis confirmed the Fe 3 O 4 composition of the iron oxide. The particles exhibited superparamagnetic characteristics at room temperature (M s = 12.6 emu/g and T B = 171 K), with excellent magnetic responsiveness but readily redispersing after removal of the magnetic field.

Figure 2 -

 2 Figure 2-38. (a) Schematic representation of the preparation of Fe 3 O 4 NPs embedded in PS microspheres by thermal decomposition of Fe(oleate) 3 at 300 °C; SEM images for: (b) the polymer seed microspheres prepared by dispersion polymerization; (c) the magnetic polymer microspheres prepared by swelling and thermolysis; (d) the outer surface of magnetic polymer microspheres; and (e) TEM image for the ultramicrotomed magnetic polymer microspheres, showing the location of the IONPs in the microspheres. Reprinted with permission from Reference 67. Copyright 2009 American Chemical Society.

Table 2 - 5 .

 25 Summary of the different organic templates used as host matrices for the synthesis of MNPs. In this review, the examples gathered from the literature were sorted according to their chemical nature and structural properties (e.g., geometry, dimensionality, size). DEX: dextran; Alg: alginate; PNIPAM: poly(N-isopropylacrylamide); PVA: poly(vinyl alcohol); PVP: polyvinylpyrrolidone; P4VP: poly(4-vinylpyridine); PMGI: poly(methyl glutarimide); DHBC: double-hydrophilic block copolymer; PGA: poly(glycerol acrylate); PAsp: poly(aspartic acid); PAEMA: poly[2-(acetoacetoxy) ethyl] methacrylate; PGMA: poly(glycerol methacrylate); PDMAEMA: poly[(N,N-dimethylamino)ethyl methacrylate] PNOR: polynorbornene; PAMPS: poly(2-acrylamido-2-methyl-1-propansulfonic acid).

-

  Natural polysaccharides: DEX, DEX sulfate, Alg, DEX-b-PMAA-b-PNIPAM Synthetic linear polymers: PVA, PVP, P4VP, PEO, PAA, PMAA, PMGI Linear DHBC or amphiphilic copolymers Hydrophilic blocks: iron-complexing (PGMA, PGA, P(norbornene-methanol or di-carboxy), PMAA, PAA, PAsp, PAEA, PAEMA) or repulsive block (PEO, PEOGMA, PDMAEMA) Hydrophobic blocks: PMMA, PNOR, PVBP Tri-blocks: Pluronics ® , PI-b-PCEMA-b-PtBA Three-dimensional scaffold macroscopic matrices: Carbon foam, sponge-like PSco-PAA, P(S-co-CBMi) networks, Alg/PNIPAM semi-IPN, PAAm, PAMPS, P(4VP-co-HEMA) hydrogels Star-like (PPO-b-PAA, PEO-bor triangle: PEGDA, PVCL-co-AAEM core-VIm shell microgels Lamellar or hexagonally ordered cylinders (PS-b-P2VP, PS-b-PEO) Membranes (PVA, Nafion ® )Microemulsions:

Figure 3 - 1 .

 31 Figure 3-1. Schematic representation of the approach of two sterically stabilized particles. The parameter  represents the thickness of the solvophilic protective polymer layer forming the shell. Reprinted with permission from Reference 1. Copyright 1983 Academic Press.

4 *

 4 The steric layer thickness was calculated based on the molar mass of the polymer as ( ̅̅̅̅̅ ) ̅̅̅̅̅ with the intramolecular expansion factor  = 1.3; the expansion factor for chains normal to the interface f = 1.3; bond length l = 0.15 nm; polymer limiting characteristic ratio C  = 7, and molar mass of the repeating unit M o = 100.

  published an informative study on water-dispersible micelles produced by the complexation in water of a strong cationic polyelectrolyte, poly(N-ethyl-4vinylpyridinium bromide) (P4VP•EtBr), and poly(methacrylic acid)-b-polyisobutylene (PMAA-b-PIB). It is noteworthy that at pH 9, PIC formation and micellization of the amphiphilic PMAA-b-PIB block copolymer occurred simultaneously. The structure of the self-assembled PMAA-b-PIB micelles allowed the complexation of P4VP•EtBr with the PMAA shell through a polyion exchange reaction. This hypothesis was verified by DLS measurements, revealing the smaller size of the PIC micelles for [N + ]/[CO 2 -] = 0.4 as compared to the self-assembled species (19 nm vs. 22.5 nm, due to the collapse of some segments of the formerly stretched PMAA blocks interacting with PVP•EtBr). An onion-like structure consisting of a PIB core, a shell of polyion-complexed P4VP•EtBr and PMAA, and

  Gohy observed nanophase separation within the corona of the spherical PIC micelles obtained by mixing a polystyrene-b-poly(2-vinylpyridine)-b-poly(ethylene oxide) (PS-b-P2VP-b-PEO) triblock copolymer with a tapered triblock of poly(ethylene oxide) methyl ether methacrylate and acrylic acid, P(PEO 11 MA) 63 -b-PAA 657 -b-P(PEO 11 MA) 63 .

Figure 3 - 2 .

 32 Figure 3-2. Schematic illustration of the formation of a polyion complex micelle by PAA-b-PHEA and G0PS-g-P2VP in water.

3. 3 . 4 3 97

 343 Synthesis of Poly(tert-butyl acrylate)-b-Poly(2-trimethylsilyloxyethyl acrylate)Poly(tert-butyl acrylate)-b-poly(2-trimethylsilyloxyethyl acrylate) or PtBA-b-P(HEATMS) was synthesized by ATRP using either P(tBA)13 -Br ( n = 1660 g/mol) or P(tBA) 27 -Br ( n = 3460 g/mol) as macroinitiator. The procedure reported here used the macroinitator with n = 3460 g/mol. In a 100-mL Schlenk flask were added CuBr (65 mg, 450 µmol, 1 equiv), anisole (10 mL), PMDETA (93 µL, 44.5 mmol, 0.99 equiv), and freshly distilled HEATMS (31.6 mL, 157 mmol, 350 equiv). The reaction mixture was stirred for 5 min before the addition of PtBA-Br (1.555 mg, 450 µmol, 1.0 equiv). The solution was then degased by three successive freeze-pump-thaw cycles, the flask was filled with N 2 , and heated to 85 °C for 4 h 20 min to reach ̅ n = 60, and 19 h to reach ̅ n = 260. The reactions were stopped by submerging the flask in liquid nitrogen, before the addition of acetone (50 mL). The solution was passed through a column (15 cm height × 2 cm diameter) of activated neutral alumina (Aldrich-Sigma, Brockmann I grade, 150 mesh size) using acetone (100 mL) as eluent. After removal of the organic solvent by rotary evaporation, the polymer was dissolved in a minimum amount of methanol (ca. 30 mL) and further purified by dialysis in a 1,000 MWCO Spectra/Por ® 7 regenerated cellulose bag against 1 L of methanol. The solvent was changed thrice within 24 h, and the polymer was collected after removal of the solvent under vacuum. Chapter PtBA 13 -b-PHEATMS 50 1 H NMR (CDCl 3 , 400 MHz): n (NMR) = 11060, ̅ n,PHEATMS = 50, δ (ppm): 0.14 (s, 450H), 1.06-1.13 (m, 3H), 1.14-1.47 (b, 145H), 1.47-1.74 (b, 70H) 1.74-2.02 (s, 32H), 2.16-2.24 (b, 65H), 3.55-3.72 (b, 103H), 3.88-4.28 (b, 100H); n (SEC-PS) = 11850, n • w -1 = 1.15. PtBA 13 -b-PHEATMS 150 1 H NMR (CDCl 3 , 400 MHz): n (NMR) = 29860, ̅ n,PHEATMS = 150, δ (ppm): 0.14 (s, 1356H), 1.06-1.13 (m, 3H), 1.14-1.47 (b, 145H), 1.47-1.74 (b, 216H) 1.74-2.02 (s, 86H), 2.16-2.24 (b, 167H), 3.55-3.72 (b, 307H), 3.88-4.28 (b, 318H); n (SEC-PS) = 37620, n • w -1 = 1.23. PtBA 27 -b-PHEATMS 56 1 H NMR (CDCl 3 , 400 MHz): n (NMR) = 14730, ̅ n,PHEATMS = 56, δ (ppm): 0.14 (s, 506H), 1.06-1.13 (m, 3H), 1.14-1.47 (b, 283H), 1.47-1.74 (b, 93H) 1.74-2.02 (s, 46H), 2.16-2.24 (b, 83H), 3.55-3.72 (b, 115H), 3.88-4.28 (b, 114H); n (SEC-PS) = 14350, n • w -1 = 1.08. PtBA 27 -b-PHEATMS 260 1 H NMR (CDCl 3 , 400 MHz): n (NMR) = 50450, ̅ n,PHEATMS = 260, δ (ppm): 0.14 (s, 2306H), 1.06-1.13 (m, 3H), 1.14-1.47 (b, 283H), 1.47-1.74 (b, 348H) 1.74-2.02 (s, 180H), 2.16-2.24 (b, 286H), 3.55-3.72 (b, 552H), 3.88-4.28 (b, 520H n (SEC-PS) = 65780, n • w -1 = 1.16. PtBA-b-PHEATMS 13 C NMR (CDCl 3 , 100 MHz), δ (ppm): -0.01 (Si-(CH 3 ) 3 ), 23.67(CH 3 -CH), 26.10 (C-(CH 3 ) 3 , 39.43 (CH 2 backbone), 58.49 (CH backbone), 64.60 (CH 2 -O), 66.03 (CH 2 -O-SiMe 3 ), 78.38 (CH 2 -O-CO), 173.07 (CO).

9 Chapter 3 98mmol

 93 -b-P(HEATMS) 260 . To a mixture of THF(83 mL) and CH 2 Cl 2 (83 mL) was added P(tBA) 27 -b-P(HEATMS) 260(16.5 g, 81.HEATMS units, 8.5 mmol tBA units, ca. 10% v/v). The flask was cooled in an icewater bath with vigorous stirring and when the polymer was dissolved, H 2 O (5 mL) was added to the solution followed by trifluoroacetic acid (TFA, 277 mL, 3.62 mol, 40 equiv) drop-wise. The solution was left to react for 24 h. The solvent was then removed under vacuum and the deprotected polymer was redissolved in ethanol (EtOH, 30 mL). Solvent exchange was performed by dialysis in a 1,000 MWCO Spectra/Por ® 7 regenerated cellulose bag against EtOH (2 L) for 24 h, followed by EtOH/H 2 O (50/50 v/v, 2 L) for 24 h, and finally against 6 changes of H 2 O (5 L) for 72 h. To accelerate dialysis, the pH of the H 2 O used in the last step was maintained at pH 7 by the addition of NaOH solution. The clear solution obtained was collected and stored at 4°C. PAA 13 -b-PHEA 50 1 H NMR (DMSO-d 6 , 400 MHz) δ (ppm): 1.01-1.11 (b, 3H), 1.37-1.70 (b, 99 H), 1.70-1.89 (b, 32H), 2.01-2.47 (b, 65), 3.49-3.65 (b, 103H), 3.82-4.24 (b, 100H), 4.58-4.88 (b, 131H). PAA 13 -b-PHEA 150 1 H NMR (DMSO-d 6 , 400 MHz) δ (ppm): 1.00-1.11 (b, 3H), 1.35-1.70 (b, 177H), 1.74-1.91 (b, 68H), 2.01-2.47 (b, 165), 3.49-3.7 (b, 303H), 3.82-4.24 (b, 300 H), 4.58-4.88 (b, 121H). PAA 27 -b-PHEA 56 1 H NMR (DMSO-d 6 , 400 MHz) δ (ppm): 1.00-1.11 (b, 3H), 1.35-1.70 (b, 134H), 1.74-1.91 (b, 41H), 2.01-2.47 (b, 81H), 3.49-3.7 (b, 116H), 3.82-4.24 (b, 113 H), 4.58-4.88 (b, 46H). PAA 27 -b-PHEA 260 1 H NMR (DMSO-d 6 , 400 MHz) δ (ppm): 1.00-1.11 (b, 3H), 1.35-1.70 (b, 379H), 1.74-1.91 (b, 104H), 2.01-2.47 (b, 283H), 3.49-3.7 (b, 523H), 3.82-4.24 (b, 521H), 4.58-4.88 (b, 241H). PAA-b-PHEA 13 C NMR (DMSO-d 6 , 100 MHz), δ (ppm): 34.35 (CH), 40.08 (CH 2 ), 58.79 (CH 2 -O), 65.63 (CH 2 -OH), 174.07 (CO).

  PAA 27 -b-PHEA 260 for f = 0.5. In a 10-mL vial, G1 (1.7 mg, 14.7 µmol of N) was completely dissolved in 300 µL of aqueous HCl solution at pH 1.4 by sonication (37 kHz, 30 min, Ultrasonic Cleaner Elmasonic SH075EL). A pH 7 aqueous solution of PAA 27 -b-PHEA 260 (8.5 mg, 5 mg/mL, 7.35 µmol of CO 2 H) was quickly added. The mixture was stirred for 1 h before the pH was adjusted to 4.7 with a 1 M NaOH solution, and stirring was continued for 1 h. The pH was further adjusted to 7 with 0.1 M NaOH and the solution was stirred for 30 min. The solution was then dialyzed (50,000 MWCO Spectra/Por ® 7 regenerated cellulose bag) against Milli-Q water (5 L) for 24 h before it was collected and stored at 4 °C.

  C experiments) Bruker Avance I NMR spectrometer with a 5 mm BBFO probe. The concentration of the samples for the analysis ranged from 10-30 mg/mL. The apparent molar mass dispersity Ð SEC = w / n of the polymers was determined by size-exclusion chromatography (SEC) analysis on a PL-GPC 50 Plus (Agilent Technologies) instrument with RI and UV detectors and TOSOH TSK gel columns (G40000HXL, G3000HXL, G2000HXL). The instrument was calibrated with low dispersity polystyrene standards, and THF (with toluene as a flow rate marker) served as eluent at a flow rate of 1.0 mL/min at 40 ºC.

  632.8 nm, 22 mW output power), a proprietary ALV optical fiber-based detector, an APD-based single photon detector, ALV/LSE-5003 Electronics, and an ALV-5000/EPP correlator. The measurements were carried out with 5 samples at 0.2-1 mg/mL in aqueous solutions at 25 °C, at angles ranging from 30 to 150°. The intensity of scattered light was measured with the photomultiplier detector, and the data were analyzed by the Zimm extrapolation technique to zero concentration and angle.

Scheme 3 - 1 .Scheme 3 - 3 .

 3133 Scheme 3-1. Synthesis of PtBA via ATRP.

Figure 3 - 3 . 1 HFigure 3 - 4 .

 33134 Figure 3-3. 1 H NMR (400 MHz) spectra for (a) PtBA, (b) PtBA-b-P(HEATMS) in CDCl 3 , and (c) PAA-b-PHEA in DMSO-d 6 .

Chapter 3 109a 5 RbFigure 3 - 5 .

 3535 Figure 3-5. Ionization levels of CO 2 H and N functional groups at different pH.

Figure 3 - 6 .

 36 Figure 3-6. Hydrogen bonding between a P2VP unit and a PAA unit.

  The efficacy of each DHBC as colloidal stabilizer was screened using dynamic light scattering (DLS) measurements. The intensity-weighted hydrodynamic diameters and the size dispersity, defined in the cumulant analysis of DLS data by the polydispersity index (PDI, defined in Section 3.3.6) vs. the f ratio were used to compare the results, which are summarized in Figure 3-7, Figure 3-8, Figure 3-9, and Table 3-2. In all cases where an intensity-weighted unimodal size distribution was observed, similar results were also obtained for number-and volume-weighted distribution curves, but at slightly different peak positions. Since the intensity-weighted z-average is obtained by cumulants analysis of the autocorrelation function of the scattered intensity and is proportional to the sixth power of the particle size, it is biased toward larger particle sizes, particularly in the case of bimodal distributions or aggregation. However when unimodal distributions are obtained, the numberweighted distribution mean diameter is customarily used to describe the size of NPs with no bias toward larger sizes. The number-weighed diameters are reported here in parallel with the intensity-weighed values, and are used to provide more realistic dimensions of the NPs for comparison with other measurements providing number-weighted results (e.g.27 -b-PHEA 56 G1@PAA 13 -b-PHEA 50 G1@ PAA 13 -b-PHEA 150 G1@PAA 27 -b-PHEA 260

Figure 3 - 7 .Figure 3 - 8 .

 3738 Figure 3-7. Intensity-weighted size distributions for G1 (pH 4) and G1@PAA-b-PHEA PIC micelles (pH 7) obtained at various f ratios at 25 °C.

Figure 3 - 9 .

 39 Figure 3-9. Intensity-or number-weighted hydrodynamic diameter and zeta potential of PIC micelles. The last points are not connected with the lines as they were off-scale.

3 118

 3 -b-PHEA 50 (l PHEA  10.1 nm), should provide unimolecular PIC micelles with D max  35 nm. Better stabilization was observed for a ratio f = 3, yielding PIC micelles with = 32 nm (smaller than D max  35 nm) and a relatively narrow size distribution (PDI = 0.18). The seemingly unimolecular character of these micelles highlights the advantages of this third polymer. The thicker PHEA shell (shell thickness  9 nm, exceeding the 5 nm effective distance of the van der Waals forces) provides enhanced steric stability for the PIC micelles, which favors the formation of unimolecular species even though the contribution from electrostatic stabilization is fairly small (ZP = -13 mV).ChapterStabilization was also investigated for f =1, yielding PIC micelles with = 29 nm, PDI = 0.14 and ZP = -15 mV. These PIC micelles are still considered unimolecular, with a shell thickness  7 nm. Reducing the f ratio from 3 to 1 decreased the density of BC in the micelles 3-fold, such that steric stabilization due to the long polymer chains likely dominated colloidal stability. These results show that by introducing more HEA units into the polymeric stabilizer, the colloidal stability of the PIC micelles changed from electrosteric (a combination of electrostatic repulsion and steric stabilization, when using short PHEA chains) to predominantly steric stabilization (when employing longer PHEA blocks).

  27 -b-PHEA 260While the goal of determining the characteristics required for a DHBC to stabilize a G1 arborescent copolymer and form unimolecular structures was already achieved to some extent, understanding and controlling the size of PIC micelles by tuning the BC chain segment lengths was also an interesting challenge. Polymer PAA27 -b-PHEA 260 (l PHEA  12.2 nm) was designed to roughly double the size of the most effective BC tested up to this point (PAA 13 -b-PHEA 150 ). The of the PIC micelles obtained for f = 3 and f = 1 was comparable with micelles formed by PAA 13 -b-PHEA 150 , but interestingly the layer thickness increased by ca. 2-3 nm, in agreement with the larger dimensions of the PHEA chains. When comparing PAA 27 -b-PHEA 260 with PAA 27 -b-PHEA 56 at the same f, for example, the G1@PAA 27 -b-PHEA 260 f = 1 PIC micelles were efficiently stabilized by the long chain BC, whilst G1@PAA 27 -b-PHEA 56 f = 1 aggregated due to insufficient steric stabilization by the short BC chains.

  3.(top).

Figure 3 - 10 . 3

 3103 Figure 3-10. Measured dn/dc of the PIC micelles as a function of composition.

Figure 3 - 11 .Figure 3 - 12 .Figure 3 - 13 .

 311312313 Figure 3-11. Zimm plots for PIC micelles G1@PAA 27 -b-PHEA 56 and G1@PAA 13 -b-PHEA 50 for f = 9.

  BC chains shielding a G1 core in the micelles. For the five samples deemed to be unimolecular in nature the number of BC chains per micelle varies from 27 PAA 13 -b-PHEA 150 surrounding one G1 core for f = 1, to 68 molecules for G1@PAA 27 -b-PHEA 260 with f = 3. The number of BC molecules per micelle increased as the amount of BC (f ratio) used in their preparation increased, which seems reasonable. It is also larger for the PAA 27 -b-PHEA 260 sample series in comparison to the PAA 13 -b-PHEA 150 samples, in spite of the longer PHEA 260 segment increasing BC bulkiness. This highlights the dominating influence of the anchoring PAA segment length in determining the number of BC chains incorporated in the PIC micelles.

Figure 3 - 14 . 4 Figure 3 - 15 .

 3144315 Figure 3-14. Dependence of A 2 on the number of -OH groups/PIC micelle.

  surfaces. The G1 substrate, deposited from acidic solutions (pH 4), exhibited ill-defined morphologies because the arborescent graft copolymer flattened extensively on mica (visible more clearly on the phase image of Figure3-16), presumably due to strong electrostatic interactions between the protonated G1 and negatively charged mica. Both the molecular diameter (ca. 43 nm, larger than the = 29 nm in acidic solutions) and the height (ca. 1.9 nm) obtained by section analysis of the height image confirm strong flattening of the molecules.

Figure 3 - 16 .

 316 Figure 3-16. 3D model, height and phase AFM images (from left to right) for the G1 substrate.

Figure 3 - 17 .

 317 Figure 3-17. 3D models (row 1), height (row 2) and phase AFM images (row 3) for G1@PAA 27 -b-PHEA 260 with f = 0.5 at set-points ratios of 0.96 (column 1), 0.92 (column 2), and 0.86 (column 3).

3 O 4

 34 NPs, before binding the PAA-b-PHEA copolymer for PIC formation to enhance their colloidal stability and biocompatibility. It is the first time that a dendritic graft (arborescent) copolymer is used to assist in MNP synthesis. Magnetic suspensions with improved colloidal stability in aqueous environments over a wide pH range were obtained by that method, as confirmed by dynamic light scattering (DLS) measurements. The magnetic micelles had a hydrodynamic diameter D h  130 nm (polydispersity index PDI = 0.136) from DLS analysis, and a core-shell morphology was observed by atomic force microscopy imaging. The Fe 3 O 4 content of the micelles was quantified by thermogravimetric analysis, and large Fe 3 O 4 crystallites were visualized by transmission electron microscopy. The biocompatible poly(2-hydroxyethyl acrylate) shell coating these MNPs should facilitate their clinical applications as MRI contrast agents and hyperthermia mediators.

Figure 4 - 1 .

 41 Figure 4-1. The different phases of iron oxyhydroxides formed in a bidimensional diagram versus the molar OH -/Fe Total hydroxylation ratio and the ferrous-ferric composition in the system. Reprinted with permission from Reference 21. Copyright 2006 Académie des Sciences.

  MNPs with a hydrophilic shell are presented in this Chapter. The first method is to use PIC micelles incorporating a welldefined branched poly(2-vinylpyridine) (P2VP) polymer structure and a hydrophilic shell of poly(2-hydroxyethyl acrylate) (PHEA) as templates for MNPs. The second pathway is to first use the G1 copolymer as a template for the Fe 3 O 4 NP synthesis, and to then bind the PAA-b-PHEA copolymer to enhance the colloidal stability and biocompatibility. The high density of 2-vinylpyridine units in the very compact structure of arborescent copolymers was expected Chapter 4 148 to assist the templating Fe 3 O 4 NPs. The polyion complexation process of G1@Fe 3 O 4 MNPs

Scheme 4 - 2 .

 42 Scheme 4-2. Synthesis of an arborescent copolymer (G0PS-g-P2VP) by successive grafting reactions. Reprinted with permission from Reference 77. Copyright 2012 The Royal Society of Chemistry 2012.

Figure 4 - 2 .

 42 Figure 4-2. Size distributions for G0-G2 graft copolymers in HCl solutions (pH 4) at 25 °C.

PAA 27 - 3 A 4 A

 2734 b-PHEA 260 for f = 0.5. Briefly, in a 10-mL vial, G1 (1.9 mg, 16.7 µmol of N) was completely dissolved in 300 µL of aqueous HCl solution at pH 1.4. A pH 7 aqueous solution of PAA 27 -b-PHEA 260 (9.6 mg, 5 mg/mL, 8.3 µmol of CO 2 H) was quickly added. The mixture Chapter 4 152 was left to react for 1 h before the pH was adjusted to 4.7 with a 1 M NaOH solution, and stirring (400 rpm) was continued for 1 h. The pH was further adjusted to 7 with a 0.1 M NaOH solution and the solution was stirred for 30 min. The solution was dialyzed against Milli-Q water for 48 h before it was collected and stored at 4 °C. stock solution of FeCl 3 (24 mg, 0.148 mmol) and FeSO 4 7H 2 O (24.2 mg, 0.087 mmol) in 2 mL of Milli-Q water acidified with HCl (pH 1.4) was prepared before use. The sulfate salt was preferred for the production of Fe 3+ ions in situ by the oxidation of Fe 2+ with H 2 O 2 , since the chloride salt will generate Cl 2 gas. In a 15 × 150 mm 2 test tube, a water solution (pH 7) of PIC micelles G1@PAA 27 -b-PHEA 260 (11.2 mg, 33.3 mol of 2VP units) in 600 µL was prepared. The PIC micelle solution was stirred vigorously in an oil bath at 80 °C and 200 µL of the iron stock solution was quickly added (Fe/2VP molar ratio = 0.7), followed by 17 µL of concentrated NH 4 OH to trigger the coprecipitation. The solution color changed from yellow to brown, probably corresponding to different precipitated phases of iron oxyhydroxides rather than magnetite (black). Sedimentation of the magnetic nanoparticles (MNPs) in the magnetic field of a NdFeB permanent magnet (Calamit magnets, Milan, Italy) and decantation allowed complete removal of the excess reagents. The MNPs were rinsed water (2 × 5 mL) and redispersed in 2 mL of H 2 O to produce an orange coloured ferrofluid. stock solution of FeCl 3 (24 mg, 0.148 mmol) and FeSO 4 7H 2 O (24.2 mg, 0.087 mmol) in 2 mL of Milli-Q water acidified with HCl (pH 1.4) was prepared before use. In a 15 × 150 mm 2 test tube, G1 (3.9 mg, 33.3 mol of 2VP units) was completely dissolved in 600 µL of HCl solution (pH 1.4) by sonication at 37 kHz for 30 min (Ultrasonic Cleaner Elmasonic SH075EL). The G1 solution was then stirred vigorously with a mechanical rotor (> 800 rpm) in an oil bath at either 50, 80 or 100 °C and 200 µL of the iron stock solution Chapter 4 153 was quickly added (Fe/2VP molar ratio = 0.7), followed by 17 µL of concentrated NH 4 OH to trigger the coprecipitation. The solution color quickly changed from yellow to black.

  -b-PHEA 150 ( n = 18340 g/mol, Đ M n  w -1 = 1.23) and PAA 27 -b-PHEA 260 ( n = 30940 g/mol, Đ M = n  w -1 = 1.16) were selected for the present investigation. Complexation of the G1-templated MNPs with various amounts of the two PAA-b-PHEA block copolymers was carried out in aqueous solutions. The parameter f = CO 2 H/N (defined above) was used to quantify the molar ratio of CO 2 H groups and 2VP units. The following procedure describes the complexation of the G1-templated MNPs G1@Fe 3 O 4 with PAA 27 -b-PHEA 260 for f = 0.5. In a 10-mL vial, G1@Fe 3 O 4 (0.99 mg Fe 3 O 4 , 1.9 mg of G1, 16.7 µmol of N functional groups) was dispersed in 1 mL of HNO 3 solution (pH 1.4). A pH 7 aqueous solution of PAA 27 -b-PHEA 260 (9.6 mg, 5 mg/mL, 8.3 µmol of CO 2 H functional groups, f = CO 2 H/N = 0.5) was quickly added, the mixture was stirred for 1 h before the pH was adjusted to 4.7 with a 1 M NaOH solution, and stirring was continued for 1 h. The pH was further adjusted to 7 with 0.1 M NaOH and the solution was stirred for 30 min. The solution was then dialyzed (50,000 MWCO Spectra/Por ® 7 regenerated cellulose bag) againstMilli-Q water (5 L) for 24 h before it was collected and stored at 4 °C.

1 : 2 : 8 :

 128 Select gas 1 (N 2 ) Equilibrate at 30 °C 3: Ramp at 10 °C/min to 140 °C 4: Isothermal for 25 min 5: Ramp at 10 °C/min to 470 °C 6: Isothermal for 20 min 7: Select gas 2 (O 2 ) Ramp at 10 °C/min to 800

Figure 4 - 3 (

 43 right), contact of the PIC micelles (stabilized at pH 7, D h = 42 nm, PDI = 0.08) with the acidified iron solution (pH 1.4) yielded a pH 2.6 solution, inducing a low level of aggregation (D h = 180 nm, PDI = 0.282), similarly to the pH response Chapter 4 157 of PIC micelles discussed in Section 3.4.2.4. The addition of NH 4 OH solution to induce alkaline coprecipitation resulted in very large particles at pH 9 (bimodal, D h  470 nm, PDI  0.456, Figure 4-3, right), unsuitable for biomedical applications. Nanoparticles with diameters below 200 nm and homogeneous in size are indeed desired for systemic circulation, i.e., long blood half-lives, especially if they are coated with a neutral polymer sheath to achieve steric repulsion towards proteins.81 Due to the poor performance of this approach both in terms of templating and stability, the direct in situ employment of PIC micelles for Fe 3 O 4 alkaline coprecipitation was abandoned. The second preparation pathway, separating the templating and stabilization steps, was rather investigated.

Figure 4 - 3 .

 43 Figure 4-3. (Left) Weak magnetization of the MNPs produced by coprecipitation of iron salts in the PIC micelle templates. (Right) Hydrodynamic size distribution by DLS at 25 °C for PIC micelles G1@ PAA 27 -b-PHEA 260 f = 0.5 (pH 7), PIC micelles mixed with the iron salt solution (pH 2.6), and the product obtained after adding the NH 4 OH solution (pH 9).

D

  TEM = 22  3 nm), the G1 arborescent polymer should be a useful platform in itself for templating iron oxide NP formation (Figure4-4).After redispersion in HNO 3 solution (pH 1.4), the MNPs prepared by in situ coprecipitation in the template, G1@Fe 3 O 4 , had better colloidal stability than bare Fe 3 O 4 NPs prepared as a control sample produced under the same conditions without G1 arborescent copolymer. The bare Fe 3 O 4 NPs clustered and aggregated after a short time period (10 min), or immediately when subjected to a weak magnetic field, while the G1@Fe 3 O 4 ferrofluid was stable in both cases (Figure4-5).

Figure 4 - 4 .

 44 Figure 4-4. (Left) Hydrodynamic size distribution by DLS for G1 (pH 4) at 25 °C. (Right) TEM image for G1 prepared by depositing a THF solution, and staining with iodine.

Figure 4 - 5 .

 45 Figure 4-5. Improved colloidal stability of templated G1@Fe 3 O 4 ferrofluid as compared with non-templated Fe 3 O 4 ferrofluid.

Figure 4 - 6 .

 46 Figure 4-6. Crystallite size of Fe 3 O 4 NPs synthesized in the presence of: linear P4VP at 50 °C, G1 at 50 °C, and G1 at 80 °C. Inset: Selected area diffraction pattern for templated G1@Fe 3 O 4 .

Figure 4 - 7 . 4 164

 474 Figure 4-7. Top: TGA curves for pure G1 template (green, dotted), a mixture of dry G1 and Fe 3 O 4 (blue, long dash), and G1@Fe 3 O 4 (red, solid). Bottom: Temperature ramp (green, broken dash), weight loss (red, solid), and differential of the weight loss curve (blue, short dash) for G1@Fe 3 O 4 .

Fe 3 O

 3 4 paste. The slightly larger weight loss at 140 °C (solid trace, inset of Figure 4-7 top) evidences the abundance of physically adsorbed water molecules and of surface hydroxyl groups, since the G1 template was protonated. An almost flat plateau was observed after the 140 °C isothermal step, as shown by the derivative of the weight change curve (Figure 4-7. bottom), which is consistent with the continuous evaporation of a small amount of physically adsorbed water and the elimination of surface hydroxyl groups. -A massive weight loss in the 300-470 °C range, attributed to decomposition of PS and P2VP in the G1 template not coordinated with Fe 3 O 4 . The rate of weight loss decreased before reaching 470 °C. This weight loss is similar to what was observed for the two other samples, where the G1 template had no chemical interactions with Fe 3 O 4 .

4 165

 4 observed a two-step weight loss when characterizing folate-tetra(ethylene glycol)-terminated polyglycerol monoacrylate (FA-TEG-PGA) Fe 3 O 4 nanoparticles. PGA, with 1,2-diols groups, was assumed to be chemisorbed onto the Fe 3 O 4 NPs. The first weight loss was attributed to decomposition of the FA-TEG blocks and the second one, occurring at a higher temperature, was assigned to the degradation of PGA bound to Fe 3 O 4 . This phenomenon also occurred with dopamine Lglutamic acid linear and dendritic ligands used to functionalize Fe 3 O 4 NPs.88 At 460 °C a portion of the ligands immobilized at the surface of MNPs decomposed, while more energy (a higher temperature) was needed to break the bonds formed between dopamine and Fe 3 O 4 .ChapterThe stepwise thermal degradation of the G1 template likewise evidences strong interactions between P2VP and Fe 3 O 4 , thus confirming the success of templating.Beyond the initial 3.1 wt% loss due to the elimination of water (T  140 °C), a 54.9 wt% loss of organic material was observed for the G1 template for T  470 °C, and a residual mass of ca. 42.0 wt% was found at the end of the TGA experiment. If this the composition of the residue were pure Fe 2 O 3 , this would correspond to 43.3 wt% Fe 2 O 3 after correction for the initial water loss. This is higher than the theoretical composition of 35.1 wt% Fe 2 O 3 calculated for a Fe/2VP molar ratio = 0.7 (Section 4.3.1.4). The Fe 2 O 3 content

Fe 2 O 3 .

 23 The total sample weight loaded for the TGA analysis was 4.126 mg, of which 3.1 wt% was water, leaving 3.998 mg of nanocomposite. The Fe 2 O 3 content based on UV-VIS analysis of the residue is therefore 1.29/3.998 = 32.2 wt%, reasonably close to the theoretical composition of 35.1 wt% Fe 2 O 3 . As compared to TGA, the UV-VIS analysis method was therefore more reliable to determine the Fe (and Fe 3 O 4 ) content of the MNPs.

Figure 4 - 8 .

 48 Figure 4-8. (a) Absorbance spectra for 5 M HCl without iron (top panel) and with 10 μg/mL iron (bottom panel). (b) Calibration curve in 5 M HCl generated for iron concentrations ranging from 0 to10 μg/mL. Preprinted with permission from Reference 96. Copyright 2007 BioTechniques. (c) Calibration curve generated in 5 M HCl at  = 350 nm (path length l = 2 mm).

  to G1 alone. As expected, stable magnetic polyion complex (MPIC) micelles were formed for the same f ratios as the bare copolymers. PAA 13 -b-PHEA 150 assembled with G1@Fe 3 O 4 f = 1 to form MPIC micelles with an intensity-weighted D h = 124 nm and a number-weighted = 66 nm (to handle with caution, since the Malvern software does not handle the Mie-scattering model for hybrid particles made from two materials with different refractive indices and optical absorption coefficients), PDI = 0.172 (Figure4-9).Increasing the BC amount further yielded MPIC micelles in the same size range PDI = 0.175.A lack of BC stabilizer at f = 0.5 almost led to doubling the size of the MPIC micelles (D h = 181 nm, = 118 nm, PDI = 0.150). Therefore f = 1 was the optimal ratio for the complexation of G1@Fe 3 O 4 with PAA 13 -b-PHEA 150 . The MPIC micelles formed for f = 1 and 0.5 were nevertheless both prepared to be tested for biocompatibility (Chapter 6).

Figure 4 - 9 .

 49 Figure 4-9. Size distributions for magnetic micelles G1@Fe 3 O 4 @PAA 13 -b-PHEA 150 at different f ratios obtained at 25 °C and pH 7.

Figure 4 - 10 .

 410 Figure 4-10. Size distributions for magnetic micelles G1@Fe 3 O 4 @ PAA 27 -b-PHEA 260 at different f ratios obtained at 25 °C and pH 7.

  f = 0.5 appeared as aggregates of Fe 3 O 4 NPs in the TEM images, with D TEM = 11.6  2.0 nm for the Fe 3 O 4 crystallites (Figure 4-11). When observed by AFM, the MPIC micelles appeared as spheres (D AFM = 58  15 nm) with a rather broad size distribution on the mica surface. A soft hydrophilic shell surrounding each MPIC micelle was visualized around a harder core on the phase image, confirming the presence of a PHEA layer (Figure 4-12).A larger phase lag in tapping mode AFM is indeed associated with a softer material.[97][98] The inner core-shell morphology of the MPIC micelles was probed using different amplitude set points (Figure4-13), i.e., different forces exerted by the cantilever on the surface.

Figure 4 - 11 .

 411 Figure 4-11. TEM images for Fe 3 O 4 crystallites in G1@Fe 3 O 4 @PAA 27 -b-PHEA 260 f = 0.5.

Figure 4 - 12 .

 412 Figure 4-12. AFM images for magnetic micelles G1@Fe 3 O 4 @PAA 27 -b-PHEA 260 f = 0.5.

  of the MPIC micelles G1@Fe 3 O 4 @PAA 27 -b-PHEA 260 f = 0.5 was found to be pH-dependent, similarly to the metal-free PIC complexes discussed in Chapter 3.Highly negative charges (ca. -20 mV) were found at pH  7 (Figure4-14) due to the presence of CO 2 -functionalities. In the highly basic range, a plateau in the zeta potential of the MPIC micelles indicates complete ionization of the CO 2 H groups. The increase in zeta potential in the acidic pH range is assigned to protonation of the carboxylate groups to produce a hydrophobic CO 2 H derivatives, while the 2VP units of the G1 substrate yield NH + moieties. The point of zero charge (isoelectric point) of the MPIC micelles was around pH 3.

Figure 4 - 13 .

 413 Figure 4-13. Magnetic micelles at various amplitude set points (the lower the value in mV, the higher the average deflection of the cantilever and thus the exerted force).

Figure 4 - 14 .

 414 Figure 4-14. Zeta potential and intensity-weighted hydrodynamic diameter D h of MPIC micelles G1@Fe 3 O 4 @PAA 27 -b-PHEA 260 at f = 0.5 as a function of pH.

  Fe 2+ /Fe 3+ salts and the PIC micelles required different pH (1.4 and 7, respectively), and an intermediate pH led to the undesirable precipitation of Fe 2+ /Fe 3+ hydroxides and the aggregation of PIC micelles; and ii) the presence of the PAA-b-PHEA block copolymer as a potentially competing ligand in the shell, interfering with the accessibility of the iron precursors to the 2-vinylpyridine moieties.

pH

  For the first time, an arborescent copolymer (G0PS-g-P2VP) was used as a polymeric template to control the size and improve the size distribution of iron oxide NPs. The presence of the G1 component led to larger Fe 3 O 4 crystallites (D TEM  12 ± 2 nm) and enhanced colloidal stability due to electrostatic repulsion by the protonated NH + groups of the P2VP

  hyperthermia efficiency measurements probed by calorimetry. The materials prepared displayed properties typical of superparamagnetic iron oxide nanoparticles (SPIONs). The influence of the Fe/N ratio, the coprecipitation temperature, and the Fe 3 O 4 NP coating on the magnetization properties was examined. Discrepancies among the NP radii determined by relaxometry, TEM and magnetometry were explained by the different nature of these characterization methods. The magnetization value obtained by relaxometry was lower than by magnetometry, due to the larger relaxometric size englobing the non-magnetic polymer coating. The relaxivities of all the uncoated and block copolymer-coated magnetic nanoparticles were measured. The coated SPIONs had better colloidal stability but displayed limited clustering (due to their multicore nature), and thus had higher transverse relaxivity r 2 = 335 (s -1 mM -1 ) and r 2 /r 1 = 31.4 values (1.47 T, 37 °C) than the uncoated SPIONs. Our MNPs fare well when compared with contrast agents commercially available and in the literature, and could be useful as negative agents for T 2 -weighted magnetic resonance imaging. The nuclear magnetic resonance dispersion (NMRD) curves for the MNPs exhibited no low-field dispersion. The divergence observed between the experimental NMRD curves and the theoretical fits was ascribed to the complicated internal structure of the MNPs in comparison to the common "Outer-Sphere" model developed for individually dispersed ultrasmall superparamagnetic iron oxide particles. The uncoated samples had low relaxation rates and flat NMRD profiles, indicating strong agglomeration and confirming the essential role of the block copolymer coating. A good specific absorption rate (SAR = 55.6 Wg -1 ) was obtained by alternating magnetic field calorimetry (755 kHz, 10.2 kAm -1

Figure 5 - 1 .

 51 Figure 5-1. Switching of the magnetization in a particle across the anisotropy barrier under a) no external magnetic field; b) external magnetic field pointing downward; and c) external magnetic field pointing upward.

  therefore the exponential factor tends to 1. The Néel relaxation time is determined by the pre-exponential term , which decreases with increasing E a . At a given temperature, if the measurement time is longer than the Néel relaxation time, the magnetization rapidly relaxes by thermal fluctuation. The system has an "unblocked" magnetization typical of the superparamagnetic state. When E a >> k B T, the Néel relaxation time is determined by the exponential factor, which rapidly rises as the anisotropy energy increases. If the measurement time is shorter than the Néel relaxation time, the magnetization relaxes slowerit seems to be "blocked". The specific temperature at which the relaxation time matches the measurement time is called the blocking temperature, usually noted T B . External fields, interactions, and especially the particle size distribution were found to affect the blocking temperature. As most magnetization measurements (VSM or SQUID) have experimental times within one minute ( ), the blocking temperature usually obeys to the phenomenological equation ⁄ , defining numerically . Experimentally, blocking temperatures are determined qualitatively at the maximum of the "zero field cooled" curve (ZFC) representing the sample magnetization vs. temperature or, for polydispere samples, through a more quantitative fitting of the ZFC curve.8

Figure 5 - 2 .

 52 Figure 5-2. a) Main parameters of interest extracted from a generic hysteresis loop; b) Room temperature magnetization curve for superparamagnetic particles.

Figure 5 - 3 .

 53 Figure 5-3. Nuclei spin oriented along B 0 , with a slight excess in its direction producing a net longitudinal magnetization, M 0 . Reprinted with permission from Reference 15. Copyright 2006 Humana Press Inc.

2 .

 2 Nuclei undergo transverse or spin-spin relaxation: They stop precessing in phase, losing their phase coherence. The transverse magnetization (M xy ) gradually decays back to zero with a characteristic decay time T 2 . Despite the variety and complexity of RF pulse sequences used in MRI to manipulate the population of spins and create MR images, the simpliest one is the spin-echo sequence, represented in Figure 5-4. According to the solutions of Bloch equations describing longitudinal and transverse relaxations, the MRI signal detected by the antenna along M xy is proportional to the density of protons and to the product ( ⁄ ) ⁄ , where TR and TE are respectively the repetition time and the echo time. Sequences with short TR and short TE are considered as "T 1 -weighted" sequences, while those with long TR and long TE are "T 2 -weighted", and sequences with long TR and short TE are considered "densityweighted" (i.e. their contrast depends only on the local concentration of protons).

Figure 5 - 4 .

 54 Figure 5-4. 180 o Refocusing pulse and the formation of a spin echo. Reprinted with permission from Reference 15. Copyright 2006 Humana Press Inc.

Figure 5 - 5 .

 55 Figure 5-5. Outer-sphere relaxation of water protons in the vicinity of a superparamagnetic CA. Reprinted with permission from Reference 19. Copyright 2009 John Wiley & Sons, Inc.

Figure 5 - 6 .

 56 Figure 5-6. (Left) Custom setup for single frequency (755 kHz) hyperthermia measurements in Bordeaux. (Right) Field lines calculated using cylindrical axi-symmetry with the finite element simulation freeware FEMM (http://www.femm.info), showing calculated B field values close to the experimentally measured ones (B = 1 mT corresponds to H  800 Am -1 ).

Figure 5 - 7 .

 57 Figure 5-7. Commercial DM3 device from nanoScale Biomagnetics (Zaragoza, Spain)

  component nomenclature system to describe the samples: first figure (0.7 or 1.0) is the molar ratio of Fe/N used in coprecipitation reaction, the second figure (80 or 100) is the Chapter 5 205 temperature (°C) of coprecipitation reaction, and the third figure (UC or BC) refers to particles that are either bare (uncoated) or coated with block copolymer PAA 27 -b-PHEA 260 .

- 8 .

 8 All the curves exhibit no hysteresis loop, zero coercivity and zero remanence, thus indicating superparamagnetic behavior for the Fe 3 O 4 nanoparticles.

   = 0.385 vs. 0.492), and a smaller volume-average diameter D v (11.1 vs. 14.2 nm) (Table5-1)..

Figure 5 - 8 .

 58 Figure 5-8. Magnetization curves (at 300 K) for the Fe 3 O 4 samples. Sample nomenclature is explained in the text, and the iron oxide concentrations (mg/mL) are listed in Table 5-1.

2 .

 2 Less efficient templated nucleation at higher Fe 2+/3+ concentrations (for Fe/N = 1.0 as compared to 0.7), due to limited accessibility of the Fe 2+/3+ ionic precursors to the anchoring moieties (N). Due to the compact structure of the G1 template, it is very unlikely that 100% of the N moieties can be targeted (especially for Fe/N = 1.0), which gives rise to partial nontemplated nucleation. The broad size distribution ( = 0.492) observed is actually analogous to what is normally achieved for non-templated alkaline coprecipitation. The narrower size distributions for 0.7-80-BC ( = 0.385) and 0.7-80-UC ( = 0.386) are evidence for Chapter 5 208

Fe 2 O 3 (

 23 The volume-weighted diameter D vwhich emphasizes the contribution from larger particles was much larger than the number-weighted D n , and the ratio of these two values can be calculated from the width of the log-normal size distribution whose standard deviation of Ln(D), , is used to compute the number-and volume-average diameters (see Section 5.2.2). For example, sample 0.7-80-BC had D v = 11.3 nm, close to D n = 6.81 nm , while sample 1.0-80-BC had D v = 14.2 nm, much more different from D n = 6.86 nm . A narrower size distribution ( = 0.385) was indeed found for 0.7-80-BC than for 1.0-80-BC ( = 0.492). The reactions with a sub-stoichiometric amount of Fe 2+/3+ precursors (i.e. Fe/N = 0.7 rather than 1.0), designed to attenuate nonhomogeneous crystal growth, resulted in narrowing of the size distribution, as apparent by a decrease in the gap between D v and D n under every condition tested. The BC-coated samples prepared at 80 °C were found to have higher magnetization values (Figure 5-8 b, c, d) as compared to samples prepared at 100 °C, while no significant change in size distribution was observed. A higher reaction temperature facilitated the oxidation reaction, converting magnetite Fe 3 O 4 (with a higher M s value) to maghemite with an M s value lower by 20%). Coprecipitation reactions performed at 100 °C usually formed larger particles (D h = 206 nm, PDI = 0.2), which would be expected to Chapter 5 209 hamper internalization of the particles by the cells, so we chose to carry out the coprecipitation reactions at 80 °C.

3 O 4 @ 4 (

 344 PAA 27 -b-PHEA 260 nanoparticles, where the Fe 3 O 4 crystallites are distributed within the G1 template and protected by block copolymer chains functioning as surface-active agents. The uncoated samples were stored and characterized in HNO 3 solution at pH 1.4, which facilitated the oxidation reaction converting magnetite Fe 3 O with a higher M s value) to maghemite Fe 2 O 3 (with a 20% lower M s value). The BC thus acts as a protecting layer minimizing the oxidation of the Fe 3 O 4 NPs.

- 1 .

 1 Then a volume fraction obtained for sample 0.7-80-BC from the scaling law was subsequently used to calculate the number of Fe 3 O 4 NPs in one MPIC micelle: 13 spherical Fe 3 O 4 NPs of 12 nm diameter coexisting in a polymer-stabilized sphere with ca. 130 nm hydrodynamic diameter, which is consistent with the TEM and AFM images obtained for the magnetic PIC micelles.

Figure 5 - 9 .

 59 Figure 5-9. (Top) Correlation between the normalized transverse relaxivity and the particle diameter at high field (≥ 1 T) and 37 °C. (Bottom) Correlation between the transverse relaxivity and the normalized diameter at high field (≥ 1 T) and 37 °C. The transverse relaxivity r 2 = 335 s -1 mM -1 measured for sample 0.7-80-BC locates it in the high range of the spectrum. Adapted with permission from Reference 88. Copyright 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Figure 5 - 10 .

 510 Figure 5-10. NMRD profiles for the various synthesized Fe 3 O 4 samples. The sample nomenclature is described in the text.

Figure 5 - 11 . 5 219

 5115 Figure 5-11. Fits to the NMRD curves for the uncoated magnetic G1@Fe 3 O 4 micelles.

Figure 5 - 12 .

 512 Figure 5-12. Fits to the NMRD curves for the coated magnetic G1@Fe 3 O 4 micelles.

  3 O 4 when the Fe precursors exceeded the encapsulating capability of the G1 template, resulting in a broad size distribution of Fe 3 O 4 crystals, as determined earlier from the large standard deviation ( > 0.45) measured by VSM. This confirms that Fe/N = 1.0 is larger than the optimal ratio. The parameters extracted from these fits ( and ) cannot have physical significance because of the existence of such a broad size distribution. Fits for the samples with Fe/N = 0.7 gave much better results, which is consistent with a narrower size distribution (as confirmed by a lower standard deviation   0.38 from VSM); The parameters obtained are thus useful to interpret the results. The larger NMRD radius values for the BC set as compared to the UC set (Table 5-4) are interpreted as being due to the desirable presence of hydrophilic block copolymers around the crystals. At very high fields, when Curie relaxation dominates over Néel relaxation, the presence of a hydrophilic layer like PHEA increases the translational diffusion correlation time , which is the predominant component contributing to the total correlation time . Sharifi found a very small (0.06 nm) increase when comparing the apparent crystal radii (from NMRD) for the bare Fe 3 O 4 NPs and Fe 3 O 4 NPs coated with carboxyethylsilanetriol,

Figure 5 - 14 .

 514 Figure 5-14. Specific absorption rate (SAR) of magnetic G1 micelles as a function of magnetic field frequency for various fixed values of magnetic field amplitude H max .

  by different authors, but our results show that this normalization must be done with caution.

Figure 5 - 15 .

 515 Figure 5-15. Specific absorption rate (SAR) of the magnetic micelles as a function of the product of squared magnetic field amplitude H max 2 and frequency f.

Fe 3 O

 3 4 nanoparticles exhibited superparamagnetic behavior, i.e. no hysteresis loop, zero coercivity and zero remanence. The templating Fe/N ratio, temperature, and the presence of coating materials were found to influence the magnetization and relaxometric properties of the Fe 3 O 4 NPs to different extents. G1@Fe 3 O 4 @BC samples prepared with a ratio Fe/N = 0.7 displayed higher magnetization than those produced with Fe/N = 1, due to the formation of larger median diameter magnetite NPs with narrower size distributions. Samples prepared at 80 °C exhibited higher saturation magnetization than samples prepared at 100 °C, while no significant change in size distribution was observed, since a higher temperature facilitated the oxidation reaction converting magnetite Fe 3 O 4 to maghemite -Fe 2 O 3 . Higher magnetization was also found for samples coated with PAA 27 -b-PHEA 260 . The observed discrepancy between diameters determined by VSM and by TEM was ascribed to spin canting occurring at the surface or within the volume of the nanoparticles.

  double-hydrophilic block copolymers (DHBC) poly(acrylic acid)-b-poly(2-hydroxyethyl acrylate) by atom-transfer radical-polymerization (ATRP) were described in Section 3.3. Briefly, low molar mass poly(tert-butyl acrylate) PtBA-Br macroinitiators synthesized via ATRP were employed in a second ATRP reaction to create poly(tert-butyl acrylate)-bpoly(2-trimethylsilyloxyethyl acrylate) PtBA-b-P(HEATMS). These block copolymers were then hydrolyzed to produce the corresponding PAA-b-PHEA copolymers, which were purified by dialysis and stored in aqueous solutions at pH 7. Two block copolymers, PAA 13b-PHEA 150 ( n = 18,340 g/mol, n . w -1 = 1.23) and PAA 27 -b-PHEA 260 ( n = 30,940 g/mol, n . w -1 = 1.16), were used in the investigation. One of the block copolymers was labeled with a fluorescent dye using 1-ethyl-3-(3dimethylaminopropyl) carbodiimide hydrochloride (EDC, Sigma-Aldrich, ≥ 99.0%) and fluoresceinamine isomer I (FA, Sigma-Aldrich, 75%). The labeled PAA 27 -b-PHEA 260 copolymer (hereinafter referred to as PAA 27 * -b-PHEA 260 ) was prepared via amidation of the carboxylic groups in the PAA block with fluoresceinamine as illustrated in Scheme S6-1. A water dispersion of PAA

3 . 6 .

 36 The following procedure describes the complexation of the G1 copolymer with PAA 27 -b-PHEA 260 for f = CO 2 H/N = 0.5 (molar ratio of CO 2 H groups on the PAA segment of the block copolymer and N moieties of the P2VP segments on the arborescent copolymer). Briefly, in a 10-mL vial, G1 (1.7 mg, 14.7 µmol of N) was completely dissolved in 300 µL of aqueous HCl solution at pH 1.4. A pH 7 aqueous solution of PAA 27 -b-PHEA 260 (8.5 mg, 5 mg/mL, 7.35 µmol of CO 2 H) was quickly added.The mixture was left to react for 1 h before the pH was adjusted to 4.7 with a 1 M NaOH solution and stirring was continued for 1 h. The pH was further adjusted to 7 with a 0.1 M NaOH solution and the solution was stirred for 30 min. The solution was dialyzed against Milli-Q water for 48 h before it was collected and stored at 10 °C.The preparation of the fluorescently labeled PIC micelles (hereinafter referred to as PIC* micelles) was performed in the dark by the same procedure, but with a mixture of 5% PAA 27 * -b-PHEA 260 (7.0% labeled) and 95 mol% of (non-labeled) PAA 27 -b-PHEA 260 .

1. 4 .For

 4 Concentrated NH 4 OH was added to trigger the coprecipitation into colloidal Fe 3 O 4 . The magnetic nanoparticles (MNPs) were washed and redispersed in HNO 3 at pH 1.4 to produce a black homogeneous ferrofluid. Complexation of the G1 templated MNPs with various amounts of PAA-b-PHEA block copolymers was carried out in aqueous solutions. The f = CO 2 H/N ratio (defined previously) was varied within the range f = 0.25-1. Briefly, the G1@Fe 3 O 4 ferrofluid in HNO 3 pH 1.4 (0.99 mg Fe 3 O 4 , 1.9 mg of G1, 16.7 µmol of N functional groups) was loaded in a 10-mL vial and an aqueous solution of PAA-b-PHEA at pH 7 (9.6 mg, 5 mg/mL, 8.3 µmol of CO 2 H functional groups) was quickly added. The mixture was left to stir for 1 h before adjusting the pH to 4.7, and stirring further for 1 h. The pH was then adjusted to 7 and the solution was stirred for 30 min, dialyzed against Milli-Q water (5 L) for 48 h, collected and stored at 10 °C. The preparation of the fluorescently labeled MPIC* micelles was performed in the dark by the same procedure, but with a mixture of 5% PAA 27 * -b-PHEA 260 (7.0% labeled) and 95% of non-labeled PAA 27 -b-PHEA 260 . cell culture, Dulbecco modified Eagle medium (DMEM, with 4.5 g/L glucose, sodium bicarbonate, without sodium pyruvate, sterile-filtered, suitable for cell culture), GlutaMAX fetal bovine serum (FBS), and 0.5% trypsin solution 10× were purchased from Gibco, Grand Island, NY. Sodium pyruvate was obtained from Biowhittaker (Walkersville, MD). PBS solution (with Ca 2+ and Mg 2+ ), PBS solution (without Ca 2+ and Mg 2+ ), penicillin and streptomycin were purchased from Lonza. The mouse fibroblast-like L929 cell line was purchased from LifeTechnologies. The U87 human glioblastoma was the line ATCC ® HTB-14 ™ obtained from the American Tissue Culture Collection (Manassas, VA) . 3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) was obtained from Promega (Madison, WI) for the cell viability tests. Paraformaldehyde (Sigma-Aldrich, powder 95%), Triton X-100 (Sigma-Aldrich, BioXtra, for molecular biology), bovine serum albumin (BSA), Alexa Fluor® 594 phalloidin (Life Technologies, 300 units), 4',6'-diamidino-2-phenylindole (DAPI, Sigma-Aldrich 98.0%), VECTASHIELD HardSet Mounting Medium (Vector laboratories) served for the cell internalization experiments.

Figure 6 - 1 .

 61 Figure 6-1. (Left) 48-well plate where L929 and U87 cells were seeded and incubated with the MPIC micelles at 3 concentrations (1250, 700, and 140 µg Fe 3 O 4 /mL) in DMEM before being rinsed, trypsinized and transferred to NMR tubes for magnetic field exposure in a 37°C thermostated bath (Right). The image also shows the two optical fibres linked to the signal conditioner (Opsens) recording the temperature profiles.

Figure 6 - 2 .

 62 Figure 6-2. (Left) Fluorescence spectra for PAA-b-PHEA, free fluoresceinamine and fluorescently labeled PAA*-b-PHEA; (Right) Linear correlation of fluoresceinamine (Ex: 488 Em: 530) emission intensity vs. concentration in PBS buffer.

Figure 6 - 3 .The

 63 Figure 6-3. (Left) Fluorescence spectra for PIC and PIC* micelles in water; (Right) Fluorescence spectra for MPIC and MPIC* micelles in PBS buffer.

  -4), despite their different sizes. In this Chapter, we will only refer to the intensity-weighted hydrodynamic diameter as D h . The G1@PAA 27 -b-PHEA 260 f = 0.5 and G1@PAA 13 -b-PHEA 150 f = 1 PIC micelles had hydrodynamic diameters below 50 nm, while the G1@PAA 27 -b-PHEA 260 f = 0.25 micelles had D h = 87 nm (PDI = 0.18), and G1@PAA 13 -b-PHEA 150 f = 0.5 had D h = 104 nm (PDI = 0.04). Even after a long incubation period (48 h) the L929 cells were well spread in the wells and some of them were still undergoing mitosis, indicating the proliferation capability of the cells after their exposure to the nanoparticles. The good cell viability is attributed to the high hydrophilicity, Chapter 6 250 biocompatibility and electrostatic neutrality of the PHEA shell. The diffrences observed among the range of conditions used were found to be insignificant using Student's t-test.

Figure 6 - 4 .

 64 Figure 6-4. Cytotoxicity profiles for the PIC micelles G1@PAA-b-PHEA at various complexing ratios f with the fibroblast-like L929 cells determined via MTS assay at different concentrations after 48 h of incubation. The cell viability values are expressed as the mean values and the standard deviations from three independent experiments, each with four replicates per condition, relatively to untreated cells (100% control).

  Cell viability furtherdropped to 40% at 2000 µg of Fe 3 O 4 SPION/mL. Similar results were published by Hussain et al.,55 reporting a 30% decrease in cell viability at a concentration of 250 µg Fe 3 . Brief (4 h) exposure to a higher concentration of 23.05 mM of SPION (ca. 5347 µg Fe 3 O 4 SPION/mL) was found to have no effect on cell viability.56 In our work, the bare G1@Fe 3 O 4 particles were also found to have dose-dependent cytotoxicity. At the highest concentration tested (1250 µg Fe 3 O 4 /mL) for a long exposure time of 48 h, L929 cell viability was 51  8% (Figure6-5, non-coated, f = 0). The relative viabilities were higher (57  3% and 81  4%) when treated with 700 and 140 µg of Fe 3 O 4 /mL solutions, respectively. These results are analogous to the toxicity of bare Fe 3 O 4

Figure 6 - 5 .Fe 3 O

 653 Figure 6-5. Cytotoxicity profiles for uncoated MNPs G1@Fe 3 O 4 (f = 0) and MPIC micelles G1@Fe 3 O 4 @PAA-b-PHEA at various complexing ratios f towards fibroblast-like L929 cells determined via MTS assay at different concentrations after 48 h of incubation. Cell viability values are expressed as the mean values and standard deviations from three independent experiments with four replicates, relatively to the untreated cells (100% control).

Figure 6 - 6 .

 66 Figure 6-6. Cellular toxicity induced by SPION (Reprinted with permission from Reference 61. Copyright 2010 Neenu Singh et al.).

  PAA 27 -b-PHEA 260 at f = 0.5 (optimal) and f = 0.25. We observed low cytotoxicity for the MPIC micelles up to the highest concentration tested (1250 µg Fe 3 O 4 /mL) after 48 h of incubation. Specifically, we observed over 91  7% cell viability for the MPIC micelles as compared with 51  8% cell viability for the bare MNPs. Even after a long incubation period (48 h), the L929 cells were well-spread in the wells and some were still undergoing mitosis, indicating their ongoing capability to proliferate after exposure to the nanoparticles. The cell loss ratios were negligible at reduced Fe 3 O 4 concentrations (700, 140, 70, 28, and 14 µg of Fe 3 O 4 /mL). The MPIC micelles formed at optimal coating ratios caused no or negligible reductions in cell viability. The MPIC micelles coated with a less dense polymeric shell showed slightly higher reductions in cell viability, but only at the higher concentrations tested. These results show that PAA-b-PHEA successfully formed a protective, biocompatible shell around the Fe 3 O 4 nanoparticles.

Figure 6 - 7 .

 67 Figure 6-7. CLSM images for the L929 cells treated with the PIC* micelles G1@PAA* 27 -b-PHEA 260 f = 0.5; 2 mg/mL, 24 h incubation.

Figure 6 - 8 .

 68 Figure 6-8. CLSM images for L929 cells treated with the MPIC* micelles G1@Fe 3 O 4 @PAA* 27 -b-PHEA 260 f = 0.5; 140 µg Fe 3 O 4 /mL, 24 h incubation.

Figure 6 - 9 .

 69 Figure 6-9. 3D construction and xz, yz sections for L929 cells internalized with MPIC* micelles G1@Fe 3 O 4 @PAA* 27 -b-PHEA 260 f = 0.5; 140 µg Fe 3 O 4 /mL, 24 h incubation.

  -11, although the formal proof of this internalization pathway would require complementary experiments with clathrin inhibitors).

Figure 6 - 10 .Figure 6 - 11 .Fluorescence

 610611 Figure 6-10. TEM images for L929 cells treated with an MPIC micelle solution (100 µg/mL,

  -13).

Figure 6 - 12 .

 612 Figure 6-12. Dependence of the mean fluorescence intensity (MFI) for L929 cells treated with 100 μg/mL solution) of the MPIC* micelles G1@Fe 3 O 4 @PAA* 27 -b-PHEA 260 f = 0.5 on the concentration (after 24 h incubation) and on the incubation time (*p < 0.002, **0.002 < p < 0.01 by the two-tailed Student's t-test, degrees of freedom = 4).

Figure 6 - 13 .

 613 Figure 6-13. Linear regressions for the MFI and % positive L929 cells vs. incubation time.

  by heating a cell pellet in a volume of 0.35 mL, containing 20 million human prostatic adenocarcinoma cells incubated with MNPs (at a Fe 3 O 4 concentration of 2.5 mg/mL, SAR = 203 W.g -1 ), to 44.6 °C after applying an AMF of 700 kHz at B 0 = 31 mT (H 0 = 24.8 kA/m) for 10 min. The maximum temperature reached showed strong dependence on the concentration of the MNPs in the cell pellet: Cells incubated at 0.41 mg Fe 3 O 4 /mL only reached a plateau temperature below 39 °C. More importantly, the lower efficiency of heating induced by intracellular MNPs as compared to freely dispersed MNPs in water was likely due to confinement of the MNPs within the intracellular vesicles, implying that Néel relaxation was the sole mechanism of heat generation by intracellular MNPs. This conclusion was later confirmed by Di Corato et al. 107 who suggested the complete inhibition of Brownian relaxation in cellular conditions on the basis of abruptly decreasing specific absorption rate (SAR) values, especially when the MNPs were attached to the cell membrane or internalized into intracellular vesicles.Magnetic hyperthermia was also applied to enhance release of the drug doxorubicin encapsulated in alginate microbeads.108 Only 5.7% of viable cells resulted when doxorubicin release was triggered by the hyperthermia of MNPs after 2 h of exposure to an AMF of 700 kHz and B 0 = 10 mT (H 0 = 8 kA/m) amplitude, which brought the cells to 45 °C. Using an AMF with doxorubicin and iron oxide dual-loaded hydrogels resulted in 60% of viable cells, although a temperature of 45 °C was also reached in that case, also contributing to cellular death. A similar approach using MNPs and doxorubicin-loaded polymersomes performed by Oliveira et al.109 resulted in an 18 % increase in cell toxicity after 72 h of post-AMF exposure (750 kHz at B 0 =14 mT (H 0 =11.2 kAm -1 ) for 10 min, 3 times a day, without macroscopic variation of temperature due to the release of doxorubicin triggered by melting of the polymersome membranes. Using a sub-lethal dose of doxorubicin encapsulated in polymersomes (12 µg/mL), the authors evidenced that cell viability for HeLa cells decreased to 55% after field treatment, which was lower less than for the controls (95% for polymersomes loaded with MNPs but without drug, and 73% for cells incubated with the drug and MNP-loaded polymer vesicles, but without magnetic field application).While reaching a lethal cell temperature is the most common goal in hyperthermia treatment, Creixell et al. reported a method that reduces cell viability without a perceptible temperature rise, by comparing the magnetic hyperthermia-induced toxicity of Fe 3 O 4 MNPs and Fe 3 O 4 MNPs grafted with antibodies against the epithelial growth factor receptor (EGFR), which is often overexpressed in tumoral vasculature.104 Only 50% of the cells incubated with Fe 3 O 4 MNPs were viable after AMF exposure (233 kHz, 37.5 kA/m, 2 h). In comparison, EGFR-targeted Fe 3 O 4 MNPs resulted in 40% of cell viability even in absence of magnetic field. Exposing the cells incubated with EGFR-targeted Fe 3 O 4 MNPs to the same AMF treatment reduced cell viability to 4-6%, evidencing the synergetic effect of specific targeting receptors with antibodies and magnetic hyperthermia. Asín et al. also demonstrated the possibility of killing cells without perceptible temperature increase by exposing cells incubated with MNP-loaded dendritic cells to an AMF (260 kHz, 12.7 kA/m).20 In that case, 100% cell death was reported based on a trypan blue staining after 15 min of AMF exposure. Sanchez et al.21 grafted iron oxide NPs with gastrin, a peptidic ligand that specifically binds to CCK2R, a G-protein coupled receptor overexpressed in several malignant cancers including endocrine pancreas cancer. Iron oxide NPs grafted with the ligand were internalized and found to accumulate in lysosomes. Exposure to an AMF (275 kHz, 52 mT, 2 h) reduced cell survival to 30%, even though the amount of internalized iron was low (2.2 pg Fe/cell). Using a confocal microscope simultaneously with AMF application, thanks to a miniaturized induction applicator, Connord et al.22 discovered that cell damage seen exclusively in cells containing MNPs exposed to AMF was induced by lysosomal membrane permeabilization and ROS generation. The self-alignment of lysosomes with internalized

Figure 6 - 14 .

 614 Figure 6-14. Temperature variation of suspensions of control cells and cells incubated with MPIC micelles during exposure to an alternating magnetic field (755 kHz, 10.2 kA/m, 1.5 or 3 h).

Figure 6 - 15 .

 615 Figure 6-15. Viability of L929 (left) and U87 (right) cells determined by the MTS assay after 24 h of incubation with MPIC micelles G1@Fe 3 O 4 @PAA 27 -b-PHEA 260 f = 0.5, followed by or without exposure to the high frequency alternating magnetic field. Cell viability values were expressed as the mean values relative to the untreated cells (control 100%; statistical analysis was done by the two-tailed Student's t-test).

6 271 2 .

 62 Local heating produced by MNPs exposed to an AMF is generally assumed as the main source of energy inducing membrane alteration, cytoskeletal damage and apoptosis in cells. Riedinger designed molecular temperature probes consisting in a fluoresceinamine dye attached at different distances from the surface of the MNPs through a thermally sensitive azo linker, using PEG spacers of different w .118 Rapid local heating with a temperature rise of up to 45 °C was measured at distances below 0.5 nm from MNPs surface, based on thermally induced cleavage of the azo molecules and the subsequent realease of the dye. The temperature decayed exponentially with distance, thus explaining the significant difference between the local temperature at the nanoparticle surface and that of the bulk. The concept of local heating was further developed by Huang and co-workers 119 who employed DyLight549 red fluorescence as a thermometer to record the temperature increase distribution in cells. After 30-45 s of exposure to an AMF, a local temperature increase of more than 15 °C (determined from the change in DyLight549 fluorescence intensity) was measured at the plasma membrane where the MNPs were attached, but no temperature change was observed at the Golgi apparatus. The local temperature increase was sufficient to open the TRPV1 channels, leading to an influx of calcium ions. After 15 s of AMF exposure, the cytosolic calcium concentration increased 16-fold, confirming the successful thermal activation of the TRPV1 channels by the MNPs and AMF exposure. High-frequency motions of the NPs may also be an energy generation mechanism killing the cells in magnetic hyperthermia experiments without leading to a perceptible temperature rise. Carrey et al. 120 exposed MNPs to an inhomogeneous AMF and found that the MNPs could mechanically oscillate and generate ultrasound waves, due to the alternating gradient. The idea of generating ultrasounds by internalized MNPs was seen as a possibility to apply ultrasound therapy at the cellular level. Chapter Why do cells die? What are the effects that the energy generated by the MNPs under AMF can bring to cells? Apoptosis has been proposed as a main cause leading to cell death. Villanueva et al. 121 reported significant cellular damage that led to cell death by an apoptotic mechanism when applying a AMF to HeLa tumour cells incubated with MNPs. Hoechst-33258 stained apoptotic nuclei were visualized and counted by fluorescence microscopy. Approximately 20% of the cells were reported to have morphological characteristics typical of apoptosis (round shape, condensation, shrinkage, and chromatin fragmentation) accompanied with cell detachment, even though the cell culture temperature increased by less than 0.5 °C.Lysosomal membrane permeabilization (LMP)[20][21]122 has been suggested more often as a mechanism causing cell death. Work by Domenect et al. thus showed that epidermal growth factor receptors (EGFR) targeting the MNPs after they were internalized into the cells could cause LMP when subjected to an AMF.122 Lysosomal rupture was confirmed with acridine orange (AO), a lysosomal dye that emits red fluorescence inside intact lysosomes and a weak green fluorescence when bound to DNA or in the cytosol. The reduction in AO intensity revealed a 4-fold increase in cells with lysosomal rupture. The origin of the LMP was assigned to heat dissipation or to the rotation of MNPs adjacent to lysosomes, that increased the production of reactive oxygen species (ROS) and resulted in the massive peroxidation of membrane lipids.122 A similar mechanism leading to cell death was proposed by Sanchez et al.21 who used LysoTracker Red as a lysosome stain to detect a decrease in the number of lysosomes. Lysosome membrane permeabilization followed by leaking of their content (lower pH, metalloproteases…) was suspected to cause tumoral cell death under AMF.

Chapter 6 273

 6 Cell internalization of the MNPs was visualized by CLSM and TEM, and quantified by FACS. In spite of being negatively charged, both fluorescently labeled PIC* G1@PAA* 27 -b-PHEA 260 f = 0.5 and MPIC* micelles G1@Fe 3 O 4 @PAA* 27 -b-PHEA 260 f = 0.5 were successfully internalized through the L929 cell membrane, as evidenced by the green fluorescence found primarily at the periphery of the nuclear membrane and in intracytoplasmic vesicles. The fast cellular uptake kinetics of both PIC and MPIC micelles showed the influence of their small size on cell internalization. However clustering of the iron oxide nanoparticles after endocytosis was observed, and has been suggested in the literature to explain the enhancement of the magnetic properties and for increased magnetic resonance imaging contrast. Higher uptake of the NPs is expected for cancer cell lines, suggesting that the NPs will be even more effective in their targeted biomedical applications (MRI contrast enhancement and hyperthermia). The appearance of iron oxide crystallites with high contrast inside large intracellular endosomes or lysosomes suggests that the MPIC micelles were internalized by a clathrin-dependent endocytosis pathway. Cellular uptake exhibited strong dependence on the NP concentration and incubation time. Linear correlations between the intensity level (MFI) and the % of positive cells vs. incubation time obtained could serve as a predictive tool for cellular uptake kinetics.An in vitro MFH assessment was conducted and exhibited strongly decreased cell viability as a function of both increased incubation concentration and exposure time. At 1250 µg/mL iron oxide the lowest cell viabilities were observed: about 46-57% after 1.5 h of treatment, and 30-35% after 3 h. The hypothesis that AMF-excitation of the intracellular MNPs was the main factor leading to cell death based on the fact that cell viability was decreased in a dose-dependent manner with the concentration of internalized MNPs in the cells, even in the absence of macroscopic heating.

  2 kAm -1 . The SAR values were also evaluated under other AMF conditions, to compare them with predictions from the linear relaxation theory. It was determined that: 1) The SAR values varied nearly with the square of the magnetic field amplitude H max for different fixed frequencies f in the high and medium ranges, consistently with the superparamagnetic nature of the MNPs and a dominant contribution from Néel relaxation to the heating process. 2) Closer-to-1 exponents for the SAR vs. H max curves at lower frequencies, which were explained by a more significant contribution from Brownian relaxation of the MNPs to the heating process, and enhanced dipole-dipole inter-crystal interactions, despite the positive influence of the hydrophilic polymer coating in separating multiple magnetic cores within the same G1 micelle. incubation time. Linear correlations between the fluorescence intensity level (MFI) and the percentage of positive cells vs. incubation time obtained could serve as a predictive tool for the cellular uptake kinetics. A clathrin-dependent endocytosis pathway was suggested.The PIC micelles were similarly tested for cytotoxicity, and their cell internalization was visualized by CLSM. None of the PIC micelles showed any significant cytotoxicity for L929 cells at concentrations of up to 10 mg/mL, in spite of their different coating densities and sizes. The good colloidal stability and non-cytotoxicity of the PIC micelles are likely due to their high biocompatibility and hydrophilicity, and to the neutrality of the PHEA shell.

Figure S3- 1 .

 1 Figure S3-1. HSQC spectra for PtBA and PtBA-b-PHEATMS in CDCl 3 , and for PAA-b-PHEA in DMSO-d 6 (from top to bottom).

25 Figure S3- 2 . 1 .Figure S6- 1 .Figure S6- 2 .

 252112 Figure S3-2. 3D models (row 1), height (row 2) and phase AFM images (row 3) for G1@PAA 27 -b-PHEA 260 at f = 0.25 (column 1), G1@PAA 13 -b-PHEA 150 at f = 0.5 (column 2) and G1@PAA 13 -b-PHEA 150 at f = 0.25 (column 3).

Table 2 -

 2 1. Influence of the PEG:H 2 O volume ratio on the identified phases and crystallite size collected from X-ray diffraction (XRD) data. All samples are prepared at 150 °C for 24 h. Reprinted with permission from Reference 20. Copyright 2008 Elsevier B.V. .. 22

Table 2 -

 2 2. Influence of reaction temperature and time on the phases of nanostructured iron oxides and crystallite sizes. All samples are prepared using PEG:H 2 O = 1:2 in volume. Reprinted with permission from Reference 20. Copyright 2008 Elsevier B.V. ..................... 22

Table 2 -

 2

3. Zeta potential and average hydrodynamic diameters of poly(acrylic acid) (PAA) or poly(styrene sulfonate-alt-maleic acid) (PSS-alt-MA)-coated IONPs with different cross-linking densities for an iron oxide concentration of 0.14 wt% at pH 8. Adapted with permission from Reference 34. Copyright 2011 American Chemical Society. ...................... 36 Table 2-4. Summary of TGA weight loss results and particle size (d TEM and d XRD ) of polymer-coated IONPs at different polymer-to-iron ratios. PAEA: phosphonic acid ethyl acrylate. Reprinted with permission from Reference 37. Copyright 2014 Royal Society of Chemistry. ........................................................................................................................... 39

Table 2 -

 2

	poly(glycerol methacrylate); PDMAEMA: poly[(N,N-dimethylamino)ethyl methacrylate]
	PNOR: polynorbornene; PAMPS: poly(2-acrylamido-2-methyl-1-propansulfonic acid)....... 79

5. Summary of the different organic templates used as host matrices for the synthesis of MNPs. In this review, the examples gathered from the literature were sorted according to their chemical nature and structural properties (e.g., geometry, dimensionality, size). DEX: dextran; Alg: alginate; PNIPAM: poly(N-isopropylacrylamide); PVA: poly(vinyl alcohol); PVP: polyvinylpyrrolidone; P4VP: poly(4-vinylpyridine); PMGI: poly(methyl glutarimide); DHBC: double-hydrophilic block copolymer; PGA: poly(glycerol acrylate); PAsp: poly(aspartic acid); PAEMA: poly[2-(acetoacetoxy) ethyl] methacrylate; PGMA:

Table 3 -

 3 1. Theoretical estimates of van der Waals attraction between sterically stabilized particles (conditions leading to dispersion instability are bolded). Adapted with permission from Reference 1. Copyright 1983 Academic Press. .......................................... 85

Table 3 -

 3 2. Hydrodynamic diameter and PDI of G1@PAA-b-PHEA PIC micelles (in water at pH 7) obtained for various f ratios at 25 °C. The optimal f values for each DHBC are bolded. .............................................................................................................................. 114

Table 3 -

 3 3. Measured (top) and calculated (bottom) refractive index increments of selected PIC micelles. ........................................................................................................ 120

Table 3 -

 3 PHEA. ..................................................................................................... Scheme 4-1. Alkaline coprecipitation and oxidation reactions. ...............................

	Scheme 3-3. ATRP synthesis of PtBA-b-PHEATMS and deprotection reaction to
	produce PAA-b-

4. Data derived from the SLS and DLS measurements on PIC micelles. .... 124 Table 4-1. Characterization data for G1-G3 arborescent copolymer templates. Adapted with permission from Reference 77. Copyright The Royal Society of Chemistry 2012. ................................................................................................................................. 149 Table 5-1. Magnetic properties of the Fe 3 O 4 samples synthesized. ......................... 207 Table 5-2. Longitudinal and transverse relaxivities of the samples at 20 and 60 MHz. .......................................................................................................................................... 211 Table 5-3. Relaxivities of 0.7-80-BC SPIONs and commercial contrast agents. ...... 212 Table 5-4. Magnetic properties of Fe 3 O 4 samples from NMRD analysis. ................ 221 xxxii List of Schemes Scheme 3-1. Synthesis of PtBA via ATRP. ............................................................ Scheme 3-2. Silylation of HEA. ............................................................................. Scheme 4-2. Synthesis of an arborescent copolymer (G0PS-g-P2VP) by successive grafting reactions. Reprinted with permission from Reference 77. Copyright 2012 The Royal Society of Chemistry 2012. ............................................................................................... xxxiii

List of Abbreviations and Symbols

Table 2 -1.

 2 Influence of the PEG:H 2 O volume ratio on the identified phases and crystallite size collected from X-ray diffraction (XRD) data. All samples are prepared at 150 °C for 24 h. Reprinted with permission from Reference 20. Copyright 2008 Elsevier B.V.

PEG:

H 2 O (in volume) Phases Crystallite size (nm)

	1:3	Fe 3 O 4 , α-Fe 2 O 3	21.8
	1:1	Fe 3 O 4 , α-Fe 2 O 3 , γ-Fe 2 O 3	24.7
	3:1	Fe 3 O 4 , α-Fe 2 O 3 , γ-Fe 2 O 3	31.6
	0:4	Fe 3 O 4 , α-Fe 2 O 3 , γ-Fe 2 O 3	42.2
	4:0	NaFeO 2	64.5

Table 2 -2. Influence of reaction temperature and time on the phases of nanostructured iron

 2

	oxides and crystallite sizes. All samples are prepared using PEG:H 2 O = 1:2 in volume.
	Reprinted with permission from Reference 20. Copyright 2008 Elsevier B.V.

Conditions:

temp. (°C)/time (h) Phases Crystallite size (nm)

	100/24	Fe 3 O 4 + NaFeS 2 (H 2 O) 2	10.6
	125/24	Fe 3 O 4 + α-Fe 2 O 3 + NaFeS 2 (H 2 O) 2	13.8
	150/24	Fe 3 O 4	30.3
	150/48	Fe 3 O 4 + α-Fe 2 O 3	24.1
	150/72	Fe 3 O 4 + α-Fe 2 O 3 + γ-Fe 2 O 3	28.4

Poly(acrylic acid) (PAA) Chains for Fe 3 O 4 Growth Inhibition 2.4.2.8

Table 2 -3. Zeta

 2

	potential and average hydrodynamic diameters of poly(acrylic acid) (PAA)
	or poly(styrene sulfonate-alt-maleic acid) (PSS-alt-MA)-coated IONPs with different cross-
	linking densities for an iron oxide concentration of 0.14 wt% at pH 8. Adapted with
	permission from Reference 34. Copyright 2011 American Chemical Society.

Cross-linking (%) Hydrodynamic diameter (DLS) (nm) Zeta potential (mV) PSS

	-alt-MA before cross-linking	44 ± 9	-48.6 ± 4.7
	PAA before cross-linking	83 ± 2	-39.7 ± 1.5
	PAA, 12.5% cross-linking	69 ± 17	-48.6 ± 1.1
	PAA, 50% cross-linking	126 ± 9	-44.3 ± 2.2
	PAA, 12.5% cross-linking	77 ± 16	-47.1 ± 2.2
	PAA, 100% cross-linking	91 ± 2	-38.3 ± 3.0

Table 2 -

 2

	4. Summary of TGA weight loss results and particle size (d TEM and d XRD ) of
	polymer-coated IONPs at different polymer-to-iron ratios. PAEA: phosphonic acid ethyl
	acrylate. Reprinted with permission from Reference 37. Copyright 2014 Royal Society of
	Chemistry.

Hybrid magnetic core- shell [Polymer]:[Fe] weight ratio Wt loss TGA d TEM (nm) d XRD (nm) Grafting density (nm -2 )

		1:10	23%	10.45	9.83	0.083 ± 0.005
	IONP@P(PAEA)-b-P(OEGA)	1:4 1:2 1:1	30% 38% 44%	9.80 9.40 8.64	9.10 8.52 7.32	0.124 ± 0.003 0.168 ± 0.008 0.192 ± 0.01
		2:1	51%	-	-	-
		1:10	16%	10.25	10.09	0.065 ± 0.001
	IONP@P(AA)-b-	1:4	22%	8.91	8.75	0.084 ± 0.003
	P(OEGA)	1:2	26%	7.83	7.44	0.091 ± 0.003
		1:1	36%	-	-	-
		1:4	6%	12.39	11.47	0.023 ± 0.001
	IONP@P(GA)-b-P(OEGA)	1:2 1:1 2:1	11% 18% 22%	10.24 9.14 8.65	10.62 9.78 8.41	0.039 ± 0.001 0.063 ± 0.002 0.073 ± 0.001
		4:1	33%	-	-	-

Spherical Micelles Loaded with IONPs 2.4.3.11

	In the same context, amphiphilic block copolymers were also used as templates by
	introducing bidentate moieties, such as in poly[2-(acetoacetoxy) ethyl] methacrylate

Table 3 - 1 .

 31 Theoretical estimates of van der Waals attraction between sterically stabilized particles (conditions leading to dispersion instability are bolded). Adapted with permission from Reference 1. Copyright 1983 Academic Press.

3.2 Synthesis of Poly(tert-butyl acrylate) Macroinitiator Low

	tert-Butyl acrylate (tBA, Sigma-Aldrich, 98%) was stirred overnight with freshly crushed calcium hydride (CaH 2 , Sigma-Aldrich, lumps, reagent grade, 95%), distilled under reduced pressure and stored under N 2 at 4 °C. 2-Hydroxyethyl acrylate (HEA, Sigma-Aldrich, 96%, with 200-650 ppm hydroquinone monomethyl ether as inhibitor) was purified by liquid-liquid extraction to remove diacrylates and acrylic acid impurities before distillation. 42 An aqueous solution of HEA (81 mL, 0.65 mol) in deionized water (27 mL, 25% v/v) was washed 10 times with n-hexanes (Sigma-Aldrich, ACS reagent, ≥ 98.5%) and saturated with sodium chloride. The HEA in the aqueous phase was then extracted with diethyl ether (Sigma-Aldrich, ACS reagent, ≥ 99.0%, 3 × 75 mL). This monomer solution was dried over anhydrous MgSO 4 powder (Fisher, Certified) before concentration by rotary evaporation. Hydroquinone (Alfa Aesar, 99.0%, 75 mg, 0.1% w/w) was added as an inhibitor and the purified monomer was stored at -15 °C until distillation under reduced pressure immediately before use. CuBr (0.252 g, 1.75 mmol, 0.53 equiv), PMDETA (0.36 mL, 1.74 mmol, 0.52 equiv), degased acetone (5 mL), and tBA (20 mL, 140 mmol, 42.4 equiv) were added to an oven-dried round-bottomed flask (rbf) and the reagent mixture was degased with 3 freeze-pump-thaw cycles. The green-colored reaction mixture (ascribed to the Cu I -PMDETA complex) was then placed in an oil bath at 60 °C for 2 h 40 min to reach ̅ n = 13, and 5 h 20 min to height × 1 cm diameter) of activated neutral alumina (Sigma-Aldrich, Brockmann I grade, 150-mesh size) using ca. 50 mL acetone as eluent. After removal of the organic solvent by rotary evaporation, the polymer was dissolved in a minimum amount of methanol (ca. 10 mL) and further purified by dialysis in a 1,000 MWCO Spectra/Por ® 7 regenerated cellulose bag against 1 L of methanol. The solvent was changed thrice within 24 h, and the polymer was collected by removal of the solvent under vacuum. PtBA 13 1 H NMR (CDCl 3 , 400 MHz): n (NMR) = 1660, DP = 13, δ (ppm): 1.06-1.13 (m, 3H), 1.14-1.65 (b, 140H), 1.65-2.02 (b, 6H), 2.01-2.34 (b, 14H), 2.34-2.62 (b, 2H), reach ̅ degased acetone (ca. 10 mL) was added. The solution was passed through a column (10 cm 3.55-3.65 (s, 3H), 3.98-4.15 (m, 1H); PtBA 27 1 H NMR (CDCl 3 , 400 MHz):

(Sigma-Aldrich, > 99.7%), dichloromethane (Sigma-Aldrich, > 99.5%), ethyl acetate (Sigma-Aldrich, > 99.8%), tetrahydrofuran (THF, Sigma-Aldrich anhydrous, ≥ 99.9%, inhibitor-free), ethanol (Sigma-Aldrich, ACS reagent, 99.8%), triethylamine (Et 3 N, Fisher, ≥ 99.0 %), anisole (Alfa Aesar, 99.0%), trifluoroacetic acid (TFA, Sigma-Aldrich, > 99.0%), methyl 2-bromopropionate (Sigma-Aldrich, 98%), and trimethylsilyl chloride (TMS-Cl, Alfa, ≥ 98.0%) were used as received from the suppliers. Milli-Q water (18.2 MΩ•cm) was obtained from a Milli-Q deionized water purification system (PURELAB Classic, ELGA LabWater).

N,N,N′,N″,N″-Pentamethyldiethylenetriamine (PMDETA, Sigma-Aldrich, 99%) was purified by distillation under reduced pressure and stored under N 2 at 4°C. Copper(I) bromide (CuBr, Sigma-Aldrich, 98%) was purified by washing twice (4 h and 24 h) with glacial acetic acid (Sigma-Aldrich, 99.75%), three times with absolute ethanol (Sigma-Aldrich, ≥ 99.8%), and finally with diethyl ether (Sigma-Aldrich, anhydrous, ≥ 99.0%). The white powder was dried under vacuum overnight and stored under N 2 .

The G1 arborescent graft copolymer used in this study was synthesized earlier by anionic polymerization and grafting.

58

It consists of a G0 or comb-branched polystyrene substrate ( n = 9.7×10 4 g/mol; M w /M n = 1.03; branching functionality f n = 17) grafted with poly(2-vinylpyridine) chains ( n = 5.5×10 3 g/mol; M w /M n = 1.15). Purification of the copolymer from linear polymer contaminants was performed with 3 cycles of precipitation/fractionation from 5 g•L -1 solutions in THF/methanol (4/1 v/v) mixtures with nhexane. The graft copolymer G0PS-g-P2VP ( n = 1.1×10 6 g/mol; M w /M n = 1.08; branching functionality f n = 182) had a 2VP units content of 91 mol%.

46

3.molar mass macroinitiators PtBA-Br were synthesized via atom-transfer radicalpolymerization (ATRP), by adapting a procedure reported by Davis and Matyjaszewski

47

as illustrated in Scheme 3-1. Briefly, methyl 2-bromopropionate (0.37 mL, 3.3 mmol, 1 equiv), n = 27. The reactions were stopped by cooling the flask in liquid nitrogen and non-n (SEC-PS) = 1724, Đ (SEC) = n / w = 1.05. n (NMR) = 3460, DP = 27, δ (ppm): 1.06-1.13 (m, 3H), 1.14-1.65 (b, 283H), 1.65-2.02 (b, 11H), 2.01-2.34 (b, 28H), 2.34-2.62 (b, 2H), 3.55-3.65 (s, 3H), 3.98-4.15 (m, 1H); n (SEC-PS) = 3524, Đ (SEC) = 1.06. PtBA 13 and PtBA 27 13 C NMR (CDCl 3 , 100 MHz) δ (ppm): 27.68 (CH 2 ), 28.08 (C-(CH 3 ) 3 ), 41.88 (CH 2 -CH-Br), 42.34 (CH-CH 3 ), 80.33 (C-(CH 3 ) 3 ), 174.18 (CO).

3.

3.3 Silylation of 2-Hydroxyethyl Acrylate Protection

  J cis = 10.41 Hz, 2 J = 1.21 Hz, 1H), 6.02 (dd,3 J trans = 17.33 Hz,3 J cis = 10.42 Hz, 1H), 6.30 (dd,3 J trans = 17.33 Hz, 2 J = 1.21 Hz, 1H).

	Chapter 3
	was purified by dissolution in ethyl acetate (300 mL) and washing with water (300 mL) three
	times. Anhydrous MgSO 4 (ca. 5 g) was then added to the solution which was decanted after
	15 min, and the solvent was removed under vacuum.
	1 H NMR (CDCl 3 , 400 MHz) δ (ppm): 0.07 (s, 9H), 3.69 (t, J = 4.8 Hz, 2H), 4.10 (t, J
	= 4.8 Hz, 2H), 5.70 (dd, 3 13 C NMR (CDCl 3 , 100 MHz) δ (ppm): -0.62 (Si-(CH 3 ) 3 ), 60.55 (CH 2 -O-SiMe 3 ),
	65.60 (CH 2 -O-CO), 128.24 (CH), 130.72 (CH 2 -CH), 166.01 (CO).
	mL, 0.652 mol, 1.0 equiv), purified as described in Section 3.3.1, dichloromethane (750 mL)
	and triethylamine (109.5 mL, 0.79 mol, 1.21 equiv) were added in a 2-L rbf and the solution
	was cooled to 0 °C in an ice bath. Trimethylsilyl chloride (91.5 mL, 0.72 mol, 1.1 equiv) was
	then added drop-wise, resulting in the formation of a white precipitate (Et 3 N•HCl). The
	solution was stirred in the ice bath overnight and allowed to reach room temperature
	gradually. Suction filtration (Whatman ® filter paper, grade 4) was employed to remove the
	precipitate, the solvent was removed by rotary evaporation, and the liquid residue was
	filtered once more. The protected monomer, 2-trimethylsilyloxyethyl acrylate (HEATMS),
	96

of the hydroxyl functionality of HEA was carried out according to a procedure reported by Mühlebach et al.,

42

illustrated in Scheme 3-2. The monomer HEA

(75

Table 3 - 2 .

 32 Hydrodynamic diameter and PDI of G1@PAA-b-PHEA PIC micelles (in water at pH 7) obtained for various f ratios at 25 °C. The optimal f values for each DHBC are bolded.

		f	9	6	3	1	0.5	0.25	0.125
	G1@PAA 27 -b-	(nm) a	67/44 84/61 184/80 1600	-	-	-
	PHEA 56	PDI	0.16	0.10	0.20	~1	-	-	-
	(l PHEA  7.3 nm,	(nm) b	15	-	-	-	-	-	-
	D max  29 nm)
		ZP (mV)	-29	-30	-28	-29
	G1@PAA 13 -b-	(nm) a	48/36 66/45 100/75 1290	-	-	-
	PHEA 50
	(l PHEA  7.1 nm,	PDI	0.07	0.10	0.07	~1	-	-	-
	D max  29 nm)	(nm) b	11	-	-	-	-	-	-
		ZP (mV)	-26	-23	-13	-34
	G1@PAA 13 -b-	(nm) a	-	-	50/32 46/29 104/83	169/98	660
	PHEA 150
	(l PHEA  10.1 nm,	PDI	-	-	0.18	0.14	0.04	0.16	0.47
	D max  35 nm)	(nm) b	-	-	9	7	-	-	-
		ZP (mV)			-13	-15	-14	-18	-31
	G1@

PAA 27 -b- PHEA 260 (l PHEA  12.2 nm, D max  39 nm)

		(nm) a	-	-	63/37 51/34 42/26	87/53	642
		PDI	-	-	0.18	0.13	0.08	0.18	0.44
		(nm) b	-	-	11	10	6	-	-
		ZP (mV)			-19	-17	-15	-18	-34
	a	and : intensity-weighted and number-weighted hydrodynamic diameters
	b Thickness of protecting layer		-	] of assumed unimolecular micelle, where D G1 
	14.6 nm for a collapsed spherical PS-g-P2VP core

Table 3 - 3 .

 33 Measured (top) and calculated (bottom) refractive index increments of selected PIC micelles.

	Samples	G1@PAA 13 -b-PHEA 150	G1@PAA 27 -b-PHEA 260
	f	3	1	0.5
	%wt G1	3.3	10.5	16.5
	dn/dc (mL/g)	0.1294 ± 0.0005	0.1367 ± 0.0002	0.1389 ± 0.0004
	R 2	0.99993	0.99999	0.99996
	Samples	G1@PAA

13 -b-PHEA 150 G1@PAA 27 -b- PHEA 260 G1@PAA 13 - b-PHEA 50 G1@PAA 27 - b-PHEA 56

	f	1	0.5	0.25	3	0.25	9	9
	%wt G1	7.9	15.3	31.1	3.4	36.8	3.4	5.9
	Cal. dn/dc	0.1334 0.1388 0.1503 0.1301 0.1545	0.1301	0.1319
	(mL/g)

Table 3 - 4 .

 34 Data derived from the SLS and DLS measurements on PIC micelles.

	G1@ PAA x -b-PHEA y	w (10 6 g/mol)	#BC /PIC	R g (nm)	R h (nm)		A 2 (10 -5 cm 3 molg -2 )
		f	Theor.	Exp.
	27-56	9	18.7	1.3	22	26.7	33.5	0.80	9.5
	13-50	9	32.5	1.4	54	45.3	24	1.89	12.1
	13-150	3	33.2	1.7	33	35.8	25	1.43	12.3
		1	14.0	1.6	27	27.7	23	1.20	1.8
		0.5	7.2	39	-	49.6	52	0.95	0.3
		0.25	3.5	170	-	124	84.5	1.47	0.2
	27-260	3	32.8	3.2	68	23.9	32	0.76	19.3
		1	10.5	2.8	51	12.2	25.5	0.48	1.9
		0.5	6.6	2.1	32	9.9	21	0.47	3.3
		0.25	2.9	9.7	-	17.4	43.5	0.40	1.7

  suggested room temperature quasi-instantaneous electron transfer between Fe 2+ and Fe 3+ as a processes leading to the crystallization of spinel. The incorporation of a small proportion (ca. 10 mol%) of Fe 2+ into the precipitate formed initially (a poorly crystalline Fe

3+ -ferrihydrite FeOOH weakly diffracting X-rays or electrons, and thus called 2-line ferrihydrite) can induce the crystallization of all the iron into spinel via fast electron hopping, causing local structure rearrangements and driving spinel ordering. The crystallization of spinel can occur by a dissolution-crystallization mechanism forming two types of non-stoichiometric spinel particles with very different mean sizes. The Fe 2+ content in the system determines which pathway dominates: only for [ ]

Table 4 - 1 .

 41 Characterization data for G1-G3 arborescent copolymer templates. Adapted with permission from Reference 77. Copyright The Royal Society of Chemistry 2012. Absolute values from SEC-MALLS. b Values estimated by combining the absolute M n of the substrate with the copolymer composition from 1 H NMR analysis. c Branching functionality: Number of branches grafted in the last cycle. d From 1 H NMR analysis. e From n and f n .

		G(n-1)PS substrate	P2VP side chains	Graft copolymer Gn = G(n-1)PS-g-P2VP
	G	n (gmol -1 ) a	w / n	n (gmol -1 ) a	w / n	n (gmol -1 ) b	w / n	f n	c	2VP (mol%) d	2VP groups e
	0 5.2×10 3	1.06	5.1×10 3	1.15	7.4×10 4	1.08	13	93	625
	1 9.7×10 4	1.03	5.5×10 3	1.15	1.1×10 6	1.08	182	91	9500
	2 1.1×10 6	1.03	6.2×10 3	1.10	8.4×10 6	1.09	1180	87	6.9×10 5
	a

Table 5 - 1 .

 51 Magnetic properties of the Fe 3 O 4 samples synthesized. S and M d : volume-and mass-average specific magnetization; D 0 : median diameter; D n and D v : numberand volume-average diameter;  : polydispersity; the sample nomenclature is described in the text.

	Sample *	(mg/mL)	M s (10 5 A/m)	M d (emu/g Fe 3 O 4 )	D 0 (nm)		D n (nm)	D v (nm)
	0.7-80-UC	3.4	2.28	44.7	6.66	0.386	7.2	11.2
	1.0-80-UC	2.9	2.70	52.9	6.20	0.457	6.9	12.9
	0.7-100-UC	2.9	2.65	51.9	6.26	0.389 6.75 10.6
	1.0-100-UC	2.9	2.36	46.3	5.96	0.447	6.6	12.0
	0.7-80-BC	1.9	3.04	59.6	6.26	0.385	6.8	11.3
	1.0-80-BC	1.9	2.49	48.7	6.08	0.492	6.9	14.2
	0.7-100-BC	1.3	2.85	55.9	6.34	0.409	6.9	11.4
	1.0-100-BC	1.6	2.64	51.8	6.01	0.458	6.7	12.5

M

  Baaziz et al.71 suggested sizedependent oxidation defects and surface or volume spin canting to explain the lower saturation magnetization observed for MNPs as compared to the bulk materials. Smaller NPs

	Chapter 5
	(< 8 nm) were indeed almost completely oxidized to maghemite, with mainly surface spin
	canting. NPs of intermediate sizes (8-12 nm) exhibited surface, but particularly also volume
	210

  83

Table 5 - 2 .

 52 Longitudinal and transverse relaxivities of the samples at 20 and 60 MHz.

			20 MHz			60 MHz
	Sample	r 1 (s -1 mM -1 )	r 2 (s -1 mM -1 )	r 2 /r 1	r 1 (s -1 mM -1 )	r 2 (s -1 mM -1 )	r 2 /r 1
	0.7-80-UC	11.1	89.3	8.1	7.3	99.2	13.6
	1.0-80-UC	11.6	60.1	5.2	6.5	38.9	6.0
	0.7-100-UC	9.6	45.8	4.8	6.5	36.3	5.6
	1.0.100-UC	11.2	69.5	6.2	6.8	68.6	10.1
	0.7-80-BC	27.3	269.0	9.9	10.7	335.8	31.4
	1.0-80-BC	23.5	200.7	8.6	8.4	239.8	28.4
	0.7-100-BC	21.1	195.1	9.3	8.4	213.9	25.5
	1.0-100-BC	22.7	205.4	9.1	9.0	263.1	29.4

Sample nomenclature is described in the text.

Table 5 - 3 .

 53 Relaxivities of 0.7-80-BC SPIONs and commercial contrast agents.

	r 1 (s -1 mM -1 )	r 2 (s -1 mM -1 )	r 2 /r 1

IOs (Resovist ® ) 86 IO-loaded PLGA-mPEG 86

		1.5 T, 37 °C	11.4	282.4	24.8
		1.5 T, 37 °C	7.5	532.7	71.0
	USPIOs
	AMI-227 (Sinerem ® ) 85	0.47 T, 39 °C	22.7	53.1	2.3
	MION46L (Clariscan ® ) 87	0.47 T, 35 °C	22.2	43.7	2.0
		1.5 T, 37 °C	3.95	19.6	4.9

  18,90

  3 suffered from partial oxidation that converted Fe 3 O 4 to Fe 2 O 3 and led to a reduction in M s values by ~ 21-26% as compared to BC samples, as expected from the respective bulk magnetization values. The complexation with BC to create a stabilizing, protecting layer for the SPIONs is thus essential to maintain high magnetization values with good potential as MRI contrast agents. A 6 % reduction in M s values was reported by Sharifi et al. when comparing magnetization values for carboxyethylsilanetriol-coated SPIONs to bare SPIONs.

Table 5 - 4 .

 54 Magnetic properties of Fe 3 O 4 samples from NMRD analysis.

	Sample	r NMR (nm)	(emu/

g Fe 3 O 4) 0.7-80-UC

		8.0	22.9	1.4	0.51
	0.7-80-BC	8.1	31.1	1.4	0.52
	0.7-100-UC	7.9	23.0	1.5	0.44
	0.7-100-BC	8.0	29.2	1.4	0.52
	The sample nomenclature is explained in the text. *The VSM radius is defined as half of the volume-
	average diameter obtained from a log-normal law:	⁄ ; D v values listed in Table 5-1.

  27 -b-PHEA 260 (50 mg, 0.162 µmol BC, 43.6 µmol of COOH groups) was prepared while fluoresceinamine (FA, 3.35 mg, 9.65 µmol, FA/COOH = 6/27 mol/mol) was dissolved in DMF (2 mL). The two solutions were combined and vigorously stirred for

	10 min before EDC (67.73 mg, 436 µmol, EDC/COOH = 270/27) was added to create the
	highly reactive O-acylisourea intermediate. The mixture was allowed to react for 24 h before
	the product was dialyzed against deionized water for 5 d with 15 changes of water. The
	PAA 27	* -b-PHEA 260 copolymer was collected and stored at 10 °C. The fluorescence emission
	of the block copolymer was monitored on a UV/Vis/Fluorescence SpectraMax ® M2e
	Multimode Microplate Reader. The number of FA molecules conjugated to each PAA 27 -b-
	PHEA 260 molecule (containing 27 CO 2 H groups), determined by fluorometry, was 1.88 
	0.15 (Section 6.4.1), corresponding to 7.0 mol%.

Table S3 - 1 .

 S31 Effective f* = CO 2 -/NH + ratios at pH 4.7 and 7.

	f pH 4.7 equiv CO 2 -	CO 2 H equiv	NH + equiv	N equiv	f* = CO 2 /NH +	-
	9	0.14	8.86	0.67	0.33	0.21
	6	0.09	5.91	0.67	0.33	0.14
	3	0.05	2.95	0.67	0.33	0.07
	1	0.02	0.98	0.67	0.33	0.02
	0.5	0.01	0.49	0.67	0.33	0.01
	0.25	0.00	0.25	0.67	0.33	0.01
	0.125	0.00	0.12	0.67	0.33	0.00
	f pH 7	CO 2 equiv -	CO 2 H equiv	NH + equiv	N equiv	f* = CO 2 /NH +	-
	9	6.84	2.16	0.01	0.99	684
	6	4.56	1.44	0.01	0.99	456
	3	2.28	0.72	0.01	0.99	228
	1	0.76	0.24	0.01	0.99	76
	0.5	0.38	0.12	0.01	0.99	38
	0.25	0.19	0.06	0.01	0.99	19
	0.125	0.09	0.03	0.01	0.99

Chapter 3 Polyion Complex Micelles

Synthesis from Arborescent Polymers

Figure 5-13. Specific absorption rate (SAR) of magnetic G1 micelles as a function of magnetic field amplitude H max at various fixed frequencies f. Smaller orders of 1.25 and 1.24 were obtained at the lower frequencies of 217 and 146 kHz, respectively. Reductions in the SAR values leading to a linear correlation with the field amplitude H max , contradicting the quadratic increase predicted by the linear response theory, have been observed by various groups, in particular for multi-core nanoflower morphologies. [56][57]99,[102][103][104][105] It is also noteworthy that the linear response theory (LRT) was proposed and used for non-interacting superparamagnetic nanoparticles in the limit of low magnetic field strengths and sample concentrations, but it failed to account for many aspects of colloidal MNP suspensions, such as interparticle interactions and particle motions. 57,98 The reduction in the SAR values demonstrates the larger contribution of Brownian relaxation to the heating process at lower frequencies as compared to medium-range frequencies. Hergt et al. also observed smaller SAR values at very low frequencies (1-10 kHz) for gel suspensions (in which the MNPs were immobilized) as compared to the original aqueous suspensions. 99 This decrease in heating power was ascribed to the reduced y = 1.013x 1.731 y = 0.488x 1.676 y = 0.379x 1.551 y = 0.337x 1.251 y = 0.246x predominates. 99 This hypothesis can be applied to our magnetic micelles to explain the closeto-first-order dependence observed at low frequencies, since the complex and bulky nanostructure of the micelles might hamper Brownian relaxation, reducing the SAR values and resulting in smaller than square dependence on H max . In the high and medium frequency ranges, where Néel relaxation dominates, restricted Brownian relaxation has less influence on the SAR values, resulting in higher values that obey the square power law. Similar reasons leading to first order laws were also suggested by Yuan and Borca-Tasciuc 106 in the form of "cluster oscillation".

For our magnetic micelles, the encapsulation of multiple Fe 3 O 4 crystals inside an uncoated G1 polymer template probably enhanced the dipole-dipole inter-crystal interactions, but this effect was counterbalanced by the hydrophilic shell in the micelles coated with The linear dependence of the SAR of magnetic micelle on the magnetic field frequency was tested for various fixed magnetic field amplitudes (Figure 567891011121314). Deviations from first order were also reported in the literature for cobalt ferrite (with Brownian relaxation), but not for -Fe 2 O 3 . 50

Overview

The properties of magnetic polyion complex (MPIC) micelles stabilized by a poly(2hydroxyethyl acrylate) (PHEA) shell were shown to be promising for applications as magnetic resonance imaging (MRI) contrast agents and for heat generation in cellular The usefulness of MPIC micelles for cellular radiofrequency magnetic field hyperthermia was also confirmed, as the MPIC micelles had a dual dose-dependent effect (concentration and duration of magnetic field exposure) on cell viability. The viability of L929 fibroblast cells incubated with MPIC micelles at 1250 µg Fe 3 O 4 /mL concentration decreased to 46% after 1.5 h and to 30% after 3 h of exposure to a high frequency magnetic field (755 kHz, 10.2 kA/m). Similar treatments on U87 human glioblastoma cells resulted in 57% and 35% cell viability after 1.5 and 3 h exposure, respectively, suggesting an effective and tunable treatment for various types of cells.

Cell Internalization Studies

Cell internalization of the PIC micelles was visualized by confocal laser scanning microscopy (CLSM). Cellular uptake of the MPIC micelles was evidenced by CLSM but also by transmission electron microscopy (TEM), since the Fe 3 O 4 crystallites showed high contrast towards electrons, and was quantified by fluorescence-activated cell sorting (FACS).

Confocal Laser Scanning Microscopy 6.4.3.1

Preliminary qualitative cellular uptake studies were done with the same cell line used for the cytotoxicity assessment. The cytoskeleton F-actin was labeled with phalloidin (red), while the nuclei were labeled with DAPI (blue). The PIC and MPIC micelles (G1@PAA-b-PHEA and G1@Fe 3 O 4 @PAA-b-PHEA) labeled with the fluorescent probe fluoresceinamine (FA, Ex: 488 nm and Em: 525 nm) were visualized as green fluorescent species.

Even though they were negatively charged, the PIC* micelles G1@PAA* 27 -b-PHEA 260 f = 0.5 were successfully internalized in the L929 cells, as evidenced by the green fluorescence found primarily in the cytoplasm at the periphery of the nuclear membrane, and probably in vesicles (Figure 67). This observation is in good agreement with the residence of nanoparticles in endosomes or lysosomes reported by other groups. 9,19,86 The ease of internalization of the NPs is attributed to the small size (D h = 42 nm, PDI = 0.08) of the PIC micelles, which is close to the 50 nm range mentioned above. The spherical shape of individual PIC* micelles was clearly visualized, the PHEA shell possibly preventing degradation induced by proteases and other enzymes existing in the low pH environment of late endosomes and lysosomes. The cells contained variable numbers of PIC micelles.

Chapter 7 Conclusions

Chapter 7 275

Summary and Original Contributions to Knowledge

The major goal of this Ph.D. research work was to synthesize superparamagnetic iron oxide nanoparticles in situ within the core of arborescent copolymer micelles bearing a stabilizing hydrophilic shell. Control over the iron oxide nanoparticle synthesis and the subsequent stabilization process was achieved to obtain a product with good potential as magnetic resonance imaging (MRI) contrast agent and for heat generation in cellular (and eventually in vivo) radiofrequency magnetic field hyperthermia (MFH) treatment. Six main research areas were investigated as detailed below.

Study and Optimization of the Polyion Complexation Process to Produce

Unimolecular Micelles

We reported the successful synthesis of poly(acrylic acid)-block-poly(2-hydroxyethyl acrylate) PAA-b-PHEA block copolymers by atom transfer radical polymerization (ATRP).

To the best of our knowledge, this is also the first report on the complexation of PAA-b-PHEA with preformed P2VP-containing nanostructures in water to produce unimolecular polyion complex (PIC) micellar species. Water was used as the solvent for complexation, allowing the formation of dynamic PIC micelles (unlike other glassy copolymer micelles formed in organic solvents, remaining frozen when transferred to aqueous environments).

Their structure was characterized in aqueous dispersions, and then in the dry state by atomic force microscopy (AFM) and transmission electron microscopy (TEM), as it was done in most PIC system investigations in the literature. We successfully performed the polyion complexation process in water by adjustment of the pH to a narrow range where both components (N and CO 2 H) can be partially present in their ionic forms, which is a requirement to form strong electrostatic interactions between polycations and polyanions. [1][2] Once formed, these PIC micelles were found to be stable in neutral pH buffers such as cell growth media.

Control over the size and the structure of the PIC micelles, from large flocculated species to stable unimolecular entities with sizes ranging from 42 to 67 nm, was Regarding frequency variations, the exponent slightly larger than unity highlights the caution needed when using the intrinsic loss power concept (the definition of ILP indeed assumes a perfect quadratic variation of the SAR with the field strength, and a linear variation with the frequency).

Biocompatibility Assessment

Biocompatibility assessments for the MPIC micelles were completed within the recommended guidelines to evaluate the biological safety of manufactured nanoparticles: first with a cytotoxicity assay, followed by cell internalization, visualized by confocal laser scanning microscopy (CLSM) and TEM, and quantified by fluorescence-activated cell sorting (FACS). The MPIC micelles were non-cytotoxic up to the highest concentration tested (1250 µg Fe 3 O 4 /mL), evidencing the efficiency of the PAA-b-PHEA copolymers as protecting agents preventing the interaction of the Fe 3 O 4 cores with the cells (potentially causing ROS production with Fe 2+ leaking through the Fenton reaction). The role of the protecting shell was further confirmed by the slightly higher cell losses observed for MPIC micelles coated with a lower amount of polymeric shell material, but only at high concentrations. In spite of being negatively charged, labeled MPIC micelles were successfully internalized through the L929 cell membrane, as evidenced by CLSM revealing green fluorescence mainly at the periphery of the nuclear membrane and in intracytoplasmic vesicles. The TEM images confirmed the presence of iron oxide crystallites with a high contrast inside large intracellular endosomes or lysosomes. Clustering of the iron oxide nanoparticles after endocytosis was observed, which has been suggested in the literature to explain enhancement of the magnetic resonance imaging negative (T 2 -weighted) contrast.

The fast cellular uptake kinetics of MPIC micelles was ascribed to their small size.

Higher NP uptake is expected for cancer cell lines, suggesting that the NPs will be even more effective in their targeted biomedical applications (MRI contrast enhancement and hyperthermia). Cellular uptake exhibited strong dependence on the NP concentration and Appendices