Prof Laurence

Prof Peter York In Crystecpharma

Frédéric

Ruxandra Gref for creating the research environment

study, and confocal experiments. I am really grateful that she is very patient to show me the experiments in detail. Ms. Katie Buxton was the first internship student in the lab and she worked with me for around half a year. It was a nice memory to work with her. I thank my colleague Ms. Elisabetta Pancani for helping me with the communication on inscription issues.

Many thanks to all the collaborators in completing this thesis. Mr. Michel Terray, Mr. Laurent Lachmanski, and Mr. Samir Safi in Malvern Instruments kindly provided the Raman microscopy (Morphologi G3-ID) for the individual particle characterization, including particle morphology and Raman spectrum. Dr. Frédéric Laurent, Dr. Jérôme Josse and Ms. Virginie Tafani in École Normale Supérieure de Lyon (ENS Lyon) performed the in vitro efficacy experiment on Amoxicillin and calvulanate loaded nanoMOF.

Last but not the least, I would like to express my profound gratitude to my parents for their unfailing support throughout the process of researching and all my life. Although my mother was discovered to have small cell lung cancer in May, 2016, she was very strong and her continuous encouragement gave me great strength to face all of the difficulties. Thanks to my father, he raised me with a love of science and supported me for all my pursuit. Most of all, I wish to thank my husband, Jingwen QIU. He came to Orsay in September, 2016, away from his friends, jobs, and life in China. His supporting made me faithful.

Regrettably, but inevitably, the list of my gratitude will be incomplete, and I hope that those who are missing will forgive me, and will still accept my sincere appreciation of their influence on my work.

General Introduction

Pharmacy has evolved regarding the specific needs of the patient. In the last five decades, the development of pharmaceutical carriers allowed the formulations to protect the fragile drugs from degradation, reduce the side effects of drugs together with allowing controlled drug release and targeted delivery [1][2][3][4][5] . Among all the drug delivery systems, "cage" particles recently drew special attention since they could act as "drug containers" which potentially load large amount of drugs, improve the stability of the drugs, and co-encapsulate synergetic drugs [6][7][8] .

Cyclodextrins (CDs) are typical "cage" molecules due to their truncated cone shape with a hydrophobic cavity and a hydrophilic outer surface 9 . These unique properties enable CDs to form inclusion complexes with a variety of active molecules. They have been used as "molecule containers" to improve the solubility and bioavailability of poorly soluble drugs, stabilize drugs from light, thermal, and oxidative degradation, and mask the bitter taste of the drugs [10][11][12] .

Therefore, self-assembled nanoparticles (NPs) composed of CDs are attractive formulations.

However, since the cavity of the CDs is small, their loadig capacity is reduced to a number of drugs or portions of drugs.

Recently, a new type of porous CD-based metal organic frameworks (CD-MOFs) was synthesized 13 . They can be imagined as buildings built using CDs which act as hollow bricks. CD-MOFs contain not only the cavities of CDs, but also the big pores built up by CDs selfassemblies (Fig. 1). Compared with CD-based NPs (CD-NPs), CD-MOFs have the possibility to load more drug payloads. Unfortunately, these particles immediately disassemble in aqueous media, which limits their application for oral or intravenous administration. Surface modification is therefore necessary to improve their stability in water. However, the process of surface modification leads to larger particles and reduced drug payloads.

Water-stable MOFs are a novel type of hybrid particles, showing a high potential as drug carriers. Iron trimesate MOFs, namely, MIL-100 (Fe) (MIL stands for Material of Institute Lavoisier) was among the first nanoscaled MOFs to be used for drug delivery 14 . It is a "cage" porous particle made of iron trimers connected by trimesic acid linkers. Because of their high pore volumes and surface areas, MIL-100 (Fe) particles were shown to load a large variety of drugs, such as antibiotics and anticancer drugs, which were able to efficiently penetrate within the porous MOF structures.

Therefore, three types of particles were successfully synthesized and used for the incorporation of active ingredients.

1. CD-NP was the first investigated system in this study. The advantages of these NPs lie on their "green" and simple preparation procedure. They could be obtained in pure water just by mixing two neutral polymers which instantaneously associate together. The host-guest interactions leading to inclusion complexes between hydrophobic molecules and the apolar concavities of CDs can be exploited for the formation of stable CD-NPs. A main objective was to use microfluidics to automatically prepare NPs in water media. Our interest was also focused on developing complementary techniques to well characterize the particles by in situ size measurements, and individual particle tracking (chapter 2), however, the drug loading capacity is not high (~ 5wt%).

2. Aiming at enhanced payload of drugs, CD-MOFs were synthesized because of their porous structure. Firstly, the synthesis of CD-MOFs was optimized to shorten the synthesis process and to obtain particles suitable as drug carriers (Chapter 3.1). Drugs of interest, such as lansoprazole (LPZ) were then encapsulated. Of note, methods were developed to study the morphology and chemical composition of individual particles (Chapter 3.2). In order to improve the stability of the loaded CD-MOFs in aqueous conditions and control the drug release, surface modification was performed (Chapter 3.3).

3. Since the surface modification was normally complicated and could probably reduce the drug payloads, MIL-100 (Fe) NPs were finally used as water-stable drug carrier in this study. It has been shown that they interact efficiently with phosphated drugs/coatings with high efficiencies. To gain new insights into the complex interactions taking place in the MOF/drug/coating systems, we developed a method based on Raman microscopy to track the morphology and chemical composition of individual particles (Chapter 4.1). Aiming at a drug combination delivery, co-encapsulation of two synergic antibiotics (Amoxicillin and potassium clavulanate) was successfully acheived (Chapter 4.2).

Chapter 1: Introduction 1. "Cage" molecules: CDs

CDs were first isolated in 1891 by Villiers from the enzymatic degradation products of starch 1 .

They are a family of cyclic oligosaccharides composed glucopyranose units. CDs are cage-like supramolecular with a hydrophilic outer surface and a hydrophobic central cavity. As a result of this cavity, CDs are able to form inclusion complexes with a variety of hydrophobic guest molecules, which can lead to advantageous changes in the chemical and physical properties of the included guest molecules. Therefore, CDs are widely used as "molecular cages" in the pharmaceutical to enhance the solubility, stability, and bioavailability of drug molecules 2 . They have been recognized as pharmaceutical excipients for the past 20 years.

Structure and properties

The most common natural CDs are α, β and γ-CDs, containing 6, 7 and 8 glucopyranose units respectively, attached by α-1,4-linkages (Fig. 1). Due to the chair formation of the glucopyranose units, CDs are shaped like bottomless nest-shaped (truncated cone) molecules with primary hydroxyl groups extending from the narrow edge and the secondary groups from the wider edge. This makes the outer surface of CD molecules hydrophilic, whereas the lipophilicity of their inner cavity is comparable to an aqueous ethanolic solution 3 . Fig. 1 Structure of α, β and γ-CDs. These unique molecular shape and structure allow CDs to act as a molecular container by trapping guest molecules in their cavity. Once the guest molecules were included, their water solubility, volatility, stability, bioavailability, and even the taste could be improved 4 .

The natural CDs, in particular β-CD, have limited water solubility (18.5 mg/mL, 25°C) 2 , which often results in precipitation of the inclusion complexes from the aqueous systems. Therefore, functionalized CDs were synthesized, including CD derivatives, and cross-linked CD polymers 5 . They are prepared by chemical or enzymatic reactions. The main objective of functionalization for pharmaceutical interest are: 1) to improve the solubility of the CDs and their inclusion complexes; 2) to reduce the toxicity of CDs, such as the nephrotoxicity of β-CD;

3) to improve the fitting and/or the association between the CD and its guest. CD derivatives. New derivatives of CDs for pharmaceutical application were synthesized and currently used in pharmaceutical products, including hydroxypropyl derivatives of β-CD and γ-CD (HP-β-CD and HP-γ-CD), sulfobutylether β-CD (SBE-β-CD), and randomly methylated β-CD (RM-β-CD) 6,7 . These CD derivatives are of very high water solubility (>500 mg/mL). The main reason for this significant solubility enhancement is due to the transformation of the crystalline forms of natural CDs into amorphous mixtures of isomeric derivatives 8 . Moreover, hydrophilic derivatives of β-CD could be used in parenteral formulations showing reduced nephrotoxicity as compared to natural CDs 9 .

Cross-linked CD polymer. Recently, cross-linked CD polymers were synthesized and widely used in polymeric systems, such as hydrogels, nano/microparticles, and micelles, etc. Crosslinked CD polymers were generally synthesized with cross-linker agents at controlled temperature 5,10 . Epoxides (e.g. epichlorohydrine (EP) or glycidylethers), isocyanates, aldehydes, and ketones are suitable cross-linking agents for CDs 11 . EP is considered as the most investigated agent. It posseses two reactive groups that can react with the hydroxyl groups of CDs or with other EP molecules under alkaline conditions. This reaction usually leads to microgels, which are composed of a mixture of cross-linked CDs joined by repeating glyceryl units of polymerized EP 12 . Hydrosoluble CD polymers can also be obtained if the cross-linking reaction is quenched at a certain stage 13 . Poly-β-CD synthesized by this way draw special attention in polymeric self-assembled systems because of its high solubility, and high capacity to form inclusion complexes with guest molecules [14][15][16] .

Safety and biocompatibility of CDs

The α, β and γ-CDs were well tolerated when administered orally only a small fraction absorbed from the gastrointestinal tract 2,7 . Some CD derivatives such as HP-CD could be used safely for intravenous administration and were mainly excreted unchanged in the urine. However, β-CD showed nephrotoxicity when administered parenterally, in part, possibly due to its low aqueous solubility 2 . Lipophilic CD derivatives, such as RM-β-CD, were absorbed to a greater extent from the gastrointestinal tract and induced a toxic effet after parenteral administration 17 . Oral administration of RM-β-CD is presently limited because of its potential toxicity. However, it is worth mentioning that the first targeted, polymer-based nanoparticle system carrying siRNA to be systemically administered to humans was based on polymeric cationic CDs 18 .

Inclusion complex formation based on host-guest interactions

The inclusion complexes between CDs and guest molecules are formed mainly by hydrogen bonding, and van der Waals interactions. The release of enthalpy-rich water molecules from the CD cavity, and electrostatic interactions also contribute to the inclusion formation. As there are no covalent bonds, guest molecules in the complex are in equilibrium with free molecules in solution (Fig. 2). Fig. 2 The conventional inclusion complex formation when one guest molecule forms a complex with one CD molecule.

The key factor for the inclusion complex formation is the thermodynamic interaction between the chosen CDs and the drug molecules. As shown in Fig. 2, the larger the KC, the more the equilibrium will shift towards complex formation. There are four energetically favorable interactions that help increase KC 19 : (i) the displacement of polar water molecules from the hydrophobic CD cavity; (ii) the increased number of hydrogen bonds formed as the displaced water returns to the larger pool; (iii) a reduction in the repulsive interactions between the hydrophobic guest and the aqueous environment; and (iv) an increase in van der Waals interactions and/or hydrogen bonds formation as the guest molecule inserts itself into the hydrophobic CD cavity. The first two interactions are similar in any case, but the last two interactions are up to the affinity between the guest molecule and the CD, which is dependent on the size, hydrophobicity and structure of both the guest molecules and the type of CDs.

Inclusion complexes could be formed at the molar ratios of 1:1 or 1:2 between the guest molecule and CD (Fig. 3). However, as shown in Fig. 3C, CDs are also known to form noninclusion complexes and complex aggregates capable of dissolving drugs through micelle-like structures 3 . Fig. 3 Types of inclusion complexes formed at the molar ratio of 1:1 (A) and 1:2 (B) between the guest molecule and CD; Non-conventional CD complexes (C).

CD-based drug-carriers

CDs are widely used in drug delivery system, including hydrogels, polymeric NPs, liposomes, niosomes, micelles, millirods, nanosponges, and supramolecular vesicles, etc 20 . They were employed almost everywhere in the pharmaceutical field, but there are only few types of drug carriers mainly composed of CDs. Herein, CD-based hydrogels and NPs driven by CD inclusion complexes and CD-based metal organic frameworks (CD-MOFs) were introduced.

CD-based hydrogels and NPs driven by CD inclusion complexes.

Supramolecular structures could be formed by simply mixing poly-CDs or polymers grafted with CDs and polymers bearing guest groups. Both hydrogels and NPs can be obtained depending on the concentration of the polymers and their architecture 21 . The inclusion complex formed between CDs in one polymer and the guest molecules in other polymer acted as knots that link the lines into nets which could further assemble into NPs or hydrogels.

The combination between β-CD-bearing polymers and adamantane (Ada)-bearing polymers were stable, due to their host-guest interaction (Ka ∼ 10 4 -10 5 M -1 ) 8,17,22 . Some other guest molecules were also used, such as alkyl chains, cholesterol, β-benzyl L-aspartate, benzimidazole, and even some hydrophobic drugs 10,22,23 . It was found that polymers grafted with alkyl chains could also form stable supramolecular structures, despite the relatively low CD-alkyl chain association constant 14 . The cooperative effect of multiple complex formation played a major role on ensuring the stability of the edifices.

Drugs could be loaded in different ways. As already described, some hydrophobic drugs could be used directly as guest molecules 5 . In this case, the drugs could be conjugated in CD-bearing polymer or in another polymer. Fig. 4 A and B show the NPs formation driven by CD-drug inclusion. Alternatively, drugs could be loaded after the hydrogels/NPs formation by forming inclusion complexes with free CDs remaining unoccupied after particle formation (Fig. 4C).

This strategy avoids the synthesis of drug-polymer conjugate, which can be deleterious for some fragile drugs 24 . However, the disadvantage of this type of particles lies on the low drug loading and the equilibrium between the free drug and included drug. When the particles were diluted, the equilibrium would shift to the free drug formation which leads to drug leakage. Fig. 4 Different models that represent how the drugs were loaded in the CD-based hydrogels/NPs driven by CD inclusion complexes. A, the drugs were conjugated to CD-bearing polymers; B, the drugs were conjugated on a different polymer; C, the drugs were loaded in free CDs that were not occupied by the guest molecules.

"Cage" particles: CD-based metal organic frameworks (CD-MOFs).

Although CDs are "cage" molecules, the cavity of the cage is very limited because of the dimension of the molecules. Therefore, porous "cage" particles, CD-MOFs, were recently synthesized in the group of Dr. Fraser Stoddart 25 . CD-MOFs were formed by reacting γ-CD and potassium ions employing a vapor diffusion method. In the originally reported synthesis 25 , "edible" CD-MOFs were prepared by dissolving commercially available food-grade KCl or potassium benzoate and food-grade γ-CD in distilled water. The aqueous solution was filtered through cotton wool, following vapor diffusion of grain alcohol into the solution over around one week. In this process, the γ-CD molecules are linked by potassium ions to form a body-centered cubic structure (Fig. 5). They are porous particles built with cubic units which have a pore of 1.7 nm. CD-MOFs greatly improved the capability of CDs to encapsulate drug molecules [26][27][28] .

Fig.5 structure of CD-MOFs

To obtain monodispersed CD-MOFs for drug delivery, the synthesis was optimized 29 . It has been discovered that addition of cetyl trimethylammonium bromide (CTAB) leads to the preparation of smaller CD-MOFs (1-10 μm). If methanol was also added together with CTAB, nanoscale CD-MOF could be obtained (200-300 nm). All these syntheses employing vapor diffusion methods took long time (26-32 h). It was recently reported that nano-CD-MOFs (100-300 nm) were successfully obtained by a microwave-assisted method in shorter time (≤ 70 min) 30 . However, the formed CD-MOFs were polydispersed. Further optimization of CD-MOFs synthesis is still needed for their biomedical applications.

CD-MOFs have shown their capability to encapsulate molecules of interest, including ibuprofen 28 , fenbufen 31 , doxorubicin 32 , curcumin 27 , sucralose 33 , ferulic acid 26 , 4phenylazophenol and rodamine B 25 . Impregnation and co-crystallization are two reported approaches for the incorporation. A high payload of 23-26 wt % was reached in the case of ibuprofen by impregnation 28 . Payload of 18.5 wt % was reached for Rhodamine B by cocrystallization 25 .

However, applications of CD-MOFs as drug carriers are severely hampered since they can be easily disassociated in aqueous environment. To address this issue, the first idea was to crosslink CDs in the framework of CD-MOFs, producing CD-MOF hydrogels 29 . However, the cross-linking reaction required long time incubation (over three days) at controlled temperature (~ 65°C), which would be harmful for many of the drugs if they are already loaded. Recently, a facile method to improve their stability has been developed via the incorporation of hydrophobic fullerene (C60) in their matrices 32 . It has been shown that C60 (0.7 nm in diameter) can be partially trapped within the cavity of a γ-CD, forming a CD-MOF/C60 composite. Waterresistance of CD-MOFs could be improved since the trapped C60 endowed CD-MOFs with enhanced hydrophobicity. When the CD-MOF/C60 composite was immersed in water, C60 layer could effectively repel water molecules from contacting MOF surface, therefore, the stability of CD-MOFs in aqueous condition was significantly improved. This study was performed with very large CD-MOFs (more than 400 µm) and the sytem still needs further investigations for its medical application.

Hybrid metal-organic framework nanoparticles (MOFs)

MOFs are an intriguing class of hybrid materials obtained by the self-assembly of metallic ions or clusters and bridging organic ligands, leading to the formation of three-dimensional networks with high and regular porosities [34][35][36][37][38] . Taking advantage of MOF important porous surface and large pore size, versatility in terms of architecture, composition and physico-chemical properties, they have been intensively used in the last several years for a number of different applications such as catalysis, separation and gas storage and more recently, in nanomedecine.

The use of MOF materials for biomedical applications, and in particular as drug nanocarriers, requires MOFs to be scaled-down to the nanometer sizes 39 using essentially bottom-up approaches, involving controlling the MOF nucleation rate and crystallization in order to limit the MOF growth to submicronic sizes.

MOFs have high porosities together with important available BET (Brunauer, Emmett and Teller) surface areas ranging generally from 1000 up to 7000 m 2 /g 38,[40][41][42][43][44][45] higher than BET surface of mesoporous silica nanoparticles (MSNs), which is up to around 1200 m 2 /g 46 . The large nanoMOFs surface areas together with their peculiar inner environments (both hydrophilic and hydrophobic) make these materials particularly appealing for the entrapment of drugs. The active molecules cross the MOF windows to get adsorbed onto the inner surfaces and interact with metal sites and/or organic constituting ligands.

When scaling down the synthesis to the nanometer range, the outer surface properties play an increasing role, as the external surface-area-to-volume ratio is increasing. Indeed, the outer surface properties determine the nanoMOFs' colloidal stability and interactions with biological environments.

Strategies to scale-down MOFs to the nano-regime.

A large variety of methods have been developed to produce MOFs with submicronic sizes (Fig. 6). They include solvothermal methods assisted or not by microwave irradiation 47,48 , solvent diffusion synthesis 25 , dry-gel conversion methods 49 , mechanochemical [50][START_REF] Pichon | Solvent-free synthesis of a microporous metal-organic framework[END_REF][START_REF] Lee | Synthesis of metal-organic frameworks: A mini review[END_REF] or sonochemical synthesis [START_REF] Carné | Nanoscale metal-organic materials[END_REF] , production by reverse-phase microemulsions [START_REF] Rieter | Nanoscale metal-organic frameworks as potential multimodal contrast enhancing agents[END_REF][START_REF] Sun | Metal-organic frameworks as potential drug delivery systems[END_REF][START_REF] Taylor | Surfactant-assisted synthesis of nanoscale gadolinium metal-organic frameworks for potential multimodal imaging[END_REF] , electrochemical synthesis [START_REF] Sun | Recent progress in the synthesis of metal-organic frameworks[END_REF] , preparation using a microfluidics device [START_REF] Juan-Alcaniz | Metal-organic frameworks as scaffolds for the encapsulation of active species: state of the art and future perspectives[END_REF] . Besides, step-by-step [START_REF] Shekhah | Step-by-step route for the synthesis of metal-organic frameworks[END_REF] and highthroughput synthesis methods [START_REF] Banerjee | High-Throughput Synthesis of Zeolitic Imidazolate Frameworks and Application to CO2 Capture[END_REF] were developed. Whatever the process, a main challenge is to control the crystallization process in order to achieve particles with uniform sizes less than one micron. Main strategies to achieve this goal consisted in adding crystal growth inhibitors, speeding the crystallization process, using nanoscale templates and downsizing the particles by mechanical or mechano-chemical methods.

Fig.6. Overview of main synthesis methods for MOFs

Blocking or inhibiting crystal growth. Various blocking agent or acido-basic or inhibiting additives such as acetic acid, hydroxybenzoic acid and pyridine were added in the MOF synthesis to retard the nucleation growth process. For example, the size of the flexible porous iron muconate MIL-89 (MIL stands for Materials from Institut Lavoisier) could be reduced down to 30 nm by adding acetate ions in the reaction process [START_REF] Horcajada | Colloidal route for preparing optical thin films of nanoporous metalorganic frameworks[END_REF] . In another study, pyridine was used as inhibitor in the solvothermal synthesis of porous indium terephthalate particles [START_REF] Cho | Growth-controlled formation of porous coordination polymer particles[END_REF] . Both the size and shape of the particles were affected by the inhibitor. In the absence of pyridine, hexagonal rods of around 1.75 μm width and 16.3 μm length were formed. Pyridine enables to reduce both width and diameters to 0.97 μm and 0.43 μm, respectively.

Luminescent nanoparticles made of terbium benzenedicarboxylate with sizes of only 4-5 nm were successfully obtained by adapting a previous method [START_REF] Reineke | From Condensed Lanthanide Coordination Solids to Microporous Frameworks Having Accessible Metal Sites[END_REF] using a poly(vinilpyrrolidone) as inhibitor of crystal growth [START_REF] Kerbellec | Luminescent coordination nanoparticles[END_REF] .

Accelerating the crystallization process. An alternative methods to the use of crystal growth inhibitors, was to speed up the crystallization process. This strategy was applied to CD-MOFs prepared by solvent diffusion (Fig. 6).

The chiral γ-CDs comprise eight α-1,4-linked D-glucopyranosyl residues disposed in a symmetric configuration. This allows the γ-CD building units to assemble with alkali metal ions (K + , Na + , Rb + , and Cs + ) in aqueous media to form body-centered cubic CD-MOFs (Fig. 5). For example, K + based CD-MOFs were obtained under the form of large particles of around 200-400 µm by reacting γ-CD with KOH in aqueous solution, followed by vapor diffusion of methanol into the solution at room temperature during 2-7 days. The addition of cetyl trimethylammonium bromide (CTAB) together with an optimization of the reaction conditions enabled to reduce the reaction times and to obtain nano-scaled CD-MOF (200-300 nm) 31 . Of note, cubic particles with homogeneous sizes were obtained by using CTAB.

The reaction time was found to dramatically influence the MOF size distribution. The diameters of Zn3(BTC)2•12H2O MOFs were reduced from 700 -900 nm to 50 -100 nm by decreasing the reaction time from 90 to around 10 min [START_REF] Qiu | Facile synthesis of nanocrystals of a microporous metal-organic framework by an ultrasonic method and selective sensing of organoamines[END_REF] .

Nanoscale templates.

Another promising strategy to obtained nanoscaled MOFs was the synthesis in confined spaces, using a template approach. For example, Gd-based MOF66 nanoparticles were obtained in reverse microemulsions prepared using mixtures of CTAB, isooctane, 1-hexanol and water. The morphology and size of nanoscale templates were dramatically altered by both the composition of the system and the presence of sodium salicylate, a hydrotrope [START_REF] Hatakeyama | Synthesis of gadolinium nanoscale metal-organic framework with hydrotropes: Manipulation of particle size and magnetic resonance imaging capability[END_REF][START_REF] Bhat | Characterization of interaction between butylbenzene sulfonates and cetyl pyridinium chloride in a mixed aggregate system[END_REF] . Downsizing. Nanosized MOFs were prepared mechanochemical synthesis, consisting in a mechanical breakage of intramolecular bonds followed by a chemical transformation. First examples of the synthesis of porous MOF by this method were reported in 2006 [START_REF] Pichon | Solvent-free synthesis of a microporous metal-organic framework[END_REF] .

Advantageously, mechanochemical reactions can occur at room temperature under solvent-free 20 conditions [START_REF] Garay | Solvent-free synthesis of metal complexes[END_REF] . Nanoparticles of HKUST-1 were successfully produced in solvent free conditions 50 . Mechanochemical synthesis of MOF nanoparticles were reviewed [START_REF] Friščić | New opportunities for materials synthesis using mechanochemistry[END_REF] . More recently, nanosized Gd-based porous MOF has been synthesized by mechanical downsizing by ball milling the as-synthesized MOF with millimeter-scaled sizes. [START_REF] Kundu | Mechanical downsizing of a gadolinium(III)-based metal-organic framework for anticancer drug delivery[END_REF] All these synthesis method have led to the obtention of a large variety of materials with rigid or flexible structures. Examples are compiled in Fig. 7.

Drug entrapment and release

Active molecules, mainly anticancer drugs have been successfully incorporated in the nanoMOFs using three main strategies schematized in Fig. 8.

The first group of MOFs considered as potential drug delivery system is the MIL family. In particular, MIL nanoMOFs build from trivalent metal centers and polycarboxylic acid bridging ligands develop outstanding surface areas (1500-5900 m 2 /g) and large pore sizes (25-34 Å) making them particularly appealing for drug entrapment 44 They were obtained by using aminoterepthalic acid instead of trimesic acid 45 . 1, a variety of active molecules have been successfully loaded into MIL nanoMOFs, including anti-inflammatory (ibuprofen, aspirin), antiviral (cidofovir, azidothimidine triphosphate (AZT-TP) and azidothimidine monophosphate (AZT-MP)), anticancer (busulfan, doxorubicin, ethoxysuccinato-cisplatin prodrug, gemcitabine-monophosphate (Gem-MP) and topotecan) drugs, nitric oxide, metallodrugs and peptides. All these molecules were loaded by impregnation using preformed NPs.

Drug loading by impregnation As shown in Table

Advantageously, hydrophilic molecules such as azidothimidine mono-and triphosphate and gemcitabine monophosphate were entrapped simply by soaking from their aqueous solutions.

The nanoMOFs acted as molecular sponges and the rapid and simple loading procedure lead to AZT-TP payloads of around 42 wt% and 21 wt% for MIL-101 and MIL-100 nanoMOFs, respectively [START_REF] Horcajada | Porous metal-organic-framework nanoscale carriers as a potential platform for drug delivery and imaging[END_REF] . Moreover, excellent loading efficiencies (> 95%) have been achieved, due to the high affinity of the phosphated molecules for the iron-based nanoMOFs [START_REF] Agostoni | Towards an Improved anti-HIV Activity of NRTI via Metal-Organic Frameworks Nanoparticles[END_REF][START_REF] Agostoni | Impact of phosphorylation on the encapsulation of nucleoside analogues within porous iron(iii) metal-organic framework MIL-100(Fe) nanoparticles[END_REF] . It has been shown by complementary techniques that coordination took place between the unsaturated Lewis acid iron sites and the phosphate groups of the drug [START_REF] Agostoni | Impact of phosphorylation on the encapsulation of nucleoside analogues within porous iron(iii) metal-organic framework MIL-100(Fe) nanoparticles[END_REF] In addition to MIL-family, a series of other MOFs were used as drug carriers, mainly Zn, Cu and Zr-based MOFs (Table 1). Several MOFs based on Ni, Gd, and In were also studied for drug encapsulation, with the advantages that some metal ions could confer also imaging properties. However, it has been pointed out that the toxicity of some metals could be a drawback for medical applications of some MOFs 118 . Drug release was found to be strongly dependent on the nanoMOF stability in the release media.

For instance, MIL-101 nanoMOFs with poor stabilities in aqueous media released AZT-TP in a fast ("burst") manner, in contrast with the more stable MIL-100 nanoMOFs which keep their integrity over longer incubation times [START_REF] Horcajada | Porous metal-organic-framework nanoscale carriers as a potential platform for drug delivery and imaging[END_REF] . As a general trend, it was found that the larger the pore size, the lower the MOF stability. Drug release from highly porous nanoMOFs appeared to be related to several factors: i) drug interaction with the metal sites and ligands; ii) drug diffusion inside the open porosity of the materials; iii) nanoMOF degradation kinetics in the release media.

Moreover, it was found that in some case, drug incorporation inside nanoMOFs enabled stabilizing the matrix upon degradation. Topotecan (TPT), a hydrophilic cytotoxic drug was efficiently encapsulated in MIL-100 nanoMOFs filling the available space inside their large pores 93 . The drug molecules formed aggregates inside the pores, which in turn strongly stabilized the crystalline tridimensional networks towards degradation in physiological media.

Interestingly, TPT release could be triggered by two-photon light-irradiation, opening new standpoints for further developments in stimuli-induced drug delivery. Further studies are on the way to understand the release mechanism.

Drug loading during particle synthesis A few recent examples show the possibility to load

drugs in MOFs during their synthesis. Curcumin was successfully incorporated in CD-MOFs without altering their crystallinity. Interestingly, when dissolving the curcumin-loaded CD-MOFs in water, unique complexes between curcumin, CD and potassium cations were found.

The stability of curcumin in these complexes was enhanced by at least 3 orders of magnitude compared to curcumin: CD complexes. It was suggested that the CD-MOFs could be useful to store and stabilize curcumin for food applications 27 .

In another study, ferulic acid (FA) was strongly associated to CD-MOFs during their preparation by methanol vapor diffusion into an aqueous solution of γ-CD, KOH and FA. Differential scanning calorimetry studies indicated the formation of the stable association between FA and γ -CD in FA/CD-MOF 26 .

BioMOF nanoMOFs were synthesized using the drug as a ligand to be directly linked to metal sites, thus building the tridimensional structures 119 . These so called bioMOFs could load up to 71.5 wt% of nicotinic acid. Disuccinatocisplatin was used as a linker to prepare NCP-1 120 (NCP stands for nanoscale coordination polymer). As most drug molecules possess metal complexing groups such as carboxylates, phosphates or amines, this approach bears a high potential for the development of biocompatible drug-delivery systems, maximizing the drug loading capabilities. Of note, bioMOFs containing therapeutically active molecules as a part of the structural framework are usually synthesized with a minimum number of synthetic steps for attaining maximum drug payloads. Some MOFs were composed of organic linkers which are not active molecules per se, but transform in physio-biological conditions to release active molecules. For instance, 1,3,5tris(1H-1,2,3-triazol-5-yl)benzene 121 and 2,5-dihydroxyterephthalic acid 122 were used as linkers releasing NO.

Moreover, bioMOFs synthesized with active molecules as linkers could also be further used to encapsulate other drugs. Olsalazine-based porous MOF was successfully synthesized reaching 51% (w/w) drug loading. Then, phenethylamine could be impregnated into the MOFs reaching a loading of 11% (w/w) [START_REF] Levine | Olsalazine-Based Metal-Organic Frameworks as Biocompatible Platforms for H2 Adsorption and Drug Delivery[END_REF] Besides, the use of biologically active metal centers as a part of the MOF structure has lately been investigated. Thus, MOFs can act as reservoirs of metal ions, providing their gradual release to achieve a sustained antibacterial action. A variety of materials have been studied so far, including as metal ions Ag 123-125 , Co 126 , Cu 127 and Zn 128 .

Surface modification

The application of nanoparticles for drug delivery is expected to make a difference in the field of biotechnology and pharmaceutical industries in the near future, as it paves the way to achieve targeted delivery in a cell-or tissue-specific manner, with the capability to bypass biological barriers (blood-brain barriers, epithelial and endothelial barriers, etc) and to visualize the in vivo fate of the nanocarriers by imaging modalities. Nanoparticle surface modification is a prerequisite to achieve these goals, as the interactions between the particles and the living media take place at their interface with the complex biological systems. However, the porous structure of nanoMOFs required the development of specific coating methods which do not alter the porosity and thus the loading capacity of the nanoMOFs.

Silica coatings have been extensively used to modify the surface of both inorganic and polymer 29 nanoparticles [129][130] . A variety of nanoMOFs have been coated with silica shells in an attempt to stabilize them, control their degradation, increase their biocompatibility and water dispersibility and enable further functionalization with a variety of silyl-derived molecules.

Silica shells were synthesized on nanoMOFs made using a variety of metals, including Gd 131 , Mn 132 , Tb 133 , Fe 100 , and Zr 134 . It was found that the silica shell significantly slowed down the nanoMOFs degradation. For example, the release of Gd 3+ ions depended on the silica shell thickness. Silica-coated MIL-101 nanoparticles with the diameter of around 200 nm had a stability of around 14 h in PBS, while the uncoated ones readily decomposed 100 . Moreover, the silica shell also allowed to control drug release depending on its thickness. Furthermore, the surface of these core-shell structures could be further modified by grafting silyl-derived molecules through surface silanol groups to impart additional functionalities, such as targeting ligands, optical contrast agents, and sensing agents. Interestingly, by grafting a cyclic arginineglycine-aspartate (targeting peptide), the anticancer efficacy against human colon cancer cells of Tb-based nanoMOFs was significantly increased as compared to the non-targeted MOFs 133 .

Reversible addition-fragmentation chain transfer (RAFT) polymerization was used to modify the surface of Gd-based NMOFs. Polymer-coating on nanoMOFs were achieved using a trithiocarbonate RAFT agent. The coatings were attached on the MOF surfaces through vacant coordination sites on the Gd 3+ ions. The resulting polymer-coated Gd-MOFs were demonstrated to be biocompatible and had cancer cell targeting abilities and bimodal imaging properties 134- 136 .

Recently, simple, fast, and ''green'' strategies for functionalization of nanoMOFs were reported. MIL-100 (Fe) was ''easily'' coated by CD molecules bearing strong iron complexing groups (phosphates) 137 or by heparin 138 . The heparin coating endowed the nanoMOFs with improved biological properties, such as reduced cell recognition, lack of complement activation, and reactive oxygen species production 138 .The stable CD-based coating was further functionalized with: i) targeting moieties to increase the nanoMOF interaction with specific receptors and ii) PEG chains to escape the immune system. These results pave the way towards the design of surface-engineered nanoMOFs of interest for applications in the field of targeted drug delivery. Polysaccharides like dextran and chitosan were studied to improve the solubility, cellular adhesion and imaging properties of native MOFs 139 .

Lipid coatings have recently been used to modify the surfaces of MIL-100 (Fe) 140 . Dye molecules were entrapped in the resulting MOF@lipid systems. It was shown that the lipid bilayer could: I) prevent the premature release of the dye, II) increase the colloidal stability of the nanoparticles and III) favored a higher uptake by cancer cells as compared to uncoated particles. Amorphous MOFs or nanoscale coordination polymers, were also coated with lipid coatings [141][142][143][144] . The pH-responsive coatings have also been developed. Doxorubicin loaded MIL-101 was functionalized with pH responsive benzoic imine bond and a redox active disulfide system. Both in vitro and in vivo results demonstrated that this system efficiently inhibited cancer cell growth while showing reduced side effects 145 . Chitosanmodified magnetic nanoMOFs loaded with doxorubicin showed pH-responsive drug release for several days. Moreover, the particles could be modified with folic acid, a target molecule, which allowed a more specific cellular internalization in folate-overexpressing cancer cells (HeLa) in comparison to normal (L929) cells 146 .

In vivo studies

For biomedical and pharmaceutical applications, MOFs are firstly required to have toxicologically compatible features. Nine representative nanoMOFs were assessed to zebrafish embryos, including barely cytotoxic (nanoUiO-66, nanoUiO-67, and nanoCo-MOF-74, and nanoMg-MOF-74), moderately cytotoxic (nanoZIF-7, nanoMIL-100, and nanoMIL-101), and highly cytotoxic (nanoZIF-8 and nanoHKUST-1). It is found that at 120 h post fertilization, the viabilities of the embryos exposed to nanoCo-MOF-74, nanoMg-MOF-74, nanoUiO-66, nanoUiO-67, nanoMIL-100, and nanoMIL-101 were not significantly different to those of the control group (without nanoMOFs), even at highest concentration (200 µM). In contrast, nanoZIF-7, nanoZIF-8, and nanoHKUST-1 provoked significant decreases in embryo survival. NanoZIF-7 was slightly toxic at 200 µM (embryo viability 79.2%); nanoZIF-8 was more toxic at the same concentration (embryo viability 33.3%); and nanoHKUST-1 was extremely toxic even at 20 µM (viability: 0%). It was shown that the leaching of solubilized metal ions played a main role on the levels of the MOF toxicity 147 .

Toxicity of iron trimesic MIL-100 (Fe) and trimesic acid were performed through intravenous administrating to rats. Around 220 mg/kg of MIL-100 and 78 mg/kg trimesic acid were intravenously injected and the results showed that the animal behavior and weight evolution of injected rats were totally normal in comparison with control group. It was concluded that this material is acceptable for drug delivery 148 . Biodistribution of MIL-100 nanoMOFs at early times (up to 24 h) or long term (from 24 h to 1 month) after administration were evaluated. The pharmacokinetic profile suggested that the nanoparticles act as a depot in the blood stream during the first hours before being cleared. Accumulation took mainly place in the liver and, in some extent, in the spleen. Histological studies demonstrated the absence of morphological alterations due to the presence of the particles in these organs. Liver function was however slightly altered as reflected by the increased plasma aspartate aminotransferase concentrations.

Finally trimesate was progressively eliminated in urine. Nevertheless, it was demonstrated that in the long-term study, these abnormalities in the iron levels were completely reversed after seven days and were not related to any toxicity signs.

Imaging with MOF

Magnetic resonance imaging (MRI). MRI is a powerful and noninvasive diagnostic imaging

technique allowing the detection of nuclear spin reorientations with high spatial resolution.

Contrast agents are used to improve the image quality by reducing the proton relaxation times.

NanoMOFs have received attention as contrast agents because of their hybrid composition consisting of metal centers and organic ligands. Moreover, as all other types of nanoparticles, they can be engineered in order to accumulate in targeted organs or tissues of interest (such as tumors) either by "passive targeting" via enhanced permeability and retention effect, or by "active targeting" using various target-ligands 150 . Firstly, Gd 3+ -containing nanoMOFs have shown high relaxivities as T1-weighted contrast agents. They possessed increased relaxivities as compared to the clinically used Omniscan. Gd-based nanoMOFs endowed with multimodal imaging properties were further developed. For instance, both a specific ligand and a fluorophore were grafted onto the nanoMOF surfaces enabling to achieve a tumor-targeted MRI effect 132 .Multifunctional MOFs endowed with bimodal imaging properties via MRI and fluorescence microscopy were synthesized for tumor targeting and diagnostic imaging 136 .A variety of MOFs endowed with imaging properties was synthesized, including as metal ions Cu 151 , Ru 134 Eu 131 , and Tb 152 . Although the relaxivities of Mn 2+ -based nanoMOFs were modest, the possibility for site-specific imaging with the help of a silica shell at the surface of nanoMOFs has been demonstrated 132 . Fe 3+ -containing nanoMOFs possessed good T2-weighted contrast enhancement. [START_REF] Horcajada | Porous metal-organic-framework nanoscale carriers as a potential platform for drug delivery and imaging[END_REF] . MRI measurements on Wistar female rats shows that the treated liver and spleen organs were darker than the normal ones, because of the preferential accumulation of the nanoMOFs in these organs. However, three months after injection, the liver and spleen returned to a similar appearance to that of the untreated rats, in accordance with the temporary accumulation of the nanoMOFs in these organs. Recently, a multifunctional nanoplatform based on isoreticular metal organic frameworks (IRMOF-3) was developed. Folic acid and the fluorescent molecule rhodamine B isothiocyanate (RITC) were both conjugated to the nanoMOF surface for cancer cell targeting and optical imaging. Paclitaxel was loaded into these magnetic nanoMOFs. The targeted nanoMOFs showed stronger T2-weighted MRI contrast towards cancer cells 153 .

X-ray Computed Tomography (CT) Imaging.

CT imaging is based on specimen attenuation of X-rays and can provide 3D images with excellent spatial resolution. Typical contrast agents are materials containing high Z number elements, including iodine, barium and bismuth. Currently, barium sulfate and iodinated aromatic are used clinically for intravenous and gastrointestinal tract imaging, respectively. The contrast agents for CT have the same drawbacks as for MRI, including a non-specific distribution and a fast clearance. Therefore, nanoMOFs have been developed as carriers for iodinated aromatic molecules 154 . They were prepared using tetra iodo benzene dicarboxylic acids as bridging ligands and Cu 2+ or Zn 2+ as metal connecting points.

The efficacy of the resulting iodinated nanoMOFs for CT has been clearly demonstrated.

All the above presented studies highlight the potential of nanoscale MOFs as platforms for MRI and X-ray CT.

Conclusion

Despite an enormous amount of research in the area of nanomedicine and advanced nanocarriers drug targeting in cancer, the transition from laboratory to the market is usually very slow and unpredictable. The majority of the problems arise from effective oversight in safetyand efficacy-by-design approach for nanomedicines. "Cage" particles based on "CD" and MOFs were shown to be biocompatible and versatile materials, adapted for drug incorporation. Moreover, their "green" scalable preparation positions these materials as good candidates for biomedical application. They are promising carriers for various drugs, including antibiotics, anticancer and anti-inflammatory drugs, etc.

CDs are recognized as "molecule containers" to form inclusion complexes with a variety of active molecules. These inclusion could not only improve the solubility and bioavailability of poorly soluble drugs, stabilize drugs from light, thermal and oxidative degradation, mask the bitter taste of the drugs, but also can be used as the driving forces to form NPs. Remarkably, the NPs spontaneously formed by CD inclusion complexes are easily formed by "green" methods, in absence of organic solvents or surfactants. While self-assembled NPs composed of CDs are attractive formulations, the stability of the formed NPs need to be studied in depth upon dilution, since the supramolecular edifices are constructed solely on the basis of noncovalent bonds. Indeed, in theory, the inclusion complexes are prone to dis-assemble upon extreme dilution, leading to the destructuring of the NPs.

CD-MOFs are a new type of porous CD-based particles, which contain not only the cavities of

CDs, but also the big pores built up by CDs self-assemblies (Fig. 1). Compared with CD based NPs, CD-MOFs have the possibility to load drugs with higher payloads. Given the fact that the particles disassemble in water, we foresee that a promising administration route could be pulmonary inhalation. However, with appropriate surface modification, the stability of CD-MOFs was significantly improved, which broadens their application also for potential oral and intravenous administration.

MOFs, especially biodegradable MOFs stable in aqueous media, have drawn a lot of attention as drug delivery systems. MIL-100(Fe) was considered among the most efficient material as drug carrier due to its large pore size, biocompatibility and biodegradability. A variety of drugs have been efficiently encapsulated, including anti-inflammatory (ibuprofen, aspirin), antiviral (cidofovir, AZT-TP and AZT-MP), anticancer (busulfan, doxorubicin, ethoxysuccinatocisplatin prodrug, Gem-MP and topotecan) drugs. There are two types of cages in the structure of MIL-100(Fe), so it could be possible to load two drugs simultaneously in the same particles in theory. To the best of our knowledge, studies so far report only the incorporation of one drug in the same particles. In this context, it would be interesting try to entrap synergic drugs in the same particles to treat complex pathologies such as cancer and severe viral or bacterial infections.

By taking advantage of their specific characters such as green preparation procedure, high drug payloads, and co-encapsulation of synergic drugs, these "cage" particles, these "cage" particles are very promising for drug delivery applications. 86. Lin

General Objectives and Author Contributions

CD-based nanotechnology has drawn special attention since it combines the advantage of selfassembled host-guest interactions and nanotechnology. Herein, polymeric nanoparticles driven by CD inclusion complexes were spontaneously formed in aqueous media, in the absence of organic solvents or surfactants. In order to prepare stable CD-based nanoparticles (CD-NPs), a highly soluble β-CD polymer (poly-CD) was used as the source of CDs, which was prepared by cross-linking β-CD with epichlorohydrin (EP). Benzophenone (Bz) was chosen as the guest molecule, because of its strong affinity with the cavity of β-CD and because it was well-studied in NPs made of poly-CD. Bz was grafted with dextran, forming a new polymer, namely, Dex-Bz. Poly-CD and Dex-Bz were used in this study as the two polymers for CD-NPs preparation.

They are both neutral polymers, therefore, the interactions between the two polymers in water are mostly due to the host-guest interactions between β-CD and Bz, without electrostatic interactions.

To prepare and characterize these particles, complementary methods were used bringing new insights on the formation and stability of the supramolecular systems:

1) Microfluidics was used to prepare the particles by mixing the two aqueous solutions directly in the microfluidic chamber. CD-NPs with narrow size distributions were immediately formed upon contact of the two polymeric solutions.

2) In situ size measurements during the particle formation showed that the CD-NPs were spontaneously formed in aqueous media.

3) Individual particle tracking analysis was used to gain insights on their size distribution and concentration. Since the CD-NPs were formed by non-covalent interaction, it is important to check if the NPs disassemble upon dilution. Individual particle tracking analysis was an adapted method to prove that the particles were stable even upon extreme dilution despite their noncovalent nature.

General Objectives and Author Contributions

In this subchapter, CD-MOFs around 6 µm with monodispersed size distribution were used as drug carrier for Lansoprazole (LPZ). The molecular dimensions of LPZ is about 4 × 17 Å, which fits to the dimensions of γ-CD cavity (7.8 Å) and the internal spherical cavities (17 Å) of CD-MOF.

In addition to drug absorption by impregnation shown in subchapter 3.1, a new approach was used in this subchapter for drug encapsulation, namely, co-crystallization. By impregnation approach, the obtained payloads were not as high as expected, at best around 12.6% (wt) in the case of captopril, possibly because the small pore windows of the CD-MOFs limit the penetration of drugs. Therefore, co-crystallization was used for LPZ, where the drug was added during particle synthesis. Remarkably, LPZ payloads reached 23.2±2.1% (wt) which corresponds to a molar ratio of 1:1 between LPZ and γ-CD.

To well characterize the LPZ loaded CD-MOFs, the study of morphology and chemical composition of individual particles was performed with Raman microscopy. It was demonstrated that there were no significant differences on Raman spectra of individual particles, indicating that each individual particle had identical chemical composition. Molecular modelling was performed for deeper insight into the interaction between the LPZ and CD-MOFs. Moreover, it was demonstrated that even after two years storage, the incorporated drug inside the CD-MOFs maintained its spectroscopic characteristics. This study lay the foundations for further application of CD-MOFs as LPZ carriers for drug delivery.

General Objectives and Author Contributions

CD-MOFs have shown their high drug loading capacity up to 23% (wt) in subchapter 3.2, however, their applications in the biomedical field are severely hampered by their physical frailness and their high solubility in aqueous media. Therefore, surface modification is needed in order to improve the stability of CD-MOFs in aqueous media and also to control the drug release. Herein, CD-MOFs were embedded in a biocompatible polyacrylic acid (PAA) polymer matrices by a solid in oil-in-oil (s/o/o) emulsifying solvent evaporation method. Briefly, several steps were included:

1) Nano-scaled CD-MOFs were firstly synthesized to obtain a good dispersion inside PAA matrices.

2) Drugs of interest, Ibuprofen (IBU) and Lansoprazole (LPZ), were loaded in CD-MOFs by co-crystallisation.

3) Morphology, size distribution, and crystallinity of CD-MOFs, loaded or not with drugs were well characterized.

4) CD-MOF nanocrystals-containing microspheres, namely drug-CD-MOF/PAA composite microspheres, were prepared by a solid in oil-in-oil emulsifying solvent evaporation method.

As controls, microspheres containing only drug, or drug-CD complex were prepared in parallel.

5) Morphology, size, and drug release of the prepared microspheres were investigated.

6) The cellular toxicities of CD-MOF nanocrystals, blank PAA microspheres, pure IBU, IBU-CD-MOF nanocrystals and the IBU-CD-MOF/PAA composite microspheres were evaluated in J774 macrophages using MTT assay.

As a result, the composite microspheres exhibit not only spherical shapes and sustained drug release over a prolonged period of time, but they also demonstrate reduced cell toxicity.

This work has been performed in collaboration with Dr. J.

General Objectives and Author Contributions

"Cage" porous particles based on MOFs have emerged as a new class of ordered porous materials for drug delivery. Iron trimesate MOFs: MIL-100 (Fe) are considered among the most efficient materials because of their biodegradability, biocompatibility, and unprecedented drug loading capability. Among all the investigated molecules, MIL-100 (Fe) interacted most efficiently with phosphated ones which could be efficiently soaked from their aqueous solutions. However, MIL-100 (Fe) rapidly degraded in phosphate buffers. It is important to understand the complex interactions taking place between MIL-100 (Fe) and different phosphated molecules. Therefore, in this subchapter, we developed a method based on Raman microscopy to track the morphology and chemical composition of individual particles.

Briefly, the degradation of nanoMOFs and microMOFs were firstly studied in phosphate buffer saline (PBS). In particular, the degradation behavior of individual particles in PBS was tracked by Raman microscopy. Of upmost interest, it was possible to detect modifications of the chemical composition on different regions on the same particle during degradation. A formation of an erosion front during MOF degradation was clearly observed with obvious modification on Raman spectra.

To understand if the phosphated molecules would lead to degradation, Gemcitabinemonophosphate (Gem-MP) and β-CD-phosphates (CD-P) were chosen as model molecules.

Gem-MP is a small molecule could be penetrated within the porous MOF structures, in contrast, CD-P is larger than the cage of MOF and could stay only on the surface of the particles as coating material. In both cases, phosphated drugs/coating materials did not contribute to particle degradation.

All the authors have contributed in this work. X. Li contributed to experiments on the nanoMOFs synthesis, characterization, surface coating, and drug loading, and wrote the main manuscript.

Introduction

Metal-organic frameworks (MOFs) are a recent class of ordered porous materials formed by the coordination of metal clusters and organic ligands. Due to their versatile chemistry, high pore volumes and large surface areas, MOFs attracted growing interest in various areas, including gas storage 1,2 , catalysis [3][4][5] , separation science 6 , sensing 7,8 , ionic conduction 9 and more recently, biomedicine [10][11][12][13] . Lately, biodegradable micro-or meso-porous iron polycarboxylate MOFs were employed as drug carriers 10,11,[14][15][16][17][18] . More particularly, iron trimesate MIL-100 and iron terephthalate MIL-101 (MIL stands for Materials of Institute Lavoisier) were considered among the most efficient materials to load drugs because of their large pore sizes (24-29 and 27-34 Å for MIL-100 and MIL-101, respectively) 19 . As an example, ibuprofen payloads reached up to 35 wt% and 140 wt% for MIL-100 and MIL-101, respectively 11 . However, nano-scaled MOFs (nanoMOFs) made of biocompatible MIL-101(Fe) suffer from a very fast degradation in phosphate buffer saline (PBS) hampering their applications for controlled drug delivery 20 . In contrast, MIL-100(Fe) nanoMOFs degraded in a more progressive manner compatible with biomedical applications and were well tolerated in vivo 21 . A variety of drugs have been successfully incorporated into MIL-100(Fe) nanoMOFs, including antiviral drugs (cidofovir 10 , azidothimidine triphosphate (AZT-TP) and azidothimidine monophosphate (AZT-MP)) 15,22 , anticancer drugs (busulfan 16 , topotecan 18 , doxorubicin 23 and gemcitabine-monophosphate (Gem-MP) 24 ), biological gases (nitric oxide) 25 , caffeine 26 , aspirin 27 and metallodrugs 28 .

The highest loading efficiencies were reported in the case of phosphated drugs, including AZT-TP, AZT-MP and Gem-MP 15,22,24 . MIL-100 were able to soak the drugs within minutes from their aqueous solution, reaching elevated drug loadings (around 30, 25 and 36 wt% for Gem-MP, AZT-TP and AZT-MP, respectively) and encapsulation efficiencies higher than 98%. It was demonstrated that coordination of the drug's phosphate groups on the iron(III) Lewis acid sites in MIL-100 nanoMOFs played a main role, leading to an efficient encapsulation 22,24 .

Indeed, the same drugs without phosphate groups were poorly incorporated 22 .

Phosphate molecules are essential components in every living organism. When non-controlled, strong interactions between phosphates and MOFs can lead to a fast degradation and thus to "burst" drug release. Therefore, it is of outmost importance in the development of MOFs drug carriers to gain deep understanding on their degradation in the presence of phosphates. It was shown that MIL-100(Fe) particles exhibit good colloidal stability and biodegradability in a series of simulated physiological fluids 29 and the degradation of nanoMOF has been studied in different cell culture medium as reported 29 . There was around 10wt% of trimesate released in Dulbecco's Modified Eagle's medium (DMEM), but only around 5% released in Minimum Essential Medium Eagle (MEM). Trimesate release was dependent on the composition of the cell culture medium, especially on its content in phosphates. Intravenously administered nanoMOFs in rats were rapidly sequestered by the liver and spleen, then further biodegraded and directly eliminated in urine or feces 30 . The trimesate linkers were progressively eliminated in urine 21 . All these studies have shown the biodegradability of nanoMOFs for potential biomedical applications, but their degradation mechanism is not fully understood yet. .

Recently, Bezverkhyy et al. 32 demonstrated that MIL-100(Fe) nanoMOFs maintained their crystalline structures while heated in water at 100 °C for 48 h, but partially degraded. On the contrary, at neutral pH the particles degraded within one hour yielding poorly crystallized iron oxide (ferrihydrite). These experiments were however conducted in media without phosphate groups, not mimicking the biological conditions. In other studies, it was shown that the MIL-100(Fe) nanoMOFs progressively disintegrated in PBS because of the strong interactions between phosphates and iron and that this degradation was well correlated to drug release 15,18,23 .

One can also tailor the MOF particle size according to the targeted application and route of administration even leading to micron-sized MOF (microMOF) particles 33 . NanoMOFs had been reported to exhibit a narrower particle size distributions than microMOFs but were more prone to faster degradation because of larger surface areas 33 . However, no study dealt yet with the influence of the MOF particle size on the degradation mechanism. Moreover, the investigations on MIL-100(Fe) MOF degradation were conducted so far using batches of particles, but never on individual particles. In addition it has not been assessed yet at the particle level if drug loading or surface modification impacts the stability of the nanoMOF. It is therefore of main interest to study the degradation of individual MOFs.

To address this goal we have used here Raman microscopy to monitor in situ the degradation in phosphate-containing media of individual MIL-100(Fe) MOF particles of different sizes.

This versatile method combines the advantages of both Raman spectroscopy and optical microscopy. The utility of Raman microscopy as a rapid, label-free, and non-destructive method was proven in many applications where chemical analysis and imaging are simultaneously required. This technique allows to qualitatively or quantitatively characterize the chemical composition and structure of a sample. In the pharmaceutical field, Raman microscopy is used mainly to characterize the distribution of molecules of interest in tablets, to determine the crystallinity of the materials, to investigate purity and chemical composition of complex devices.

In this study, MOF batches were analyzed in terms of particle size distribution and shape.

Raman spectra of individual particles were recorded, enabling to assess the homogeneity of the MOF population in terms of chemical composition and morphology. Of note, the degradation of each particle in PBS was monitored up to 8 days, enabling to detect eroded and intact zones in the same particle. Finally, we showed that the integrity of the MIL-100(Fe) MOFs was maintained after drug loading and surface modification.

Results and discussion

Synthesis and characterization of nanoMOFs. Nano-scaled MIL-100(Fe) particles were successfully prepared as reported by a solvent-free and fluoride-free "green" hydrothermal microwave assisted method using as reactants iron chloride and trimesic acid 34 . As schematized in Fig. 1A, the spontaneous coordination of Fe(III) trimers and trimesic acid generates hybrid supertetrahedra which further assemble into a zeotypic architecture consisting of: i) small mesoporous cages (Ø =25 Å), delimited by pentagonal openings (5.6 Å) and ii) large mesoporous cages (Ø =29 Å) accessible through both pentagonal and larger hexagonal windows (8.6 Å) 11 . The nanoMOFs' Powder X-Ray Diffraction (PXRD) patterns and nitrogen porosimetry BET surface areas (SBET = 1690 ± 50 m 2 /g) are in agreement with previously published data 15,19 .

As illustrated in Fig. 1B, the nanoMOFs appear as small crystals with a faceted morphology and rhombic structures. The Transmission Electron Microscopy (TEM) analysis of more than 8000 nanoMOFs particles allowed estimating their average diameter of 187 ± 55 nm and a mean circularity of around 0.89 (Fig, 1C) in agreement with the sharp-edged shapes observed by TEM In a complementary approach, the size of individual nanoMOFs was determined by nanoparticle tracking analysis (NTA), a method which combines an optical microscope and a laser illumination unit 35 . The mean diameter of each particle was calculated from its trajectory in Brownian motion, using the Stokes-Einstein equation. The obtained mean diameter of 196 ± 59 nm (Fig. 1D) was in agreement with those obtained by both DLS and TEM studies. These findings support the fact that the nanoMOFs have a relatively narrow particle size distribution.

Synthesis and characterization of MIL-100(Fe) microMOFs

MicroMOFs were synthesized by a similar method as nanoMOFs, but with longer reaction times to allow the crystals to grow larger. The microMOFs displayed a crystalline structure according to PXRD patterns and nitrogen porosimetry BET surfaces of around 1700 ± 60 m 2 /g.

More than 2000 individual particles were observed by Raman microscopy. Typically, the microMOFs appeared as crystals with irregular shapes (Fig. 1E). Statistical analysis showed that microMOFs possess a mean circularity (0.84) slightly lower than the nanoMOFs (Fig. 1F).

MicroMOF samples contained: i) a main population with an average diameter of around 3 µm and ii) a smaller population of much larger particles of size around 60 µm (Fig. 1G). Contrary to nanoMOFs which had a narrow size distribution, microMOFs possess thus a bimodal size distribution, possibly because of the difficulty to control crystal nucleation and growth, a process dependent on many experimental parameters. These findings highlight the importance to perform individual particle analysis to gain insights into the degradation of MIL-100(Fe) particles of different sizes.

Degradation of nanoMOFs

To study the effect of phosphates on nanoMOF degradation, the crystalline particles were incubated in PBS with different phosphate concentrations and in water, as a control. NanoMOFs incubated in PBS (1.19 mM) at 37°C experienced a rapid degradation, releasing large amounts (34 ± 3 wt %) of their constituting ligand, trimesate, after only 6 h. These data are in agreement with previously reported studies, where 29.9 ± 2.1 wt% trimesate was released under similar conditions 28 . Comparable amounts of ligands (31 ± 3 wt %) were released but after two days of incubation in PBS when diluted ten times (0.12 mM)(for details see SI section). In both cases, the concentration of nanoMOFs was 0.5 mg/mL, corresponding to 0.1 mM Fe. This highlights the influence of the concentration of phosphates on the degradation mechanism: the higher the concentration of phosphates, the faster the degradation of nanoMOFs in PBS.

In contrast, nanoMOFs released negligible amounts (less than 2 wt %) of their trimesate constituting ligand within two days incubation in pure water at 37°C, in agreement with previous investigations 15,22 , where less than 1 wt% trimesate was released after incubation for three days. This is in agreement with the nanoMOFs' diameter that remained practically unchanged over two days incubation in water (224 ± 25 nm).

Note that nanoMOFs lost their crystallinity in PBS within only 4 h, whereas they maintained an intact crystallinity in water, as indicated by PXRD results (Fig. S1).

These observations were well corroborated by TEM investigations, evidencing the nanoMOF morphological aspects before (Fig. 2A) and after (Fig. 2B) degradation in PBS. Noteworthy, degradation in PBS leads to a progressive rounding (Fig. 2B) of the nanoMOF sharp initial edges (Fig. 2A), in agreement with a progressive disorganization of the crystalline 3D structure.

Interestingly, DLS measurements showed that despite the degradation, the nanoMOFs maintained practically the same mean diameter (Fig. 2C), with a moderate decrease in size from 221 ± 24 nm to 187 ± 22 nm after two days incubation in PBS, despite the fact more than 30 % of their constitutive ligand has left the particles, as shown above.

In a nutshell, it confirms that these iron carboxylate based nanoMOFs are remarkably stable in water, but degrade in PBS with a progressive departure of the constitutive organic linkers associated with an amorphization. However, it is remarkable that despite their dramatic morphological and structural changes in PBS, nanoMOFs maintain practically unchanged diameters over two days. Based on these findings, it was interesting to further investigate the degradation of larger particles (microMOFs) in PBS.

Degradation of MIL-100(Fe) microMOFs

As shown in Fig. 2D, MIL-100(Fe) microMOFs are red-orange crystals with irregular shapes.

The red color is attributed to Fe 3+ species in the [Fe3O(OH)(H2O)2] 6+ clusters linked by carboxylate anions 27 . Contrary to nanoMOFs, microMOFs degrade very slowly and therefore concentrated PBS (11.9 mM) was used to accelerate the process. After 8 days of incubation in this medium, the particles changed color (Fig. 2E). However, surprisingly, the size of the particles monitored by Raman microscopy remained unchanged (less than 0.5 % variation in all dimensions) (Fig. 2F) as it was previously shown in the case of nanoMOFs. Grey regions (arrows in Fig. 2E) were clearly observed, whereas the core of the particles maintained its initial red color.

In a nutshell, MOF particles degraded with the formation of a grey shell delimiting a red-colored core. Raman microscopy was further used to study the composition of these different regions in the same particle.

Individual MIL-100(Fe) particle degradation

Typical Raman spectra of microMOFs show the specific MIL-100(Fe) peaks in the 100-1900 cm -1 region (Fig. 3). The peak A at 210 cm -1 is attributed to the ordered crystalline iron-based structure, whereas the B bands from 450 cm -1 to 600 cm -1 are related to lattice vibrations and network binding modes. The principal peaks in Fig. 3 correspond to the fingerprints of the trimesate linker, the aromatic ring peaks at 800 cm -1 (peak C) and 1000 cm -1 (peak D). The E peaks around 1200 cm -1 can be assigned to the C-O-Fe stretching of Fe-trimesate, whereas F bands ranging from 1400 cm -1 to 1600 cm -1 are related to the H-O-H bonding vibrations, which indicate that the Fe-trimesate network contains coordinated water. 32 . This is also in agreement with the disappearance of peak F in the eroded region II, indicating that coordinated water faded away.

Fig. 3 Raman spectra of a single microMOFs particle of around 50 microns before (brown) and after (red and blue) degradation for 8 days in PBS (11.9 mM). Raman spectra were recorded on the same particle, both in the red core (region I) and in the degraded grey shell (region II).

In conclusion, Raman spectra gave useful insights regarding MOF degradation in PBS, suggesting that the most probable degradation mechanism is the competitive replacement of trimesate by phosphate ions in PBS solution. Advantageously, Raman microscopy enabled obtaining spectra in different regions on the same particle. To gain further insights on the degradation mechanism, single particles were tracked over their degradation in PBS during 8 days.

Automated imaging was used to gain a deeper understanding of the kinetics of the degradation process of each selected particle, since it can provide a direct measurement of the size, shape, color, surface area, and circularity of each particle. Images were captured every half an hour during 8 days to track the degradation process. Selected images illustrate the typical morphologies of one single particle during degradation (Fig. 4). Although shells (eroded zones) with a grey color were clearly formed and progressively penetrated into the particles, no significant variation in size could be detected.

Additionally, the variation of mean intensity and surface area was also investigated. Mean intensity is a precise parameter to evaluate the color of a particle, based on the amount of light reflected by a particle. The darker the particle, the less is the reflected light, resulting in lower intensities. Figure 4 shows a sharp increase in the intensity of a MOF particle in the first 6 hours, indicating a rapid color change, which was too subtle to be observed by the naked eye. This fast phase was followed by a slower increase in mean intensity, accompanied by no detectable surface area variation (Fig. 4, progressive phase). It can be speculated that in the first hours after incubation, a fast coordination reaction occurred between phosphates in the incubation medium and accessible iron sites at the MOF surface. At 300K, the hyperfine structures of the nano-and microMOF samples after degradation was modified (Fig. 5). Before degradation, the spectrum was well described by means of 3 quadrupolar components (Fig. 5), in agreement with the crystallographic MIL-100(Fe) structure, while after degradation, quadrupolar doublets with broadened lines were observed.

The best physical description requires a discrete distribution of quadrupolar doublets suggesting a structure close to that of a typical amorphous structure. The Mössbauer spectra recorded at 77K are similar to those obtained at 300K at low velocity scale and their corresponding hyperfine structures confirm previous conclusions (Fig. S2). A deep analysis of the 12 mm/s spectra at 77K allows a small magnetic component (estimated at about 2% in at Fe) to be observed, as displayed on the insets of Fig. S2, where relative transmission scale has been strongly extended. It corresponds clearly to hematite in the case of the degraded microMOFs while it could be attributed to poorly crystallized hematite (including traces of ultra-fine grains of goethite). Taking into account the presence of phosphates in the media surrounding the microMOF, it is plausible that a highly disordered FeP based skeleton phase was formed, corroborating our previous hypothesis. Energy dispersive X-ray analysis (EDX) was performed in order to obtain the Fe/P ratios of the intact MOFs, half degraded microMOFs, and totally degraded microMOFs. The results showed that there is a significant reduction of Fe/P ratios from 1000 (intact MOFs) to 7.6 (half degraded microMOFs) and 0.9 (totally degraded microMOFs), confirming that P was coordinated inside the porous MOF structures.

To demonstrate the progressive coordination of phosphates during the degradation process, Infra-Red (IR) spectroscopy was performed out on micro-MOFs at different degradation times (see Fig. S3). In addition to the decrease in intensity of the carboxylate stretching bands at ca. 1390 and 1580 cm -1 , one could observe the progressive increase of intensity of the phosphate bands centered at 1050 cm -1 . Although one cannot exclude that amorphous Fe oxide is also formed during the degradation process, this confirms the progressive incorporation of phosphates throughout of the particles when dispersed into PBS. The broadness of the phosphate bands is also in agreement with the amorphous character of the degradation product.

MOF stability during drug loading

Gem-MP has been successfully loaded in MIL-100(Fe) MOFs, following the previous methodology, which acted as efficient "nanosponges", soaking the drugs from their aqueous solutions to reach within minutes unprecedented amounts (up to 30 wt%) of drug with loading efficiencies higher than 98% 24 . This is due to the coordination of the phosphate group of the drug on the Fe metal sites. In this study, the results from Gem-MP quantification by HPLC showed a high payload of 25 wt%. It is therefore important to investigate if drug loading induces a degradation of the MOF structure as it was the case when the MOFs were incubated in the presence of phosphates in PBS.

The drug loaded particles were studied by Raman microscopy. Gem-MP did not present characteristic Raman peaks in the studied region, facilitating the studies. Fig. 6 does not reveal any significant differences in the MIL-100(Fe) microMOFs Raman spectra before and after drug loading. These studies are in agreement with HPLC investigations, showing less than 2% of loss of the constitutive trimesate. Of note, the microMOFs maintained their crystalline structure after Gem-MP loading 24 . In a nutshell, the MOF 3D structure was maintained after drug loading with no impact of the drug on the MOF long-range or local structure. MOFs particles before (red) and after (purple) Gem-MP loading.

Previously we showed that MOFs immersed in PBS degraded with kinetics dependent upon the phosphates concentration. When Gem-MP was loaded in MOFs at the maximum payload of 30 wt%, the equivalent amount of phosphates was 0.038 mM, whereas the molar ratio P/Fe was only 0.38, which could explain why MOFs did not degrade during drug entrapment.

MOF stability during coating

Cyclodextrins (CDs) bearing several phosphate anchoring groups (CD-P) were successfully used to functionalize the surface of MOF particles 33 . Taking advantage of the fact that the size of CD-P is larger than the MIL-100(Fe) windows (Fig. 7) the coating could be achieved by a simple way consisting in incubating the MOFs with CD-P in water. This "green" method allowed the formation of CD-P shells within minutes and the coating was stable, even in biological media 33 , due to the cooperative coordination between phosphates on CD-P and Fe sites at the MOF's surface.

In this study, CD-P coatings were produced on microMOFs following the previous protocol 33 .

As reported in the previous work 33 , elemental analysis was used to evaluate the coating efficacy of grafting CD-P, showing that around 17 wt% of CD-P was associated to the MOFs. The particles, before and after coating, were studied by Raman microscopy. Of note, CD-P did not present any specific peak in the observed region. It was found that after coating, the particles maintained same morphology, color and Raman spectra, indicating that CD-P surface modification did not induce particle degradation. Similarly as in the case of drug loading, the P/Fe ratio in CD-P coated microMOFs was lower than 1 (only 0.6), which could explain why the microMOFs did not degrade after coating. The good stability of the particles after CD coating offer thus promising possibilities to load drugs of interest and graft ligands for targeting purposes.

Conclusion

Whatever their size, MIL-100(Fe) MOFs degrade in PBS losing their constitutive trimesate linkers and eventually became amorphous with a clear impact of the phosphate concentration and the particle size: the higher the phosphate concentration and the lower the particle the faster the degradation of the MOF particles. Interestingly, nano-and microMOFs both kept their initial sizes during degradation but transformed into inorganic edifices. Raman microscopy enabled gaining insights on the degradation mechanism and showed that intact cores were separated from the eroded regions by clearly visible erosion fronts. Iron phosphates were identified by Mössbauer spectrometry (and IR spectroscopy) as main components in the eroded regions. In contrast, neither drug entrapment and nor particle coating did affect the integrity of the tridimensional MOF network due to the more diluted conditions. These findings are of interest in the development of stable drug-loaded MOF particles. Further studies will be focused on the degradation of other engineered MOF materials of interest in biomedicine.

Materials and Methods

Chemicals. Iron (III) chloride hexahydrate (98%, Alfa Aesar, Schiltigheim, France), 1,3,5benzenetricarboxylic acid (BTC, 95%, Sigma-Aldrich, Saint-Quentin-Fallavier, France) and absolute ethanol (99%, Carlo Erba, Val-de-Reuil, France) were used for MOF synthesis. PBS (11.9 mM, pH 7.4 ± 0.1) was purchased from Life Technology Co., Ltd. (Saint Aubin, France).

It contains 11.9 mM phosphates, 137 mM NaCl and 2.7 mM KCl. Gem-MP (CH12F2N3OP) was purchased from Toronto Research Chemicals, Canada. Phosphated β-cyclodextrin sodium salt (CD-P, Cyclolab, CY-2017.1, molecular formula: C42H70O47P4Na4) was used for coating. Milli-Q water was obtained from a Millipore apparatus equipped with a 0.22μm filter. Reagents and solvents were used without further purification.

Nano-and micro-MOFs synthesis. MIL-100(Fe) iron trimesate nanoMOFs were synthesized by adapting a previously described method 1 . Briefly, nanoMOFs were obtained by microwaveassisted hydrothermal synthesis from a mixture of iron chloride (8.97 mmol) and BTC (4.02 mmol) in 20 mL of deionized water. The mixture was heated for 6 min at 130 o C under stirring.

The power applied was 800 Watts (Mars-5, CEM, US) with a power maximum output 1,600 ± 240 Watts and frequency at full power 2,450 mHz. The resulting nanoMOFs, recovered by centrifugation, were washed with absolute ethanol 6 times to remove the residual non reacted organic acid. A last centrifugation at 5500 g was performed during 1 min in absolute ethanol to sediment the largest particles and recover the supernatants as a suspension of monodisperse nanoparticles. Nanoparticles were stored wet in ethanol until final use.

To prepare microMOFs, a reaction mixture composed of 2.7 g iron (III) chloride hexahydrate (10.00 mmol) and 2.1 g trimesic acid (10.00 mmol) in 50 mL of water, was placed in a large Teflon bomb under magnetic stirring for 15 minutes. The Teflon reactor was encased in a metal bomb with controlled pressure, before being placed in an autoclave with a 1 hour heating ramp to 130 °C, and held at this temperature for 72 hours. The product was then cooled before being filtered and was then washed by heating under reflux, first in 700 mL of ethanol at 75 °C for 2 hours and finally in 700 mL of water at 90 °C for 2 hours. The product was then collected by filtration under vacuum and stored as powder after being dried in air.

MOFs

Statistical analysis of MIL-100(Fe) degradation. The size distribution and morphology of

MOFs after degradation in PBS were characterized. Briefly, Raman microscopy was applied for microMOFs to investigate their size and circularity before and after incubation in PBS (11.9 mM) for 8 days. TEM was used for nanoMOFs to observe the morphological variation after incubation in PBS (1.19 mM) for 6 hours. DLS was used to characterize the mean diameter variation during degradation of nanoMOFs in PBS with different concentrations (11.9 mM, 1.19 mM and 0.12 mM).

HPLC determination of the Gem-MP loading and trimesate release. The detection of Gem-

MP and trimesate was carried out in HPLC (Agilent 1100, USA) connected to a Phenomenex C18 column (4.6 × 250 mm, 5 µm) at a flow rate of 1.0 mL/min. For the analysis of Gem-MP, a mobile phase consisting of 84% buffer (0.2M TEAA):16% MeOH was used. It was detected at 254 nm with an injection volume of 20 µl. Analysis of trimesate was performed with a mobile phase containing 90% buffer (5.75 g/L of NH4H2PO4): 10% Acetonitrile (5 mM TBAP). The injection volume was 5 µl and the detection wavelength was set at 220 nm.

Individual analysis of MIL-100(Fe) degradation. Individual particles were observed by

Raman microscopy and Raman spectra were obtained for degraded and non-degraded particles.

Microparticles were kept in PBS (11.9 mM) for 8 days under the monitor of Raman microscopy.

Images were captured every 30 min to track the degradation process using automated imaging.

Surface areas and intensity mean were extracted from the software and kinetic analysis was carried out in function of surface areas and intensity mean. Raman spectra were recorded at the laser wavelength of 785 nm. The laser spot was 3 μm in diameter with power output as 15 mW.

Transmission 57 Fe Mössbauer spectrometry was performed using a conventional electromagnetic transducer with a constant acceleration and a [START_REF] Sun | Recent progress in the synthesis of metal-organic frameworks[END_REF] Co/Rh γ-ray source. Mössbauer spectra were recorded at 300 K and 77 K using a bath cryostat on samples which consist in a powdered layer containing about 5 mg of Fe/cm 2 . The hyperfine structure was modeled by a least-square fitting procedure involving quadrupolar doublets and/or Zeeman sextets composed of lorentzian lines using unpublished program 'MOSFIT'. The isomer shift values (IS) are referred to that of -Fe at 300 K, used as a standard to calibrate the spectrometer.

EDX analysis.

The chemical compositions of the samples were determined by EDX performed on a FEI Magellan 400 Scanning Electron Microscope. Three samples were analyzed: i) intact MOFs; ii) half degraded microMOFs; iii) totally degraded microMOFs. Twelve spectra were obtained for each sample and the content of P, Fe, and Cl was calculated by getting the average of the 12 spectra. Finally, Fe/P ratios were calculated for each sample.

PXRD analysis. PXRD patterns were collected in reflection mode using a PANalytical Empyrean Series 2 diffractometer with an average wavelength of 1.541 Å Cu Kα (45 kV and 40 mA). Measurements were performed at room temperature between 3° and 100° in 2θ, using a step size of 0.026° step, and a counting time per step of 200 s.

Investigation of drug loaded MIL-100(Fe).

Gem-MP was loaded in microMOFs by adapting a previously described method 24 . Briefly, MOFs in ethanol were re-dispersed in water under gentle magnetic stirring overnight. Then Gem-MP loaded MOFs were prepared by soaking for one week microMOFs in Gem-MP aqueous solution (0.5 mg/mL) using a weight ratio 1:2 (Gem-MP: microMOFs) to reach maximal loadings of 30 wt%. Afterwards, Raman spectra of Gem-MP loaded MOFs were obtained by Raman microscopy.

Investigation of CD-P coated MIL-100(Fe). MIL-100(Fe) MOFs were coated with CD-P as previously described 33 . Briefly, MOFs were suspended in a CD-P aqueous solution under gentle stirring at room temperature for 24 hours. The initial MOFs/CD-P weight ratio was 1:0.

Supplementary Results

NanoMOFs degraded quickly in concentrated PBS (11.9 mM), with their crystallinity lost (Fig. S1) and 32 ± 3.0 wt % of trimesate released after 4 hours incubation (Table S1). When PBS was diluted to 1.19 mM, it took 6 hours to get totally degraded, releasing 34 ± 2.6 wt % of trimesate. NanoMOF can also be degraded even in extremely diluted PBS (0.12 mM), releasing 31 ± 2.8 wt % after two days incubation. Whereas, there was no degradation observed in water after 2 days incubation, with less than 2 wt % of their trimesate released and the crystallinity well preserved.

Fig. S2 Mössbauer spectra at 77K of degraded nanoMOF and microMOF

Energy dispersive X-ray analysis (EDX) was performed in order to obtain the Fe/P ratios of the intact MOFs, half degraded microMOFs, and totally degraded microMOFs. The results showed that there is significant reduce of Fe/P ratios from 1000 (intact MOFs) to 7.6 (half degraded microMOFs) and 0.9 (totally degraded microMOFs), confirming that P was coordinated inside the amorphous matrices of MOF structure during the degradation process.

General Objectives and Author Contributions

Because of their high pore volumes and surface areas, MIL-100 (Fe) was shown to load a series of drugs, such as antibiotics and anticancer drugs, which are able to efficiently penetrate within the porous MOF structures. However, to the best of our knowledge, there is no report on synergic drugs co-encapsulation in MOF particles yet. Combinatorial drug therapies emerge among the most promising strategies to treat complex pathologies such as cancer and severe infections. Herein, Amoxicillin (AMOX) and Potassium Clavulanate (CL), the typical synergic antibiotics, were chosen for drug co-encapsulation in MIL-100 (Fe).

As described before, MIL-100 (Fe) contains two types of cages: small cages (24 Å) with pentagonal (5.6 Å) window and large cages (29 Å) with hexagonal windows (8.6 Å). The dimension of AMOX (12×9×4 Å) allows it penetrate only within the large cages. CL, with a significant smaller dimension (8×5×4 Å) than AMOX, is expected to travel through both the large and small cages. Interestingly, AMOX has larger affinity than CL, making it occupy the large cages and CL in the small cages.

Briefly, this study included four parts:

1) AMOX or CL was encapsulated as single component by by a simple "green" methods, where the as-synthesized nanoMOFs were impregnated the in aqueous drug solutions. The encapsulated AMOX and CL loadings plateaued at 13 and 27 wt%, respectively.

2) Co-encapsulation of AMOX and CL was achieved by impregnating the as-synthesized nanoMOFs in aqueous drug combination solutions. High payloads for both drugs were obtained: 13 wt% for AMOX and 22 wt% for CL, which is quite similar to the individual drug loadings.

3) Molecular simulations showed that each drug located in a separate compartment within the interconnected 3D structure of nanoMOFs.

4) In vitro study on infected macrophages indicated that the drug loaded nanoMOFs were efficiently internalized in the cells, and surprisingly, could co-localize with the bacteria and act synergistically with the entrapped drugs to kill intracellular bacteria.

All the authors have contributed in this work. X. Li performed the experiments on the nanoMOFs synthesis, characterization and drug loading, and also contributed to the analysis of the data and writing of the main manuscript.

The treatment of complex pathologies such as cancer and severe viral or bacterial infections involves taking into consideration multiple molecular and genomic pathways. In this context, combinatorial drug therapies are considered among the most effective strategies [1][2][3][4] and a recent trend is to co-incorporate synergic drugs in nanoparticles (NPs) able to target them to the diseased sites. Indeed, nanotechnology revolutionizes drug delivery by achieving: i) drug targeting; ii) transcytosis of drugs across biological barriers; iii) delivery of drugs to intracellular targets and iv) visualization of sites of drug delivery (theranostics).

The treatment of intracellular infections is particularly challenging, requiring the drugs to bypass cellular membranes to reach their target, the pathogens located in intracellular niches.

Unfortunately, many drugs poorly penetrate inside cells, necessitating to increase the administered doses with might lead to side effects and drug resistance. Ideally, drugs could be loaded in NPs, which would act as "Trojan horses" to co-localize with the pathogens and release their drug cargo. Amoxicillin (AMOX) and potassium clavulanate (CL) are typical examples of drugs used in tandem to treat a variety of bacterial infections, mainly rhinosinusitis, ear infections, pharyngolaryngitis, tonsillitis, bronchitis and urinary tract infections 5 . However, these polar drugs are poorly permeable across the cell membranes and lack efficacy in intracellular infections. [6][7][8] There are still few examples of NPs with different compartments engineered for drug coincorporation. Anisotropic Janus NPs [9][10][11][12] , nanocapsules 13 and polymersomes 14,15 are composed of several macroscopic compartments, each one being adapted to accommodate a specific drug.

Nevertheless, the preparation methods of anisotropic NPs are relatively complex and require the use of organic solvents and surfactants. Another example is porous NPs in which drug combinations were physically adsorbed 16,17 . However, as a general trend, when drugs were coencapsulated, they impeded each other leading to dramatically reduced loadings. For example, the loading of gemcitabine monophosphate in NPs made of coordination polymers was drastically reduced from 57 ± 2 wt% to 12 ± 2 wt% when oxaliplatin was co-encapsulated 18 .

NanoMOFs based on porous iron (III) trimesate were used here to address the challenge of coincorporating drugs with high loadings. NanoMOFs have emerged as a new class of versatile, biodegradable and non-toxic drug nanocarriers [19][20][21] . Because of their high pore volumes and surface areas, nanoMOFs were shown to load unprecedented amounts (within the 20-150 wt % range) of a series of drugs able to efficiently penetrate within the porous MOF structures 19,20,22- 25 .

Here we show that AMOX and CL can be efficiently co-incorporated in nanoMOFs following a solvent-free "green" procedure. Each drug was located in a specific compartment inside the porous nanoMOF structure, following a distinct incorporation mechanism. Remarkably, molecular simulations predicted the location of each drug as well as the maximal loadings, in good agreement with the experimental results. Advantageously, a high number of cellinternalized nanoMOFs were found to co-localize with bacteria and to degrade within hours.

Contrary to free drugs, drug-loaded nanoMOFs efficiently reduced the intracellular bacterial loads, in vitro in a model of macrophages infected with Staphylococcus aureus (S. aureus).

Results and discussion

NanoMOFs synthesis and drug incorporation

NanoMOFs made of non-toxic mesoporous iron (III) trimesate MIL-100(Fe) (MIL standing for Material from Institute Lavoisier) are of particular interest for drug encapsulation. MIL-100(Fe) nanoMOFs are built up from iron (III) octahedra trimers and trimesate linkers (1,3,5-benzene tricarboxylate) that self-assemble to build a porous architecture delimiting two types of compartments: large (29 Å) and small (24 Å) mesoporous cages (Fig. 1). The two types of cages are accessible for drug adsorption inside the crystalline MOF structure through microporous pentagonal (~5 Å) and hexagonal windows (~9 Å) 26 . ± 100 m 2 .g -1 were successfully prepared by a solvent-free "green" hydrothermal method. They displayed a crystallized structure in agreement with previously reported data 22,27 and a facetted morphology (Fig. 2a). In a first step, AMOX and CL were encapsulated as single components by impregnating the assynthesized nanoMOFs in aqueous solutions of each drug. The molecular dimensions of AMOX (12×9×4 Å) (Fig. 1) are very similar to the ones of azidothimidine-triphosphate (AZT-TP) which was previously shown to exclusively access the large cages of MIL-100(Fe) through the hexagonal windows 26 . Given its dimensions, AMOX is expected to cross only the large hexagonal MOF windows to access the large cages. In contrast, CL, with significant smaller dimensions (854 Å) than AMOX (Fig. 1), is able to cross both the pentagonal and the hexagonal windows of the interconnected 3D structure allowing the occupancy of both small and large mesoporous cages.

In drug loading experiments, nanoMOFs suspensions were incubated with aqueous drug solutions with increasing concentrations to estimate the maximal loadings for each drug (Fig. 3a). At weight ratios nanoMOF: drug of 32:1, the encapsulation efficiencies (EE) were higher than 98%, showing that the drugs were efficiently soaked by the MOF "nanosponges" from their aqueous solutions (for EE calculations see methods I.3). This clearly shows the excellent affinity of nanoMOFs for the studied antibiotics and is in line with recent findings showing that MOFs can be used to remove trace antibiotics as pollutants in wastewater.

Maximal AMOX and CL loadings were 13 and 27 wt%, respectively. Even at these maximal loadings, the morphology and crystalline structure of the nanoMOFs were unaffected, as revealed by: i) identical powder X-ray diffraction patterns before and after encapsulation (Fig. S1) and ii) similar shape and facetted morphologies of the pristine and the drug-loaded nanoMOFs (Fig. 2b). The nanoMOFs loaded with maximal amounts of AMOX (13 wt%) had similar mean diameters (284 ± 18 nm) as the unloaded ones (273 ± 23 nm). However, an aggregation was observed with CL-loaded nanoMOFs at their maximal loading (27 wt%). In this case, the mean diameters reached 457±46 nm after CL incorporation. Possibly, a fraction of CL located in the nanoMOF top layers induced a bridging effect, leading to their aggregation.

The proof that the drugs were mainly located inside the nanoMOF cages and not at the external surface came from BET experiments. After encapsulation of maximal amounts of AMOX and CL, BET surfaces decreased from 1490 m 2 .g -1 to 994 and 643 m 2 .g -1 for AMOX and CL at maximal loadings, respectively. These findings are consistent with an efficient pore filling by the encapsulated molecules, without altering their tridimensional crystalline structure, as previously reported with other drugs 10,22,23 .

loaded without impeding each other. The combinational payload reached at maximum 35 wt%, including 22 wt% of CL and 13 wt% of AMOX.

As in the case of single drugs, co-incorporation of AMOX and CL did not alter the nanoMOF crystalline structure (identical X-ray diffraction patterns, Fig. S1) nor their morphology (Fig. 2b). Interestingly, after co-incorporation of both drugs at their maximal loadings (13 wt% and 22 wt% for AMOX and CL, respectively) there was less than 10% variation of the mean diameters (273 ± 23 nm and 298 ± 9 nm, before and after drug loading, respectively). The nanoMOFs loaded with both drugs were stable upon storage and in cell culture media.

As previously, GCMC simulations were performed to gain insights into the possible locations of co-incorporated AMOX and CL. A careful analysis of the GCMC runs evidenced that each drugs followed a distinct pore filling mechanism. The AMOX molecules were almost exclusively incorporated in the large cages with an uptake of 15.5 wt%, identical to the one simulated above in the case of AMOX adsorption as single component. In contrast, CL was able to accommodate both small and large cages. Interestingly, almost all the entire fraction of the encapsulated CL molecules occupied the small cages with less than 5% CL located in the large cages near the windows. The simulated CL uptake in these compartments was 20 wt%, in excellent agreement with the experimental results (22 wt%). In conclusion, GCMC simulations were a powerful tool to predict the competitive adsorption of AMOX and CL in MIL-100(Fe) nanoMOFs.

Furthermore, the density probability plot reported in Fig. 4 clearly showed that the drugs were characterized by totally different distributions inside the MOF. The two distinct preferential adsorption regions (AMOX, green and CL, red) suggest independent incorporation mechanisms. The preferential arrangements of AMOX and CL in the large and small cages, respectively, were determined by Monte Carlo simulations (see methods I.4). Figure 5a shows that in the large cage, AMOX adapted its conformation to establish relatively strong interactions with the cage walls, displaying several interacting pairs characterized by distances below 3.0 Å. More precisely, the carbonyl and the hydroxyl functions of AMOX interacted with the Fe metal sites and the carbonyl groups of the MIL-100(Fe) framework, respectively. As illustrated in Fig. 5b, CL showed similar interactions as AMOX with the small cage walls, although this molecule contains a lower number of active sites as compared to AMOX. The calculated interacting distances were significantly longer, suggesting a weaker host/drug interaction. These results were confirmed by simulated adsorption enthalpies, which were in the rage of -224 kJ/mol and -150 kJ/mol for AMOX and CL, respectively. In a nutshell, simulation studies clearly demonstrated that AMOX has a stronger affinity than CL for the MIL-100(Fe) framework. This supports the previous experimental findings, which showed that once incorporated in the large cages, AMOX couldn't be displaced by CL, a drug with lower affinity than AMOX for the nanoMOFs. Given the capacity of MIL-100(Fe) nanoMOFs to incorporate high amounts of both AMOX and CL (total loading 35 wt%), these nanocarriers were further studied in vitro on infected cells to test their antibacterial properties.

Uptake of nanoMOFs by macrophages and effects on intracellular S. aureus

S. aureus possesses several virulence mechanisms to accommodate a diversity of niches. On one side, S. aureus can be the production of toxins that favor cell lysis and tissue destruction in acute infections. On the other side, it can invade and survive in host cells such as epithelial cells, osteoblasts, neutrophils or macrophages and be responsible for chronic infections. Indeed, several main antibiotics such as β-lactams and aminoglycosides lack of efficacy to eradicate intracellular pathogens 26,28,29 , as they can poorly bypass cell membranes and/or are rapidly cleared off by efflux pumps (Fig. 6a). Contrary to free drugs, nanoMOFs are able to efficiently penetrate inside infected macrophages and to release their drug cargo near pathogens could be powerful alternatives to treat intracellular infections 26,30,31 . The ability of nanoMOFs loaded with AMOX and CL to address this important goal was tested here. Firstly, the cytotoxicity of nanoMOFs at various concentration was tested (data not shown). The nanoMOFs were found to be non-cytotoxic in J774 macrophages at concentrations up to 50 µg/mL. Indeed, after 24 h of contact with nanoMOFs, cell viability was higher than 90%. The internalization of nanoMOFs by macrophages was studies at concentration of 25 µg/mL.

Efficient internalization of nanoMOFs is crucial to eliminate intracellular pathogens. The presence of nanoMOFs was first confirmed by optical microscopy investigations (Fig. 2d). It was found that nanoMOFs were efficiently taken up by J774 cells, mainly by phagocytosis and in a lesser extent by a clathrin-dependent pathway (Fig. S2). In agreement with these findings, a large number of nanoMOFs were found in endosomal or/and phagolysosomal compartments of non-infected cells (Fig. 2e). After 6 h incubation, the nanoMOFs degraded inside these compartments, showing a blurry transparent structure (Fig. 2f) similar to the previous one reported in in vitro degradation studies in PBS 32 . Finally, we investigated the effect of nanoMOFs on intracellular S. aureus in in vitro cell infection models using J774 murine macrophages. Cells were first pre-incubated with nanoMOFs, loaded or not with drugs, during 2 h. Then, cells were washed and infected with S.

aureus for 2 h. At the end of the infection time, extracellular bacteria were eliminated and cells were incubated with gentamicin for 1 h or 6 h. This experimental protocol allowed us to focus on killing of intracellular staphylococci by internalized nanoMOFs without dealing with what happens in the extracellular environment. First, we evaluated the effect of empty nanoMOFs and AMOX-loaded nanoMOFs on penicillin-susceptible S. aureus ATCC 55585. As we observed on TEM images, several nanoMOFs were found to co-localize with intracellular S.

aureus in the phagosomes of infected cells, even after 1h post incubation (Fig. 6b). After 6 h incubation, intracellular nanoMOFs structure became blurred indicating their degradation as it was observed in non-infected cells (Fig. 2f, Fig. 6b). Quantification of intracellular staphylococci reported a decrease of 80% after 6h incubation with AMOX-loaded nanoMOFs.

As expected, free AMOX known to poorly penetrate inside the cells 33,34 had no effect on intracellular bacteria. Interestingly, a decrease of intracellular bacteria number was also reported with empty nanoMOFs (Fig. 6d). The bactericidal effect of this cell-internalized free nanoMOFs was reported proportional both to the amount of nanoMOFs initially in contact with the cells and the incubation time (Fig. S3). A significant bactericidal effect of empty nanoMOFs was observed when the amount of nanoMOFs initially in contact with the cells was higher than 12.5 μg/mL and after 6 h of incubation.

To confirm our results and test the co-encapsulation biologic activities, we evaluated the intracellular efficacy of nanoMOFs loaded with AMOX and CL on a clinical penicillin-resistant S. aureus. We still observed a decrease of the number of intracellular staphylococci with empty nanoMOFs, but this effect was not significantly enhanced by the incorporation of AMOX or of AMOX and CL (Fig. 6e). Experiments with longer incubation times (up to 24 h) gave similar results. The antibacterial effect of empty nanoMOFs on intracellular bacteria overwhelmed that of the incorporated drugs. Of note, nanoMOFs had no direct effect on S. aureus (MIC > 250 µg/mL). Further studies will be needed to decipher the bactericidal effect of nanoMOFs which could be related to reactive oxygen species (ROS) production 35,36 , without toxic effects of the J774 macrophages.

Conclusion

Here we showed for the first time that two drugs can be efficiently incorporated in MOFs, each one being preferentially adsorbed in the large or small mesoporous cages. The drugs followed individual incorporation mechanisms without impeding each other, resulting in total loadings as high as 35 wt%. Molecular modelling corroborated well the experimental findings and predicted the preferential location of each molecule. It was discovered that, nanoMOFs were prone to co-localize with intracellular pathogens and had an intrinsic bactericidal action, contributing, together with the incorporated drugs, to reduce the bacterial loads. These results pave the way towards the design of engineered NPs in which each component (drugs and nanocarrier) plays a role in fighting intracellular infections.

Methods

Methods, including statements of data availability and any associated accession codes and references, are available.

Materials and Methods

I.1 Materials

Chemical materials. Iron (III) chloride hexahydrate (98%, Alfa Aesar, Schiltigheim, France), 1,3,5-benzenetricarboxylic acid (BTC, 95%, Sigma-Aldrich, Saint-Quentin-Fallavier, France)

and absolute ethanol (99%, Carlo Erba, Val-de-Reuil, france) were used for the synthesis of nanoMOFs. AMOX and CL were purchased from Sigma-Aldrich. All the chemicals were used without further purification. Bacterial culture. The beta-lactam sensitive S. aureus (ATCC55585) stock strains were maintained in 20% glycerol at -80°C. Before experiments, the bacteria were transferred onto fresh Brain Heart Infusion (BHI)/ agar (Difco, Invitrogen, Cergy-Pontoise, France) and incubated at 37°C for 24 h. For each experiment, bacteria were subcultured in BHI (medium) at 37°C for 18 h. The day of the experiment, the bacteria were washed twice with sterile PBS, counted in a Salumbini chamber, and adjusted to 5×10 6 bacteria/mL in RPMI-1640 medium supplemented with 10% v/v FBS.

I.2 MIL100 (Fe) nanoMOFs synthesis and characterization

NanoMOFs synthesis. MIL100 (Fe) iron trimesate nanoMOFs were synthesized by adapting a previously described method 26 . Briefly, nanoMOFs were obtained by microwave-assisted hydrothermal synthesis from a mixture of iron chloride (8.97 mmol) and BTC (4.02 mmol) in 20 mL of deionized water. The mixture was heated 6 min at 130 o C under stirring. The power applied was 400 Watt (Mars-5, CEM, US) with a power maximum output 1,600 ± 240 Watts and frequency at full power 2,450 mHz. The resulting nanoMOFs were recovered by centrifuging 10 min at 10,000 ×g and washed with absolute ethanol 6 times to remove the residual non reacted organic acid. A last centrifugation at 5500 ×g was performed during 1 min in absolute ethanol to sediment the largest particles and recover the supernatants as a suspension of monodisperse nanoparticles. NanoMOFs were stored wet in ethanol until final use.

NanoMOFs

I.3 Drug incorporation

Drugs were loaded within MIL-100 (Fe) nanoMOFs simply by impregnation of well defined amounts of nanoMOFs and drug(s) solutions in water. Practically, 1 or 4 mg of nanoMOFs were centrifuged for 10 min at 10,000 g and re-suspended in 1 mL of aqueous drug solutions (concentrations from 0.125 to 1 mg/mL) or water as a control. After incubation for 12 h under gentle stirring, the nanoMOFs were recovered by centrifugation (10,000×g, 10 min) and the non-encapsulated drug in the supernatant was quantified by adapting a previously described HPLC method 37 . Specifically, HPLC analysis was performed on a Waters TM system using a

Waters TM 486 tunable UV absorbance detector. Eluant flowed at a rate of 0.5 mL/min through a C18 Silica 150 × 3.0 mm column (Interchim, Montluçon France) maintained at 30°C. The injection volume was 20 μL which ran for 8 min after each injection. The mobile phase consisted of 5.2 g/L of sodium dihydrogene phosphate monohydrate in 30% (v/v) methanol.

The pH was adjusted to 5 using dilute ortho-phosphoric acid solution. CL and AMOX were detected at 247 nm and retention times were 2.1 and 4.6 minutes, respectively.

The encapsulation efficiency (EE) was calculated as Equation (1):

100 ) ( ) ( (%)   mg rug Initial D mg ed Drug Encapsulat EE (1)
where "encapsulated Drug" represents the amount (mg) of drug incorporated in nanoMOFs and "Initial drug" represents the amount (mg) of drug initially added in the experiment.

The drug payload was calculated as Equation ( 2):

100 ) ( ) ( (%)   mg nanoMOFs mg ed Drug Encapsulat Payload ( 2
)
Drug release was studied in different media (water, cell culture media). After incubation at 37°C during different lapses of time, nanoMOFs were recovered by centrifugation (10 min, 10000×g) and supernatants were assessed by HPLC to determine the amounts of released drug(s).

I.4 Molecular simulations

Simulation of the accessible surface area and free volume.

The accessible surface area and free volume were calculated using a geometric computational approach. Two cases were considered with the accessibility of (i) both small and large spherical mesoporous cages and (ii) only the large cages, the accessibility of the small cages being prohibited by the presence of a dummy atom with a van der walls radius which fits with the diameter of the small cage.

The theoretical accessible surface area of nanoMOFs powder was estimated using the method previously reported by Düren et al 38 . This surface was calculated from the center of a nitrogen probe molecule rolling across the surface. The diameters of each atom constituting the nanoMOFs were taken from the Universal Force Field (UFF) 39 whereas the diameter of the nitrogen probe molecule was considered to be 3.60 Å. 40 The calculations were performed using the crystal structure of MIL-100(Fe) 41 with 2 iron centers per trimer unsaturated and the third one coordinated by an -OH group with a corresponding bond length of 0.97 Å. Two cases were considered with the accessibility of (i) both small and large spherical mesoporous cages and (ii) only the large cages, the accessibility of the small cages being prohibited by the presence of a dummy atom with a van der walls radius which fits with the diameter of the small cage. These predicted accessible surface areas were then compared with the BET experimental values obtained before and after encapsulation of AMOX and CL.

The free volume corresponding to the volume not occupied by the atoms of the MIL-100(Fe) nanoMOFs was also calculated using a similar method of trial insertions within the entire volume of the unit cell considering a probe size of 0 Å 42 . The experimental pore volume (0.66 cm 3 /g) was lower than the theoretical free volume (0.99 cm 3 /g) calculated using a perfect and infinite crystal. Indeed, small sized nanoMOF crystals are far from being perfect and the void space of the nanoMOFs is only partially available for the drugs. Therefore, a scaling factor of 1.5 was applied for the GCMC simulations, corresponding to the ratio (free volume/experimental pore volume).

Structural features were calculated using Materials Studio software. The predictions of the optimal encapsulation uptake for AMOX and CL as single component and binary encapsulation in the MIL-100(Fe) were performed using Grand Canonical Monte Carlo (GCMC) simulations at 303 K. The MIL-100(Fe)/drugs and drugs/drugs interactions were treated as a sum of a Lennard-Jones (LJ) and a coulombic contribution. The microscopic models selected for both the drugs and the MIL-100(Fe), the associated LJ parameters and partial charges are described in SI section I.4. Additional Monte Carlo simulations were conducted to reveal the preferential arrangements of both drugs in the pores of the MOFs. All the computational details are provided in SI section I.4.

Prediction of AMOX and CL loadings and energetic considerations.

The predictions of the optimal encapsulation uptake for AMOX/CL in the MIL-100(Fe) were performed using Grand Canonical Monte Carlo (GCMC) simulations at 303 K with the CADSS software (Complex Adsorption and Diffusion Simulation Suite) 43 . The crystal structure of MIL-100(Fe) was considered with one iron metal per trimer coordinated by an -OH group while the two other metal sites are coordinated by water molecules, to mimic the fact that encapsulation is achieved in aqueous media. The partial charges carried by each atom of the frameworks were extracted as a first approximation using the qEq charge equilibration approach 44 . This protocol has been revealed to be sufficiently accurate for screening the properties of a series of MOFs for carbon dioxide capture 45 . The partial charges of AMOX and CL were extracted by DFT calculations using the B3LYP functional associated with the 6/311++G (d,p) basis set 46 as implemented in GAMESS 47 . The Lennard-Jones (LJ) parameters for all atoms of the MIL-100(Fe) frameworks and the drug molecules were taken from UFF 4 and the general Amber Force Field (GAFF) 48 respectively. The MIL-100(Fe)/drugs and drugs/drugs were treated as a sum of a LJ and a coulombic contribution. The Lorentz-Berthelot mixing rule was employed for all cross LJ terms

General Discussion and Perspectives

Although people have already been using medicinal substances as far as there have been people, the history of pharmacy as an independent science dates back to the 19th century when chemical companies established their research labs for medical applications of their products. The last two centuries witnessed the outstanding progress of pharmaceutical science. In 1970s, microparticles (MPs) and NPs were applied as drug carriers, which are recognized as the first pioneering works for the further development of functionalized formulations. Recently, formulations for targeting 1 and personalized treatment 2 are attracting increasing attentions aiming to deliver the drug directly to the diseased cells and tissues of the body, and therefore enhance the drug's efficacies, decrease the treatment's side effects, and improve the patient's life quality.

The use of nanotechnologies for the treatment of diseases has led to new therapeutic strategies.

Many functionalized NPs developed in the lab allow the efficient targeting, as well as the controlled drug release in these specific biological targets. However, the journey of a nanomedicine from lab to shelf is quite costly and time consuming 3 . Statistically, the research and development of a new drug to the market takes around 12 years and costs around £1.15 billion, while tens of thousands of candidate drugs fail. One of the main reasons that limits drug commercialization is the fact that their preparation needs to employ large amounts of potentially toxic organic solvents and surfactants, which is often not acceptable, at least for parenteral administration. For instance, a variety of polymer NPs were prepared by the well-known solvent evaporation method using volatile organic solvents to solubilize the polymer. Traces of solvents are very difficult to remove even using very complicated and time consuming methods such as ultradialysis, ultrafiltration or ultracentrifugation.

Therefore, there is an urgent need to develop particle preparation procedures avoiding the use of organic solvents and surfactants. Herein, CD-based NPs were prepared in pure water just by mixing two neutral polymers which instantaneously associate together by a "lock and key" mechanism (Chapter 2). In this chapter, we show not only the concept that host-guest interactions between hydrophobic molecules (Bz moieties) and the apolar cavities of CDs can be exploited for the formation of stable NPs, but also that this preparation can be successfully scaled up by microfluidics, an automatic technique. Briefly, two polymers for NPs preparation were firstly synthesized: i) a poly-CD polymer and ii) Dex-Bz polymer where Bz moieties were grafted with dextran. Bz acts as both the hydrophobic moiety that interacted with CDs and also the active ingredient. By mixing the aqueous solutions of these two polymers with microfluidics, CD-NPs of around 100 nm were spontaneously formed with narrow size distribution. Although CD-NPs could also be prepared manually with pipette, it is very difficult to well control the procedure for NPs preparation, whereas the microfluidic chamber allows for exquisite control over the mixing profile of the solutions containing the NPs forming agents 4,5 .

Parameters, including ratio between the different compounds, flow rate as well as temperature can be easily and accurately controlled, which make possible to prepare NPs in a wellcontrolled, reproducible and high-throughput manner. Moreover, the microfluidic device could allow a continuous NP production at a rate of tens to hundreds of microliters per minute. Studies in the literature 6 describe the utility of microfluidic devices to scale up the NP production.

The other novelty in this study is the use of NTA to evaluate the NPs' stability in terms of size distribution and concentration upon extremely dilution. As the inclusion complexes were formed by host-guest interaction, instead of covalent bonds, an equilibrium exists all the time between the components in the system, as described in introduction (section 1.3). When the system is extremely diluted, the equilibrium will shift to the disassociation of the inclusion complexes, which would induce the disassembly of the CD-NPs. NTA allows to measure the size distribution and concentration at extremely diluted condition by individually following the CD-NPs in their Brownian motions. Remarkably, CD-NPs were very stable even when their concentration was as low as 100 ng/mL.

The solvent-free "green" procedure and the remarkable stability of CD-NPs make them promising as drug carrier for hydrophobic drugs which fits with the cavity of CDs. However, considering the payload of active ingredient, which is normally less than 5 wt%, their application for many drugs is limited, especially when large doses are needed for the diseases, as it is the case with antibiotics. But they are still good candidates for drugs with small dosages such as anticancer drugs.

To improve the drug payload, the next strategy that we explored is to use "cage" porous particles. CD-MOFs were the first particles tried in this study since it is built with the "cage" molecules (CDs). The use of CD-MOF materials for biomedical applications requires them to have very homogeneous sizes. Before their applications as drug carriers, the optimization of the synthesis and the evaluation of their crystalline structure were performed. (Chapter 3.1). In this subchapter, a fast solvent evaporation approach was developed to synthesize and tailor the CD-MOFs to a controlled size, with the synthesis time shortened from the reported 26 h 7 to only 6 h. The size of CD-MOFs were well controlled by adjusting the reactant concentrations and molar ratios, temperature and reaction time, without any significant changes in crystallinity and porosity. A modified hydrothermal protocol has been established which produces efficiently at 50 °C in 6 h micron (5-10 µm) and nanometer (500-700 nm) diameter CD-MOF particles of uniform size with smooth surfaces and powder X-ray diffraction patterns that are identical with those reported in the literature 7 .

CD-MOFs experienced degradation at high temperature (100 °C), high humidity (92.5%) and with polar solvents (methanol and DMF). More importantly, it was found that γ-CD was released and characteristic peaks in PXRD patterns disappeared when immersing the CD-MOFs in polar solvent whilst the morphology remained unchanged. After the stability study, the preliminary experiments on drug adsorption were performed with more than 20 drugs, the results shows that CAP has high affinity with CD-MOFs reaching payloads as high as 19.3 wt%, which corresponding to a molar ratio between CAP and γ-CD close to 1.6:1. These results confirm the high drug loading capacity of CD-MOFs.

LPZ was then used as drug of especial interest, as it is a proton-pump inhibitor used to reduce the production of acid in the stomach and recently identified as an antitubercular prodrug 8 . CD-MOFs of around 6 µm in diameter were used for LPZ encapsulation. LPZ-loaded CD-MOFs were successfully synthesized by a co-crystallization method, obtaining cubic crystals with the uniform size of around 6 µm. High drug payloads of 23.2 wt% were obtained, and calculations indicated that each CD in the CD-MOFs was able to host one LPZ molecule. Moreover, it was demonstrated that even after two years storage, the incorporated drug inside the CD-MOFs maintained its spectroscopic characteristics. Compared to other CD-based drug carriers, CD-MOF appears as one of the best systems in term of drug payload.

The CD-MOFs loaded or not with LPZ were fully characterized to gain a deep insight of the system. XRPD patterns of LPZ loaded CD-MOFs show that the peaks corresponding to the LPZ were not observed, indicating that LPZ was incorporated in CD-MOFs in amorphous state.

As a crystalline drug, incorporation of LPZ is very challenging since the drug might form crystals which are not well encapsulated and difficult to be separated from the drug-loaded particles, hampering the medical applications. However, we demonstrated that in CD-MOF crystals, all the LPZ molecules located in each cavity of CDs, which possibly prevented the LPZ crystallization. Molecular modelling gave deeper insight into the interactions between LPZ and CD-MOFs showing that LPZ molecules preferred to insert in the γ-CD cavities of the CD-MOFs instead of locating in the hydrophilic large pores. LPZ molecules were bended interacting strongly with the CD cavities mainly through their benzo imidazole and pyridine moieties. This result surprisingly indicated that the inclusion complexes formed between LPZ and CDs could self-assemble with coordinated K + ions into the cubic CD-MOF crystals.

Furthermore, we set up a novel characterization method based on Raman microscopy allowing to gain information on the structure and chemical composition of individual particles.

Thistechnique combines the advantages of Raman spectroscopy and optical microscopy.

Individual CD-MOF crystals could be analyzed in terms of morphology (particle size, shape)

and chemical composition, enabling to confirm the homogeneity of the CD-MOF population.

Morphologies of more than 2500 particles were analyzed, showing almost perfect cubes with diameters around 6 µm, before or after drug loading. Raman investigations confirmed the effective LPZ incorporation in CD-MOFs and suggested an interaction between γ-CD and LPZ, more specifically between the CDs and the benzo imidazole and pyridine moieties of LPZ, which agrees with the theoretical molecular modelling results. Of note, it was found that Raman spectra of different individual particles were the same, showing a remarkable homogeneity in terms of drug loading.

These results pave the way towards the use of CD-MOFs for drug delivery purposes, especially for pulmonary administration. Considering the size of CD-MOFs and the antitubercular effect of LPZ, the LPZ loaded CD-MOF crystals are quite promising as inhalation for tuberculosis, an infectious disease that occurs the lungs. Compared with other diseases caused by a single infectious agent, tuberculosis is the second biggest killer, globally. Pulmonary administration has recently gain more attention as it enables to deliver the drugs directly to lung both for local and systemic treatment 9 . For further study, the interactions between pulmonary surfactant and CD-MOFs would be interesting to investigate. Afterwards, in vitro evaluation of the drug loaded particles to kill tuberculosis could be performed in macrophage.

For other routes of administration except pulmonary inhalation, the drawback of CD-MOFs lies on the fact that they disassemble immediately in water, leading to burst release before the particles reach the target tissues or organs. In order to enlarge the scope of their application for more routes of administration, surface modification was designed to prepare CD-MOF/polymer composites, which are emerging as a new class of carriers for sustained drug delivery. In the literature, most of the MOFs and the polymers used so far in these composites are not pharmaceutically acceptable. Herein, biocompatible CD-MOFs were embedded inside polyacrylic acid (PAA) polymer matrices by a solid in oil-in-oil emulsifying solvent evaporation method. Since the size of the solid dispersed in the internal phase needs to be as small as possible, hence, nanometer-sized CD-MOFs (around 650 nm in diameter) were selected in order to prepare CD-MOFs/PAA composite microspheres. Ibuprofen (IBU) and LPZ, both insoluble in water and lacking in stability, were entrapped with high drug loading in nano-scaled CD-MOFs by co-crystallization without causing MOF crystal degradation during the loading process. Nano-scaled CD-MOF crystals dispersed well inside PAA matrices leading to their uniform distribution in the microspheres, which further allowed the obtention of composite microspheres with spherical shapes. On the contrary, microspheres prepared with drug-γ-CD complexes have non spherical shapes since the size of the drug-γ-CD complexes was larger and not uniform when compared with the drug-loaded CD-MOF nanocrystals, even after sieving. Particle aggregation occurred during microsphere preparation with drug-γ-CD complexes.

Burst release is considered to be one of the main drawbacks hampering the NP applications.

Achieving sustained drug release is important for drug formulations. In vitro cumulative drug release profiles showed sustained drug release from CD-MOF/PAA microspheres. IBU was released slowly over 48 h, whereas LPZ enabled a sustained release also during 48 h with an almost linear profile. However, microspheres containing IBU-γ-CD and LPZ-γ-CD complexes both showed a very fast burst and uncontrolled releasei.e., 70% in 30 min for IBU and 80% in 2 h LPZ. With no doubt, this observation reflected the good dispersion of drug-loaded CD-MOF nanocrystals in the microspheres and the homogeneous distribution of the drug molecules within these crystals, resulting from the efficient co-crystallization loading process.

Cytotoxicity is one of the most crucial parameters to evaluate the drug carrier. The cellular toxicity study showed that CD-MOFs were not toxic (cell viability > 90%) even at concentrations as high as 300 µg•mL -1 . At concentrations up to 1.4 mg•mL -1 , the cellular viability was still high (~ 80%). Interestingly, the toxicity of IBU loaded CD-MOF/PAA composite microspheres was reduced dramatically. The cell viability was around 90% even for samples at high concentrations of 17.5 mg•mL -1 , corresponding to a CD-MOF nanocrystal concentration of 1.4 mg•mL -1 and IBU concentration of 0.2 mg•mL -1 . These results showed that composite of CD-MOF nanocrystals in PAA microspheres could to be used as safe carriers for sustained drug delivery.

Taking into account the bio-adhesive properties of PAA and its excellent biocompatibility, the composite microspheres have potential applications for biomedical applications and, in particular, for oral drug delivery. However, a limitation of the CD-MOF/PAA microspheres is that their drug loading is relatively low (about 1%, w/w) and so this system would be better adapted for the delivery of drugs requiring lower doses, such as anti-tumor drugs.

While CD-MOFs seem very promising for pulmonary administration, their applications for other routes of administration are limited by their physical frailness and their high solubility in aqueous media before surface modification or the low drug payloads after modification.In this context, our alternative strategy was to use stable porous particles to obtain both high payloads and high stability in water. However, although stability is very important, drug carriers need to be degraded inside the body for excretion. MIL-100 (Fe) MOFs are stable in water and could be degraded in a series of simulated physiological fluids. They are also considered among the most efficient materials for biomedical applications because of their unprecedented drug loading capability and biocompatibility 10 . In this view, MIL-100 (Fe) was chosen as the third drug delivery system.

Firstly, the degradation mechanism of MIL-100 (Fe) was investigated. As reported, MIL-100 (Fe) interacted efficiently with phosphate groups. Phosphated drugs, such as Gem-MP, were soaked from their aqueous solutions with high payloads (> 25wt%) and efficiencies (> 98%), and phosphated coatings, such as CD-P, were attached to the outer surfaces by incubation just in water. Unfortunately, MIL-100 (Fe) readily degraded in phosphate buffers leading sometimes to uncontrolled drug release. It is therefore necessary to have a deep understanding of the MIL-100 (Fe) degradation mechanism in complex media containing phosphates to be able to control it.

To better understand their degradation mechanism in PBS and the complex interactions taking place in the MOF/drug/coating systems, we developed a method based on Raman microscopy to track the morphology and chemical composition of individual particles. It was shown that MIL-100 (Fe) MOFs degraded in PBS releasing their constitutive trimesate linkers and leading to the formation of amorphous material, whereas the particle sizes remained unchanged. It was extremely interesting and fascinating to observe that despite the fact that more than 80% of the connective linkers were released, the particles perfectly maintained their initial morphology.

Remarkably, Raman spectra of different regions on the same particle could be recorded, evidencing the formation of an erosion front during MOF degradation. This front clearly separated an intact non eroded crystalline core from an amorphous shell made essentially of an inorganic network. To gain a deeper understanding of the kinetics of the degradation process of single particles, automated imaging was used track the degradation process by capturing MOFs' images every half an hour during 8 days degradation.

Of interest, the method provided a direct measurement of the size, shape, color, surface area, and circularity of each particle. A fast degradation phase was observed in the first 6 hours with a rapid color change and increase of surface areas, which were too subtle to be observed by the naked eye. A second phase occurred in the following 8 days with the formation of an eroded zone with a clear color change (progressive phase). It can be speculated that in the first hours after incubation, a fast coordination reaction occurred between phosphates in the incubation medium and accessible iron sites at the MOF surface. This phase was followed by a slower diffusion of phosphates in the eroded region. The advancement of the degradation front was presumably limited by the diffusion of the reacting species (phosphates) from the media into the MOFs and by the diffusion of released trimesate out of the matrix.

To investigate the chemical composition of the eroded zone, [START_REF] Sun | Recent progress in the synthesis of metal-organic frameworks[END_REF] Fe Mössbauer spectrometry was applied to MIL-100 (Fe) particles before and after degradation. Before degradation, the spectrum was well described by means of 3 quadrupolar components, in agreement with the crystallographic MIL-100 (Fe) structure, while after degradation, quadrupolar doublets with broadened lines were observed. The best physical description requires a discrete distribution of quadrupolar doublets suggesting a structure close to that of a typical amorphous structure.

Although the hyperfine structures of the nano-and microMOF samples after degradation was modified, the refined values of the isomer shift indicate unambiguously the presence of only Fe 3+ species whatever the sample (nano-and microMOFs) and their degradation state. It could be concluded that no Fe reduction occurred during degradation. Taking into account the presence of phosphates in the media surrounding the MOF, it is plausible that a highly disordered Fe-P based skeleton phase was formed.

In contrast, neither drug entrapment nor particle coating did affected the integrity of the tridimensional MOF network. As descripted, MOFs degraded in PBS with kinetics dependent upon the amount of phosphates in the suspension media. When Gem-MP was loaded in MOFs, the equivalent amount of phosphates was up to 100 times lower than that in diluted PBS (1.19 mM), which could explain the reason why MOFs did not degrade during drug entrapment.

When MOF particles were coated with CD-P, which could not penetrate inside the tridimensional MOF network, no degradation was induced. The good stability of the particles after Gem-MP loading and CD-P coating offer promising possibilities to load drugs of interest and graft ligands for targeting purposes. These findings are of interest in the development of stable drug-loaded iron trimesate particles.

Combinatorial drug therapies are considered among the most effective strategies for the treatment of complex pathologies such as cancer and severe viral or bacterial infections involving multiple molecular and genomic pathways. However, co-administration of synergic drugs is challenging because of the differences in pharmacokinetics and biodistribution of each individual drug. Nanotechnology revolutionizes drug delivery in many aspects, but there are still few examples of NPs for drug co-encapsulation, such as Janus NPs, nanocapsules and polymersomes which are composed of several macroscopic compartments, each one being adapted to accommodate a specific drug. However, the preparation of anisotropic NPs are relatively complex and require the use of organic solvents and surfactants.

Taking advantage of the two types of "cages" present in MIL-100 (Fe) nanoMOFs, synergic antibiotics AMOX and CL were co-entrapped inside the porous "cage" nanoMOFs. AMOX and CL are typical drugs used in tandem to treat a variety of bacterial infections, mainly rhinosinusitis, ear infections, pharyngolaryngitis, tonsillitis, bronchitis and urinary tract infections. By using a simple "green" strategy consisting of incubating the nanoMOFs with aqueous solutions of both drugs, AMOX and CL were easily co-encapsulated. As a general trend, when drugs are co-encapsulated, one drug in the combination reduces the entrapment of another. Interestingly, the AMOX and CL were co-incorporated inside nanoMOFs without impeding each other. The combinational payload reached at maximum 35 wt%, including 22 wt% of CL and 13 wt% of AMOX. Molecular modelling gave a clear picture of location for the two drugs, showing that AMOX occupied mainly the large cages and CL the small cages.

Considering their excellent drug encapsulation properties, nanoMOFs were further studied in vitro on S. aureus infected cells. NanoMOFs were able to penetrate inside infected macrophages, whereas free drugs could poorly bypass cell membranes and/or are rapidly cleared off mainly because of efflux pumps. NanoMOFs, loaded or not with drugs, were nontoxic tested by MTT assays. Surprisingly, empty nanoMOFs co-localized with bacteria in macrophages and had bactericidal effect, proportional to the amount of nanoMOFs initially in contact with the cells. AMOX-loaded nanoMOFs were successful to show synergic effect with empty nanoMOFs, whereas no bactericidal effect was found with free AMOX, which is reasonable due to its poor intracellular penetration. When using a resistant S. aureus strain, the bactericidal effect due to internalized nanoMOFs was so strong (85% bacteria were destroyed), that it was comparable to that of (co)encapsulated drugs. (Need to be adapted according to the ongoing study). In conclusion, intracellular nanoMOFs synergistically contribute with encapsulated drugs to eradicate intracellular bacteria. They are efficiently taken up inside macrophages essentially by phagocytosis and advantageously co-localize with bacteria, to eventually deliver their drug cargo. These results pave the way towards the design of engineered nanocarriers in which each component synergistically plays a role in fighting the disease.

For future study, it would be very interesting to deeply investigate the mechanism of antimicrobial effect of empty MIL-100 (Fe) nanoMOFs. The growing resistance of pathogens has become a public health problem. New effective antimicrobial materials are necessary and urgent for resistant pathogen treatment. It was discovered that nanoMOFs, entirely different to antibiotics, played a role as antibacterial materials providing promising novel solution to the problem of drug resistance. There are already some reports on antimicrobial MOFs based on Cu, Co, Zn, and Ag, but no reports on MOF based on Fe yet, to the best of our knowledge.

Understanding their bactericidal mechanisms could help not only to explain the obtained results, but would be also beneficial to design new antimicrobial materials.

The current antibacterial mechanisms of reported MOFs involves 11 : 1) release of metal ions, such as Ag + 12 ; 2) reactive oxygen species (ROS) formed by metal oxides, especially ZnO, NiO, CoO, CuO, and Cu2O 13 ; 3) antibacterial autophagy 14

Fig. 7

 7 Fig.7 Overview of some representive examples of flexible and rigid of MOFs. The breathing MOFs: MIL-53-It (It=low temperature), MIL-53-Ibu (Ibu= Ibuprofen), and MIL-53-ht (ht=high temperature) are from reference 87; The swelling MOFs are from reference 45. The series of MIL-88 were prepared with iron trimer and different ligand: fumaric acid (MIL-88A), terephthalic acid (MIL-88B), 2,6-naphthalenedicarboxylic acid (MIL-88C) and 4,4′biphenyldicarboxylic acid (MIL-88D).

  . For example, rigid mesoporous MIL-100 nanoMOFs, built up from trimers of iron(III) octahedra and trimesate anions, show two types of mesoporous interconnected cages accessible through microporous windows: i) large cages (~ 29 Å) delimited by hexagonal (~ 8.6 Å) and pentagonal (~ 4.8 × 5.8 Å) windows and ii) small cages (~ 25 Å) accessible exclusively by pentagonal windows 71 . MIL-100(Fe) MOFs possessed the same architecture but with larger pore openings and adsorption surfaces.

Fig. 8

 8 Fig.8 Drug association to hybrid MOF nanoparticles.

  Despite the paramount importance of in vivo experiments, there are only scarce examples showing in vivo data, certainly because of nanoMOFs' novelty in the biomedicine domain. Simon-Yarza et al. reported the first in vivo results with MIL-100 nanoMOFs loaded with the antineoplastic drug busulfan. Drug-loaded nanoMOFs were administered to rats and compared with the commercial Busilvex®. Large differences in serum concentration of both busulfan and the constitutive ligand (trimesate) revealed the great impact of drug encapsulation both on the drug and on nanoparticle pharmacokinetics during the first 24 h of administration 149 . Tumor targeting MOFs (TTMOFs) based on MIL-101 were successfully developed via surface modification with polymers comporting pH responsive benzoic imine bonds and redox responsive disulfide bonds. In vivo experiments showed that doxorubicin-loaded TTMOFs could effectively inhibit tumor growth with minimal side effects 145 .

(

  Fig 1B). The mean diameter of the nanoMOFs measured by Differential Light Scattering (DLS, number values) is 216 ± 21 nm with a polydispersity index (PdI) of 0.131.

Fig. 1 .

 1 Fig.1. Schematic representation of the "green" hydrothermal synthesis of MOFs (A) and their morphological characterization, including TEM images of nanoMOFs (B) and optical images of microMOFs (E); circularity for nanoMOFs (C) and microMOFs (F) calculated from more than 8000 particles for nanoMOFs and 2000 particles for microMOFs, respectively, as well as size distributions for nanoMOFs (D) and microMOFs (G). Iron polyhedra, oxygen, carbon atoms are in orange, red and black, respectively.

Fig. 2

 2 Fig.2Morphology of MIL-100(Fe) nano-and microMOFs and size variations after incubation in water or PBS. TEM images of nanoMOFs before (A) and after (B) degradation in PBS (1.19 mM) for 6 hours. Size variation (C) of nanoMOFs (100 µg/mL) in MilliQ water (orange) and PBS (0.12 mM, blue) for 2 days. Images of microMOFs observed by Raman-microscopy before (D) and after (E) degradation in PBS (11.9 mM) for 8 days; size distribution (F) of microMOFs before (green) and after (red) degradation.

Figure 3

 3 Figure3presents a typical image of a microMOF particle before (left) and after 8 days degradation in PBS (right). The morphology was dramatically changed with the appearance of a sharp transition delimiting a red-colored core (region I) from a grey shell (region II). Of note, Raman microscopy enabled monitoring Raman spectra in both I and II regions. The spectrum of region I consisted of peaks very similar to those characteristic of non-degraded MOFs, with the exception of peak D which was broadened at its base. On the contrary, in region II, all peaks disappeared and only a large peak D could be detected. The disappearance of band A indicates an amorphous phase formation. Peaks B shifted to lower wavelengths, become much broader, and less resolved, suggesting a change in coordination environment. The disappearance of the well resolved peaks C could be interpreted as a proof of trimesate release. The band D which was broadened but did not disappear could be ascribed to symmetric and asymmetric stretching vibrations of Fe-O-P bonds, strongly indicating the formation of phosphate complexes by coordination with phosphate ions32 . This is also in agreement with the disappearance of peak

Fig. 4

 4 Fig.4 Kinetic study of the degradation of a microMOFs (size around 50 µm in length) in PBS (11.9 mM) at room temperature. Particle surface area and mean intensity were recorded each 30 min up to 8 days. A fast degradation phase was observed in the first 6 hours with a rapid increase in mean intensity. A second phase occurred in the following 8 days with the formation of an eroded zone with a clear color change (progressive phase).

  This phase was followed by a slower diffusion of phosphates in the eroded region. The advancement of the degradation front was presumably limited by the diffusion of the reacting species (phosphates) from the media into the MOFs and by the diffusion of released trimesate out of the matrix.Mössbauer spectrometry[START_REF] Sun | Recent progress in the synthesis of metal-organic frameworks[END_REF] Fe Mössbauer spectrometry was applied to MIL-100(Fe) particles to probe several chemical features of interest, such as the Fe oxidation state, the type of Fe species, the possible presence of Fe-containing phases different from of MIL-100(Fe) and any Fe oxide resulting from degradation. Mössbauer spectra were recorded at 300 and 77K with different velocities and only typical Mössbauer ones are illustrated in Fig.5. The hyperfine structures result essentially from the presence of quadrupolar components. The refined values of the isomer shift indicate unambiguously the presence of only high supersaturation (HS) Fe 3+ species whatever the sample (nano-and microMOFs) and their degradation state. It could be concluded that no Fe reduction phenomenon occurred in PBS.

Fig. 5 .

 5 Fig. 5. Mössbauer spectra of nano-and microMOF obtained at 77 K and at 300K. Spectra of degraded nanoMOF (Dnano) and microMOF (Dmicro) obtained at 300K.

Fig. 6

 6 Fig.6 Effect of loading of Gem-MP in MIL-100(Fe) on the Raman spectra of the particles.

Fig. 7

 7 Fig.7 Raman spectra of individual MIL-100(Fe) MOF particles, coated or not with P-CD. Schematic representation of the coating procedure (A) and experimental results (B) for MOF particles before and after CD-P coating.

  characterization. The crystallinity and purity of MIL-100(Fe) particles were assessed by PXRD patterns which were collected in a conventional high resolution (-2) D5000 Bruker diffractometer (Cu K, K2) from 1º to 20º (2) using a step size of 0.02º and 4° per step in continuous mode. The morphologies of microMOFs were investigated by Raman microscopy (Malvern ® Morphologi G3-ID). Briefly, optical images of more than 2000 particles were captured and statistical analysis of size and circularity were carried out.Dynamic light scattering (DLS) and Nanoparticle tracking analysis (NTA) were performed by a Zetasizer (Malvern ® Nano-ZS90) and Nanosight (Malvern ® LM10), to characterize the size distribution of nanoMOFs. Zeta potential of nanoMOFs was characterized by a Zetasizer (Malvern ® Nano-ZS90). Nanosight analysis is a combination of a conventional optical microscope and a laser to illuminate the nanoparticles introduced into the visualizing unit with a 1.0 mL syringe system. Individual particles could be visualized as point-scatter moving under Brownian motion. Measurements were performed for 5 times over a period of 60 s at 25°C. The particle size and concentration were analyzed using the NTA software. Transmission electron microscopy (TEM) images for nanoMOFs were collected using a JEOL ® JEM-2200FS microscope. The circularity of nanoMOFs was obtained from TEM images and analyzed by Origin 9.0 software.

Fig. S3

 S3 Fig. S3 FTIR spectra of microMOFs during their degradation in PBS

Figure 1 .

 1 Figure 1. Schematic representation of drug incorporation in nanoMOFs. AMOX, bulkier than CL, can only cross the large hexagonal windows, whereas CL can penetrate through both pentagonal and hexagonal windows. Therefore, AMOX and CL are prone to locate in large or in large and small cages, respectively

Figure 2 .

 2 Figure 2. Microscopic investigation of nanoMOFs before and after their internalisation in J774 macrophages. TEM images of nanoMOFs: empty (a), loaded with AMOX and CL (b) and after 4 h degradation in PBS (c). J774 cells before (d, insert) and after (d, black arrows) nanoMOF uptake (optical microscopy). TEM images of cellular compartments containing nanoMOFs after 1 h (e) and 6 h (f) incubation with cells.

Figure 3 .

 3 Figure 3. (Co)-incorporation of drugs as a function of the NanoMOF: drug ratio used in the preparation procedure (green: AMOX; red: CL). (a) Incorporation of individual drugs; (b, c) Co-incorporation of AMOX and CL

Figure 4 .

 4 Figure 4. Density probability plots for AMOX (green) and CL (red) in the cages of MIL-100(Fe). Data were extracted from the GCMC simulations conducted for the co-encapsulation of the two drugs (uptakes of 15.5 wt% and 20 wt% for AMOX and CL, respectively).

Figure 5 .

 5 Figure 5. Preferential geometries of AMOX (a) and CL (b) in the large and the small mesoporous cages of MIL-100(Fe), respectively. Calculations were issued from Monte Carlo simulations performed with one drug molecule per cage. For clarity reasons, the hydrogen atoms of the framework are not represented. The characteristic drug/MOF interacting distances are reported in Å.

Figure 6 .

 6 Figure 6. The internalization of drug loaded nanoMOFs in infected macrophages and their antibacterial effects. Schematic representation (a); TEM images of J774 macrophages showing nanoMOFs co-localized with S. aureus after 1 h incubation (b, red arrows) and partially degraded after 6 h (c, red arrows); Antibacterial effects of free AMOX, empty and drug-loaded nanoMOFs on S. aureus (ATCC55585) (d) and of AMOX and CL on a clinical

Cell culture.

  Murine macrophages J774 were grown in RPMI-1640 medium (BioWhittaker, Walkerville, MD, USA) supplemented with 10% v/v decomplemented fetal bovine serum (FBS) (Cambrex BioSciences, Verviers, Belgium) at 37°C in humidified air containing 5% CO2. Lactate dehydrogenase leakage (LDH, Sigma-Aldrich) were used for toxicity evaluation of nanoMOFs. NanoMOFs internalization assay. Cytochalasin D 5µg/mL, Latrunculine A 400 nM, Wortmannin 300 nM, Chlorpromazine 20 µM, Ikarugamycin 4 µM, methyl-β-cyclodextrin (Mβ-CD 1 mM, Genistein 100 µM, Fillipin III 1 µM (Sigma-Aldrich) were used as pharmacological inhibitors to investigate the nanoMOF mechanisms of cell internalisation. Tiron (Sigma-Aldrich) was used to inhibit the ROS effect. Prussian blue staining (Sigma-Aldrich) was used as Iron staining kit to visualise the intracellular nanoMOFs.

  characterization. The cristallinity and purity of MIL-100 (Fe) nanoMOFs were assessed by X ray powder diffraction (XRPD) patterns which were collected in a conventional high resolution (θ-2θ) D5000 Bruker diffractometer (λCu Kα, Kα2) from 1º to 20º (2θ) using a step size of 0.02º and 4° per step in continuous mode. The size distribution and Zeta potential of nanoMOFs were characterized by dynamic light scattering (DLS; Malvern ® Nano-ZS) nanosight (NTA, LM10 Nanosight), and using a Zetasizer (Malvern ® ZSP, UK) respectively. Nanosight analyses combines a conventional optical microscope with a laser to illuminate nanoparticles in Brownian motion. Particles could be visualised one by one as moving pointscatters. Each measurement was repeated 5 times, at room temperature. The particle size distribution was determined using the NTA software. Transmission electron microscopy (TEM) images were collected in a Darwin 208 Philips microscope (60-80-100 KV; Camera AMT). The nanoparticles porous surface was measured by nitrogen sorption experiments at -77 K on a ASAP 2020 (Micromeritics) after sample's outgassing at 150°C for 18 h under secondary vacuum.

  Cytochalasin D, Latrunculine A Wortmannin, Chlorpromazine, Ikarugamycin, methyl-βcyclodextrin (Mβ-CD), Genistein, and Fillipin III were used as pharmacological inhibitors to investigate the mechanisms of internalisation of nanoMOFs inside J774 cells. The inhibitors were incubated with the cells for 1 h, previously to adding the nanoMOF suspensions. Results are gathered in Fig.S2.The internalization of nanoMOFs was reduced by 35% in the presence of Cytochalasin D 1 and Latrunculin A 2 , which block actin polymerization and hence disrupt microfilament-mediated internalization, manifesting the role of phagocytosis. Wortmannin 1 , Ikarugamycin 3 , and Chlorpromazine4 , used as inhibitors of clathrin-dependent endocytosis, didn't significantly lessen the uptake of nanoMOFs, indicating that clathrin-pathway hardly played a part in the internalization. Genistein 4 and Filipin III 5 , which block caveolin-mediated endocytosis, didn't significantly affect the internalisation. Mβ-CD, used in previous studies4 to induce cholesterol depletion of cellular membrane, had no influence on the nanoMOF internalisation, neither. Furthermore, it was found that the nanoMOF incubation temperature plays a major role. The uptake of nanoMOFs was reduced by 60% when the temperature was decreased to 4 o C, suggesting an energy-dependent internalization mechanism. As a conclusion, nanoMOFs were mainly internalised in J774 macrophages by phagocytosis.

Fig. S2 .

 S2 Fig. S2. Effect of different pharmaceutical inhibitors on the internalisation of nanoMOFs in J774 macrophages. The cells were pre-incubated for 1 h at 37°C with different inhibitors

5 .

 5 . It would be very interesting to understand the main antibacterial mechanisms for MIL-100 (Fe) nanoMOFs.MOFs are an emerging class of materials suitable for infection treatment by encapsulation of antibiotics and their intrinsic antimicrobial properties. The two types of MOFs used in this study were able to efficiently load large amount of drugs for infection treatments. LPZ loaded CD-MOFs are very promising particles as pulmonary inhalation for tuberculosis treatment, whereas AMOX and CL loaded nanoMOFs could be used for many infections using different routes of administration.sustained drug release over a prolonged period of time (over 48 h). No burst release of IBU (and LPZ) was observed from the CD-MOF/PAA composite microspheres, suggesting the homogeneous distribution of CD-MOF nanocrystals inside PAA matrices as well as strong drug carrier interactions inside the CD-MOF. Sustained release was achieved for IBU (and LPZ)-CD-MOF/PAA composite microspheres (ca. 50% in 24 h). The cellular toxicity study shows that IBU loaded MOF/PAA composite microspheres are not toxic (cell viability ~ 90%) even at very high concentrations of 17.5 mg•mL -1 . In conclusion, MOF/PAA composite microspheres constitute an efficient and pharmaceutically acceptable MOF-based carrier for sustained drug release. Biodegradable iron trimesate MOFs, MIL-100 (Fe), degraded in PBS losing their crystallinity and constitutive trimesate linkers, whereas, they kept their morphology intact. The degradation mechanism was revealed by Raman microscopy via recording spectra in different regions on the same particle, showing that intact cores were separated from the eroded regions by clearly visible erosion fronts. Mössbauer spectroscopy supported the hypothesis that Iron phosphates were formed in the eroded regions. In contrast, the integrity of the tridimensional MOF network was not affected by drug entrapment nor by particle coating.6. Co-encapsulation of two synergic antibiotics (AMOX and CL) in MIL-100 (Fe) nanoMOFs was achieved following a simple and "green" procedure consisting of incubating the nanoMOFs with aqueous solutions of both drugs. Molecular modelling showed that each drug preferentially located in a separate compartment (large or small mesoporous cage) of nanoMOFs. Surprisingly, nanoMOFs were prone to co-localize with bacteria once internalized in infected macrophages. NanoMOFs acted synergistically with the entrapped drugs to kill intracellular S. aureus, in vitro. These results pave the way towards the design of engineered nanocarriers in which each component synergistically plays a role in fighting the disease.These studies unravel the potential of "cage" nanoparticles for efficient drug entrapment and release. They open new routes of investigation especially in the field of the treatment of severe intracellular infections. Moreover, it was shown that "cage" particles are not just simple "carriers" but could play a role in killing bacteria. particules, parfaitement stables dans l'eau, se dégradent dans des milieux contenant des phosphates (PBS) en perdant rapidement leur caractère cristallin et leurs ligands constitutifs. De façon étonnante, nous avons constaté que malgré leur dégradation, ces MOFs conservent leur taille intacte. Une analyse approfondie basée sur la microscopie de Raman a permis d'obtenir des informations pertinentes sur la morphologie et la composition chimique de particules individuelles. Ainsi, il a été montré qu'un front d'érosion délimitait nettement un coeur intact et une coquillé inorganique érodée. Cependant, ni l'encapsulation ni la modification de surface des MOFs n'altérait leur intégrité. Enfin, nous avons étudié la co-encapsulation de deux molécules actives utilisées en combinaison (Amoxicilline et Clavulanate de potassium) dans les nanoMOFs stables à base de MIL-100 (Fe). Les antibiotiques ont été incorporés par imprégnation et chaque molécule s'est localisée préférentiellement dans un compartiment (large ou petite cage) corroborant parfaitement les simulations par modélisation moléculaire. De plus, il a été découvert, de manière surprenante, qu'un grand nombre de nanoMOFs se localisait au voisinage des bactéries (Staphylococcus aureus) dans des cellules infectées. En se dégradant dans ces cellules, les nanoMOFs contenant les antibiotiques ont réduit de manière importante la charge bactérienne intracellulaire. Ces études révèlent le potentiel des particules de type "cage" pour une incorporation efficace de molécules actives et leur libération contrôlée et ouvrent de nombreuses possibilités d'application. Espace Technologique / Immeuble Discovery Route de l'Orme aux Merisiers RD 128 / 91190 Saint-Aubin, France Titre : Particules de type "cage" pour des applications biomédicales Mots clés : particule "cage"; MOF ; médicament; dégradation; antibiotique Résumé: Les systèmes à délivrance de médicaments sont des technologies conçues pour administrer des molécules actives de façon optimisée afin d'améliorer leurs effets thérapeutiques tout en minimisant les effets secondaires. En effet, ces systèmes permettent une libération au niveau d'une cible thérapeutique. Les particules de type «cage» ont récemment attiré une attention particulière en raison de leur capacité accrue à (co)incorporer et à protéger des molécules actives vis-à-vis de dégradations in vivo. Les cyclodextrines (CDs) sont des exemples type de molécules "cage", possédant une cavité hydrophobe et une surface extérieure hydrophile. Nous avons élaboré tout d'abord des assembages supramoléculaires à base de CDs d'environ 100 nm par une méthode douce consistant à mélanger deux solutions aqueuses de polymères neutres: 1) polymère de β-CD et 2) dextrane greffé avec la benzophénone, molécule invitée formant des complexes d'inclusion avec les CDs. La procédure de préparation «verte» en une seule étape rend la formulation attractive, malgré sa relativement faible capacité d'encapsulation (5%pds). Afin d'améliorer cete charge, nous avons élaboré des particules hybrides organiques-inorganiques (MOFs) à base de CDs. Avantageusement, les CD-MOF comportent non seulement des cavités de CD, mais aussi de larges pores engendrés lors l'auto-assemblage de CDs. Le lansoprazole a été incorporé avec sucès (23%pds) dans les CD-MOFs et nous avons montré que chaque CDs était capable d'accueillir une molécule de principe actif. Cependant, l'inconvénient majeur des CD-MOFs est leur faible stabilité en milieu aqueux, limitant leur domaine d'aplication. Une modification de surface est apparue donc nécessaire pour améliorer leur stabilité. Notre statégie a été d'incorporer les CD-MOFs dans des matrices d'acide polyacrylique (PAA). Des microsphères composites d'environ 650 nm ont été élaborées avec succès et ont permis une bone stabilité et une libération prolongée sur plus de 48 h. Avantageusement, ces particules composites n'étaient pas toxiques in vitro même à des concentrations élevées. Ces microsphères apparaissent comme des systèmes efficaces pour assurer une bonne stabilité dans de milieux biologiques et une libération prolongée, mais leur synthèse est laborieuse et leur taille ne peut pas être réduite en dessus de 50nm. Ainsi, nous nous sommes orientés vers l'étude comparative de MOFs plus stables dans l'eau, à base de trimesate de fer. Les MIL-100 (Fe) (Material of Institute Lavoisier) figurent parmi les premiers MOF étudiés en tant que nanomédicaments (nanoMOFs). Ces particules, pafaitement stables dans l'eau, se dégradent dans des milieux contenant des phosphates (PBS) en perdant rapidement leur caractère cristallin et leurs ligands constitutifs. De façon étonnante, nous avons constaté que malgré leur dégradation, ces MOFs conservent leur taille intacte. Une analyse approfondie basée sur la microscopie de Raman a permis d'obtenir des informations pertinentes sur la morphologie et la composition chimique de particules individuelles. Ainsi, il a été montré qu'un front d'érosion délimitait nettement un coeur intact et une coquillé inorganique érodée. Cependant, ni l'encapsulation ni la modification de surface des MOFs n'altérait leur intégrité. Enfin, nous avons étudié la co-encapsulation de deux molécules actives utilisées en combinaison (amoxicilline et clavulanate de potassium) dans les nanoMOFs stables à base de MIL-100 (Fe). Les antibiotiques ont été incorporées par imprégnation et chaque molécule s'est localisée preferentiellement dans un compartiment (large ou petite cage) corroborant parfaitement les simulations par modélisation moléculaire. De plus, il a été découvert, de manière surprenante, qu'un grand nombre de nanoMOFs se localisait au voisinage des bactéries (S.aureus) dans des cellules infectées. En se dégradant dans ces cellules, les nanoMOFs contenant les antibiotiques ont réduit de manière importante la charge bactérienne intracellulaire. Ces études révèlent le potentiel des particules de type «cage» pour une incorporation efficace de molécules actives et leur libération contrôlée et ouvrent de nombreuses possibilités d'application.

Table 1 .

 1 . Drug encapsulation in MOFs by impregnation

	MOF	Metal Ion Organic Linker	Drug	Drug Loading (%, w/w)
	CD-MOF (Cs) 75	Cs	β-CD	5-FU	151
	CD-MOF (Cs) 75	Cs	β-CD	MTX	122
	CD-MOF (K) 31	K	γ-CD	fenbufen
	CD-MOF (K) 31	K	γ-CD	captopril
	CD-MOF (K) 31	K	γ-CD	flurbiprofen
	CD-MOF (K) 33	K	γ-CD	sucralose
	CD-MOF (K) 28	K	γ-CD	ibuprofen
	CD-MOF (Na) 76	Na	α-CD	5-FU	9
	CD-MOF (Na) 76	Na	α-CD	MTX
	CD-MOF (Na) 76	Na	α-CD	silybummarianum
	CD-MOF (Na) 76	Na	α-CD	ferulic acid
	CD-MOF (Na) 76	Na	α-CD	quercetin
	Co-BBDA 77	Co	H4BBDA	5-FU
	CNZTDH 78	Zn	TATAT	5-FU
	CPO-27-Ni 79	Ni	DHTPA	NO
	Cu-BTC 80	Cu	BTC	5-FU
	Gd-pDBI 81	Gd	pDBI	Doxorubicin
	HKUST-1 82	Cu	BTC	ibuprofen
	HKUST-1 82	Cu	BTC	anethole

  , S. et al. Porous Iron-Carboxylate Metal-Organic Framework : A Novel Bioplatform with Sustained Antibacterial Efficacy and Nontoxicity. 53, 19248-19257 (2017). Facile fabrication of magnetic metal-organic framework nanocomposites for potential targeted drug delivery. J. Mater. Chem. 21, 3843-3848 (2011). 91. Gao, X. et al. Controllable Synthesis of a Smart Multifunctional Nanoscale Metal -Organic Framework for Magnetic Resonance / Optical Imaging and Targeted Drug Delivery. Relaxometry studies of a highly stable nanoscale metal-organic framework made of Cu(II), Gd(III), and the macrocyclic DOTP. J. Am. Chem. Soc. 135, 17711-17714 (2013). 152. Yang, W., Feng, J. & Zhang, H. Facile and rapid fabrication of nanostructured lanthanide coordination polymers as selective luminescent probes in aqueous solution. J. Mater.

	Chem. 22, 6819-6823 (2012).
	153. Ray Chowdhuri, A., Bhattacharya, D. & Sahu, S. K. Magnetic nanoscale metal organic
	frameworks for potential targeted anticancer drug delivery, imaging and as an MRI
	contrast agent. Dalton Trans. 45, 2963-73 (2016).
	154. Dekrafft, K. E. et al. Iodinated nanoscale coordination polymers as potential contrast
	agents for computed tomography. Angew. Chemie -Int. Ed. 48, 9901-9904 (2009).

87.

Horcajada

, P. et al. Flexible porous metal-organic frameworks for a controlled drug delivery. J. Am. Chem. Soc. 130, 6774-6780 (2008). 88. Gu, Z.-Y., Chen, Y.-J., Jiang, J.-Q. & Yan, X.-P. Metal-organic frameworks for efficient enrichment of peptides with simultaneous exclusion of proteins from complex biological samples. Chem. Commun. (Camb). 47, 4787-4789 (2011). 89. Chalati, T. et al. Porous metal organic framework nanoparticles to address the challenges related to busulfan encapsulation. Nanomedicine 6, 1683-1695 (2011). 90. Ke, F. et al. ACS Appl. Mater. Interfaces, 9, 3455-3462 (2017). 92. Gaudin, C. et al. A quantitative structure activity relationship approach to probe the influence of the functionalization on the drug encapsulation of porous metal-organic frameworks. Microporous Mesoporous Mater. 157, 124-130 (2012). 93. Di Nunzio, M. R., Agostoni, V., Cohen, B., Gref, R. & Douhal, A. A 'ship in a bottle' strategy to load a hydrophilic anticancer drug in porous metal organic framework nanoparticles: Efficient encapsulation, matrix stabilization, and photodelivery. J. Med. Chem. 57, 411-420 (2014). 94. Anand, R. et al. Host-guest interactions in Fe(III)-trimesate MOF nanoparticles loaded with doxorubicin. J. Phys. Chem. B 118, 8532-8539 (2014). 95. Rodriguez-Ruiz, V. et al. Efficient 'green' encapsulation of a highly hydrophilic anticancer drug in metal-organic framework nanoparticles. J. Drug Target. 23, 759-67 (2015). 96. Singco, B. et al. Approaches to drug delivery: Confinement of aspirin in MIL-100(Fe) and aspirin in the de novo synthesis of metal-organic frameworks. Microporous Mesoporous Mater. 223, 254-260 (2016). 97. Horcajada, P. et al. Metal-organic frameworks as efficient materials for drug delivery. Angew. Chemie -Int. Ed. 45, 5974-5978 (2006). 98. Cunha, D. et al. Rationale of drug encapsulation and release from biocompatible porous metal-organic frameworks. Chem. Mater. 25, 2767-2776 (2013). 99. Rojas, S., Carmona, F. J., Maldonado, C. R., Barea, E. & Navarro, J. A. R. RAPTA-C incorporation and controlled delivery from MIL-100(Fe) nanoparticles. New J. Chem. 40,.5690-5694 (2016). 100. Taylor-Pashow, K. M. L., Della Rocca, J., Xie, Z., Tran, S. & Lin, W. Postsynthetic modifications of iron-carboxylate nanoscale metal-organic frameworks for imaging and drug delivery. J. Am. Chem. Soc. 131, 14261-14263 (2009). 101. Hu, Y., Song, C., Liao, J., Huang, Z. & Li, G. Water stable metal-organic framework packed microcolumn for online sorptive extraction and direct analysis of naproxen and its metabolite from urine sample. J. Chromatogr. A 1294, 17-24 (2013). 151. Carné-Sánchez, A. et al.

  5, and the final MOFs concentration was 4 mg/mL. The coated MOFs were recovered by centrifugation (5000 g for 5 min) and were washed with deionized water in order to remove the excess CD-P which was not associated to the MOFs surface. Finally, Raman spectra and images of CD-P coated MOFs were recorded by Raman microscopy. incubated in 50 mL of PBS (11.9 mM) at 37 °C with stirring speed at 100 rpm. The samples were collected by centrifugation after incubation for 1 h, 2 h, 4 h, 6 h, 12 h, 24 h, 3 d, 5 d, and 7 d. The samples were dried at 100°C overnight. IR spectra were recorded in KBr disk on a Nicolet 205 FTIR.

Table S1 .

 S1 Trimesate release of nanoMOF in PBS (11.9 mM, 1.19 mM, and 0.12 mM) and in

		-12		-6	0	V (mm/s)	6		12
		1,00
		0,95
		0,90				1.002
	Relative Transmission	0,75 0,80 0,85 0,90 0,95 1,00	Degraded microMOFs Degraded		-12 0.996 0.998 1.000 1.000 1.002	-6	0	V (mm/s)	6	12
			nanoMOFs		0.998
						0.996
		0,85
						-12	-6	0		6	12
									V (mm/s)
	water during incubation at 37°C. -12	-6	0			6		12
					V (mm/s)
	PBS concentration (mM)	Incubation time (h)				Trimesate release (wt%)
	11.9				4					32 ± 3.0
	1.19				6					34 ± 2.6
	0.12				48					31 ± 2.8
	0 (water)			48					≤ 2 ± 0.2

Table S2 .

 S2 Fe/P ratios of the intact MOFs, half degraded microMOFs, and totally degraded microMOFs.

	Type of MOFs	Fe (%)	P	Fe/P
	intact	92.55 ± 1.43	0.09 ± 0.41	~ 1000
	Half-degraded	74.94 ± 3.10	9.89 ± 3.64	~ 7.6
	Totally degraded	42.60 ± 11.98	44.57 ± 13.26	~ 0.9

Tong, M, Brown, O.S., Stone, P.R., Cree, L.M., & Chamley, L.W. Flow speed alters the apparent size and concentration of particles measured using NanoSight nanoparticle tracking analysis. Placenta 38,29-32 (2016).

Acknowledgments

We acknowledge support from the European Marie Curie network CycloN Hit (N° 608407) and from the French National Research Agency (ANR) ANR-14-CE08-0017. This work was supported by ANR as part of the "Investissements d'Avenir" program (Labex NanoSaclay, reference: ANR-10-LABX-0035). X. Li acknowledges support from China scholarship council (N° 201408330166). Prof. J. Zhang is grateful for the support from the National Natural Science Foundation of China (N°81430087). We thank Katie Buxton for help with the synthesis of microMOFs. We are grateful to Drs. Sergio Marco and Sylvain Trepout (Curie Institute, Orsay, France) for help with TEM and statistical studies. We acknowledge Dr. Farid Nouar for his help with EDX experiment.

Acknowledgments

This research was funded by European Union through Cyclon Hit project (People-2013-ITN), Grant Agreement 608407. We acknowledge support from the French ANR projects AntiTBnano (ANR-14-CE08-0017) and Virmil (ANR-2010-RMNP-004-01). We thank Dr. H. Willaime, Dr. P. Horcajada and Dr. C. Serre for helpful discussions. X. Li acknowledges support from China scholarship council (CSC, n° 201408330166). G.M. thanks Institut Universitaire de France for its support.

honor to witness how the laboratory was set up in Orsay, Paris-Sud Université. to thank Prof. Jiwen Zhang in Shanghai Institute of Materia Medica

Abbreviations

General Objectives and Author Contributions

As previously described, CD-MOFs have gained increased popularity as drug carrier since it is environment-friendly biocompatible and even "edible" MOFs. It is widely recognized that size distribution and stability are crucial parameters for drug delivery system.

In the originally reported protocols for their synthesis, the size of CD-MOF crystals was around 200-400 µm. It was then discovered that addition of cetyltrimethyl ammonium bromide (CTAB) in the preparation enabled the formation of smaller crystals (1-10 µm). However, the synthesized crystals had a large polydispersity. Moreover, the syntheses were carried out at room temperature over long time periods of time (> 24 h).

Herein, we optimized the synthesis procedure by heating at 50 °C within 6 h with pre-addition of the reaction solvent to obtain uniform cubic γ-CD-MOF crystals. The factors that affect the size of CD-MOFs were systematically investigated, including the reactant concentrations, temperatures, time, γ-CD ratios to KOH and surfactant concentrations. Subsequently, the stability of CD-MOFs were studied under conditions of high temperature (100 °C), high humidity (92.5%) and polar solvents (e.g., MeOH and DMF). Finally, drug absorption of CD-MOF was evaluated with different kinds of drugs. It is discovered that most of the drug molecules containing carboxyl groups showed relatively high adsorption (> 5%), while low adsorption (< 5%) was found for drugs with nitrogen-containing heterocyclic rings. This study pave the way towards the use of CD-MOFs for drug delivery purposes.

This study has been performed in collaboration with Dr. J. Zhang in Shanghai Institute of Materia Medica in China. X. Li contributed to perform the experiments on CD-MOFs synthesis.

Additional Information

Supplementary information accompanies this paper at..

Competing financial interests:

The authors declare no competing financial interests.

Supplementary Information

New insights into the degradation mechanism of metal-organic frameworks drug carriers X. Li 1 , L. Lachmanski 2 , S. Safi 2 , S. Sene 3 , C. Serre 3 , J.M. Greneche 4 , J. Zhang 5 , R. Gref

Supplementary Methods

Trimesate release from nanoMOFs in PBS. NanoMOFs were incubated in PBS (11.9 mM, 1.19 mM and 0.12 mM) at 37°C. The samples were centifugated and the amount of trimesate in the supernatant was assessed by HPLC. Analysis of trimesate was performed with a mobile phase containing 90% buffer (5.75 g/L of NH4H2PO4): 10% Acetonitrile (5 mM TBAP). The injection volume was 5 µl and the detection wavelength was set at 220 nm. As controls, MOFs were incubated in water instead of PBS under the same conditions and trimesate release was monitored.

PXRD analysis.

The crystallinity and purity of MIL-100 (Fe) nanoMOFs before and after degradation were assessed by PXRD. PXRD patterns were collected using a high resolution (-2) D5000 Bruker diffractometer (Cu K, K2) from 1º to 90º (2) using a step size of 0.02º and 4° per step in continuous mode. Four samples were used for the analysis: i) intact nanoMOFs; ii) totally degraded nanoMOFs after incubation in PBS (11.9 mM) for 4 h; iii) halfdegraded microMOFs after incubation in PBS (11.9 mM) for 3 days, and iv) totally degraded microMOFs after incubation in PBS (11.9 mM) for 1 month.

Fourier transform infrared spectroscopy (FTIR) analysis. 100 mg of microMOFs were The Mössbauer spectra at 77K of degraded nanoMOF and microMOF are illustrated in Fig. S2.

Experiments were performed at 2 and 12 mm/s. The low velocity scale spectra are similar to those obtained at 300K and their corresponding hyperfine structures confirm previous conclusions. A deep analysis of the 12 mm/s spectra at 77K allows a small magnetic component (estimated at about 2% in at Fe) to be observed, as displayed on the insets of Fig. S2, where relative transmission scale has been strongly extended. It corresponds clearly to hematite in the case of the degraded microMOFs while it could be attributed to poorly crystallized hematite (including traces of ultra-fine grains of goethite).

Subchapter 4.2 Two drugs in two different mesoporous cages inside the same nanoparticle: design and applications

In preparation

Abstract

Combinatorial drug therapies emerge among the most promising strategies to treat complex pathologies such as cancer and severe infections. Metal-organic frameworks nanoparticles (nanoMOFs) were used here to address the challenging aspects related to the co-incorporation of a drug combination. Amoxicillin (AMOX) and potassium clavulanate (CL), a typical example of drugs used in tandem, were efficiently co-incorporated with payloads up to 36 wt%.

Each drug adopted a different penetration pathway to eventually locate in a separate compartment within the porous interconnected frameworks. Molecular simulations predicted drug loadings and locations and corroborated well the experimental findings. Furthermore, nanoMOFs were efficiently internalized in macrophages infected with S. aureus, were found in high amounts nearby the pathogen and contributed together with the entrapped drugs to eradicate it. These results pave the way towards the design of engineered nanocarriers in which both the loaded drugs and their carrier play a role in fighting intracellular infections.

Molecular simulations of drug incorporation

Molecular simulations were performed to gain further understanding on the possible location of AMOX and CL and their interactions with the host MOF matrices. Grand Canonical Monte Carlo (GCMC) simulations were conducted first to predict the optimal uptake for both drugs as single components in MIL-100(Fe), taking into account the NP available porous volume (for details see methods I.4).

The simulated AMOX uptake in the nanoMOFs of 15.5 wt% was in good agreement with the experimental data (13 wt%). This is in good agreement with the experimental N2 adsorption data which showed that the BET surface area evolved from 1490 m 2 .g -1 (empty nanoMOF) to 994 m 2 .g -1 (nanoMOF loaded with AMOX at their maximal capacity). These data support the assumption that AMOX was exclusively incorporated in the large cages of MIL-100(Fe) since the volume ratio between large cages and small cages is 1:2.

Contrary to AMOX (12×9×4 Å), CL, with significant smaller dimensions (854 Å) (Fig. 1), is expected to penetrate through the entire MOF 3D structure, allowing the occupancy of both small and large mesoporous cages. Indeed, this assumption was confirmed experimentally by a drastic reduction of the BET surface after CL incorporation (643 m 2 .g -1 ), as compared to AMOX-loaded nanoMOFs (994 m 2 .g -1 ) and empty ones (1490 m 2 .g -1 ). Moreover, the simulated CL uptake of 30.6 wt% was in good agreement with the experimental loading of 27 wt%.

Altogether, these data unambiguously demonstrate that when adsorbed as a single component, CL accommodates in both types of MIL-100(Fe) cages.

Co-incorporation of AMOX and CL

Furthermore, AMOX and CL were co-incorporated in the nanoMOFs by the same "green" strategy used for single drugs and consisting in incubating the nanoMOFs with aqueous solutions of both drugs. Extensive studies were carried on, by varying the molar ratio AMOX:

CL in an attempt to gain insights on the competition between the two drugs for the nanoMOF cages. Fig. 3b shows that when AMOX was loaded at 13 wt%, i.e. the maximal nanoMOF capacity previously determined for this drug, it was still possible to load additional 22 wt% CL in the MOFs. More precisely, Fig. 3b shows that whatever the AMOX content in the nanoMOFs (from 2 to 13 wt%), the amount of CL which could additionally be loaded was practically constant (20 ± 2 wt%). This overrules the possibility that AMOX impedes CL incorporation in the nanoMOFs. Moreover, the AMOX loadings were unaffected by CL loading (13 ± 0.9%, 13 ± 0.8% for AMOX with or without CL). In conclusion, AMOX and CL could be simultaneously

Additional information

Supplementary information is available. Correspondence and requests for materials should be addressed to R.G.

Competing financial interests

There is no competing financial interest.

Dedication

This paper is dedicated to the memory of Dr. Christian GREF. and a cut-off radius of 14 Å was used. The long-range electrostatic interactions were handled by the Ewald summation technique. The GCMC simulations were performed for a series of fugacity for the drug up to 5000 kPa in order to first estimate the saturation capacity for each single drug. The simulations consisted of 10 6 steps to ensure the equilibration, followed by 2 × 10 7 steps to calculate the ensemble averages. Additional GCMC calculations were performed to probe the co-encapsulation of the two drugs as well as their sequential uptakes. The predicted loadings were then converted into weight/weight encapsulation percentages for direct comparison with experimental DL values. The adsorption enthalpy for each drug was further calculated using the revised Widom's test particle insertion method. 49 Further, additional Monte Carlo simulations were performed with a crystal structure of MIL-100(Fe) containing one iron metal per trimer coordinated by an -OH group while the two others are unsaturated to probe the preferential sitting sites of 1 molecule of AMOX and CL acid in the large and small cages respectively.

I.5 NanoMOFs toxicity assays

Cytotoxic effects of nanoMOFs were investigated using LDH assay. Following exposure to various concentrations of nanoMOFs as previously described, cells were washed and lysed. The activity of LDH was determined using a commercially available kit from Sigma. LDH reduces nicotinamide adenine dinucleotide (NAD), a coenzyme found in all living cells, to NADH, which is specifically detected by colorimetric (450 nm) assay. The absorbance was recorded using a microplate spectrophotometer reader system.

I.6 Internalization of nanoMOFs

Quantification of NanoMOFs internalized in the cells. J774 cells were seeded in 24-wellplates (3×10 5 cells/wheel) and then incubated with nanoMOFs at a final concentration of 100 µg/mL for 4 h in RPMI 1640 supplemented with 10% of FBS at 37°C, 5% CO2. They were washed thrice with cell culture medium, dried at 60°C for 2 h. Subsequently, 300 µL of hydrochloric acid (5M) was added and then incubated for 2 h at 60°C. Afterwards, 100 µL of each well were withdrawn and mixed with 100 µL of 4% potassium ferrocyanide in a 96-wellplate and incubated at room temperature in the dark for 30 min. The absorbance was read at 690 nm to quantify the amounts of internalized nanoMOFs.

The absorbance was read at 690 nm to quatify the amounts of internalized nanoMOFs.

NanoMOFs within cells were visualized by optical microscopy and TEM. Different pharmacological inhibitors (Cytochalasin D, Latrunculine, Wortmannin, Chlorpromazine, Ikarugamycin, methyl-β-cyclodextrin) were used to investigate the nanoMOF cell internalisation mechanism (phagocytotic/endocytotic pathways). Practically, the cells were preincubated 1 h with the above mentioned inhibitors before further incubation with nanoMOFs.

Intracellular visualisation of nanoMOFs. NanoMOFs within cells were visualised by optical microscopy and TEM. Prussian blue staining (Sigma Iron staining kit) was applied for optical microscopy. After different incubation times with or without nanoMOFs, J774 cells were washed with complete medium and two times with PBS. Cells were fixed with 4% paraformaldehyde, washed and incubated for 10 minutes with 2% potassium ferrocyanide in 0.6 mM hydrochloric acid. Cells were washed again, counterstained with pararosaniline hydrochloride (0.02%) and evaluated for iron staining using light microscopy.

I.7 Intracellular antimicrobial activity of nanoMOFs.

Intracellular antimicrobial activity of nanoMOFs. The antimicrobial activity of nanoMOFs was investigated against S. aureus ATCC55585 and against a clinical penicillin-resistant S. aureus.

It was carried out in a quantitative assay based on dilution method, using the Mueller-Hinton medium and the Minimum Inhibitory Concentration (MIC) was determined. Briefly, 2.5×10 5 J774 cells were seeded in 24 well plates and incubated overnight (37°C, 5% CO2). The cells were washed with cell culture medium (RPMI 1640 containing 10% v/v decomplemented FBS) and then incubated for 2 h with or without different amounts of nanoMOFs, loaded or not with drugs. The cells were washed twice with complete cell culture media, to eliminate extracellular nanoMOFs and drugs. Then, they were infected with 500 µl of S. aureus suspension (ATCC55585) at 5×10 6 bacteria/mL (multiplicity of infection, MOI=10) and incubated for 2h.

After washing the cells, gentamicin was added to kill extracellular bacteria. The ROS effect was inhibited at this moment with Tiron 10 mM. After 1, 6 and 24 h of incubation, cells were washed again with cell culture media and lysed by treatment with ice-cold water during 1 h.

The lysates were plated at serial dilutions in sterile phosphate buffered saline (PBS, pH=7.4) on agar plates. The plates were incubated at 37°C overnight and the number of formed colonies (CFU) was evaluated 50 .

Supplementary Information

Two drugs in two different mesoporous cages inside the same nanoparticle: design and applications N. Semiramoth

I.1 X-ray powder diffraction (XRPD) characterization

XRPD experiments were performed to investigate the effect of drug loading and nanoMOF incubation in various media on their crystalline structures. or not (orange) with drugs, were incubated in the cell culture media (RPMI, 2 h, 37 °C).

As shown in Fig. S1, the XRPD patterns of the drug loaded MOFs were very similar to the ones of empty nanoMOFs, suggesting that the incorporation of AMOX and CL didn't affect the crystallinity of the nanoMOFs. However, the incubation of nanoMOFs, containing or not drugs, in cell culture medium (RPMI) for 2 h lead to a diminution of the intensity of the peaks in the region 3°-4°, showing nanoMOF degradation.

I.2 Effect of different pharmaceutical inhibitors on the internalisation of nanoMOFs in J774 cells

The cell viabilities were higher than 90% when the nanoMOFs concentration was lower than 50 μg/mL. A concentration of 12.5 μg/mL was chosen for the cell internalization studies. G: methyl-β-cyclodextrin; H: Genistein; I: Fillipin III) before further incubation with nanoMOFs. A: the cells were incubated with the nanoMOFs at 4°C instead of 37°C. All data are normalized as compared to the uptake of nanoMOFs at 37°C in the absence of any inhibitor.

I.3 Bactericidal effect of empty nanoMOFs

Interestingly, cell internalized empty nanoMOFs exhibited a bactericidal effect which was proportional both to the amount of nanoMOFs initially in contact with the cells and the incubation time (Fig. S4). A significant bactericidal effect of empty nanoMOFs was observed when the amount of nanoMOFs initially in contact with the cells was higher than 12.5 μg/mL and after 6 h of incubation.

Fig. S3. Effect of nanoMOFs concentration on their bactericidal effect. (light blue: 1 h after infection; dark blue: 6 h after infection).

There is a significant difference when the concentration of nanoMOFs is higher than 12.5 μg/mL (P< 0.05, *).

Supplementary References General Conclusion

In this study, three different types of "cage" particles were prepared in view of possible biomedical application.

1. CD-NPs of around 100 nm were prepared by a "green" and simple preparation procedure consisting in mixing poly-CD aqueous solution and Dex-Bz aqueous solution using microfluidics or just pipettes. The host-guest interactions between β-CD in poly-CD polymer and Bz in Dex-Bz contributed to the spontaneous formation of stable CD-NPs.

CD-NPs were instantaneously produced by mixing 2 aqueous solutions of neutral polymers. Despite their non-covalent nature, the CD-NPs were extraordinarily stable in terms of concentration and size distribution, even on extreme dilution (concentrations as low as 100 ng/mL).

2. In order to improve the drug loading in CD-based drug delivery systems, uniform cubic CD-MOFs were successfully synthesized. An optimized vapor diffusion method was developed at elevated temperature (50 °C) within 6 h, which is much shorter than the reported time (>24 h). Both microcrystals (~ 6 µm) and nanocrystals (~ 650 nm) were obtained by adjusting the amount of the pre-added methanol to the aqueous solution of γ-CD and KOH. Title : "Cage" nano and micro-particles for biomedical applications Keywords : "cage" particles; drug carrier; degradation; antibiotic Abstract : Drug delivery systems are engineered technologies to administer pharmaceutical ingredients to improve their therapeutic effects, aiming at minimizing their side effects by means of targeted delivery and/or controlled release. "Cage" particles recently drew special attention since they could act as "drug containers" which potentially load large amount of drugs, improve their stability and offer the possibilities to co-encapsulate synergetic drugs. Cyclodextrins (CDs) are typical "cage" molecules with a hydrophobic cavity and a hydrophilic outer surface. Taking advantage of the host-guest interactions between β-CD and benzophenone (Bz), CD based nanoparticles (CD-NPs) were the first formulation investigated. CD-NPs of around 100 nm were instantaneously produced by mixing two aqueous solutions of neutral polymers : 1) poly-CD containing β-CDs, and 2) Bz grafted Dex (Dex-Bz). The "green" and facile preparation procedure makes it attractive formulation, whereas its limitation lies on the low drug payloads (~ 5 wt%). In order to improve the drug loading capacity of CDs, porous CD based metal orgainc frameworks (CD-MOFs) were synthesiezed, which contain not only CD cavities, but also large pores built up by CDs self-assembly. Lansoprazole (LPZ) was incorporated in CD-MOF microcrystals (~ 6 µm) reaching payloads as high as 23.2 ± 2.1% (wt). Remarkably, each CD cavity was able to host a drug molecule, offering new opportunities for the use of CD-MOFs for drug delivery purposes. However, these particles disassembled in aqueous media, which limits their application for oral and intravenous administration. Surface modification is therefore necessary to improve their stability in water. The drug loaded CD-MOF nanocrystals (~ 650 nm) were successfully embedded in polyacrylic acid (PAA) polymer matrices. The composite microspheres exhibited spherical shapes and sustained drug release over a prolonged period of time (over 48 h). Drug loaded MOF/PAA composite microspheres were not toxic in vitro (cell viability ~ 90%) even at very high concentrations up to 17.5 mg/mL. MOF/PAA composite microspheres constitute an efficient and pharmaceutically acceptable MOF-based carrier for sustained drug release. However, the process of surface modification was complicated and lead to larger particles and reduced drug payloads. Water-stable MOFs are a novel type of hybrid particles, showing a high potential as drug carriers. Iron trimesate MOFs, namely, MIL-100 (Fe) (MIL stands for Material of Institute Lavoisier) was among the first nano-scaled MOFs used for drug delivery. These particles were stable in water but degraded in phosphate buffer saline (PBS) losing their crystallinity and constitutive trimesate linkers. However, it was discovered that they kept their morphology intact. A thorough analysis based on Raman microscopy was carried on to gain insights on both the morphology and chemical composition of individual particles. It was evidenced the formation of a sharp erosion front during particle degradation. Noteworthy, the MOFs did not degrade during drug loading nor surface modification. Co-encapsulation of two synergic antibiotics (amoxicillin and potassium clavulanate) in MIL-100 (Fe) nanoMOFs was achieved following a "green" procedure by soaking nanoMOFs in aqueous solutions of both drugs. Molecular modelling showed that each drug preferentially located in a separate nanoMOF compartment. Surprisingly, nanoMOFs were prone to colocalize with bacteria once internalized in infected macrophages. NanoMOFs acted synergistically with the entrapped drugs to kill intracellular S. aureus, in vitro. These results pave the way towards the design of engineered nanocarriers in which each component synergistically plays a role in fighting the disease. These studies unravel the potential of "cage" particles for efficient drug entrapment and controlled release and open numerous possibilities for applications.