Pr Frederic Dominique Hapiot

Pr Pierre Hourdet

Schaaf

Imre Daria Kaczmarek

Amar, Beatrice, Céline, Jakob Julia Akanksha

Kathrin

Joanna Raymond

Céline Gaelle

Cédric Charbonneau

Clémence Lorthioir

Le Coeur

Thank you for the continuous guiding and support for more than 3 years of my PhD project and especially during the last couple of months of writing process

List of abbreviations

Introduction

The phenomenon of hierarchical self-assembly is traditionally defined as the non-covalent organization of molecules and macromolecules which takes places over distinct increasing length scales, whereby the non-covalent interactions at each subsequent length scale gradually decrease in strength. 1 Hierarchical self-assembly was one of the main nature's tools to ensure the life emergence ca. 4 billion years ago. In the last 20-30 years a fast development of the supramolecular chemistry area has occurred; the latter exploits weak non-covalent interactions, e.g. van der Waals forces and hydrogen bonds, to link molecules together. Given this, along with establishing deeper understanding of the processes taking place in nature, a lot of effort has been made to construct multi-level smart hierarchical systems, using synthetic and semisynthetic soft matter building blocks.

In the first part of this literature survey we will describe the general principles and scope of artificially constructed hierarchical materials. It will be followed by a discussion of the recent advances in the preparation and biomedical applications of cyclodextrin-based hierarchically structured systems, while dividing the latter into two distinct classes, i.e.: 1) supramolecular nanoparticles and related nanoscale systems; 2) host-guest mediated supramolecular hydrogels.

Part of this work, entitled "Cyclodextrin-mediated hierarchical self-assembly and its potential in drug delivery applications", has been recently accepted as a review in the Journal of Pharmaceutical Sciences with minor corrections required.

Driving forces, building blocks and variety of architectures of hierarchically self-assembled systems

Hierarchical self-assembly might take place over a wide range of length scales and involves various types of interactions (usually non-covalent). Those include, but are not limited to: electrostatic interactions, hydrogen bonding and van der Waals forces. In addition to that, hostguest complexation and associations based on biological complementarity are often considered as two separate classes, though in fact they arise as a result of synergy between more basic interactions sterically arranged in a specific way. A classic example from nature illustrating the scope of length scales covered by hierarchical self-assembly is the formation of collagen fibers, Three individual polypeptide chains built up of covalent amide bonds self-assemble into a triple helix, held together by intermolecular hydrogen bonds. The latter are 300 nm long and 1.5 nm in diameter and in a subsequent step self-assemble to produce elongated collagen microfibrils of 30 to 300 nm in diameter. 2,3 Finally, the complex pattern of hydrogen bonds, van der Waals interactions and sparse disulfide bonds between cysteine residues leads to formation of cordshaped collagen fibers about 1 to 20 microns in diameter.

Inorganic and hybrid hierarchically self-assembled materials

The construction elements used in hierarchical self-assembly are not limited to biopolymers and soft matter materials. There are a lot of examples of systems containing inorganic building blocks arranged in a specific way from nanometer to macroscopic length scales. Those are encountered both in living matter (e.g. human bone tissues and their mimics by biomineralization of hydroxyapatite with organic templates) 4,5,6 and in purely inorganic materials with double porosity. 7,8 Given that these classes of hierarchically organized systems are out of the scope of the present work, a few examples will be given to highlight the recent advances.

In the field of zeolite catalysts considerable effort has been made to prepare hierarchical zeolites with dual micro/mesoporosity, as in so doing one can reduce diffusion limitations and enhance mass transport of substrate molecules, making the catalytic process faster and more efficient. 9,10,11 The bottom-up methods to generate mesoporosity in intrinsically microporous synthetic zeolite were shown to allow for a better control over the pores parameters and rely either on precise tailoring of zeolites hydrothermal crystallization process 12 or on the usage of pore-forming templates, such as surfactant micelles. 13 The template-free methods are favorable in terms of avoiding of multistep synthesis and purification procedure. In a recent example Fan et al. have reported a facile one-step pathway to hierarchical ZSM-5 zeolites. 14 By carefully controlling the Si/Al ratio and the amount of base in the reaction mixture the self-assembling of microporous 30 nm precursor particles into 500 nm spherical aggregates was achieved, thus generating micro/mesoporosity of the final material (Figure I.2).

Two other classes of purely inorganic materials constructed via hierarchical self-assembly are environmental photocatalysts and active elements in photovoltaics. In the first case controlled hierarchical arrangement of the primary photocatalyst nanoparticles allows for more efficient mass transfer of organic pollutants 15,16 while in the case of TiO 2 -based photovoltaic cells it facilitates the interparticle transfer of photogenerated electrons. 17 15 Due to the flexibility and variety of soft matter building blocks, during the last 2 decades a lot of effort has been dedicated to the development of hierarchical inorganic-organic hybrids which now constitute a vast class of materials. The most prominent examples include metal-organic frameworks with double porosity 18 , graphene-based hybrids for energy storage devices 19 and self-healing hybrid hydrogels reinforced with inorganic nanoparticles. 20 Metal-organic frameworks (MOF) are materials constructed via formation of coordination bonds between metal ions or clusters as nodes and organic ligands as spokes, possessing inherent microporous structure. 18 The wide choice of transition metals and molecular components allows for tuning of microspores size, shape and surface functionality. As in the case with microporous zeolites higher-order hierarchical structuring of MOF at meso/macroscopic leads to the synergy between the different length scales, thus boosting their performances as drug delivery vehicles 21,22 , catalysts 18 or integrated parts of chemical sensors. 23 According to Furukawa et al. hierarchically structured MOF may be categorized into four groups depending on their dimensionality: (a) zero-dimensional (0D) architectures including hollow capsules or microspheres, (b) one-dimensional (1D) architectures such as nanorods or nanofibers, (c) two-dimensional (2D) architectures i.e. thin films, membranes or patterns, and (d) three-dimensional (3D) architectures that consist of extended in space systems (Figure I.4). 18 The synthetic strategies for hierarchical structuring of metal-organic frameworks rely both on adjusting of the primary MOF nanocrystals parameters and on spatial control over their secondary self-assembly and imply usage of hard templates (macrostructural templating with e.g. inorganic NPs or nanofibers), soft templates (molecular templating) or reaction confinement in biphasic systems.

Figure I.4:

Illustration of different dimensionalities of hierarchical structuring of MOF nanocrystals at meso/macroscale. Reprinted from Furukawa et al. 18 Another recent example of inorganic-organic hierarchical hybrid comes from the field of high performance energy storage materials. Yin et al. have elaborated the procedure to assemble graphene sheets into macroporous honeycomb structure by using cationic surfactants as spacers between the neighboring graphene layers and water droplets as macroporous templates (Figure I.5). 19 Once again, bioinspired porous framework provided significantly decreased diffusion distances of lithium ions, making the prepared material promising for high performance Li-ion batteries. 24 Figure I.5: Preparation of multi-layered honeycomb-shaped graphene for Li-ion energy storage devices. Reprinted from Yin et al. 19

Soft matter hierarchically self-assembled materials

Biopolymers and soft matter in general are major construction materials in biological systems.

The living matter being the main source of inspiration, there is no surprise that soft matter building blocks are prevalent in artificially prepared nanoarchitectures. Despite an immense variety and complexity of such systems created so far some basic classification appears to be possible.

Peptides and peptide-based polymeric materials outperform their synthetic counterparts in a number of parameters, such as: 1) multifunctionality/tunability, 2) adaptability/stimuliresponsiveness, 3) synthesis and processing under ambient and aqueous conditions, and 4) recyclability and biodegradability. 25 The chemical information encoded in peptide chains allows them to self-assemble across multiple length-scales in a modular, hierarchical fashion. Hence, during the last decade a lot of work has been dedicated to the preparation of hierarchically selfassembled nanoarchitectures constructed from peptide-based modular blocks tailored so as to increase the final material performance as scaffolds in regenerative medicine 26 , templating vessels for biomineralization 27 and drug or gene delivery vehicles. 28,29 Figure I.6: Schematic illustration of the hierarchical self-assembly of poly(L-lysine) dendrimers and linear poly(L-leucine) into hierarchical peptidesomes. Reprinted from Xu et al. 29 X. Xu and coworkers have successfully prepared biomimetic capsid-like nanoarchitectures of sub-micron size called peptidosomes. 29 They used a two-step strategy involving the formation of peptide amphiphiles via electrostatic interactions and/or hydrogen bonding between poly(Llysine) dendrimers with polyhedral oligomeric silsesquioxane cores and hydrophobic linear poly(L-leucine)-based peptide as the first step. Upon nanoprecipitation from DMF in water peptide amphiphiles self-organized into stable peptidesomes (Figure I.6). The hierarchical peptidesomes exhibited high gene-transfection efficiency as non-viral gene vectors.

In another recent example interpeptide charge complementarity has been used to construct cage-within-cage Matryoshka-like architectures between two oppositely charged peptides of different sizes. 30 By carefully regulating ionic strength of the medium cage-within-cage structures were possible to obtain by two pathways: a) direct loading of intact capsid-like 30 Another class of valuable building blocks for construction of hierarchical architectures is nucleic acids. 31,32 Self-assembly properties of nucleic acids such as DNA and RNA are based on weak but specific hydrogen bonding between four distinct naturally occurring nucleotides. Artificial modifications of nucleotide sequences allow hybridization of nucleic acids into secondary structures unknown in nature, e.g. tiles further self-propagating into 2-D lattices. 33 However this approach to construction of hierarchical nanomaterials is limited from practical point of view due to its complexity and time-consuming purification of cohesion blocks. 31 Preparation of DNA block copolymers (DBCs), which combine nucleic acid strands as hydrophilic blocks with various hydrophobic synthetic polymers, proved to be a more robust and versatile way to exploit the patterning properties arising due to the DNA hybridization. Highly hydrophobic polymers such as polystyrene (PS) 34 , polypropylene oxide (PPO) 35 or poly(butadiene) 36 were conjugated with DNA-oligonucleotides by solid phase synthesis in organic media. Likewise conventional amphiphilic block copolymers, when dissolved in water DBCs hydrophobically self-assemble into various nanometric architectures such as micelles 35 , cylinders 37 or polymersomes 38 with DNA strands facing the aqueous medium. Their size and morphology can be tuned by adjusting the length of the DNA sequence and/or polymer molecular weight. 39 The DNA corona on top of DBC-based nanostructures allows for their functionalization and extension with complementary nucleic acid strands attached to nanoparticles, drugs or dyes.

Using this strategy various high-order hierarchical structures have been prepared. For instance, Wang et al. have decorated nanocylinders self-assembled from amphiphilic DNA-dendron with complementary modified gold nanoparticles. 37 In another example even more complex superstructures were prepared: semiconductor single-walled carbon nanotubes (SWCNT) were first sterically stabilized with PPO-b-DNA copolymers and then attached via nucleic acid hybridization either to DNA-modifies gold NPs or DNA-covered gold electrodes (Figure I.8). 39 A vast family of approaches to construct soft polymeric materials structured across multiple length scales is based on cooperative electrostatic interactions or simply ionic self-assembly (ISA) of polyelectrolytes (PE) with surfactants, amphiphilic block copolymers containing PE charge-carrying blocks 40 or polymeric ionic liquids. 41 A wide range of materials which are prepared by hierarchical ISA include: spanning liquid-crystalline (LC), porous, hybrid organic-inorganic 42 , and electroactive materials. 40 Ahmed et al. have described a pathway to hierarchically organized organic-metalorganic hybrids via combination of hydrophobic self-assembly of block copolymers (takes place on a length scale of 10-100 nm) and self-assembly between PE-amphiphilic surfactant. 43 They prepared polystyrene-b-poly(ferrocenylmethyl-(trimethylammoniumpropynylsilane)) DBCs of varying block ratios. Further, by synthetic variation of the block volume ratios, along with complexation of PE block with oppositely charged amphiphilic surfactants (hydrogenated or perfluorinated), which also changed the volume fractions in a predictable way; they ended up with a formation of long-range well-ordered phase structures, consisting of aligned cylinders of PS (with dspacing of 35 nm) in a lamellar PFS-surfactant matrix extending over many micrometers (Figure I.9). In addition, by oxidative etching of polystyrene part thin metalorganic membranes with 34 nm holes were obtained. 43 Yuan et al. have explored the unique ability of poly(ionic liquid)s (PILs) based on 3-n-alkyl-1vinylimidazolium bromide to self-assemble in one step during the polymerization process. 44 Upon varying the alkyl chain length from C12 to C18 for lower polymerization concentrations

Hierarchical self-assembly mediated by Host-guest interactions

Inclusion complexation, wherein usually macrocyclic "host" binds hydrophobic "guest" in a reversible manner constitutes a basis of a separate family of non-covalent interactions. Hostguest interactions arise as a result of synergy between non-polar van der Waals forces, hydrogen bonding and precise matching in size between the guest molecule and macrocycle hydrophobic internal cavity. The size and sterical requirements to the guests lead to the socalled "molecular recognition" phenomenon, and consequently to high specificity and controllability of host-guest interactions as opposed to their simple non-covalent counterparts used in supramolecular chemistry. 45,46,47 In addition to "molecular recognition" controlling tool, host-guest complexes formation and dissociation is sensitive to environmental changes such as temperature, light, mechanical pressure or competitive guests binding. 45 Due to these prominent features host-guest complexation was successfully applied in the construction of smart hierarchically structured architectures such as supramolecular polymers 47 , molecular machines 48 and supramolecular responsive gels. 45,49,50 A vast variety of both naturally derived and synthetic "host" macrocycles was described and used in supramolecular chemistry. Those include, but not limited to: porphyrins 51 , calixarenes 52 , crown-ethers 53 and cryptands 54 , pillarenes 55,56 and cucurbiturils. 57 In this account, however, we will limit our focus to cyclodextrins -without doubt the most widely exploited in supramolecular self-assembly class of macrocycles.

Cyclodextrins (CDs) are naturally occurring cyclic oligosaccharides commonly composed of six, seven, or eight (α, β and γ-cyclodextrins respectively) D-glucose units connected by α-1,4glucosidic linkages. 49,58,59 Due to the intramolecular network of hydrogen bonds between the secondary hydroxyl groups of D-glucose, CDs assume a quite rigid toroidal shape. The primary hydroxyl groups are found on the narrower, primary face, of the resulting toroid, while the secondary hydroxyls are located on the wider, secondary face. 49,58,60 The exterior of the CDs cavities is polar due to the hydroxyl groups sticking out, while the interior is nonpolar due to the -CH 2 -groups and ether-like oxygens. Besides their rich host-guest chemistry and easiness of modification, cyclodextrins also show excellent biocompatibility. 45 So far, CDs are the only class of macrocycles approved by the Food and Drug Administration (FDA) for usage in food, personal care products and drug formulations. Some notable examples of their application include the first siRNA delivery vehicles studied in humans, composed of βCD-based nanoassemblies containing RNA and sterically stabilized by PEG-Ada via host-guest interactions. 61 All these features make CDs indispensable building blocks for the construction of hierarchically structured systems with potential biomedical applications.

Nanoscale hierarchical systems based on cyclodextrins

Gene delivery systems

One of the most prominent directions in the biomedical application of cyclodextrin-based supramolecular systems is gene delivery. After a short period of popularity of the viral gene vectors in the early 90s, the associated risks and pitfalls of the latter such as toxicity, immunogenicity and difficulty of scale-up production quickly became obvious 62,63 . Thus, in the last two decades a big deal of effort has been dedicated to the development of synthetic nonviral gene carriers, usually belonging to one of the following classes of compounds: cationic polymers [START_REF] Park | Current status of polymeric gene delivery systems[END_REF] or cationic amphiphiles. [START_REF] Li | Lipid-based nanoparticles for nucleic acid delivery[END_REF][START_REF] Srinivas | Cationic amphiphiles: promising carriers of genetic materials in gene therapy[END_REF] In the presence of polyanionic nucleic acids these compounds undergo self-assembly into nanoscale particles, known as polyplexes and lipoplexes.

In this way, the transported genetic material can be protected from the immune system response and targeted to the place of action in an organism 62 . However the transfection efficiency of such systems is usually inferior to that of viral carriers and because of their heavily charged nature they demonstrate high toxicity. [START_REF] Wagner | Strategies to improve DNA polyplexes for in vivo gene transfer: will "artificial viruses" be the answer?[END_REF] To overcome the high charge-related issue, "steric stabilization" has been used as a means of protecting the positively charged surface of polyplexes and preventing their non-specific interactions with blood proteins. Carbohydrates and cyclodextrins in particular proved to be efficient sterical stabilizers of polyplexes due to the versatility of the benefits they bring: a) enhancement of the membrane adsorption of the polyplexes; b) protection from non-specific interactions; c) opportunity to further functionalize gene-carrying nanoparticles by exploiting the CD inclusion chemistry. The most successful examples using CDs for gene vectors formulations include the work of Mark E. Davis and coworkers. In the 1 st step they prepared the so-called polyCDplexes -nanoparticles formed by electrostatic self-assembly between plasmid DNA and positively charged linear βCDpolymer (pβCD) and sterically shielded them from non-specific interactions and aggregation in the salty physiological medium by the inclusion of adamantane-modified poly(ethylene glycol) (Ada-PEG) into the superficial CD-cavities of the polyplexes. [START_REF] Mishra | PEGylation significantly affects cellular uptake and intracellular trafficking of non-viral gene delivery particles[END_REF] They further developed the concept by using bifunctionalized Ada-PEG-Tg, additionally modified with active targeting agents (Tg). Thus, modification with galactosylated Ada-PEG-Gal selectively brings the pDNAcontaining polyCDplexes to hepatocytes with overexpressed galactose membrane receptors. [START_REF] Pun | Development of a nonviral gene delivery vehicle for systemic application[END_REF] When galactose is replaced by transferrin, the genetic material, in this case siRNA, is efficiently targeted to cancer cells characterized by upregulated transferrin receptors. 61,[START_REF] Bartlett | Physicochemical and biological characterization of targeted, nucleic acid-containing nanoparticles[END_REF] The resulting four component transferrin-targeted formulation under the name CALAA-01 (Figure I.11) has been the first targeted delivery of siRNA tested in humans. The CALAA-01 nanoparticles demonstrate similar to conventional PEI-based carriers siRNA transfection efficiency while being more selective to various cancer cell lines and far less toxic. [START_REF] Hu-Lieskovan | Sequence-specific knockdown of EWS-FLI1 by targeted, nonviral delivery of small interfering RNA inhibits tumor growth in a murine model of metastatic Ewing's sarcoma[END_REF][START_REF] Heidel | Administration in non-human primates of escalating intravenous doses of targeted nanoparticles containing ribonucleotide reductase subunit M2 siRNA[END_REF][START_REF] Davis | Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles[END_REF] In another example Park et al. proposed a way to spatially control CD-PEI DNA-polyplexes delivery by immobilizing them onto solid surfaces patterned with adamantyl groups. [START_REF] Park | Supramolecular assembly of cyclodextrin-based nanoparticles on solid surfaces for gene delivery[END_REF] Polyplexforming PEI was modified with βCD moieties, ionically self-assembled with negatively charged pDNA and adsorbed onto Ada-modified gold surface via inclusion complexation (Figure I.12).

Due to the multivalency of the HG-interactions between βCDs on a single nanoparticle and the surface-linked Ada-functions the binding strength was enhanced such that nanoparticles are not detached even at >10 000-fold concentrations of competitor monomeric βCD. The pDNA could be released from the surface on-demand by addition of polyanionic heparan sulfate which competitively disassembles the nanoparticles. [START_REF] Park | Supramolecular assembly of cyclodextrin-based nanoparticles on solid surfaces for gene delivery[END_REF] Amiel's group developed a macromolecular DNA vector based on epichlorohydrin/βCD copolymer (pβCD). [START_REF] Burckbuchler | Development and characterization of new cyclodextrin polymer-based DNA delivery systems[END_REF][START_REF] Burckbuchler | DNA compaction into new DNA vectors based on cyclodextrin polymer: Surface enhanced Raman spectroscopy characterization[END_REF][START_REF] Wintgens | Smart DNA vectors based on cyclodextrin polymers: compaction and endosomal release[END_REF] Inclusion complexation between initially neutral pβCD and a series of amphiphilic cationic connectors, consisting of adamantane group, spacers with different chemical nature and length and a mono-or bicationic polar head, resulted in formation of reversible pseudo-polycation. The latter proved to hierarchically self-assemble to yield though not able to compact DNA itself, when used in combination with cationic Ada5, proved to act as an efficient endosomal escape enhancer due to its "proton sponge" properties. [START_REF] Varkouhi | Endosomal escape pathways for delivery of biologicals[END_REF] Moreover, the positive charge density of the vector and the polyCDplexes characteristics such [START_REF] Wintgens | Smart DNA vectors based on cyclodextrin polymers: compaction and endosomal release[END_REF] Threading of cyclodextrins along the hydrophobic polymer chains or blocks, dubbed as rotaxanation, is widely known to endow the resulting supramolecular rotaxanes with higher hydrophilicity, flexibility and bioavailability. 46,62,[START_REF] Wenz | Cyclodextrin rotaxanes and polyrotaxanes[END_REF] enhanced, compared to PEI-b-PEG diblock copolymers, gene transfection efficiency. [START_REF] Shuai | Novel biodegradable ternary copolymers hy-PEI-g-PCL-b-PEG: synthesis, characterization, and potential as efficient nonviral gene delivery vectors[END_REF] However, at higher PCL block lengths and -PCL-b-PEG grafting densities the DNA binding rate and watersolubility of the resulting polyplexes was drastically decreased due to the PCL hydrophobicity and its hydrogen binding with positively charged PEI. The solution to this problem consisted in including the PCL block inside the α-cyclodextrin (α-CD) cavities. [START_REF] Shuai | Supramolecular gene delivery vectors showing enhanced transgene expression and good biocompatibility[END_REF] Being able to form inclusion In a number of recent studies CD-based hierarchical gene delivery systems with the so-called "biological stimuli-responsiveness" were reported. [START_REF] Dan | Biological stimuli-responsive cyclodextrin-based host-guest nanosystems for cancer therapy[END_REF] For instance, intracellular milieu is known to be more reducible comparing to extracellular matrix due to its 100-fold higher concentration

(2-10 mM) of glutathione (GSH) -thiol-containing antioxidant tripeptide. [START_REF] Dan | Biological stimuli-responsive cyclodextrin-based host-guest nanosystems for cancer therapy[END_REF][START_REF] Hogg | Targeting allosteric disulphide bonds in cancer[END_REF] This considerable GSH concentration difference may be used to trigger rapid disassembly of gene delivery carriers, followed by the release of genetic material at the site of action. The strategy usually involves linking CD-containing moieties to nucleic acid-condensing cationic polymers via easily reducible disulfide S-S bond. The polymer responsible for sterical stabilization is modified with hydrophobic guest functions and linked to CD-containing gene vector via inclusion complexation prior to polyplex formation. Once inside the cell, the protective guest polymer corona is removed by S-S bond reduction and the genetic material is released. This approach with βCD cationic star polymer as a host and adamantyl end-capped zwitterionic phosphorylcholine based polymer as a guest was successfully realized by Liu et al. for p53 anti-cancer gene delivery in MCF-7 cells. [START_REF] Wen | Highly Efficient Multifunctional Supramolecular Gene Carrier System Self-Assembled from Redox-Sensitive and Zwitterionic Polymer Blocks[END_REF] Within the same strategy Ping et al. reported a gene delivery carrier with redox-detachable PEG corona. [START_REF] Ping | FGFR-targeted gene delivery mediated by supramolecular assembly between β-cyclodextrin-crosslinked PEI and redox-sensitive PEG[END_REF] In their case S-S bond was introduced into the structure of Ada-ended PEG (Ada-SS-PEG) guest polymer. A host segment consisted of βCD-crosslinked low molecular weight PEI conjugated with a targeting peptide, able to condense DNA into 100-200 nm polyplexes. The formulation could be selectively delivered to carcinoma cells, followed by glutathione-induced PEG-corona detachment making the carried DNA available.

Various drug delivery-oriented nanoscale systems

Multivalent inclusion complexation between host polymers, modified with cyclodextrin moieties, and guest polymers, bearing hydrophobic groups, proved to be a powerful strategy to prepare supramolecular host-guest nanoparticles with interesting drug delivery profiles. [START_REF] Gref | New self-assembled nanogels based on host-guest interactions: characterization and drug loading[END_REF][START_REF] Daoud-Mahammed | Cyclodextrin and polysaccharide-based nanogels: entrapment of two hydrophobic molecules, benzophenone and tamoxifen[END_REF][START_REF] Nielsen | Formation of nanoparticles by cooperative inclusion between (S)-camptothecin-modified dextrans and β-cyclodextrin polymers[END_REF] Amiel and coworkers have shown the importance to carefully control the physico-chemical properties of the utilized host-guest polymers such as molecular weight, substitution degree and chemical nature of the spacer; by doing so stable in aqueous medium nanoassemblies with tailorable sizes could be obtained without using toxic organic solvents and/or surfactants.

Furthermore, free β-cyclodextrin cavities of the nanoassemblies were exploited to load them with hydrophobic drugs such as tamoxifen, which was showed to be released in a sustained manner. [START_REF] Layre | Tuning the interactions in cyclodextrin polymer nanoassemblies[END_REF][START_REF] Wintgens | Size-Controlled Nanoassemblies Based on Cyclodextrin-Modified Dextrans[END_REF] In order to make such polymeric nanoassemblies stable in salty physiological medium the same authors proposed a hierarchical strategy, which involves coating the nanoassemblies with a bifunctional dextran bearing adamantyl anchoring groups and hydrophilic "corona"-forming poly(ethylene oxide-co-propylene oxide) (PEPO) side chains to increase the steric repulsion between the nanoassemblies. [START_REF] Wintgens | Cyclodextrin Polymer Nanoassemblies: Strategies for Stability Improvement[END_REF] M. Zan et al. have recently described a host-guest nanogels-based drug delivery system with dual pH-responsiveness. [START_REF] Zan | Dual pH-triggered multistage drug delivery systems based on host-guest interaction-associated polymeric nanogels[END_REF] They used random copolymers of two types: adamantyl (Ada)- An example of hierarchical core-shell host-guest nanoparticles with promising drug delivery profile was reported by Ma and coworkers. They synthesized a hydrophilic-hydrophilic diblock copolymer comprising of a PEG block (M w ~ 5000) and a polyaspartamide block (M w ~ 1500), the latter carrying βCD units on the side chain (PEG-b-PCD). [START_REF] Zhang | Polymeric core-shell assemblies mediated by host-guest interactions: Versatile nanocarriers for drug delivery[END_REF] This host copolymer was further used for self-assembly with various monomeric and polymeric hydrophobic guests, resulting in core shell nanoparticles with the sizes in the range 50-120 nm. For instance, when pyrene is inserted into the βCD, due to its large dimensions, a part of the molecule protrudes from the βCD cavities inducing a localized hydrophobicity in the polyaspartamide block. The resulting pseudoamphiphilic copolymer further self-assembles into core-shell aggregates with the cores made up from βCD/pyrene complexes. By using negatively charged 1-adamantanecarboxylic acid in place of pyrene they obtained a pseudo-polyelectrolyte able to form core-shell micelles with positively charged PEI. In both cases hierarchical micelles are stabilized by hydrophilic PEG shell.

The concept was further extended by preparation of thermoresponsive nano-assemblies between PEG-b-PCD and poly(N-isopropylacrylamide) (PNIPAM), using the inclusion complexation interaction between the hydrophobic cavity of βCD and isopropyl group of PNIPAM. [START_REF] Zhang | Spontaneous formation of temperature-responsive assemblies by molecular recognition of a β-cyclodextrin-containing block copolymer and poly (N-isopropylacrylamide)[END_REF] The particle size of the assemblies was tunable and could be decreased by dropping Voltage-responsive cyclodextrin-containing hierarchical vesicles were also described and

showed to have a high potential as controllable drug delivery vehicles by Yan et al. [START_REF] Yan | Voltage-responsive vesicles based on orthogonal assembly of two homopolymers[END_REF] In the first step they prepared an amphiphilic pseudo-copolymer by linking together ferrocene end-capped poly(ethylene oxide) (PEO-Fc) and poly(styrene)-β-cyclodextrin (PS-βCD) via inclusion complexation between Fc and βCD. Upon reaching the critical aggregation concentration ~0.28 mg/mL, PS-βCD/PEO-Fc hydrophobically self-assembles into ~102 nm hierarchical vesicles with the wall thickness ~19 nm. The loss of affinity to βCD in oxidized form of ferrocene (Fc + ) was exploited to achieve reversible assembly/disassembly of the vesicles by alternately exposing them to oxidative and reductive 1.5V voltages respectively.

The same group has further developed their system by using PNIPAM-βCD/PEO-Fc pseudocopolymer, where PS-βCD was replaced by thermoresponsive PNIPAM-βCD as a host segment. [START_REF] Kang | Dual-Stimuli-Responsive Nanoassemblies as Tunable Releasing Carriers[END_REF] This way, via host-guest self-assembly they prepared a supramolecular amphiphile simultaneously containing redox (Fc) and temperature-sensitive elements (PNIPAM). Containing both redox (Fc) and temperature-sensitive elements (PNIPAM), the resulting micelles turned out to be dual-responsive, i. al. [START_REF] Liu | Redox-responsive supramolecular amphiphiles constructed via host-guest interactions for photodynamic therapy[END_REF] Zhang and colleagues have recently described an example of hierarchically organized photodynamic therapy system taking advantage of βCD/adamantane inclusion complexation.

Their strategy involved the preparation of a redox-responsive supramolecular amphiphile between the photosensitizer -adamantane-terminated porphyrin derivative bearing a disulfide bond (TPPC6-SS-Ada) and PEGylated βCD (PEG-βCD). [START_REF] Liu | Redox-responsive supramolecular amphiphiles constructed via host-guest interactions for photodynamic therapy[END_REF] [START_REF] Ma | A multistimuli-responsive supramolecular vesicle constructed by cyclodextrins and tyrosine[END_REF] The diameters of the vesicles range from 90 to 140 nm and their structure could be disrupted "on-demand" either by addition of a competitive guest, 1-hydroxyadamantane, or copper (II) ions. In the latter case Cu(II)-ions mechanism of action is based on their ability to form coordination complexes with tyrosine, thus preventing the inter-tyrosine hydrogen bonding, responsible for holding the vesicular membrane together. Triggered disassembly and nontoxicity of the developed vesicles makes them attractive for drug delivery applications. nanoMOF windows and interfering with the drug loading process.

Host-guest mediated supramolecular hydrogels

Hydrogels are cross-linked three-dimensional networks with porous structure made of various hydrophilic building blocks. 111 Due to their high water swellability and structural similarity to the human's body extracellular matrix, hydrogels have found a great deal of applications in a range of biomedical applications such as tissue engineering, regenerative medicine, storage and sustained release of therapeutics and smart chemical sensors. 49,112,113 Hydrogels may be divided into two main classes depending on the nature of the used cross-links: covalently crosslinked chemical gels and non-covalently constructed physical gels. In addition to their inherent stimuli-responsive nature 114 , physical hydrogels possess a plethora of other advantageous features, e.g. compositional flexibility, tailorability and self-healing properties. 115,116 The introduction of cyclodextrins as structural units of both chemical and physical hydrogels proved to be beneficial given the ease and versatility of CD functionalization (via multiple primary and secondary hydroxyls), their high biocompatibility and the opportunity to exploit the cyclodextrin-hydrophobic guest inclusion chemistry. 117 In order to get a general insight into the advances in the domain of CD-containing hydrogels an interested reader is encouraged to refer to a recently published review by Tan et al. 49 Herein, though, we will concentrate on the most significant examples exploiting hierarchical self-assembly strategy to construct non-covalent CDhydrogels with interesting biomedical profiles.

Hierarchical hydrogels formed from cyclodextrin-based polypseudorotaxanes

The first example of physical gel resulting from the non-covalent interactions between multiple CD-based polypseudorotaxanes (CD-PPRTX) was reported back in 1994 by Harada and coworkers. 118 In view of their thermoresponsive thixotropic properties and high biocompatibility, CD-PPRTX gels rapidly attracted a lot of interest as potential injectable drug delivery systems. 49,119 The possibility to inject a drug-loaded hydrogel through a syringe allows minimizing the damage to living tissues as a result of the administration procedure. 120,121 The CD-PPRTX hydrogels formation process is thought to proceed in a hierarchical manner and It was shown that relatively high PEG molecular weights (at least 10 kDa) are required to achieve the resulting hydrogel stability and long drug release terms. 122 However, being nonbiodegradable, only PEG polymer chains or copolymer blocks with M n below 5 kDa are efficiently eliminated from the body via renal clearance pathway. Moreover, the mechanical stability of CD-PPRTX gels is still unsatisfactory even when > 10 kDa PEG are used as guests. The typical solution to these problems consists in using linear, grafted or star-shaped copolymers consisting of PEG block with M n around 5 kDa and higher molecular weight biodegradable polymer blocks such as polyesters, cationic polymers 123 or polypeptides instead of linear PEG.

For instance, amphiphilic block copolymers containing 5 kDa PEG and either biodegradable polyester poly(3-hydroxybutyrate) (PHB, Figure I.23) 124 , or PCL 125 were used as apolar blocks.

The usage of biodegradable hydrophobic blocks was found to be doubly beneficial to the mechanical properties of the gel due to: 1) removal of the restriction on the copolymer overall molecular weight, as long as PEG blocks are reasonably small; 2) additional physical cross-linking via poly(3-hydroxybutyrate) and PCL blocks hydrophobic microdomains formation. Furthermore, both CD-PPRTX hydrogels were non-cytotoxic, injectable through a small-diameter aperture and demonstrated a sustained in vitro release of fluorescein isothiocyanate-labeled dextran Adapted from Li et al. 124 In an attempt to produce stiffer CD-PPRTX hydrogels, while decreasing the total weight concentrations of the starting components, the researchers also used interpeptide hydrogen bonding as an additional associative force. 126,127 For example Dong and coworkers demonstrated that usage of diblock copolymers containing PEG with molecular weight around 5 kDa and biodegradable polypeptide poly(L-glutamic acid) (PLG) leads to αCD-PPRTX hydrogels with remarkably high elastic storage modulus (G ' ~ 10 4 Pa at 10 wt% vs. 10 3 Pa at 15 wt% for conventional amphiphilic hydrogels) and long loaded doxorubicin release times. 126 This gel strengthening phenomenon was ascribed to extra hydrogen bonding between the PLG blocks under acidic pH and its synergy with polypseudorotaxane hydrogen bonds driven associations.

In addition, the presence of polypeptide blocks endows the resulting hydrogels with pHresponsiveness. 126 Following the same logic, PPRTX hydrogels were mechanically reinforced with various inorganic nanomaterials such as silica 128 , silver nanoparticles 129 or multi-wall carbon nanotubes (MWCNT). 130 In this case the strategy usually involves physical or chemical anchoring of PEG or PEG-containing copolymers onto inorganic scaffolds prior to polypseudorotaxane formation, so that they play the role of additional network junctions. 122 Li and colleagues used a αCD/PEG PPRTX-based hydrogel as a gene transfection system. Vancomycin, an antimicrobial agent active against Gram-positive bacteria. 133 The formulations contained 6.5 to 20% w/v of Poloxamer F127 and 0 to 9.7% of αCD. At the αCD content above 5% w/v fast gel formation was observed with G ' exceeding G '' already at room temperature (~1×10 3 vs. 1×10 2 Pa at 10°C and 5% αCD). The moduli values could be increased at higher temperatures due to the hydrophobic associations between PPO blocks. The PPRTX gels were easily injectable, colloidaly stable for more than 2 months and provided sustained release of Vancomycin for several days. In spite of its advantages, simple Poloxamer/αCD system still suffers from quite weak consistency under the stress because of its shear-thinning and thixotropic properties. Recovery of the initial G ' and G '' after syringe administration takes enough time to favor a rapid release of the drug before the hierarchical network is restructured. Therefore, the same research group has recently attempted to reinforce the Poloxamer/αCD system by addition of hydroxyethyl cellulose (HEC) as the third thickening component. 134 Such ternary systems were loaded with griseofulvin, a highly hydrophobic antifungal drug used for treatment of dermatophytosis. Due to the synergy between the Poloxamer-P123 and HEC apparent solubility of griseofulvin in water is significantly enhanced and αCD, being able to thread along both 2-hydroxy ethyl radicals of HEC and PEO blocks of poloxamer, prevents the formulation from phase separation. To summarize, despite the obviously high potential of PPRTX hydrogels as injectable sustained release systems, examples of real applications of these systems have only recently begun to emerge. 49,125,134,136,137 Thus, this field is expected to attire a great deal of researchers' attention in the years to come.

Cyclodextrin-based hierarchical hydrogels with external stimuli responsive properties

Besides CD/PEG PPRTX hydrogels, many examples of hierarchically organized physical gels with CD/guest inclusion complexation as one of the driving forces of the 3D-network formation have been reported. In addition to intrinsically high internal dynamics and self-healable properties 46 , these systems can also be imparted with stimuli responsiveness by introducing thermo-114, 138 , light-139 or redox-sensitive 140 elements. In this way, reversible on-demand sol-gel transitions and payload release are achieved by simply alternating an appropriate external stimulus. In the following section we will look at some of the recent advances in the preparation of stimuliresponsive CD-based hierarchical hydrogels with attractive biomedical profiles.

Figure I.25:

Illustration of the "star-star" supramolecular complex formation via βCD/Ada inclusion complexation, followed by its temperature-triggered hierarchical hydrogelation.

Reproduced from Zhang et al. 138 For example, Zhang and coworkers described a so-called "star-star" supramolecular architecture obtained through a host-guest self-assembly between a star-shaped adamantylterminated 8-arm PEG and a star-shaped PNIPAM containing a βCD core (Figure I.25). 138 Being encircled by thermoresponsive corona, the star-star aggregate could be converted into a 3D hydrogel network by increasing the temperature above the LCST value of PNIPAM. Rheological studies showed a highly reversible and repeatable character of hydrogelation with G' reaching initial values (G'~ 100 Pa, G"~70 Pa) after up to 6 cycles of cooling to 25°C and heating back to 37°C. Furthermore, due to the low viscosity of the star-star complex solution at 25°C, authors suggested that therapeutic agents could be mixed with it and conveniently injected into the body. The subsequent temperature triggered in vivo hydrogelation is aimed to encapsulate and sustain the release of the therapeutic agents. Applying a similar strategy, several years earlier, Ming Jiang and coworkers developed an approach to hybrid thermo-114, 141 or dual stimuli-responsive 142 hierarchical hydrogels containing inorganic nanoobjects as supramolecular cross-linking points. The strategy involved covalent modification of inorganic nanomaterials such as graphene oxide (GO) 141 or CdS quantum dots (QD) 114,142 with βCD, as the first step. Then, the resulting GO-βCD or QD-CD were selfassembled into hybrid inclusion complexes (HIC) with diblock copolymers containing a thermoresponsive PNIPAM block and poly-(N,N-dimethylacrylamide) (PDMA) block, the latter being end-modified with azobenzene 114, 141 or ferrocene 142 guest functions. The HIC superstructures were hierarchically and reversibly converted to hydrogels upon heating, while the LCST responsible for the sol-gel transition could be controlled by varying the ratio of degree A few examples of photo-responsive CD-based hierarchical hydrogels were reported in the past years. As it was mentioned, such systems are of particular interest in drug delivery applications due to their non-destructiveness, remote controllability and time-/site-specific features. Its sol-gel transitions can be induced by reversible trans/cis photoisomerization of azobenzene upon irradiation with UV light (λ 365 nm ) and visible light (λ 430 nm ). Furthermore, the hydrogel possessed low cytotoxicity and could be used for cells culturing.

A remarkable example of photocontrolled CD-hydrogel based on mesoporous silica nanoparticles (Si-MPs) was reported by Park et al. 139 Their system consisted of Si-MPs with 2 nm pores as a reservoir for guest molecules and βCD "gatekeeper" linked to the Si-MPs through a photocleavable o-nitrobenzyl ester linker acting as a nanovalve blocking the loaded molecules inside the silica pores. Loaded with a model fluorescent dye, calceine, Si-MPs were then used as building blocks to form physical hydrogels via molecular recognition-based interaction with sixarm poly(ethylene glycol)with dodecyl end groups (6-PEG-C12). The resulting hydrogel could be switched to a sol either by addition of αCD as a competitive host or by UV irradiation induced photolysis of the o-nitrobenzyl ester linker. In the latter case the disruption of the gel network was accompanied by "on demand" release of the loaded calceine.

Finally In a more recent example Himmelein and coworkers successfully used cyclodextrin vesicles (CDV) as multivalent 3D cross-linking points to prepare a chemically-responsive hydrogel. 116 The vesicles were hydrophobically self-assembled from synthetic βCD amphiphiles and their gelation was achieved by the interaction with adamantane-modified hydroxyethylcellulose (HEC-Ad) (Figure I.28). Once again, the gel network could be readily disrupted by the addition of competitors. Interestingly, the system showed shear-thinning/self-healing properties and was injectable through a syringe. This, as well as high content in free βCD cavities able to accommodate hydrophobic drugs, makes CDV-based hydrogel an interesting candidate for sustained release applications. At the same time, the drawback of chemically-responsive CD-based hydrogels comparing to their thermo-, redox-or photo-responsive counterparts lies in the irreversibility of the gel-to-sol transition in this case; i.e. the gel network becomes problematic to restore after the competitor addition, which is especially true when the manipulations are done in vitro or in vivo.

Moreover, small synthetic competitors often might be toxic and non-biocompatible.

Conclusions

In summary, we have highlighted the basic principles and recent advances in the field of artificial hierarchically self-assembled materials.

Introduction

The focus of this chapter lies on the synthesis of dextran-based host and guest polymers with hydrophilic poly(ethylene glycol) (PEG) spacers and the study of host-guest nanoassemblies formed between them.

Cyclodextrins have proven to be beneficial in solving a number of food and pharmaceutical industries issues, such as formulation of poorly water-soluble drugs 1 , masking odors 2 and several other purposes. Indeed, the inclusion of "guest" hydrophobic actives into the hydrophobic interior of the CD cavities usually allows to increase their apparent solubility 3 , chemical stability and bioavailability. 1,4 Given this, in the last years the researches are oriented toward the elaboration of colloidal or nanoscale carrier systems containing CDs, most usually based on cyclodextrin polymers (CD-polymers). 5 Such systems combine the advantages of CDs and polymers such as nanoscale size 6 and the possibility to assemble in the polymeric structure several functional elements, providing them with additional useful properties. 7 For instance, active targeting agents, fluorescent labels, "stealth" and stimuli-responsive blocks can be introduced in resulting nanocarrier structure.

Among the polymers used for drug delivery purposes, biopolymers and polysaccharides in particular are especially attractive. This is due to their biocompatibility, biodegradability and high content of easily modified hydroxyl groups. In recent years a handful of polysaccharide based CD-polymers were synthesized by various approaches such as βCD-linked chitosan by carbodiimide-mediated amidation 8 or hyaluronic acid 9 and dextran 10,11 CD-polymers via reductive amination. Pendant CD moieties in such polymers were shown to preserve the conformation and good complexing characteristics of native cyclodextrins. 8,12 On the other hand, in the last years "click" chemistry has become an increasingly popular approach in organic and polymer synthesis. It involves the use of a bunch of effective, selective and adaptive to a wide range of functional groups, reactions. 13 Copper (I) catalyzed Huisgen azide-alkyne cycloaddition (CuAAC) 14,15 and thiol-ene click chemistry 16,17 appear to be by far the most prominent among them. CuAAC "click" chemistry has been already used for the synthesis of βCD-grafted polymethacrylates 18 and linear dextran-βCD polymers. 12 Linear dextran-βCD polymers have shown excellent, compared with the branched pβCD, binding properties. When mixed in water media with their adamantane-grafted counterparts, dextran-βCD yield spherical nanoassemblies with controlled size. The latter, however, were found to lose their colloidal stability upon salt addition. 19 Being inspired by these examples, we were seeking to further advance the properties of both "host" and "guest" polymers by introducing the flexible hydrophilic spacer between the polymer backbone and βCD or adamantyl moieties.

We choose the thiolated dextran as a water-soluble, biocompatible backbone which is modified with bifunctional short PEG-acrylates by means of thiol-Michael addition click chemistry.

Depending on the omega-end functionalization of PEG-acrylate, grafted dextrans bearing either hydrophobic adamantyl functions (guest polymer) or terminal alkynes are obtained. Further "clicking" of 6-monodeoxy-6-monoazido-βCD to the alkyne terminated dextran-g-PEG copolymer by copper(I)-catalyzed azide-alkyne cycloaddition is used to obtain βCD-bearing host counterpart. The binding properties of the polymers are studied in solution by isothermal titration microcalorimetry (ITC) and on the gold surface by surface plasmon resonance (SPR).

The formation of host-guest nanoparticles upon mixing the host and guest counterparts' solutions in water is further studied by dynamic light scattering (DLS), static light scattering (SLS)

and small-angle X-ray scattering (SAXS). The impact of the presence of the hydrophilic PEG spacer between the dextran backbone and the host/guest functions is estimated by comparing their binding properties with those of previously described dextran-based host-guest polymers with short hydrophobic spacers. 19 Part of the work described in this chapter has been published in the Journal of Inclusion

Synthesis of thiolated dextrans (DT40-SH)

The thiolation of dextran was chosen due to the vast synthetic possibilities provided by -SH groups. In this work it was used to graft monosubstituted short PEG chains bearing electron deficient C=C bonds using rapid and efficient thiol-ene "click" chemistry approach. 16 Thiol functionalized dextrans were obtained using the 2-step procedure reported by Hiemstra et al (Scheme II.1). 21 The required [4-NC]/[AHG] (AHG -anhydroglucosidic ring) ratios were typically slightly higher than one given in the original article (0.56 for DS 15%). 22 As can be seen from the graph (Figure

II.
3), when going below the 0.3 ratio only negligible quantities of grafted 4-NP were found.

Synthesis of host polymers with hydrophilic PEG spacer (DT40-g-PEG-βCD)

The synthetic pathway to host polymers with PEG spacer implied preparation of heterobifunctionalized poly(ethylene glycol) acrylate bearing terminal alkyne (PEG-acryloyl-5hexynoate) and 6-monodeoxy-6-monoazido-βCD (βCD-N 3 ) synthetic modules at the first stage.

In the following step PEG-acryloyl-5-hexynoate is grafted to DT40-SH by means nucleophilemediated thiol-Michael chemistry. Finally DT40-g-PEG-βCD is obtained by copper(I)-catalyzed 1,3-dipolar cycloaddition "click" reaction between DT40-g-PEG-hex-5-ynoate and βCD-N 3 . The obtained bifunctional PEG-acryloyl-5-hexynoate was used in the next step without extensive purification from the slight excess of 5-hexynoic acid, the latter being non-active in thiol-Michael addition reaction.

Steglich esterification of poly(ethylene glycol) acrylate with 5-hexynoic acid

Nucleophile-mediated thiol-Michael click of DT40-SH and PEG-acryloyl-5-hexynoate

In the next step PEG-acryloyl-5-hexynoate was attached to DT40-SH by means of nucleophilemediated thiol-ene click chemistry.

The reactions of thiols with carbon-carbon double bonds have been attracting a lot of researchers' attention during the last decades due to the fact that they meet most of the requirements to the so-called "click" reactions conceptualized by B. Sharpless et al. in 2001: a) high yields with by-products (if any) that are removable by nonchromatographic processes, b) regioselectivity, c) insensitivity to oxygen or water, d) mild, solventless (or aqueous) reaction conditions, e) orthogonality with other common organic synthesis reactions, and f) amenability to a wide variety of readily available starting compounds. 24,25 Two main types of thiol-ene click reactions are commonly distinguished: thiol-ene free-radical addition to electron-rich carbon-carbon double bonds, and the catalyzed thiol Michael addition to electron-deficient carbon-carbon double bonds (Scheme II.3). Adapted from Hoyle and Bowman. 25 The suitability of thiol and C=C substrates for either free radical-mediated or Michael reactions is defined by their molecular structure with the general tendency for electron-deficient carboncarbon double bonds and electron-rich thiols to be more active in Michael additions and reverse situation for free radical reactions. Given the commercial availability of monosubstituted short PEG acrylate bearing electron-deficient terminal C=C bonds, the latter had been used for preparation of bifunctional PEG-acryloyl-5-hexynoate in the previous step and defined our choice of Michael addition pathway for the preparation of alkyne-terminated DT40-g-PEG-hex- 16 It was demonstrated that primary and secondary aliphatic amines and especially phosphines, despite their lower comparing to tertiary amines basicity, provide dramatically increased rates of model reaction of hexyl acrylate and hexanethiol. 26 Based on this, authors concluded that, contrary to the accepted belief, the hydrothiolation process is mediated by nucleophilic addition of primary amine (or phosphine) to the activated C=C bond instead of Thus, the initial thiol-ene click trials were performed in water with hexylamine as nucleophilic catalyst and proved to be failed: no or negligible quantities of PEG-acryloyl-5-hexynoate were "clicked" to the DT40-17SH (DS = 17 mol.%) backbone. Given the above, DMF was applied as an alternative of water, leading to dramatic improvements in the reaction profile. Nearly quantitative conversions of -SH groups were observed both for unmodified PEG methyl ether acrylate and for PEG-acryloyl-5-hexynoate within reasonable time (∼24h) under mild conditions.

The DS of DT40-g-PEG-hex-5-ynoate was calculated from 1

Synthesis of 6-Monodeoxy-6-monoazido-βCD (βCD-N 3 )

Monosubstituted at primary side βCD-N 3 was prepared via slightly modified version of a twostep procedure found in literature (Scheme II.6). 12,27 Scheme II.6: Reaction route for the azide-substituted βCD (βCD-N 3 ) Monotosylated, selectively on the primary side, βCD (βCD-OTs) was synthesized using 1-(ptoluenesulfonyl)imidazole which was reported to give better, compared to p-toluenesulfonyl chloride (TsCl), results. 27 The low content of disubstituted product was achieved at the cost of relatively low yield (23%) and controlled by thin layer chromatography (TLC). In the second step the obtained βCD-OTs was converted into azide-substituted βCD (βCD-N 3 ) with a yield of 84% by reaction with the excess of NaN 3 in water media. The βCD-N 3 was characterized by 1

Synthesis of DT40-g-PEG-βCD by copper(I)-catalyzed azide-alkyne cycloaddition

To "click" βCD-N 3 to alkyne terminated DT40-g-PEG-hex-5-ynoate, Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) in pure water or water-DMSO 1:1 mixtures with CuSO 4 /Na-Asc as a catalytic system was tested first. The obtained values turned out to be not more than 25% of the theoretical ones (P0 in the Table II.1). In addition, a significant part of 5-hexynoate was not consumed as it is obvious from the splitting of the 5-hexynoate βCH 2 -protons at 1.6-1.9 ppm (Figure II.9). This failure might be related to two principal reasons: a) oxidation of non-stabilized Cu(I)-catalyst; b) alkyne crosscoupling by Glaser, Straus or Erlington mechanisms. 15 The latter possibility appears probable, as in several cases of CuAAC carried out in DMSO partially gelly products were formed. Polytriazoles, and especially TBTA, were reported to lead to significant improvements in Huisgen cycloadditions on alkyne scaffolds when used as Cu(I)-stabilizing ligands. 12 Given this, the latter was synthesized following the previously described two-step procedure 28 and used in the next step. Regarding the rather hydrophobic character of TBTA, Cu(MeCN) 4 PF 6 /Na-Asc as a new catalytic system and pure DMSO as a reaction medium were applied. Despite being higher compared to P0, the DS CD of the obtained product (P1) was still much lower than expected from the DS alkyne of the starting DT40-g-PEG-Hex-5-ynoate (Table II.1). The βCD content could also be found from the ITC data using the approach described in greater detail in the 2. Water was shown to be the most favorable solvent for CuAAC and tends to be used where appropriate regarding the solubility of the reaction components. 14,15 Based on this we went back to the initially applied CuSO 4 /NaAsc catalytic system, while using TBTA and DMSO/water 2:1 mixture as a reaction medium (Scheme II.7). 1 H-NMR spectra of the products obtained by this approach (P2, P3 in Table II.1) showed complete disappearance of the peaks of terminal alkyne and -CH 2 -in β-position to carbonyl of "unclicked" 5-hexynoate (at 2.79 and 1.69 ppm respectively) (Figure II.10). However, the apparent grafted amounts of βCD were still lower than theoretical ( ~ 84%), which must be related to: a) inaccuracies in phase calibration and baseline correction of the saturated spectra leading to overestimation of the integrals in the middle chemical shift range; b) alkyne cross-coupling still occurring to some extent due to higher reactivity of the triple bonds at the end of flexible PEG spacers. Once again, the DS CD of successful P2 and P3 samples deduced from ITC measurements are slightly lower than those obtained by NMR due to the adsorbed water contained in the freezedried products (Table II.1). The P2 and P3 polymers are relatively well soluble in water (s = 15 An inversion phenomenon of β-cyclodextrin dimers prepared by CuAAC was recently described by Menuel et al. 29 In particular, it was found that triazole-substituted on the primary side Furthermore, it was demonstrated that the extent of the inversion in water is strongly influenced by the nature of the bridge (or spacer) between the βCD cavities. 30 Thus, increased proportions of dimers with only one out of two βCDs available were found in the cases where long and/or hydrophobic spacers had been used. At the same time, emergence of distinct secondary singlets, corresponding to the triazole protons included in the βCD cavities, could be detected for the dimers undergoing inversion. 29 Nor could we detect any reliable cross-peaks between the triazole protons and inner anomeric protons of βCD in the 2D NOESY spectrum of

P2.

The failure of our attempts to find the confirmation of the inversion phenomenon is most likely related to the polymeric structure of the investigated product, leading to lower sensitivity and intrinsic peak broadening in water, thus making separate triazole proton signals undistinguishable.

Synthesis of guest polymers with hydrophilic PEG spacer (DT40-g-PEG-Ada)

Hydrophobically modified with adamantyl groups (Ada) guest polymers were prepared following the similar, as in the case with their host counterparts, strategy. Briefly, monosubstituted poly(ethylene glycol) acrylate was functionalized directly with Ada-groups by esterification and subsequently grafted to DT40-SH by nucleophile-mediated thiol-Michael chemistry. a tertiary amine in dichloromethane (DCM) -no product formation was observed even after 24h of stirring at room temperature in the case of PhNCS. 32 In our case the failure might be due to the relatively low effective concentration of the alcohol groups at the ends of bulky PEG chains; indeed, a second order dependence of the rate of the reaction on the alcohol concentration was found in the work of Wiberg et al. 32 Given the above, in the next step we tested the 4-dimethylaminopyridine (DMAP)-catalyzed esterification of PEG-acrylate with 1-adamantanecarbonyl chloride in CHCl 3 (Scheme II.8, b).

Significantly improved results were achieved after refluxing the reaction mixture with catalytic amounts of DMAP and pyridine for 3h: emergence of a new triplet at 4.17

Binding properties of host-guest polymers with hydrophilic PEG spacers

The binding properties of the polymers were investigated by ITC and SPR, aiming to compare them to those with short hydrophobic spacers and gain insight into the influence of the flexible hydrophilic PEG spacer. Comparing the values of the binding constants (K a in Table II.3) of DT40-g-PEG-Ada with those of the previously described Ada-substituted dextrans with adamantyl groups attached directly to the backbone via ester bond (Figure II.16a), 19 one can clearly see the beneficial impact of the PEG spacer on Ada groups' availability. It leads to an average 5-fold increase of K a in polymers with close DS values (Table II.3). Interestingly, the gain in Gibbs free energy (𝛥𝐺) comes from less negative values of entropy factor TΔS for the guest polymers with PEG spacer, whereas both types of polymers show very similar large |𝛥𝐻| of interaction with βCD. This gain in entropy for DT40-g-PEG-Ada is most likely due to the enhanced mobility of the Ada-groups located at the end of the flexible PEG spacers.

Surface plasmon resonance studies

To evaluate binding properties of the guest polymers on the surface, an SPR adsorption experiment was performed. Quaternary-ammonia modified branched βCD-polymer (pβCDN+;

58.2 wt.% of βCD; 1.62 positive charges per βCD) was used as an anchor to deposit on the golden surface of the sensor. The pβCDN+ was prepared according to the previously described procedure 34,35 and its structural formula is given in Figure II.17. As explained in greater detail in the Experimental section, from the refractive index variation data one may estimate the adsorbed amounts of the polymers (𝑄 𝑎𝑑𝑠 ), provided that their differential refractive indices (dn/dc) are known. 36 And that is by using the equation below:

𝑄 𝑎𝑑𝑠 = ∆𝑅𝐼 • 𝑙 𝑑 2𝑚 • (𝑑𝑛 𝑑𝑐) ⁄ 𝑝𝑜𝑙 (II.1)
where 𝑙 𝑑 is the depth of penetration of the evanescent electromagnetic field (typically 25-50% of the probing light wavelength); for this experiment it was roughly estimated as 37% (310.8 nm). 37 We assumed 𝑙 𝑑 to be constant upon each subsequent polymer adsorption, given the low density of highly hydrated layers and their thickness being much lower than 310.8 nm. The calibration coefficient m of the sensor was estimated as 1. For simplicity the dn/dc of the polymers was considered equal to that of dextran and native βCD (0.145 mL/g), given the proximity of the latter with the dn/dc of PEG (0.135 mL/g), according to the American Polymer Standards Corporation data (http://www.ampolymer.com/dn-dcValues.html).

The 𝑄 𝑎𝑑𝑠 values calculated in this way (Table II.4) were comparable to those found by Amiel and

Wintgens for alkyl-and adamantyl-substituted poly(N-isopropylacrylamide)s (pNIPAm) adsorption on neutral pβCD layers. 37 Difficulty was encountered in achieving exactly equal adsorbed amounts of the anchor pβCDN+ layer (ΔRI 1 ) for further parallel experiments with D40Ada9 and DT40-gPEG-Ada2; in addition, the studied polymers are characterized by a significant difference in DS (Table II.3). Given this, the most correct approach to compare their binding efficiencies should imply calculation of the number of βCD and adamantyl moieties per unit area in the 1 st and 2 nd adsorbed layers respectively. From the 𝑄 𝑎𝑑𝑠 [mg/m 2 ] of the polymers and the weight percentage (wt%) of βCD or adamantyl groups in them one can determine the 𝑄 𝑎𝑑𝑠 and number of moles of βCD/Ada groups. Then, using the Avogadro constant (𝑁 𝐴 ), the number of βCD/Ada groups per unit area can be estimated. The results of these calculations are reported in Table II.4. In agreement with the ITC data, considerably stronger interactions arise between pβCDN+ and the guest polymer with PEG spacer, as it might be concluded analyzing the Ada/CD ratios in the deposited bilayers. The |𝛥𝐻| values are of 17-23 kJ•mole -1 and the complex formation is overall enthalpy driven (Table II.5). However, the gain in entropy factor due to the presence of flexible PEG spacer for DT40-g-PEG-βCD is even more pronounced than for the guest polymers described in the previous section. Indeed, the TΔS of DT40-g-PEG-βCD acquire positive values (3.6-8.2 kJ •mole -1 ) (Table II.5). In contrast, slightly negative values of TΔS are observed all the same for native βCD 38 , epichlorhydrin pβCD 38 and linear dextran-βCD polymers. 12 The binding constants (K a ) are of 5.04•10 4 (P2) and 3.45•10 4 L•mole -1 (P3), which is significantly higher than for epichlorhydrin pβCD (~1•10 4 L•mole -1 ) but, yet, lower than those of previously reported by our group linear dextran-βCD polymers (DT70GPβCD) with βCD linked to dextran via short ether bond (ca. 1-2•10 5 L•mole -1 , Table II.5). 12

Surface plasmon resonance studies

To verify the unexpected results described above, in the next step we looked into the binding properties of the host polymers of different types on the surface. The SPR binding experiments were structured in a more complex way compared to the one utilized for the guest polymers.

Namely, the same quaternary-ammonia modified pβCDN+ (Figure II.17 As in the previous case, the adsorbed amounts of the polymers 𝑄 𝑎𝑑𝑠 were calculated using the equation (II.1), assuming the evanescent field penetration depth 𝑙 𝑑 to be 310. Thus, the ITC and SPR studies of DT40-gPEG-βCD and DT40-gPEG-Ada show that the initial strategy of rendering the host and guest moieties more accessible for inclusion complexation by grafting them to a polymer backbone through flexible hydrophilic PEG spacer works in a desired way in the case of Ada-substituted guest polymers but proves to be less successful in the case of their βCD-substituted host counterparts.

Nanoassemblies prepared from host-guest polymers with hydrophilic PEG spacers

During the last decade size-controlled host-guest nanoassemblies based on cyclodextrinmodified polymers were reported in our group. 19,39,40 These had been obtained by simply mixing in pure water hydrophobically substituted (with alkyl chains or adamantyl groups) dextrans with either βCD-epichlorohydrin branched copolymers 39,40,41 or βCD-substituted linear dextrans with short spacers between the backbone and grafted βCD-groups 19 . Regarding their excellent biocompatibility profile together with avoiding the use of organic solvents and surfactants during the preparation stage, promising drug delivery applications of the host-guest nanoassemblies (HG nanoassemblies) were considered. However, they are characterized by a number of limitations, such as low stability over long periods of time and fast aggregation upon addition of salt.

One of the goals to prepare host and guest polymers with hydrophilic PEG spacer was to understand how the presence of the latter might affect the mobility and accessibility of grafted Ada and βCD groups in order to address the mentioned drawbacks of the existing HG nanoassemblies. Thus, in the following part of the chapter we will focus on self-assembly of DT40-gPEG-βCD and DT40-gPEG-Ada in water. The obtained nanoassemblies will be characterized by various techniques, such as dynamic light scattering (DLS), small-angle X-ray scattering (SAXS) and static light scattering (SLS). Their size, structure and stability will be compared with those of the HG nanoassemblies prepared from the polymers with no PEG spacers and mixed nanoparticles where either host or guest polymer with PEG spacer is used.

Size and stability of HG-nanoassemblies by DLS

Interpolymer host-guest interactions, when sufficiently large in strength, might lead to associative phase separations in a certain concentration range of the components. 42 The cohesion of the polymers happens in a "zip fastener" manner due to the establishment of numerous inclusion complexes between the hydrophobic groups (alkyl, Ada) and the βCD cavities acting as physical crosslinks between the chains of the two polymers.

To achieve the associative phase separation and formation of HG-nanoassemblies between DT40-gPEG-βCD and DT40-gPEG-Ada we worked with the stock polymer solutions at fixed Ada groups and βCD molar concentrations (2.5-5.0•10 -4 mole•L -1 ). Thus, different βCD/Ada molar ratios were achieved by simply varying the volumes of the stock solutions during the mixing process. The total polymer concentrations in the resulting nanoassemblies typically lied in the range of 0.50-1.36 g/L.

In the first experiment freshly prepared solutions of DT40-gPEG-βCD (P3, DS = 16.5 mol.%) and DT40-gPEG-Ada2 (DS = 10.5 mol.%) were mixed while varying βCD/Ada molar ratios (Table II.7).

Surprisingly, with a slight excess of Ada groups and, even at equimolar ratios, formation of large, However, when excess of βCD with respect to Ada was used the resulting nanoassemblies were smaller and more stable. At the same time, the experimental data for other types of HG nanoassemblies indicate that equimolar βCD/Ada ratios typically lead to the best results, which is explained by the opportunity to form the maximal number of physical links between the polymer chains in this case. 19,40 On the other hand, it was noticed that when the guest polymer solution used for self-assembly is stored for longer periods of time, nanoparticles with better characteristics are obtained.

rapidly
Given that, the DT40-gPEG-Ada2 stock solution was aged for 30 days at 4°C and used for analogue self-assembly experiment with freshly prepared DT40-gPEG-βCD solution. The results reported in Table II. 8 show a significant decrease in hydrodynamic diameters and improved stability of the resulting nanoassemblies for all βCD/Ada molar ratios. Moreover, the maximum in the scattered light intensity is observed for βCD/Ada = 1:1 in this case, which should be related to both the higher particle number and increase of their compactness ( Despite nanoassemblies with the lowest average D h are still obtained for the βCD/Ada ratios higher than 2:1, their polydispersity is considerably increased comparing to those prepared at equimolar ratios (Table II.8).

Interestingly, aging of the host polymer stock solution along with the guest leads to further, though much less pronounced, decrease in D h (green curve in One possible cause of the observed phenomenon could be the polymer inter-or intra-polymer association into transient hydrophobic agglomerates, as it was previously observed by Ravoo et al. for Ada-substituted poly(isobutene-alt-maleic acid) (PIBMA). 43 If the latter is true, to ensure the interaction with the host polymer, the agglomerates would require to be disrupted either by aging or by addition of the excess of βCD groups with respect to adamantanes. One clearly observes a maximum in D h of the nanoassemblies at βCD mon /Ada = 1:1, correlating with the precipitation phenomenon, expressed as a drop in scattering intensity. Most likely the presence of βCD mon leads to a competition with βCD pol for available Ada-groups and affects the growth mechanism of the nanoparticles. This, in turn, leads to coalescence of more loose interpolymer complexes into larger aggregates. Thus, the method of monomeric βCD addition is not applicable to the preparation of the nanoassemblies as it influences the nanoassemblies' growth mechanism.

SAXS and SLS investigation of the individual polymers and HG-nanoassemblies of different types

The Adapted from Patterson et al. 44 Some basic characteristics of the individual polymers and the nanoparticles, such as radius of gyration (𝑅 𝑔 ) and molecular weight, might be determined using SAXS and SLS. 45,46 One may also draw information on the shape of the scatterers in a model-free way, by combining the SAXS (or SLS) data with the DLS and calculating the 𝑅 𝑔 𝑅 𝐻 ⁄ ratios (Figure II.27). 44 However, more definite conclusions on the nano-objects shape, internal structure and aggregation state can be made by fitting the SAXS data to different structural models. 47,48 The typical values of scattering vector lengths 𝑞 covered in SAXS lay in the range 0.003-0.35 Å -1 .

According to the Fourier transformation sampling theorem, it means that the maximum overall size, that can be determined by SAXS is ~100 nm. 48 As described in the previous section, hostguest nanoassemblies typically surpass this size limit, depending on mass concentration, βCD/Ada molar ratio etc. Hence, for the proper modelling of SAXS data for such bulky nanoobjects it is often beneficial to use complementarity between SAXS and SLS, the latter giving the opportunity to extend the range of available 𝑞 down to 0.00035 Å -1 . 48 The theoretical background on SAXS and static light scattering, as well as the details on the experimental set-up can be found in the Experimental Section.

Individual host-and guest polymers

The

𝑅(𝑞) = 𝐾𝐶𝑀 • exp (- 1 3 𝑞 2 𝑅 𝑔 2 ) (II.2)
where 𝐾 is the optical constant (the details on the 𝐾 calculation may be found in the Experimental section), 𝐶 -concentration of the sample [g/mL], 𝑀 -molecular weight [g/mole].

The exponential part in the expression (II.2) is the Guinier approximation for 𝑃(𝑞) -SLS form factor. 46 Taking the logarithm on both sides of (II.2) and subsequent plotting the Rayleigh ration 𝑅(𝑞) as a function of 𝑞 2 leads to a straight line with a slope coefficient 𝑎 = -

1 3
𝑅 𝑔 2 and an intercept with the y-axis equal to ln (𝐾𝐶𝑀). This procedure also provides a simple way to calculate the molecular weight, without the need for determination the partial specific volumes of the polymers required for the 𝑀 𝑤 determination from SAXS data.

The DLS experiments performed in parallel with SLS showed the presence of two populations of scatterers in the solutions of all polymers with the 1 st one corresponding to the individual polymer molecules (𝑅 𝐻 = 6-12 nm in Table II.9) and the 2 nd one to the aggregates or cross-linked polymers (𝑅 𝐻 ca. 100 nm). However, converting the DLS data into Size-Volume distributions using the Mie theory showed that the bigger scatterers typically constitute not more than 0.5

Vol.% for all polymers but for DT40-gPEG-βCD, where their amount is one order of magnitude higher (5 Vol.%). Given this, before the determination of 𝑅 𝑔 of DT40-gPEG-βCD, the SLS data were treated so as to eliminate the contribution from the slow relaxation mode (bigger scatterers) to the scattering intensity at all 𝑞-values. Nevertheless, the obtained 𝑅 𝑔 were a way higher than the 𝑅 𝐻 of the 1 st peak in DLS for all the polymers (Table II.9), indicating that the impact of aggregation and/or cross-linking on the SLS results is still substantial and difficult to fully get rid of.

Though the obtained 𝑀 𝑤 are considerably higher than the theoretical ones for all investigated polymers (Table II.9), the most drastic discrepancy was found, once again, for DT40-gPEG-βCD (4908 kDa instead of expected 116 kDa), which is consistent with the DLS results. When constructing the larger structure, the substructure is connected at the ends of the red chain. All data were modelled assuming theta solvent conditions. The quality of a fit may be estimated from the Chi-square ( 2 ) parameter which is equal to 1 when a perfect model is chosen. 49 The proximity of the  2 values to unity (Table II.11) indicates that the applied models are appropriate for describing the studied host-guest polymers.

Furthermore, the 𝑅 𝑔 values derived from modelling are in a very good agreement with those obtained by Guinier approximation and inverse Fourier transformation of the scattering curves (Table II.10).

Host-guest nanoparticles of different types

Host II.12).

From the SLS-data, sizes (𝑅 𝑔 ) and molecular weights of the HG-nanoassemblies were calculated using the Guinier approximation, in a way similar to that described in previous section for individual polymers (Table II.12). DLS experiments on the same samples were conducted in parallel. Due to the high scattering intensity from the nanoassemblies, we didn't detect any impact from aggregates or/and cross-linked molecules as it was the case with the individual polymers. In the "𝑅 𝐻 -intensity" and "𝑅 𝐻 -volume" distributions single peak was observed for all types of nanoassemblies. By combining the SLS and DLS data we could estimate the 𝑅 𝑔 /𝑅 𝐻 ratios which turn up to lie in the range 0.54-0.59 (Table II.12). It is significantly lower than 0.775 -the indicated in literature criterion for homogenous sphere. 44 The disparity between the 𝑅 𝑔 and 𝑅 𝐻 , might be related to non-uniform radial density distribution in highly water-swollen polysaccharide-based HG-nanoassemblies. Thus, a lot of loosely attached pending polymer chains should be present on the nanoparticles surface. (19), in agreement with the assumption that the packing density is lower in the mixed system than in the system without PEG spacer.

HG nanoassemblies for the SAXS investigation were prepared at the same as for SLS C CD/Ada = 5 Unfortunately, no definitive conclusions on the nanoassemblies shape could be made based on the 𝑅 𝑔 /𝑅 𝐻 ratios derived from the SAXS data. Indeed, they lie in the range of 0.24-0.41 for all investigated cases (Table II.13), which is much lower than the previously mentioned criterion for hard homogeneous sphere. In addition to the reasons given above for SLS, that is a non-uniform density distribution in the particles, this disparity can be also related to the fact that the hydrodynamic diameters of the nanoassemblies are considerably higher than 100 nm -the size corresponding to the 𝑞 𝑚𝑖𝑛 limit of SAXS.

The modelling was performed by fitting the SAXS scattering data for the nanoassemblies using a model developed by Stieger, Richtering, Pedersen, and Lindner 50 for pNIPAm microgel particles.

The internal structure of the nanoassemblies, resulting from the host and guest polymer associations, could be related to the one of a hydrogel with a correlation length  l . The 51 For instance, the SAXS-derived correlation length values were used to estimate the size of the inhomogeneities in the structure of thermosensitive pNIPAm microgels; the results were consistent with those obtained by cryo-TEM. 52 In our case, the substitution of DT40Ada9 (P2) by the guest polymer with PEG spacer DT40-gPEG-Ada2 (P5) leads to a moderate increase in correlation length from 2.1 to 4.6 nm (Table II.14), which is coherent with the less compact structure of P5 evidenced by the SLS data (Table II.12). In the case of "mixed" particles containing host polymer with PEG spacer (P6) the increase of  l is much more pronounced, i.e. from 2.1 to 10.5 nm. Finally, the nanoassemblies composed of both guest and host polymers with PEG spacer (P4) show the highest correlation length of 16.9 nm. The results clearly demonstrate the impact of the PEG spacers on the internal structure of the nanoassemblies.

The more PEG spacers are used, the less compact are the internal structures, as evidenced by the values of correlation lengths (samples P3, P4 and P6 in Table II.14).

To summarize, by the combination of SLS/DLS and SAXS modelling we proved that the nature of the individual host-guest polymers has a strong impact on the size and internal structure of the resulting HG nanoassemblies. The nanoassemblies composed of the polymers with PEG spacer have lower sizes and significantly lower compactness comparing to their PEG-free counterparts, as evidenced by their large correlation length values. At the same time, all types of nanoassemblies appear to have non-uniform radial density distribution with denser cores and pending polymer chains at the periphery.

Conclusions

In this chapter, we have proposed a synthetic pathway to novel type of host-guest polymers with hydrophilic PEG spacer. It relies on the use of thiol-substituted dextrans as a unique platform, which is subsequently transformed into either PEG-βCD grafted host or PEG-Ada grafted guest polymers via combination of nucleophile-mediated thiol-click and CuAAC reactions.

The polymers were characterized by NMR, light scattering techniques and SAXS. We performed modelling of the SAXS data for various types of host and guest polymers. All of them except DT40-gPEG-βCD show comb-like structure in water solutions. The latter could be modelled as a star-like structure composed of at least 4 comb-like branches. It might serve as an indication that cross-linking of the alkyne-modified dextran scaffolds takes place during the CuAAC stage of the synthesis. It can be related to the fact that alkynes in DT40-g-PEG-hex-5-ynoate precursor, being located at the ends of flexible PEG chains, are more likely to approach each other and undergo acetylenic cross-coupling.

The binding properties of the host and guest polymers in solution and on solid surface were studied by ITC and SPR respectively and compared to those of previously reported βCDand Ada-substituted dextrans with short hydrophobic spacers. The latter was done with the aim of understanding the impact of flexible PEG on the availability of grafted groups for the inclusion complexes formation. Both ITC and SPR results were coherent, indicating a significant improvement of the binding properties of DT40-gPEG-Ada compared to DT40Ada (5-fold increase of the affinity constant K a for comparable substitution degrees). On the other hand, the presence of PEG spacer proved to be less beneficial in the case of host polymers, where DT40-gPEG-βCD showed in average 4 times lower K a than those of DT70GPβCD.

At the next stage we investigated the formation of nanoassemblies between various types of host-guest polymers by combination of light scattering and SAXS. The nanoassemblies were formed spontaneously upon mixing the host and guest polymer solutions and we found that their structure was not influenced by the mixing process. The low values obtained for the R g /R h ratio (<0.55) were interpreted as the result of non-uniform radial density distribution, i.e. with denser core and looser periphery parts.

In order to get insight into the internal structure of the nanoassemblies, we used a simplified model of spherical gel-like particles with uniform density distribution, characterized by a R g and a correlation length  l . The nature of individual polymers proved to have a significant influence on the size and internal organization of the nanoassemblies. In general, particles containing DT40-gPEG-βCD are smaller and have less compact structure (larger  l ) than those containing DT70GPβCD. The same effect, though to a much lesser extent, holds true for the particles composed of DT40-gPEG-Ada as a guest polymer.

The obtained results contribute to design of host-guest polymers with improved properties, i.e.

polymers capable of self-assembling into more stable, biodegradable nanoparticles with "stealth" properties and attractive drug delivery profiles.

Experimental section

Materials and reagents

Pyridine (99.9%, anhydrous), 4-nitrophenyl chloroformate (4-NC, 96%), cysteamine (98+ %), 54 and tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA) 28 were synthesized using the literature data. Monoazidated on the primary side βCD (βCD-N 3 ) was prepared using a previously reported 2-stage procedure, which may be found elsewhere.

Fourier transform infrared spectroscopy (FT-IR): FT-IR spectra were recorded on a Bruker

Tensor 27 FT-IR spectrometer in the attenuated total reflection (ATR) mode using 32 or 64 scans at a resolution of 4 wavenumbers.

Ultrafiltration: Ultrafiltration was carried out under N 2 atmosphere using Amicon equipment and regenerated cellulose membranes with MWCO 10,000.

Dialysis: Dialysis was performed using Spectra/Por molecular porous tubing with MWCO 6,000-8,000.

Freeze-drying: Solutions of the water-soluble polymers typically purified by ultrafiltration or dialysis were freeze-dried on CHRIST ALPHA 1-2 LD plus lyophilizator. where λ -laser wavelength, 𝑛 -dispersant refractive index and 2θ is the detection angle. By fitting the autocorrelation function with a suitable algorithm one can determine the diffusion coefficient 𝐷. The hydrodynamic radius 𝑅 ℎ can be then calculated using the Stokes-Einstein equation:

Isothermal titration microcalorimetry

𝑅 ℎ = 𝑘 𝑏 𝑇 6𝜋𝜂𝐷 (II.5)
where T is the temperature, 𝜂 is the viscosity of the solvent and 𝑘 𝑏 is the Boltzmann constant (1.23•10 -23 J/K).

If determined the solution has a rather narrow Gaussian-like distribution of sizes, Cumulants analysis of the data is appropriate; the latter determines the mean size (Z-average) and PdI values of the particles. If more than single exponential decay rates are observed (as it was the case for the individual polymers, Table II.9), the autocorrelation function is treated by Distribution analysis which relies on non-negative least squares (NNLS) algorithm and calculates the distribution of sizes by their scattering intensity. Finally, the Size-Intensity distribution might be converted into Size-Volume distribution by a built-in software, which applies Mie theory. 55 Static light scattering: SLS measurements were done using an ALV-CGS3system operating with a vertically polarized laser with wavelength λ = 632 nm. Individual polymer solutions in water used for SLS measurements or for nanoassemblies formation were filtered with a 0.40 μm syringe filters prior to use. The measurements were done at 20°C over a wide range of scattering wave vectors 𝑞 (0.00046-0.00249 Å -1 ). The normalized electric field autocorrelation functions (𝑔 1 (𝑡)) obtained from the multi-angle DLS measurements were analyzed in terms of a relaxation time (τ) distribution using the REPES routine 56 : 𝑔 1 (𝑡) = ∫ 𝐴(𝜏)exp (-𝑡 𝜏) ⁄ 𝑑𝜏.

For individual polymers, in addition to fast relaxation mode (short 𝜏) we observed a slow relaxation mode (longer 𝜏), which was particularly the case for the host polymer with the PEG spacer (DT40-gPEG-βCD). The slow relaxation mode has two possible origins. The first, trivially, is the presence of spurious big scatterers problematic to remove by filtration. The second origin consists in possible partial aggregation of the polymers or/and cross-linking issues. Hence, in the case of DT40-gPEG-βCD for the construction of Rayleigh ratio dependency on 𝑞 and 𝑅 𝑔 determination we were considering only fast relaxation mode by eliminating the slow mode contribution to the scattering intensity determined from multi-angle DLS.

To establish the values of the detector constants an absolute calibration with toluene was used. 46 The Rayleigh ratios (𝑅(𝑞)) for different scattering angles were calculated based on the scattering intensity:

𝑅(𝑞) = 𝐼 𝑠 (𝑞) • 𝑟 2 𝐼 0 • 𝑉 𝑠 (𝑞) (II.6)
where 𝐼 𝑠 (𝑞)the intensity of the scattered light is measured at the scattering wave vector 𝑞, 𝐼 0 is the intensity of the used laser, 𝑉 𝑠 (𝑞) is the scattering volume and 𝑟 is the distance from the scattering volume to the detector. Rayleigh ratio may be expressed through the Guinier approximation of the form factor:

𝑅(𝑞) = 𝐾𝐶𝑀 • exp (- 1 3 𝑞 2 𝑅 𝑔 2 ) (II.2)
where 𝐾 is the optical contrast constant, 𝐶 -concentration of the sample [g/mL], 𝑀 -molecular weight [g/mole]. The exponent part in the expression (II.2) is the Guinier approximation for the form factor of a scatterer𝑃(𝑞) = exp (-

1 3 𝑞 2 𝑅 𝑔 2 )
. 46 Taking the logarithm on both sides of (II.2)

and subsequent plotting the Rayleigh ration 𝑅(𝑞) as a function of 𝑞 2 leads to a straight line with a slope coefficient 𝑎 = -

1 3
𝑅 𝑔 2 and an intercept with the y-axis equal to ln (𝐾𝐶𝑀). The optical contrast constant was calculated according to the equation:

𝐾 = 4𝜋 2 𝑛 0 2 (𝑑𝑛 𝑑𝐶 ⁄ ) 2 𝑁 𝐴 𝜆 0 4 (II.7)
where 𝑛 0 is the refractive index of the solvent, 𝜆 0 is the wavelength of the laser beam in vacuum, 𝑁 𝐴 is the Avogadro number and 𝑑𝑛 𝑑𝐶 ⁄ the differential refractive index increment of the dissolved molecules. We were using 𝑑𝑛 𝑑𝐶 ⁄ = 0.145 mL•g -1 reported as the average value for most polysaccharides. 46 Small-angle X-ray scattering, General: SAXS data were recorded at a wavelength of 1.54 Å, on a flux and background optimized camera from Bruker AXS. 57 The instrument uses a rotating Cu anode as a source and is equipped with Montel multilayer optics, and a home-built (J.S.

Pedersen, Aarhus University) compact "scatterless" slit is placed before the samples, which were measured in quartz capillaries of 2 mm diameter. A so-called low-q instrumental setup with 1061 mm sample-to-detector distance was applied. Individual polymer solutions in water used for SLS measurements or for nanoassemblies formation were filtered with a 0.40 μm syringe filters prior to use. In the case of HG nanoassemblies two ways of formulation were investigated: a) successive injection of the host and guest polymer solutions into the capillary while keeping the βCD/Ada = 1:1; b) injection of the nanoassemblies, preliminary formulated by stirring the mixture of the polymers at 800 rpm. The acquisition time for the data was 1800 s. All the measurements were performed at 25°C. Individual polymer and particle scattering patterns were obtained by subtracting from the raw curves the background from Milli-Q water measured with approximately homogenous internal density distribution), the 𝑅 𝑔 can be used for calculation of the particle dimensions. For instance, the average radius of compact spherical particles is known to be:

𝑅 = 𝑅 𝑔 • √ 5 3 (II.11)
The first way to obtain the 𝑅 𝑔 values consists in constructing a so-called Guinier plot where the natural logarithm of the intensity ln 𝐼(𝑞) is plotted versus 𝑞 2 :

lnΔ 𝐼(𝑞) = ln 𝐼(0) - 𝑅 𝑔 2 𝑞 2 3 (II.12)
The 𝑅 𝑔 is obtained from the slope of the straight-line fit to the Guinier plot, which is equal to

- 𝑅 𝑔 2 3
. As it was mentioned above, the plot should be constructed using the data points from the low 𝑞 range, where 𝑞𝑅 𝑔 < 1 criterion holds.

The alternative approach to the calculation of 𝑅 𝑔 from the SAXS data relies upon the Fouriertransformation of the oscillating part of the form factor in the middle-𝑞 range, based on the equation:

𝑃(𝑞) = 4𝜋 ∫ 𝑝(𝑟) sin (𝑞𝑟) 𝑞𝑟 𝑑𝑟 ∞ 0 (II.13)
The resulting curve 𝑝(𝑟) is a so-called "pair-distance distribution function" or PDDF and is essentially a histogram of distances that could be found inside the scatterer. Parameters such as the particle maximal dimension and 𝑅 𝑔 might be then evaluated from a PDDF-curve. 61

SAXS data modelling: a) Form factor for comb-like polymers and for stars with comb-like structured arms of Gaussian chains

We use the formalism of Svaneborg and Pedersen 49 and by this reproduce the results of Casassa and Berry 62 during the

L s l/2 l/2
X derivation. The basic substructure and the contour length of the cub chains are given in the figure. The X indicates the point for which the scattering amplitude is calculated. When constructing the larger structure, the substructure is connected at the ends of the red chain.

The form factor of the subunit is: with 𝑥 = 𝑞 2 𝑏𝑙/6, where q is the modulus of the scattering vector and b is the Kuhn length.

𝑃 𝑠𝑢𝑏 (𝑞) =
Furthermore:

𝐴(𝑞, 𝑙) = (1 -exp(-𝑥)) 𝑥 (II.16)
The amplitude calculated with respect to X is:

𝐴 𝑋 (𝑞) = ( 𝑙 2 ) 𝐴 (𝑞, 𝑙 2
) + (𝑞, 𝑙/2) ((𝑙/2)𝐴 (𝑞, 𝑙 2 ) + 𝐿 𝑠 𝐴(𝑞, 𝐿 𝑠 )) (II.17 where:

(𝑞, 𝑙) = exp (-𝑥)

(II.18)
For a chain or arm consisting of N substructures (one comb-like) one has, according to Svaneborg and Pedersen 49 :

𝑃 𝑎𝑟𝑚 (𝑞) = 1 𝑁 𝑃 𝑠𝑢𝑏 (𝑞, 𝑙) + 2 1 𝑁 2 (𝑞, 𝑙) 𝑁 -𝑁(𝑞, 𝑙) + 𝑁 -1 ((𝑞, 𝑙) -1) 2 𝐴 𝑋 (𝑞) 2 (II.19)
Similarly, the scattering amplitude from the end point is:

𝐴 𝑎𝑟𝑚 (𝑞) = 1 𝑁 (𝑞, 𝑙) 𝑁 -1 (𝑞, 𝑙) -1 𝐴 𝑋 (𝑞) (II.20)
These last two expressions can now be combined in the star form factor 63 for a structure with f arms:

𝑃 𝑠𝑡𝑎𝑟 (𝑞) = 𝑃 𝑎𝑟𝑚 (𝑞, 𝑙) + (𝑓 -1)𝐴 𝑎𝑟𝑚 (𝑞) 2 (II.21)

b) Form factor for gel-like spherical particles

The particles were fitted with a polydisperse spherical form factor with a Lorentzian function added, similar to the form factor used by Stieger, Richtering, Pedersen, and Lindner. 50 The Lorentzian term accounts for the fluctuations of the polymer network:

𝐼(𝑞) = 𝐼 𝑠𝑝ℎ (𝑞) + 𝐼 𝑓𝑙𝑢𝑐𝑡 (𝑞) (II.22)
with 𝐼 𝑠𝑝ℎ (𝑞) equal to:

𝐼 𝑠𝑝ℎ (𝑞) = 𝑁 • (𝛥𝜌) 2 • 𝐹(𝑞, 〈𝑅〉, 𝜎 𝑝𝑜𝑙𝑦 ) • 𝑆(𝑞, 〈𝑅〉) (II.23)
𝛥𝜌 is the difference in electron density between the nanoparticles and the solvent, 𝑁 denotes the number density of the nanoparticles. Due to the relatively low concentrations of the samples the structure factor 𝑆(𝑞, 〈𝑅〉 was approximated to 1 while fitting the data.

The form factor 𝐹 is expressed as:

dR qR R R D R q F poly poly 2 0 int ) ( ) , , ( ) , , (       (II.24)
Where R is the average size,

             3 ) ( ) ( cos ) ( sin 3 ) ( x x x x x (II.25)
The intensity size distribution is related to the number size distribution ) , , (

poly n R R D  by: ) , , ( ) , , ( 2 int poly n poly R R D V R R D    (II.26)
where V is the volume of the particle with radius R. A Gaussian function was used for the intensity size distribution:

           2 2 2 2 2 int 2 ) ( exp 2 1 ) , , ( R R R R R R D poly poly poly    (II.27)
The fluctuation term, 𝐼 𝑓𝑙𝑢𝑐𝑡 (𝑞), is a lorentzain:

𝐼 𝑓𝑙𝑢𝑐𝑡 (𝑞) = 𝐼 𝑓𝑙𝑢𝑐𝑡 (0) 1 +  2 𝑞 2 (II.28)
where  is a correlation length.

Surface plasmon resonance: SPR measurements were carried out with a Spreeta SPR-EVM-BT from Texas Instruments. The light of an infrared light-emitting diode (LED; λ = 840 nm) reaches the sensor surface at a range of angles above the critical angle. A reflectivity-versus-angle spectrum is obtained, and an apparent refractive index (RI) is derived from it by the Spreeta software. The sensor surface is a gold layer (ca. 50 nm). In the case of adsorption on the golden surface the RI changes provide information on the adsorbed layer parameters. The sensor is integrated in a flow cell and the measurements were carried out at 23 °C using a continuous water flow (3 mL/h) which was delivered by a syringe pump (Kd Scientific), and a Rheodyne injection valve to switch to the sample solution.

The sensor surface was cleaned with a 4 % CrO 3 solution in water and rinsed with MeOH before each experiment. After settling the sensor into the flow cell, an in-situ cleaning was performed with one injection (0.1 mL) of a solution of 0.1 M NaOH and 1 % Triton X-100 in water followed by 3 injections (0.1 mL) of 10 % EtOH in water. The sensor was calibrated in pure water, assuming a RI value of 1.33300, and the RI variations after the different polymer injections were monitored as a function of time.

After the cleaning and calibration the adsorption experiments were performed as follows. Three to four consecutive injections of 0.1 mL of pβCDN+ in 0.5 M NaCl (at 2 g/L) were carried out to saturate the gold surface of the sensor. This was followed by 3-4 consecutive injections (0.1 mL) of the host-guest polymers of different types (1 g/L). Then the ΔRI 2 and ΔRI 2 were measured as the deviations of refractive index due to the 2 nd and 3 rd layer deposition respectively 20 min after the first injections.

From the values of ΔRI the adsorbed amounts of the polymers may be estimated based on the following equation 36 :

𝛥𝑅𝐼 = 𝑚(𝑛 𝑝𝑜𝑙 -𝑛 𝑠 )[1 -𝑒𝑥𝑝(-2𝑑 𝑝𝑜𝑙 𝑙 𝑑 ⁄ )] (II.29)
where 𝑛 𝑠 and 𝑛 𝑝𝑜𝑙 are refractive indexes of the solvent and the polymer respectively, 𝑑 𝑝𝑜𝑙 is the thickness of deposited polymer layer and 𝑙 𝑑 is the depth of penetration of the evanescent electromagnetic field (typically 25-50% of the probing light wavelength). In the present work it was roughly estimated as 37% (310.8 nm), according to the literature data. 36,37 We assumed 𝑙 𝑑 to be constant upon each subsequent polymer adsorption, given the low density of highly hydrated layers and their thickness being much lower than 310.8 nm. The calibration coefficient m of the sensor was estimated at 1.

Since 𝑑 𝑝𝑜𝑙 ≪ 𝑙 𝑑 , ΔRI is directly proportional to the layer thickness and the equation ( 1) may be rewritten as:

𝛥𝑅𝐼 = 𝑚(𝑛 𝑝𝑜𝑙 -𝑛 𝑠 ) (2𝑑 𝑝𝑜𝑙 𝑙 𝑑 ⁄ ) (II.30)
The deposited polymer being highly swollen with the solvent leads 𝑛 𝑝𝑜𝑙 ~ 𝑛 𝑠 which means that:

𝑛 𝑝𝑜𝑙 -𝑛 𝑠 = 𝐶 𝑠𝑢𝑟𝑓 (𝑑𝑛 𝑑𝑐) ⁄ 𝑝𝑜𝑙 = (𝑑𝑛 𝑑𝑐) ⁄ 𝑝𝑜𝑙 • (𝑄 𝑎𝑑𝑠 𝑑 𝑝𝑜𝑙 ) ⁄ (II.31)
where 𝐶 𝑠𝑢𝑟𝑓 is the surface concentration and 𝑄 𝑎𝑑𝑠 is the adsorbed amount of the polymer;

𝑑𝑛 𝑑𝑐 ⁄ is the RI increment of the polymer with respect to water. In this work it was assumed as 0.145 mL/g for both pβCDN+ and dextran-based host-guest polymers 37 . Combining (II.28) and

(II.29) one obtains already mentioned in the main part equation ( 1) to calculate the adsorbed amounts:

𝑄 𝑎𝑑𝑠 = ∆𝑅𝐼 • 𝑙 𝑑 2𝑚 • (𝑑𝑛 𝑑𝑐) ⁄ 𝑝𝑜𝑙 (II.1)

Synthetic procedures Thiolated Dextran (DT40-SH):

In the first stage the dextran hydroxyl groups were activated with 4-NC. 22 Typically dextran (4.0 g, 24.6 mmol of AHG) was dissolved in 120 ml of anhydrous DMF containing 2 w/v% of LiCl at 90°C, under vigorous stirring. The reaction mixture was cooled to 0°C and pyridine (1.5 ml, 20 mmol, molar ratio pyridine/AHG = 0. (s, anomeric proton), 5.17 and 5.31 (s, glucosidic protons at positions bearing thiol groups).

PEG-acryloyl-5-hexynoate: 5-hexynoic acid (0.69 ml, 6.2 mmol) was dissolved in 15 ml of DCM.

A catalytic amount of DMAP (87 mg, 0.72 mmol) was added followed by PEG acrylate (1.60 ml, 4.8 mmol). The solution was cooled until 3-4°C and DCC (1.09 g, 5.3 mmol) was added as a solid.

The reaction mixture was stirred at 3°C under argon for 3h and then at room temperature overnight. The formed N,N´-dicyclohexylurea was filtered off through a sintered glass funnel.

After removing the DCM under reduced pressure the obtained product was dried under vacuum and used in the next step without purification. The mixture which gets turbid almost immediately after the complete dissolving of NH 4 Cl was concentrated to approximately 3/5 of the initial volume by blowing the air over its surface. The obtained precipitate was filtered off and washed two times with 65 ml of ice cold water and 130 ml of acetone. After drying in vacuum the product was purified by dissolving it in 10 ml of DMSO and precipitation in 140 ml of water. In the following step the product was dissolved in 100 ml of boiling water and cooled in an ice bath. Finally the formed precipitate was filtered off, washed with 30 ml of water and 60 ml of acetone and isolated by drying in vacuum overnight at 60°C, over P 2 O 5 . The TLC of the obtained 6A-O-p-toluenesulfonyl-βcyclodextrin (βCD-OTs)

showed no residual native βCD and bitosylated product. Yield: 6.54 g (22.6%). 1 H-NMR (DMSOd 6 ) δ = 2.43 (s, 3H), 3.17

Introduction a. PEG and PEGylation. General information

Nowadays, poly(ethylene glycol) (PEG) is by far the most commonly used non-ionic hydrophilic polymer in cosmetic and health care industries. 1,2 The most basic advantageous properties of PEG include:  Easiness of preparation of polymers with a wide range of molar masses, from 400 g•mole -1 up 4000 kg•mole -1 and higher  The most common way of PEG synthesis, i.e. anionic polymerization of ethylene oxide allows preparing PEGs with the polydispersity indexes (PDIs) as low as 1.01 1 ; this feature is of high importance in biomedical applications to gain the reproducibility of the bioavailability and immunogenicity profiles of PEG-containing formulations  PEG has an amphiphilic character, i.e. shows a high solubility both in water and in organic solvents; the latter leads to easiness of its end-modifications with various hydrophobic functionalities, such as targeting groups, fluorescent labels etc.

 Very low toxicity (LD 50 = 50 g•kg -1 body weight for oral administration in mice and guinea pigs)

The covalent or non-covalent coupling of drugs or drug-containing nanoscale carriers with PEG is referred to as PEGylation. The first coupling of PEG to a protein was reported back in the 1970s by Abuchowski et al., and the resulting PEGylated albumin exhibited improved pharmacokinetic profile, i.e. non-immunogenicity and increased blood circulation time from 12h to 48h. 3 Since then, a great number of PEG conjugates with proteins, polypeptides, DNA, RNA and small molecule therapeutics were found to show significantly improved, as compared to non-PEGylated formulations, performances, with some of them getting the FDA approval and reaching the market. As for 2011, there was nine FDA-approved PEGylated protein drugs already on the market and much more on the last stages of clinical trials. 4 In the case of such PEG-drug conjugates there are a few main reasons behind the beneficial impact of PEG: o Increase of their molar mass leads to reduced rates of kidney excretion and results in a prolonged blood circulation time of the drug. For this reason proteins and small molecule drugs are usually coupled with relatively high molecular mass PEGs (20-50 kg•mole -1 ) o Due to the high flexibility of PEG chains, the latter exist in a form of so-called "conformational cloud" 1 , its polymer chains experience fast transitions between a large number of possible conformations in water. "Conformational cloud" creates an effect of sterical hindrance on the surface of a PEGylated drug carrier, thus preventing their enzymatic cleavage, as well as opsonization by blood proteins 5 and uptake by reticuloendothelial system (RES) 6,7 This phenomenon is also known as "stealth" effect. o In the case of charged nanomedicines such as PEI-based gene carriers, PEGylation allows shielding their positive charges, thereby diminishing non-specific interactions with blood components and reducing toxicity 8,9 The initial works describing the PEGylation of nanoparticulate drug carriers date back to the beginning of 1990s. At the first stage, PEG brush layers were attached to liposomes, which resulted in sterical stabilization and increased blood circulation times of the latter. 10 Further, in 1994, Gref et al. reported reduced rates of liver clearance for poly(lactide-co-glycolide) (PLGA) nanospheres coated with 20 kg•mole -1 PEG. 11 These early studies became the basis for the development of the only FDA-approved particulate drug delivery system on the market -Doxil/Caelyx, composed of PEGylated liposomes loaded with anticancer doxorubicin. 1 Beneficial impact of PEG coating on the nanoscale drug carriers is due to the similar reasons as those listed above for protein-, gene-and small molecule-based drugs. In the case of intravenous administration PEGylated nanocarriers are sterically stabilized against aggregation and uptake by the blood mononuclear phagocyte system (MPS) 7,12,13 ; PEG "corona" leads to higher apparent sizes, thus increasing the efficiency of nanocarriers accumulation in cancer and/or inflamed tissues due to the enhanced permeability and retention effect (EPR). Altogether these effects allow for lower frequencies of administration and decreased payloads of drugs in nanoparticulate carriers, which in turn improves the life quality of the patients. It should be noted that lower PEG molar masses (1-5 kg•mole -1 ) are typically used for modification of nanoparticles, as compared to proteins and small molecules. This is explained by the possibility of linking multiple PEG grafts to a single nanoparticle, thus still significantly increasing its molar mass and hydrodynamic diameter. 2 In addition, after the therapeutic function of a nanocarrier is completed 1-5 kg•mole -1 PEGs can be much easily excreted from the body though the renal clearance.

Besides all the aforementioned positive qualities of PEG, its biomedical use can also be associated with a number of drawbacks and unfavorable effects. 1,14 For instance, PEGylated liposomal carriers such as Doxil/Caelyx were reported to provoke immunogenic hypersensitivity reactions (HSR) that can provoke an anaphylactic shock in up to 25% of patients. 15 However, to date there is no clear evidence as to whether the HSR is caused by PEG alone or by a complex combination of several factors. The second important drawback of PEG consists in its nonbiodegradability. Therefore, the highest molar mass appropriate for in vivo applications cannot exceed the renal clearance threshold, the latter being around 60 kg•mole -1 (corresponds to hydrodynamic radius of 3.5 nm). 5,16,17 In several studies researchers observed accelerated blood clearance of PEGylated nanomedicines occurring after repeated administrations. 18,19 Though the mechanism behind it is not fully understood, it was suggested that anti-PEG antibodies might be formed in spleen. Finally, as compared to polymers with carbon backbone, PEG is more susceptible to undergo peroxide-mediated degradation at increased temperatures and under stress, leading to formation of toxic byproducts. 1 The PEGylation might be achieved using both covalent and non-covalent modification methods.

To date, covalent PEGylation techniques are more numerous and usually rely either on preparation of PEG-containing block copolymers with subsequent drug-loaded polymeric micelles formation 13 or on covalent post-modification of small molecule drugs, proteins or nanocarriers. 16,20,21 For solid NPs, such as gold, thiol-chemistry is the classic approach for PEGylation, where a sulfhydryl-capped PEG chain adheres to the gold surface. 2 Non-covalent techniques are more commonly used for PEGylation of nanoparticulate carriers and include: coating the hydrophobic nanoparticle surface with lipid-PEG conjugates (was applied for stabilization of single-walled carbon nanotubes) 22 ; simple inclusion of lipid-PEG conjugates in the lipid mixture during the preparation of liposomal systems 23 ; formation of coordination complexes between Ni 2+ or Cu 2+ chelate-functionalized PEGs and nitrogen of histidine side chains in a protein 24 ; physical adsorption by "host-guest" interactions. 25,26,27 Noncovalent approach can avoid some important shortcomings of covalent PEGylation such as decrease of activity in case of protein-based drugs and tedious purification steps that decrease the yield of the final product.

b. βCD host-guest chemistry and PEGylation

During the last two decades a high number of researches were aiming at diminishing the phenomenon of initial "burst release" of actives from nanoscale drug delivery carriers. 28 Along with superficial cross-linking, adsorption or impregnation of drug carriers with CD-containing materials was found to be an efficient strategy to address this issue. For instance, Quaglia et al.

found that co-encapsulation of hydroxypropyl-βCD into PLGA microspheres leads to a significant decrease of the initial "burst" of loaded insulin. 29 El Fagui and Amiel reported a procedure of benzophenone-loaded polylactic acid (PLA) nanoparticles coating with neutral epichlorohydrincrosslinked poly-βCD and showed it beneficial impact on the kinetic profile of benzophenone release, governed by the inclusion complexation of the latter with the βCDs in the shell of the nanocarriers. 30 In a more recent example membrane fusion between liposomes and cyclodextrin vesicles was exploited to prepare hybrid assemblies composed of liposomal core covered with layers of βCD amphiphiles. 31 Yet another way of introducing βCD in nanoscale drug carriers consists in assembling βCD polymers with "guest"-labeled amphiphilic copolymers. 32,33 Although a number of works has already reported host-guest anchoring of PEG chains onto βcyclodextrin containing surfaces 9,25 , to the best of our knowledge, only few studies of the multivalence effect, when multiple poly(ethylene oxide-co-propylene oxide) (PEPO) grafts and guest groups were attached to a polymer backbone, on the PEGylation efficiency have been done so far. 34 Cooperative character of multivalent host-guest interactions leads to strong interpolymer binding, which might be successfully exploited for the construction of various supramolecular architectures. For instance, Dubacheva et al. 35 used ferrocene (Fc)-guest and βCD-host derivatives of the same polymer, poly(N-hydroxypropylmethacrylamide (PHPMA), to construct polymeric multilayer films with no limitation of the number of layer-by-layer (LbL) deposition steps. More recently, the same researchers observed a phenomenon of so-called "superselectivity" in the multivalent interactions between self-assembled monolayers (SAMs) functionalized with the Fc-guest and hyaluronic acid (HA) modified with βCDs. 36 It implies that the surface density of bound macromolecules increases faster than linearly with the density of surface binding sites. In this work we present a possible path for combination of the benefits of PEGylation and βCD-containing materials. We report a synthesis of novel (PEG, Ada)-grafted dextrans, prepared by copper-catalyzed azide-alkyne cycloaddition (CuAAC). These bifunctionalized "guest" polymers are bearing various amounts of 5000 g/mol PEG chains and hydrophobic adamantyl (Ada) groups at the same time. The ability of (PEG, Ada)-grafted dextrans to form inclusion complexes in solution and on surface with native βCD and βCDpolymers are investigated with the purpose of their further use for facile PEGylation of interfaces and nanoparticles via multivalent host-guest interactions.

This work has been published as an original article in Carbohydrate Polymers in 2015. 37

Synthesis of PEGylated guest polymers -D40-XGP-YAda-ZPEG

Copper-catalyzed azide-alkyne cycloaddition (CuAAC) was chosen as an efficient tool to couple both adamantyl and MeOPEG moieties to the dextran backbone. 38,39 Azide-substituted adamantane derivatives, azide-terminated MeOPEG and alkyne-substituted dextrans were chosen as building blocks for CuAAC. Azide-substituted adamantane derivative (Ada-N 3 , Scheme III.1a) was prepared according to the modified 2-step procedure used by Suzuki et al. 40 It involves tosylation of 1-adamantaneethanol in pyridine, followed by nucleophilic substitution with NaN 3 in DMSO. It should be noted that the obtained 1-adamantaneethyltosylate was not exposed to tedious column chromatography purification and after filtration on silica pad it contained some traces of native 1-adamantaneethanol. However, it was not a problem due to its inability to react with NaN 3 in DMSO during the second step.

Azide-terminated MeOPEG (MeOPEG-N 3 ) was synthesized by a two-step procedure (Scheme III.1b). The terminal hydroxyls of PEG were first converted to chlorides with SOCl 2 , according to a previously described method. 41 The chlorides were subsequently converted into azides by S N 2 substitution with an excess of NaN 3 in DMF. During the purification stage washing with water proved to be crucial for the full elimination of unreacted NaN 3 .

Dextran with a molecular weight of 40 000 g•mole -1 was modified with alkynes using the basic epoxide opening of glycidyl propargyl ether in water, according to the procedure described in literature (Experimental section, Scheme III.2). 42 Choice of the ether bond was made considering its hydrolytic stability and a relatively long resulting spacer between the dextran backbone and grafted alkynes. The latter proved to be important for the preservation of good inclusion properties of subsequently grafted host-guest functions.

Scheme III.2: Synthesis of terminal alkyne-substituted dextrans (D40-GP).

At the next stage, D40-GP-Ada-PEG with various DS were prepared by one-pot CuAAC (Scheme III.3). Pure DMSO as a solvent and Cu(MeCN) 4 PF 6 /Na-Asc as a catalytic system were chosen because of the poor solubility of Ada-N 3 in water. A Cu(I)-stabilizing polytriazole ligand TBTA was applied to prevent any undesirable acetylenic cross-couplings often encountered when dealing with alkyne polymeric scaffolds. 42,43,44 Typically, in the first step the aimed amount of Ada-N 3 was "clicked" quantitatively; then 1.6-1.8 equivalents of MeOPEG-N 3 were added to the reaction mixture to consume the residual alkynes.

When performed under classical heating, due to the bulkiness of MeOPEG-N 3 , the CuAAC required very long reaction times (3 days) for the second step completion. Microwave-assisted heating was reported to significantly increase the reaction rates of CuAAC, especially when the reactants are bulky or polymeric units 45,46 . Indeed, when the same one-pot procedures were To evaluate binding properties of D40-GP-Ada-PEG polymers we studied their interaction in solution and on the surface with neutral epichlorhydrin-interconnected pβCD and positively charged pβCDN+, modified with quaternary ammonia, host polymers. The latter were prepared according to the previously reported by our scientific group procedure (described in the Experimental section). 47,48 These polymers have branched structure where βCD moieties may be thought of as balls interlinked by poly(2-hydroxypropyl)ether sequences of different lengths (the structural formulas can be found in Chapter 2, Figure II.17). Due to the seven-time excess of epichlorohydrin with respect to βCD, a part of it is consumed on the formation of polytails along with polybridges. The sample used in this work has a cyclodextrin weight ratio (g/g) of 58.2%, as determined by 1 H-NMR, a molecular weight M w of 208 kg•mole -1 , and a polydispersity index of 1.81, which were determined by size exclusion chromatography (SEC). To obtain positively charged host polymer, pβCD was modified with quaternary ammonia by reaction with 2,3-epoxypropyltrimethylammonium. The characteristics of all the host and guest polymers are reported in Table III

Binding properties of D40-GP-Ada-PEG in solution. ITC studies

The ability of D40-GP-Ada-PEG to form inclusion complexes with βCD and βCD-polymers was examined by isothermal titration microcalorimetry (ITC). The interactions of the polymers with monomeric βCD are highly exothermic (Table III.2) as expected, given the perfect matching in size between grafted Ada and βCD-cavities, leading to strong van der Waals interactions between them. The experimental titration curves (Figure III.3a) were fitted to a simple 1:1 interaction model, which assumes that each of the Ada groups grafted on one chain (12 to 25 per chain) acts independently from the others. Basic thermodynamic parameters derived from these experiments are shown in Table III.2. The association constants are as high as 4.0-6.0 • 10 4 L/mole, which is superior to previously described dextrans modified with Ada-groups via simple esterification by 1-adamantanecarbonyl chloride. 33 It might be explained by the presence of relatively long and hydrophilic spacer in D40-GP-Ada-PEG, the latter making Ada groups more loosely attached and available for complexation. As in the work by Wintgens et al., 33 a slight decrease of K a is observed when the DS by Ada-groups is increased from 5 to 10 mol%, which could be attributed to a gradual loss of their accessibility due to sterical hindrance effects.

At Likewise for monomeric βCD, the fits are done assuming a simple 1:1 interaction model. The imperfect matching between the experimental points and the fitting curves could be attributed to slight correlations between the interaction sites within a same chain. Nevertheless, the fits allowed us to estimate thermodynamic parameters of the interactions. The interactions strength is strongly dependent on the adamantyl content of the guest polymers. In the case of titration by pβCD, K a becomes almost one order of magnitude higher in favor of D40-28GP-10Ada-18PEG -more densely substituted by Ada groups and less by PEG chains. Such behavior might be attributed to interplay between the cooperativity effects of neighboring adamantyl groups and steric hindrance arising from PEG grafts. One can suppose that D40-28GP-5Ada-23PEG is less likely to approach and form stable complexes with pβCD due to a lower number of linking points (5 mol. % Ada) and higher density of bulky PEG grafts. The evolution of the TΔS values is in agreement with this assumption: pronouncedly negative TΔS values at 5 mol.% Ada, which could correspond to anti-cooperative binding, switch to positive TΔS values at 10 mol. % Ada, giving an indication of more cooperative binding (Table III The same tendency was observed in the case of titration by charged pβCDN+. Interestingly, the binding constants for the polymer bearing 5 mol.% of Ada groups are of the same order for pβCD and pβCDN+; on the other hand for the 10 mol.% polymer, K a is pronouncedly higher when titrated by pβCDN+.

Given the results above, in the next step we were interested in evaluation and comparison of the binding properties of (PEG, adamantyl)-grafted dextrans on the surface by SPR.

Binding properties of D40-GP-Ada-PEG on the surface. SPR studies

To perform SPR binding experiments, βCD-polymers were deposited as a 1 st layer on the golden surface of SPR sensor. When either pβCD or pβCDN+ solution is flowed over the sensor surface, the RI increases due to the physical adsorption of CD-polymers, driven by non-specific and electrostatic interactions between the polymers and the gold surface. In general, the experiment design was very similar to that used in Chapter 2 for the comparison of binding properties of guest polymers with and without PEG spacer, and is schematically illustrated in The adsorbed amounts of the polymers were estimated using the Equation II.1 (described in Chapter 2 and Experimental Section). Calculations showed that the adsorbed amount is roughly 1.4 times higher for the neutral pβCD -1.37 mg/m 2 -than for the charged pβCDN+ -0.97 mg/m 2 . Poorer adsorption characteristics of pβCDN+ might be related to strong hydration and spatial extension of the charged molecules, which prevents them from forming a strong linkage to the surface. In support of this, it should be noted that when pβCDN+ solution in pure H 2 O is Interestingly, the adsorption pattern on pβCDN+ is changed as well. No or little decrease in refractive index is observed straight after each consecutive injection. One can assume that due to the higher strength of interpolymer interaction with pβCDN+, there are less of loosely anchored D40-XGP-YAda-ZPEG molecules which are washed out after the deposition. Contrarily to the ITC study, where no interaction could be detected between D40-18GP-18PEG

and pβCDN+ in solution, the same polymer (D40-18GP-18PEG) exhibits non-negligible interaction with the gold/pβCDN+ surface. It might be related to a contribution from the gold surface which is less saturated with pβCDN+ than with pβCD. However, this cannot fully explain quite large adsorbed amount of free from Ada-groups D40-18GP-18PEG. As polysaccharides and polysaccharide-based nanoassemblies are typically characterized by slightly negative values of zeta potential 33 , non-specific electrostatic interactions could also be at the origin of the enhanced interpolymer interaction. Given that, the adsorption behavior of D40-GP-Ada-PEG on the positively charged pβCDN+-layer might be related to interplay between the host-guest and reinforced non-specific interactions in the system. As a result, the adsorbed amounts of D40-GP-Ada-PEG on pβCDN+ are roughly 3 times higher compared to the neutral pβCD (Figure III.7).

Figure III.7:

The adsorbed amounts of D40-GP-Ada-PEG with various DS (Ada): on pβCD (black bars, horizontal pattern); on pβCDN+ (red bars, vertical pattern).

Adsorption kinetics by SPR

The kinetics of the interaction between the pβCDN+ adsorbed layer and D40-28GP-10Ada- Since the obtained sensograms contain continuous real-time data, one can extract information on the kinetics of interpolymer interaction from them. For the first part of the sensorgram (until RI max is reached) the interaction might be schematically written as:

[R -Ada] sol. + [R ′ -CD] surf. ⇄ [Complex] surf. (III.1)
In this case the variation of RI versus time, due to complexation, is expressed by the following equation 49 :

𝑑RI 𝑑𝑡 ⁄ = 𝑘 𝑎 𝐶 𝐴𝑑𝑎 (RI 𝑚𝑎𝑥 -RI) + 𝑘 𝑑 (III.2)
where 𝐶 𝐴𝑑𝑎 is the concentration of adamantyl groups in the injected solution. The The average values of k a and k d obtained from the sensograms recorded at polymer concentrations varying in the range 0.08-0.20 g/L are of 7.0•10 2 mol -1 s -1 and 1.1•10 -3 s -1 (Table III.3). From this one can calculate the association constants K a using the following equation: The estimated average K a = 6.4•10 5 L/mole is more than one order of magnitude higher than the one determined by ITC for the interaction in solution (K a = 3.5•10 4 L/mole, Table III.2); it is also roughly 3 times higher than the K a between native βCD and monomeric adamantane derivatives previously. 42 The phenomenon of substantial increase of K a on the surface compared to the one in solution has been reported previously for polymeric guests and is attributed to increased cooperativity of interactions, the host moieties being constrained in close proximity on the surface. 50 Moreover, the K a is one order of magnitude higher than that reported for the interaction between pβCD and Ada-substituted pNIPAm (pNIPAm-Ada) on gold surface 50 , thus indicating an excellent affinity of D40-GP-Ada-PEG to pβCDN+. More specifically, association rate constants k a (700 mol -1 s -1 ) are around 3 times higher than the ones determined for PNIPAM-Ada/pβCD 50 or for adamantyl carboxylic acid/βCD systems. 51 At the same time, the dissociation rate constants k d (1.1•10 -3 s -1 ) are more than one order of magnitude lower than those for monomeric host-guest interactions (adamantyl carboxylic acid/βCD) and 3 times lower than for PNIPAM-Ada/pβCD multivalent interactions case. The observed improvement of surface binding properties can be related to the previously mentioned additional non-specific interactions arising between D40-GP-Ada-PEG and pβCDN+ layers, leading to larger adsorption and slower desorption rates.

𝐾 𝑎 = 𝑘 𝑎 𝑘 𝑑 ⁄ (III.4)

Conclusions

This study illustrates the potential of multivalent interpolymer host-guest interactions as a tool for efficient PEGylation of interfaces and colloidal particles.

A series of new (PEG, Ada)-grafted dextrans have been prepared by CuAAC click reaction, using alkyne modified dextrans and azide derivatives of adamantane and 5000 g•mole -1 PEG. These polymers have similar DS by PEG chains and the DS by Ada-groups varying from 0 to 10 mol.%.

Due to the presence of longer and more flexible spacer between the Ada-groups and the dextran backbone, the polymers show significant improvement of their surface binding properties comparing to the previously described Ada-modified pNIPAm. Moreover, their ability to form inclusion complexes with monomeric βCD, neutral pβCD and cationic pβCDN+, both in solution and on the surface, proves to be strongly dependent on the degree of substitution by Ada-groups. Thus, for the interaction with monomeric βCD the association constants K a determined by ITC show similar values for D40-GP-Ada-PEG with different DS(Ada).

However, in the case of interaction with neutral or positively charged βCD-polymers, one order of magnitude higher K a is observed for more densely substituted by Ada D40-28GP-10Ada-18PEG. The latter gives an indication of strong cooperativity effects between the Ada-groups in synthesized polymers. SPR study of the adsorption kinetics by SPR showed that the D40-28GP-10Ada-18PEG affinity to pβCDN+ is further increased when the latter is surface-anchored.

Given the above, (PEG, Ada)-grafted dextrans should be auspicious candidates for applications, where fast and reversible PEGylation of surfaces or nanoparticulate carriers is required. Their potential will be illustrated on a practical example in Chapter 4, where D40-28GP-10Ada-18PEG is used for sterical stabilization of βCD-coated pNIPAm-based microgels.

Two types of experiments were run. In the first type, three consecutive injections of 0.1 mL of pβCD in water or pβCDN+ in 0.5 M NaCl (both at 2 g/L) were carried out to saturate the gold surface of the sensor, similarly to the way that was described in the Experimental section of Chapter 2. This was followed by 3-4 consecutive injections (0.1 mL) of one of the PEG-containing dextrans in water with various DS by adamantyls (1 g/L). Then the RI variation due to the 2 nd layer deposition was measured 20 min after the first injection.

In the second type, 1 mL of pβCDN+ solution at 0.2 g/L was injected to saturate the gold surface of the sensor; then an injection of 1 mL of the D40-28GP-10Ada-18PEG solution at various concentrations (0.01-0.1 g/L) was performed, and the kinetics of the adsorption were recorded and analyzed.

The adsorbed amounts of the polymers were estimated from the values of ΔRI, using the Equation II.1; its derivation is described in the Experimental section of Chapter 2:

𝑄 𝑎𝑑𝑠 = ∆𝑅𝐼 • 𝑙 𝑑 2𝑚 • (𝑑𝑛 𝑑𝑐) ⁄ 𝑝𝑜𝑙 (II.1)
where 𝑙 𝑑 is the depth of penetration of the evanescent electromagnetic field (310.8 nm), 𝑚 is the calibration coefficient of the sensor (𝑚= 1) and (𝑑𝑛 𝑑𝑐) ⁄ 𝑝𝑜𝑙 is the refractive index increment of the polymers. The latter was taken as 0.145 mL/g for pβCD and pβCDN+ host polymers. 50 The 𝑑𝑛 𝑑𝑐 ⁄ of PEG-containing dextrans was considered to be equal to that of pure PEG, 0.135 mL/g (http://www.ampolymer.com/dn-dcValues.html). Given that D40-GP-Ada-PEG contain 80-85 wt% of PEG, this simplification appears to be fair.

Synthetic procedures

The syntheses under microwave irradiation (MW) were performed using a Monowave-300

reactor from Anton Paar. The microwave source was a magnetron with a 2.5 GHz frequency powered by a 900 W power generator, which could be operated at different power levels. The reaction mixtures were loaded inside quartz glass tubes; the tubes were closed with silicon caps using a pressure monitor unit, and placed into the reactor. The microwave reactor was programmed to maintain a constant temperature by adjusting the applied power; the desired temperature was reached gradually using an initial 3 min temperature ramp. After the end of the reaction, solutions were cooled to room temperature with compressed air. When necessary, ultrafiltration was carried out under N 2 atmosphere using Amicon equipment and regenerated cellulose membranes with MWCO 30,000. Dialysis was done with Spectra/Por molecular porous tubing MWCO 6,000-8,000. The ultrafiltrated and dialyzed solutions were freeze-dried on CHRIST ALPHA 1-2 LD Plus lyophilizator.

Short names of the polymers were attributed using the following pattern: D40-XGP-YAda-ZPEG with X, Y and Z indicating the molar degree of substitution (DS) of anhydroglucosidic rings (further AHG) by different substituents. pβCDN+: To prepare a cationized pβCDN+, neutral epichlorohydrin crosslinked pβCD (208 kg•mole -1 , 67 wt% of βCD) prepared according to previously described procedure 47,54 was modified with glycidyltrimethylammonium chloride. In a typical procedure, 4 g of pβCD was added to an alkaline solution containing 0.4 g of NaOH in 20 mL of water and mixed for 24 h at room temperature. It was followed by addition of 10 mL of aqueous solution of glycidyltrimethylammonium chloride (3.20 g, 18.95 mmol) and the reaction bath was heated at 50 °C for 24 h. The solution was then neutralized by addition of 6 N HCl to adjust the pH to 6.

Introduction

The work described in the 4 th chapter was done in collaboration with our colleagues from ELTE University (Budapest, HU) Daria Kaczmarek, Imre Varga and Attila Kardosh. We present a new approach to the synthesis and non-covalent sterical stabilization of hierarchically structured poly(N-isopropylacrylamide) (pNIPAm) microgels coated with positively charged poly-βcyclodextrin. We also show the opportunity to prepare a supramolecular macrogel composed of hierarchical hydrogels interconnected via host-guest interactions.

Microgels are chemically cross-linked polymer networks with typical sizes in the range from 50 nm to 2 μm. 1 A special family of the microgel particles is stimuli responsive microgels that can undergo what is called volume phase transition (VPT) -non-linear, reversible change in their swelling degree in response to an external stimulus. 2,3 One of the most well-known and widely employed polymers to produce responsive microgels is pNIPAm. Typically pNIPAm microgels are prepared by precipitation polymerization of N-isopropylacrylamide (NIPAm) with a bifunctional cross-linker comonomer, methylenbisacrylamide (BA), in the presence of sodium dodecyl sulfate (SDS). 3,4 This method yields nanoparticles with low polydispersity and allows copolymerizing various hydrophobic and hydrophilic monomers, while microgel size can be tuned by adjusting the amount of SDS in the reaction mixture. Synthesis and characterization of pNIPAm-based responsive microgels have attracted a significant interest in the last decades. They are considered as promising candidates for various applications ranging from heavy metal scavenging and diagnostic applications, to controlled drug delivery systems. 2,5 However, microgels composed of homopolymer pNIPAm are limited in their application in that their thermally induced VPT takes place at a fixed temperature of 33-34°C. This limitation might be overcome either by randomly introducing hydrophilic or polyelectrolyte comonomers in the structure of microgels or by preparing hydrophilic copolymer shells on top of pNIPAm core. 6 In addition to VPT temperature regulation opportunity, multiresponsive core-shell microgel architectures with complex phase transition behavior might be obtained when NIPAm is combined with another environmentally sensitive comonomer (e.g. pH-responsive AAc). However, with conventional precipitation polymerization the upper content of hydrophilic copolymers in such microgels is restricted by the necessity to satisfy the condition: VPT temperature < reaction temperature. Kardos et al. have recently developed a novel approach to prepare core-shell pNIPAm-g-pAAc microgels with unrestricted shell composition, containing up to 100% w/v of AAc (Unpublished results). Purely polyelectrolyte shell composition was aimed at facilitating post-modification of the particles with various cationic agents and at making pH-induced changes in phase transition behavior more pronounced. Hence, pNIPAm-g-100%pAAc were used in further described experiments.

Further in the text we will always refer to microgels with 100% pAAc shell as pNIPAm-g-pAAc for simplicity reasons.

Due to the carboxylic groups being deprotonated at physiological pH, negatively charged pNIPAm-g-pAAc could be used as nanocarriers for oppositely charged molecules -surfactants, polymers, etc. However as stoichiometric binding is approached, the surface charge density of the microgel/oppositely charged molecule complexes diminishes resulting in a loss of their colloid stability. Furthermore, in the context of their potential usage as drug delivery carriers, a number of significant issues should be addressed, such as limited colloidal stability, poor biocompatibility resulting in fast opsonization by blood serum proteins 7 and burst release of actives after thermally triggered VPT.

A number of recent evidences in support of superficial functionalization of drug nanocarriers with CD-containing materials are found in literature. For instance, El Fagui et al. have reported β-cyclodextrin polymer (pβCD) layer to play the role of a barrier slowing down the release kinetics of triclosane from hydrophobic PLA nanoparticles. 8,9 Another way to exploit superficial CD shell consists in post-modification of drug delivery vehicles via reversible and competitor binding-responsive host-guest complexation of molecules of interest usually pre-functionalized with Ada-groups. Using this approach, various drug/gene nanocarriers could be non-covalently functionalized with fluorescent dies 10 , targeting peptides 11 and sterically stabilizing hydrophilic polymers. 10,12,13 a. Sterically stabilized pNIPAm-g-pAAc/pβCDN+ hierarchical microgels

Given the above, in this work we aimed to develop a reliable and straightforward approach to βCD-coated pNIPAm-g-pAAc microgels, which would allow preserving thermoresponsiveness of pNIPAm cores. To achieve this we use ionic self-assembly between the negatively charged at pH 7 pAAc shell and pβCDN+ (a neutral epichlorohydrin cross-linked pβCD modified with quaternary ammonia groups). This approach was chosen since it should provide an easy way to control the degree of coating and the overall charge of the resulting pNIPAm-g-pAAc/pβCD1.62N+ microgels (for simplicity reasons they will be mostly referred to as pNIPAm/βCD1.62N) by adjusting the +/-molar ratio during the mixing stage.

The pNIPAm/βCD1.62N have potential to be used as drug nanocarriers with improved release kinetics. However, for any biomedical application the particles are expected to have neutral overall charge, whereas in our case colloidal stability of pNIPAm/βCD1.62N is lost at zero-charge point (+/-= 1). Hence, there is a need to sterically stabilize them. Traditionally it is achieved via post-modification of the shell with non-charged hydrophilic polymer chains e.g. PEG, polyoxazolines, polysaccharides etc.; though tedious and time-consuming synthetic and purification steps are required in this case. 14,15 Non-covalent supramolecular self-assembly appears to be a fast, facile and sustainable alternative. Therefore, we took advantage of a part of βCD-cavities from the pβCDN+ shell to attach a sterical stabilizer via inclusion complexation.

It was achieved in 2 steps: 1) formation of a supramolecular precursor between pβCDN+ and (PEG, Ada)-grafted dextrans described in Chapter 3; 2) addition of negatively charged microgels to the precursor solution at stoichiometrical +/-= 1 ratio. This strategy allowed obtaining stable pNIPAm-g-pAAc/pβCDN+/DT40-28GP-10Ada-18PEG hierarchical microgels with neutral overall charge (for simplicity reasons they will be referred to as pNIPAm/βCD1.62N/PEG).

b. Supramolecular hydrogels based on pNIPAm/βCD1.62N hierarchical microgels Hydrogels are cross-linked 3D-networks composed of hydrophilic polymeric chains or low molecular-mass gelators. 16 Due to their structural and functional resemblance to extracellular matrix, hydrogels find a lot of applications in biomedical industry as scaffolds in regenerative medicine and depos for sustained release of therapeutics. 17,18,19 Depending on the nature of cross-links two main classes of hydrogels are distinguished: covalently cross-linked chemical hydrogels and non-covalently constructed physical hydrogels. Comparing to their covalently cross-linked counterparts, physical hydrogels possess a number of advantages such as:

immanent stimuli-responsive nature 20 , reversibility 21,22 and compositional flexibility. The ability of physical gels to reform themselves after damage, known as self-healing, emerges as a consequence of the reversible nature of physical cross-links. The reversibility, in turn, is due to common physical interactions exhibiting much lower bond energies (8-35 kJ•mol -1 for ionic interactions and hydrogen bonding) comparing to the dissociation energy of covalent C-C bond (347 kJ•mol -1 ). 23 Incorporation of particulate drug delivery vehicles, and thermoresponsive microgels in particular, in the hydrogels matrix might increase the pharmacological performance of both components. 19 The resulting composite or "plum pudding" hydrogels ( Hydrogel matrix might play the role of a diffusion barrier able to diminish the "burst release" of drug issue observed for nanoscale carriers. 24 At the same time, fixation of the nanoparticles in the matrix prevents their migration or washing away from the site of action in vivo. Finally, multiple types of microgels carrying different payloads can be entrapped in the same gel matrix and simultaneously delivered. 25 Richtering and Saunders distinguish three types of composite hydrogels containing microgels 23 :

(i) microgel-filled hydrogels, (ii) microgels-reinforced hydrogels and (iii) doubly crosslinked carboxymethyl cellulose hydrogel. 26 The hydrogels showed capacity to facilitate temperaturetriggered long-term release of bupivacaine hydrochloride, a cationic drug. Interestingly, both increase of internal cross-link densities and covalent linking of microgels to the matrix led to lower overall rates of drug release, which was ascribed to reduced kinetics of microgel deswelling at higher temperatures (37°C). Similar microgel-reinforced systems were reported, composed of: hyaluronic acid-glycidyl methacrylate (HA-GMA) matrix and covalently linked ethylene-oxide (EO)-based microgels 27 ; HA hydrogel network and covalently integrated surfacemodified HA-microgels. 28,29 However, mechanical properties of the hydrogels didn't benefit from the presence of microgels in these cases, which is apparently related to quite high distance between the embedded particles. Hence, their contribution to the overall network strength was rather disruptive.

Temperature-induced mechanical properties improvement was found for polyacrylamide (PAAm)-based composite hydrogels non-covalently filled with pNIPAm microgels. 30 One can see that at low temperatures (15-22°C) the degree of swelling of the microgels strongly depends on pH, i.e. their D h increases from 190 to 300 nm when pH is increased from 2 to 7. Moreover, the temperature at which VPT occurs is shifted from 23°C to 36°C for pH 7 as compared to pH 2, indicating the synergetic effect of between the temperature and pH on the deswelling of pNIPAm-g-pAAc. The increase of D h at pH 2 at T > 30°C can be explained by the low solvent quality for the protonated pAAc shell at these conditions. Indeed, the hydrogen bonding of non-dissociated carboxylic groups of pAAc with water is expected to be hampered at increased temperatures.

Evidence of βCDN+ coating

The pNIPAm-g-pAAc microgel suspensions at 0.145 mM of (-) charges were mixed with pβCDN+ solutions with (+) charges concentrations in the range from 0.02 to 0. The electrostatic stabilization of overcharged pNIPAm/βCD1.62N was confirmed by cryotransmission electron microscopy (cryo-TEM) experiments. In recent year cryo-TEM electronic microscopy has become an important tool for unveiling the structural details of soft matter nanostructures. 35,36 In a typical cryo-TEM experiment, an aqueous solution of the sample is vitrified by instantaneous shock-freezing in liquid ethane prior to recording the TEM This result seems to be logical, given that cationic pβCDN+ molecules should approach already positively overcharged microgels and their adsorption should thus be less and less favorable.

The relative amount of unbound pβCDN+ has been estimated at each C(+)/C(-) ratio from the TC measurements and reported in Table IV.2. In the C(+)/C(-) range 0 -2.0, the unbound pβCDN+ fraction is of 40 % while it increases up to 54% for larger C(+)/C(-) ratio. Although these last results are rather qualitative, another important conclusion that can be drawn from the TC experiments is that for the whole C(+)/C(-) range we deal with samples containing significant amounts of non-bound pβCDN+ in the medium. At the same time, the interpretation of these data obtained for the samples at pH 2.5 should be done with caution. Partial desorption of pβCDN+ might occur during the centrifuging due to the protonation of AAc-groups (pH = 2.5), thus contributing to the TC signal of the supernatants.

Influence of βCDN+ charge density

We also have studied the influence of the charge density of pβCDN+ with equal molecular weights (see Table IV

Estimation of surface PEG chains conformation

We also tried to estimate the conformation of PEG chains in sterically stabilized pNIPAm/βCD1.62N/PEG microgel particles. De Gennes proposed the concept which distinguishes two main conformations of polymer chains anchored to surfaces, based on the grafting density: mushroom-like state and brush-like states. 38,39 If the grafting density is low, the PEG chains are likely to be found in the mushroom-like conformation, when the polymer is spread around the anchoring site into a compact structure. In contrast, if the grafting density is high, the chains are forced to be stretched out perpendicularly from the surface into a brushlike conformation. Quantitative criterion used to determine the conformational regime is based on the relation between the average distance between two adjacent PEG chains (𝐷) and their Flory dimension (𝑅 𝐹 ). The chains are considered to be in the brush-like regime when 𝐷 < 𝑅 𝐹 , whereas the polymer folds into a mushroom-like conformation when 𝐷 > 𝑅 𝐹 , and intermediate structures are observed at 𝐷~ 𝑅 𝐹 .

The Flory dimension of 5000 g/mole PEG in a good solvent (water) is given as: It is known that the lower critical solution temperature (LCST) of pNIPAm may be shifted to lower or higher values by copolymerizing it with more hydrophobic or hydrophilic co-monomers respectively. 40 Furthermore, when NIPAm is copolymerized with acid or basic co-monomers, such as acrylic acid, the overall hydrophilicity and LCST of the polymers can be tuned by adjusting pH of the medium or by counter-ion binding. The reported in Figure IV.18 results are in good agreement with this. Indeed, at +/-= 1.1 due to the charge compensation the apparent hydrophilicity pNIPAm/βCD1.62N/PEG should be lower than that of overcharged pNIPAm/βCD1.62N at +/-= 2.2, leading to a decrease in VPT of the former.

𝑅 𝐹 =

Hierarchical hydrogels based on pNIPAm/βCDN through host-guest interactions 2.1 Hydrogels based on host-guest interactions between pβCD1.62N+ and DT110Ada

In the next step we aimed to exploit the superficial βCD groups in pNIPAm/βCDN and prepare a DX microgels network cross-linked via host-guest interactions. As it was recently demonstrated by Himmelein et al. on the example of βCD vesicles, the self-assembly of the latter into a supramolecular hydrogel can be directed by addition of Ada-modified hydroxyethylcellulose. 22 Being inspired by the success of their strategy, herein we will use Ada-substituted dextrans (DT- At first we attempted a direct gelation approach, i.e. by mixing positively overcharged pNIPAm/βCD1.62N at +/-= 2 with DT110-Ada3 (Table IV.1). However, an issue with pNIPAm/βCD1.62N colloidal stability at higher weight concentrations was encountered.

Typically, precipitation of the microgels was observed at C(AAc-) > 8 mM (3.2 g•L -1 ) and C(+) > 16 mM (19.2 g•L -1 ) corresponding to a combined weight concentration of 2.25 wt% and, thus, limiting the possibility of direct hydrogel preparation.

To circumvent this limitation, an indirect gelation approach was used. In the first step, stable pNIPAm/βCD1.62N complexes at lower concentrations (typically C(AAc-) = 6 mM, C(+) = 4.8-12 mM) were prepared and mixed with DT110Ada at stoichiometrical Ada/CD molar ratios. The resulting mixtures were concentrated in 2.3-3.5 times under vacuum at 50°C. After cooling down to room temperature and reswelling of pNIPAm cores, transparent hydrogels with ~10-11 wt% dry weight of the components were formed. The characteristics of so prepared samples are given in Table IV.3. Two conclusions could be drawn from these data: 1) to avoid phase separation during concentrating one should use well-overcharged pNIPAm/βCD1.62N with +/molar ratios far enough from the stoichiometrical ratio; 2) 11 wt% by dry weight of the components seems to be required to obtain stable non-flowing hydrogels. Lower than 0.8 +/ratios were not tested since in this case the host polymer coverage of the particle surface is not full and the resulting microgels are less prone to be cross-linked via host-guest interactions.

The necessity for higher +/-molar ratios entails relatively low contributions of microgels (0.5-0.6 wt%) to the total dry weight concentration (11.6-11.9 wt%) in successful gel samples (Table IV.3). According to Senff and Richtering, rheological and phase behavior of BA-crosslinked pNIPAm microgel suspensions are strongly dependent of their effective volume fraction (𝜙 𝑒𝑓 ). 41 For instance, by measuring zero shear viscosity of microgels at different temperatures below LCST as a function of 𝜙 𝑒𝑓 , they observed a sharp increase in viscosity at 𝜙 𝑒𝑓 > 0.5. 42 The phenomenon was ascribed to the microgel soft spheres being close to contact and interacting with each other at these concentration conditions. Hence, it was important to estimate the 𝜙 𝑒𝑓 of pNIPAm-g-pAAc microgels at C μgel = 0.6 wt% (6 g•L -1 ) in order to understand the nature of the obtained hydrogels (G3, G4).

The molar weight of the used microgels M μgel ~ 10 8 g•mole -1 was determined by static light scattering. From this, the number of particles per L, 𝑁 𝑝 , may be calculated as: From this, the 𝜙 𝑒𝑓 can be estimated as:

𝜙 𝑒𝑓 = 𝑉 ℎ • 𝑁 𝑝 = 0.5 (IV.4) which coincides exactly with the threshold value required for the close contact of the microgels mentioned above. 42 From this one can assume that for G3 and G4 samples, the hydrogels exhibit structures rather similar to that of DX microgels than simply microgel-reinforced hydrogels. 23

Rheological properties

The viscoelastic properties of the G3-G4 hydrogels were studied by oscillatory rheological measurements, which are described in greater detail in the Experimental section. Frequency G'' values prove to be frequency-dependent in all cases, which was previously reported as a typical behavior for physical gels. 43,44,45 At higher frequencies and shorter observation times materials show gel character with the elastic component G' dominating the viscous component G''. At lower frequencies the system starts to behave as a viscous liquid with G'' > G'. In a number of earlier studies on host-guest polymeric hydrogels such behavior is explained by pNIPAm-g-pAAc microgels don't form gels even at concentrations as high as 10.1 wt% (G6 sample). It should be related to the difficulty of achieving the close-packing due to the repulsive forces between the negatively charged particles. The frequency sweep data for the G samples were attempted to fit to the Maxwell model. This basic viscoelasticity model was widely used for describing the behavior of entangled physically bonded polymer networks 49,50 and polymeric host-guest hydrogels. 47 The Maxwell model implies that storage G' and loss G'' moduli of a viscoelastic body vary as a function of the strain angular frequency 𝜔 according to the following equations:

𝐺 ′ (𝜔) = 𝐺 0 (𝜔𝜏) 2 1 + (𝜔𝜏) 2 (IV.6)

𝐺 ′′ (𝜔) = 𝐺 0 𝜔𝜏 1 + (𝜔𝜏) 2 (IV.7)

where 𝜔 is the angular frequency [rad•s -1 ], 𝜏 -is the relaxation time and 𝐺 0 is the G' at high frequencies where it typically reaches a stable plateau value. From the equations above it also follows that 𝐺′ 𝐺′′ ⁄ = 𝜔𝜏. In our case 𝜏 was obtained from the G' and G'' cross-over frequency value and was set as a fixed parameter during the fitting. Since the G' didn't reach the plateau in the available range of angular frequencies, 𝐺 0 was set as a free optimized parameter. Despite βCDand cholesterol-groups. 47 However, they found that the fitting to Maxwell model was no longer possible for more structurally complex hydrogels composed of 8-arm PEGs with the same end-functionalities. They ascribed it to the presence of broader range of relaxation mechanisms with different relaxation times involved in the stress relaxation in the latter case. We assume that the same argumentation holds for the rather structurally complex pNIPAm/βCD1.62N/DT110Ada3 doubly cross-linked microgels.

Since the obtained hydrogels are composed of cross-linked thermoresponsive pNIPAm beads,

Hydrogels based on host-guest interactions between pβCD3.24N+ and DT500Ada

Analyzing the composition of the successful hydrogel samples prepared from pβCD1.62N+ and DT110Ada (Table IV.3), it becomes obvious that, despite their volume fraction being ~ 0.5, the pNIPAm-g-pAAc microgels contribution to the total weight concentration of the samples is fairly low (4.2 and 5.2% for G3 and G4 respectively). Hence, the properties of thermoresponsive microgel "bricks" in such gels are likely to be dominated by the properties of the host-guest polymers "mortar". One way to increase the weight contribution from microgels while using the same +/-and Ada/CD molar ratios during the formulation of the samples consists in increasing the positive charge density of the host polymers. In an attempt to overcome this problem we used a guest polymer with an increased molecular weight (DT500Ada2 in Table IV.1) as a cross-linker. This approach proved successful: a stable transparent gel was formed at C tot = 11.0 wt% (E1 in Table IV.5). Positive impact of higher M w of the guest polymer is apparently related to the higher number of Ada groups attached to a single polymer chain in this case (200 vs. 32 Ada per chain for DT500Ada2 and DT110Ada3 respectively). As a result, a DT500Ada2 molecule has a potential to form a higher number of host-guest cross-links between the neighboring microgel particles.

corresponding to the VPT of microgels (Figure IV.24,a). Also, G3 and E1 have close T co values (37 and 41°C respectively). In the case of E2 the moduli values decrease in a less steep way, which results in a significantly higher gel-to-sol transition temperature (T co = 51°C, Figure IV.24,a). This is in a good agreement with the literature data for host-guest mediated polymer networks. 44 Given the enthalpy-driven nature of the βCD-Ada inclusion complexes, the number of cross-links gradually decreases as the temperature is raised. The activation energies found for all three samples (Table IV.6) lie in the range reported in literature for reversible polymeric networks interconnected through βCD-Ada cross-links (18-41 kJ•mole -1 ). 45,52 As expected from the appearance of the Arrhenius plots, pNIPAm microgelcontaining hydrogels (G3, E1) exhibit very close values of 𝐸 𝑎 , although they differ significantly in the sum of network forming host and guest polymers concentrations, C(pβCDN+DT-Ada) (Table IV.6). At the same time, the "blank" hydrogel sample E2, having the same as E1 C(pβCDN+DT-Ada), exhibits a substantial drop of 𝐸 𝑎 , i.e. from 26 to 18 kJ•mole -1 . Such a change in the network breaking kinetic parameters wouldn't be expected in the case simple microgel-filled hydrogels, where the microgel particles are randomly dispersed without any chemical or strong physical bonding with the network and/or the other particles. 23 Indeed, in the latter case the microgels presence would act in a similar way as a decrease in network forming polymers concentration, without strongly affecting the 𝑘 and 𝐸 𝑎 values. Thus, the obtained results might indicate that the pNIPAm-g-pAAc microgels embedded in the hydrogel preserve their pβCDN+ coating. The latter makes microgels firmly connected with the rest of the network and between each other through the combination of electrostatic and host-guest interactions. Furthermore, as showed by the 𝜙 𝑒𝑓 calculations, microgels are expected to be in close contact, thus indicating their DX microgels-like character.

Freeze-drying pathway for the preparation of pNIPAm/βCD3.24N/DT500Ada2 hydrogels

The hydrogel samples described so far were prepared by concentrating the mixtures of βCDN+coated microgels with guest polymers under vacuum at 55°C. Given the rather tedious character and not always good reproducibility of this procedure we were looking for an alternative indirect gelation approach.

203

A full analogue of the E1 hydrogel in terms of the weight concentrations and molar ratios of the components was prepared using a freeze-drying pathway. Stable pNIPAm/βCD3.24N coated microgels at low concentration (C(AAc-) = 3 mM, C(+) = 6 mM) were freeze-dried and redissolved in DT500Ada2 solution in water (Ada/CD = 1). The strategy proved successful (FR1 in Table IV.4). However, it should be noted that at equal C tot the FR1 hydrogel has a somewhat

Conclusions

This study demonstrates the versatility of core-shell pNIPAm microgels non-covalently coated with βCD polymer as potential drug delivery vehicles and building blocks for hierarchically structured hydrogels with multi-responsiveness profile.

The microgels used are composed of crosslinked thermoresponsive pNIPAm core and shell with unrestricted composition, containing up to 100% w/v of pAAc in the form of non-crosslinked densely grafted chains. We have taken advantage of the negative surface charge of the microgels at pH 7 to coat them with positively charged pβCDN+ host polymers via electrostatic self-assembly. The overall charge of the resulting pNIPAm/βCDN microgels can be tuned from negative to positive and the βCD content in the shell at a fixed +/-ratio can be varied by simply using pβCDN+ with different quaternary ammonia N+ modification levels. Positively overcharged pNIPAm/βCDN suspensions were stable in water over long periods of time.

However, the overall neutral microgels, which represent the highest interest for potential The host-guest interactions prove to be at the origin of the gel formation as evidenced by the "blank" experiments with the samples non-containing microgels. However, the activation energies 𝐸 𝑎 of the network breaking are strongly influenced by the presence of pNIPAm-g-pAAc.

Moreover, calculations of the effective volume fraction 𝜙 𝑒𝑓 of microgels show that the latter are expected to be at a close contact in the successful hydrogel samples. This indicates that the pβCDN+ coating is preserved and the structure of the hydrogels is close to that of doublycrosslinked microgels. To the best of our knowledge, this is the first example of DX microgels non-covalently crosslinked via host-guest interactions. Furthermore, the hydrogels exhibit fully reversible temperature-induced gel-sol transition due to the synergetic effect between shrinking of the microgels and dissociation of βCD-Ada crosslinks at higher temperatures. Due to the presence of microgels the gel-sol transition temperature of pNIPAm/pβCDN/DT-Ada is significantly shifted down to the physiological temperature range (37-41°C) as compared to uniform pβCDN/DT-Ada host-guest hydrogels (51°C). It opens up attractive prospects of their potential use in biomedical applications.

Abstract

This PhD work is based on the development of new architectures of polysaccharide-based host and guest polymers and their application in the design of hierarchically structured soft nanoparticles and supramolecular hydrogels with interesting drug delivery profiles. In the first section of the manuscript we describe a synthetic pathway to host and guest polymers with hydrophilic poly(ethylene glycol) PEG spacer between the dextran backbone and either β-cyclodextrin (βCD) host or adamantane (Ada) guest grafted groups. The presence of the PEG spacer led to a substantial improvement of the availability of Ada groups of the guest polymer as compared to its counterpart, where Ada are linked to the backbone with a short hydrophobic spacer. This was followed by the study of nanoassemblies formation between the different types of host and guest polymers. Once again, PEG spacer had a significant impact on the size and internal structure of the resulting nanoassemblies. The second part of this work describes synthesis of a series of new (PEG, Ada)grafted dextrans prepared by copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). The degrees of substitution (DS) by PEG grafts (5000 g/mole) are close to 20 mol% while the DS by Ada-groups are varied from 0 to 10 mol.%. The affinity of these polymers to monomeric βCD, as well as their ability to form superficial layers with β-cyclodextrin polymers (pβCD, pβCDN+), are strongly dependent on the DS by Ada, giving an indication of cooperativity effects between them. In the last part we described a strategy to a non-covalent modification of thermoresponsive poly(N-isopropylacrylamide) (pNIPAm)based microgels with a pβCDN+ host polymer shell. It uses on electrostatic self-assembly between pβCDN+ and negatively charged poly(acrylic acid) (pAAc) chains grafted to the surface of microgels. The resulting pNIPAm/βCDN microgels with neutral overall charge could be colloidally stabilized with (PEG, Ada)-grafted dextrans via a hierarchical self-assembly procedure. Finally, using Ada-modified dextrans (DT-Ada), pNIPAm/βCDN microgels could be physically cross-linked to yield hierarchical 3D hydrogels (at 10 wt%). Their gel-sol transition temperature is shifted down to the physiological temperature range (37-41°C) as compared to uniform pβCDN/DT-Ada host-guest hydrogels (51°C).

Keywords: soft matter, host-guest interactions, cyclodextrins, hierarchical self-assembly

Résumé

Ce travail de thèse concerne le développement de nouvelles architectures de polymères hôte et invité à base de polysaccharide et leur application dans la conception de nanoparticules molles à structure hiérarchique et d'hydrogels supramoléculaires pour l'encapsulation et la libération de médicaments. Dans la première partie du manuscrit, nous décrivons une voie de synthèse de polymères hôtes et invités comprenant des chaînons espaceurs poly(éthylène glycol) hydrophile (PEG) entre le squelette de dextrane et soit le groupement β-cyclodextrine (βCD)(polymère hôte) ou le groupement adamantane (Ada) (polymère invité). La présence des bras espaceurs PEG a conduit à une amélioration substantielle de la disponibilité des groupes Ada du polymère invité par rapport à la situation avec un bras espaceur court et hydrophobe. Nous avons ensuite étudié la formation de nanoassemblages entre les différents types de polymères hôtes et invités. Une fois de plus, les espaceurs PEG ont eu un impact significatif sur la taille et la structure interne des nanoassemblages. La deuxième partie de ce travail décrit la synthèse d'une nouvelle série de dextranes greffés par des chaînons PEG et Ada, préparés par réactions de cycloaddition d'azoture-alcyne catalysées le cuivre (I) (CuAAC). Les degrés substitution (DS) en chaînons PEG greffés (5000 g/mole) sont de l'ordre de 20 mol.% tandis que les DS par les groupements Ada sont variés de 0 à 10 mol.%. L'affinité de ces polymères pour la βCD native, ainsi que leur capacité à former des couches superficielles avec des polymères de β-cyclodextrine (pβCD et pβCDN+), s'avérent dépendre fortement du DS en groupements Ada, ce qui résulte de la coopérativité des interactions impliquées. Dans la dernière partie, nous avons décrit une stratégie de modification non covalente de microgels sensibles à la temperature à base de poly (N-isopropylacrylamide) (pNIPAm), pour les recouvrir d'une couronne de pβCDN+. Cette stratégie s'appuie sur l'autoassemblage électrostatique entre pβCDN+ et les chaînes de poly(acide acrylique) chargés négativement (pAAc) et greffées à la surface des microgels. Dans le cas d'une charge globalement neutre des microgels pNIPAm/βCDN, la stabilisation colloïdale a pu être réalisée à l'aide de dextranes greffés (PEG, Ada) en utilisant une procédure d'assemblage hiérarchique. Enfin, à l'aide de dextrane modifé par des groupements Ada (DT-Ada), les microgels pNIPAm/βCDN ont pu être associés pour produire des hydrogels 3D hiérarchiques (10wt %). Leur température de transition sol-gel est décalée vers le bas pour atteindre la gamme des températures physiologiques (37-41°C) par rapport à celle observée dans un hydrogel hôteinvité uniforme βCDN/DT-Ada (51°C).

Mots clés: matière molle, interactions hôte-invité, cyclodextrins, auto-assemblage hiérarchique

1 H 1 . 2 .

 112 -NMR -nuclear magnetic resonance of protons 2-PrOH -propan-2-ol 4-NC -4-nitrophenyl chloroformate 4-NP -4-nitrophenyl AdaNCS -1-adamantyl isothiocyanate AdaTMA -2-(1-adamantyl)ethyl trimethylammonium bromide AHG -anhydroglucosidic ring APS -ammonium persulfate ATR -attenuated total reflection AZT-TP -azidothymidine-triphosphate BA -methylenbisacrylamide βCDβ-cyclodextrin CDs -cyclodextrins CDV -cyclodextrin vesicles cryo-TEM -cryogenic transmission electronic microscopy CuAAC -copper(I)-catalyzed Huisgen azide-alkyne cycloaddition DBCs -DNA block copolymers DBTDL -dibutyltin dilaurate DCC -dicyclohexylcarbodiimide DCM -dichloromethane DLS -dynamic light scattering DMAP -4-(dimethylamino)pyridine DMSO-d 6 -dimethyl sulfoxide-d 6 DNA -deoxyribonucleic acid DS -degree of substitution DX microgels -doubly crosslinked microgels EO -ethylene-oxide EPR -enhanced permeability and retention effect EtOAc -ethyl acetate Fc -ferrocene FDA -Food and Drug Administration GMA -glycidyl methacrylate GO -graphene oxide GSH -glutathione HA -hyaluronic acid HSR -hypersensitivity reactions ISA -ionic self-assembly ITC -isothermal titration microcalorimetry LbL -layer-by-layer LC -liquid crystalline LCST -lower critical solution temperature MOF -metal-organic frameworks MPS -mononuclear phagocyte system MWCO -molecular weight cut-off NDIR -nondispersive infrared NOESY -nuclear Overhauser effect spectroscopy NPs -nanoparticles pAAc -poly(acrylic acid) PAAm -polyacrylamide PBS -phosphate-buffered saline PCL -poly(ε-caprolactone) PDDF -pair-distance distribution function PE -polyelectrolyte PEG -poly(ethylene glycol) PEI -polyethylenimine PEPO -poly(ethylene oxide-co-propylene oxide) PhNCO -phenyl isocyanate PhNCS -phenyl isothiocyanate PHPMA -poly(N-hydroxypropylmethacrylamide PIBMA -poly(isobutene-alt-maleic acid) PILs -poly(ionic liquid)s PLA -polylactic acid PLGA -poly(lactide-co-glycolide) pNIPAm -poly(N-isopropylacrylamide) PPO -polypropylene oxide PS -polystyrene pβCD -poly-β-cyclodextrin pβCDN+ -positively charged poly-β-cyclodextrin RES -reticuloendothelial system RI -refractive index RNA -ribonucleic acid SAMs -self-assembled monolayers SAXS -small-angle X-ray scattering SDS -sodium dodecyl sulfate Si-MPs -mesoporous silica nanoparticles siRNA -short interfering ribonucleic acid SLS -static light scattering SPR -surface plasmon resonance SWNT -single-wall carbon nanotubes TEM -transmission electron microscopy TLC -thin layer chromatography TMV -tobacco mosaic virus TC -total carbon TsCl -p-toluenesulfonyl chloride VPT -volume phase transition Table of contents Introduction ................................................................................................................................... Driving forces, building blocks and variety of architectures of hierarchically self-assembled systems ........................................................................................................................................... 1.1 Inorganic and hybrid hierarchically self-assembled materials ............................................. 1.2 Soft matter hierarchically self-assembled materials............................................................ Hierarchical self-assembly mediated by Host-guest interactions ........................................... 2.1 Nanoscale hierarchical systems based on cyclodextrins ..................................................... 2.1.1 Gene delivery systems .................................................................................................. 2.1.2 Various drug delivery-oriented nanoscale systems ...................................................... 2.2 Host-guest mediated supramolecular hydrogels ................................................................. 2.2.1 Hierarchical hydrogels formed from cyclodextrin-based polypseudorotaxanes.......... 2.2.2 Cyclodextrin-based hierarchical hydrogels with external stimuli responsive properties ................................................................................................................................................ Conclusions .................................................................................................................................... References .....................................................................................................................................

  represented in Figure I.1.

Figure I. 1 :

 1 Figure I.1: Levels of hierarchical self-assembly of collagen. Reprinted from Elemans et al.3

Figure I. 2 :

 2 Figure I.2:Self-assembly of precursor zeolite nanoparticles into 500 nm beads with hierarchical micro/mesoporous internal structure. Reprinted from Wang et al.14

Figure I. 3 :

 3 Figure I.3: Stages of growth of hierarchical flower-like bismuth tungstate (Bi 2 WO 6 ) hollow microspheres visualized by SEM. Reprinted from Zhang et al.15

  negatively charged enzyme lumazine synthase from Aquifex aeolicus bacteria (AaLS) with positively supercharged human ferritin; b) using positively supercharged human ferritin to template the self-assembly of negative AaLS capsid (Figure I.7). Moreover, three component Matryoshka-like structures loaded with iron NPs were prepared.

Figure I. 7 :

 7 Figure I.7: Schematic illustration of the strategies for cage-within-cage peptide hierarchical structures preparation: a) direct loading of positively charged ferritin (blue) into intact negative AaLS capsids (red); ferritin-templated self-assembly of 13 AaLS fragments into closed-shell complexes. Reprinted from Beck et al.30

Figure I. 8 :

 8 Figure I.8: Strategy for preparation of hierarchical nanohybrids via complementary hybridization between PPO-b-DNA -stabilized SWCNTs and DNA-modified Au NPs (a) or DNA-covered gold electrodes (b). Adapted from Kwak et al.39

Figure I. 9 :

 9 Figure I.9: Schematic representation of the formation of hierarchical organic-metalorganic nanoarchitectures via combination of ISA and hydrophobic self-assembly (a); their visualization by TEM (b). Adapted from Ahmed et al.43

(

  50 g/L) they observed formation of multilamellar vesicular structures for shorter alkyl chains and unilamelar vesicles for C16-18 (Figure I.10; a, b). This was attributed to crystallinity in the longer alkyls' self-assembled layers and, thus, their lower bendability. At higher polymerization concentrations (100 g/L) the multilamellar PILs vesicles further self-organized into 1Dsuperstructure of nanoparticles with extended multilamellar substructure via synergy between electrostatic and hydrophobic interactions (Figure I.10; c); this higher level of hierarchy was not achieved in crystalline unilamelar vesicles.

Figure I. 10 :

 10 Figure I.10: Poly(ionic liquid)s nanoarchitectures on different length scales by varying the polymerization concentration and monomer architecture. Adapted from Yuan et al.44

44

 44 Figure I.10: Poly(ionic liquid)s nanoarchitectures on different length scales by varying the polymerization concentration and monomer architecture. Adapted from Yuan et al.44

Figure I. 11 :

 11 Figure I.11: Components and formulation principle of the siRNA targeted delivery system CALAA-01. The supramolecular host-guest complexes are first formed between linear βCDP and bifunctionalized Tf-PEG-Ada in the Vial 2; upon mixing with the siRNA (Vial) one they hierarchically self-assemble into sterically stabilized targeted nanoparticles. Adapted from Davis. 61

Figure I. 12

 12 Figure I.12: a) Formulation of CD-PEI nanoparticles by condensation between plasmid DNA and positively charged βCD-grafted PEI; b) Immobilization of the resulting polyCDplexes onto Adamodifies surface via multivalent inclusion interactions. Adapted from Park et al.[START_REF] Park | Supramolecular assembly of cyclodextrin-based nanoparticles on solid surfaces for gene delivery[END_REF]

  polyCDplexes (Figure I.13). The pβCD-based vector showed high gene transfection efficiency in the experiments on HepG2 and HEK293 cell lines, as it was evidenced by luciferase protein activity monitoring. Interestingly, the imidazole-containing Ada6 connector (Figure I.13; b),

  as size and DNA compaction degree could be tuned by simple change of the connector/βCD molar ratio (Figure I.13; c).

Figure I. 13

 13 Figure I.13: a) Schematic illustration of the polyCDplexe hierarchical self-assembly between pβCD, cationic connector and DNA; b) Chemical structures of various Ada-connectors; TEM images illustrating the evolution of plasmid DNA compaction degree as a function of the amount of cationic connector Ada5 associated with prior to polyCDplexe formation (pβCD/Ada5/DNA = 20/X/1). The first image (X = 0) represents free pDNA in water. Adapted from Wintgens et al.[START_REF] Wintgens | Smart DNA vectors based on cyclodextrin polymers: compaction and endosomal release[END_REF]

  An original way to exploit this property in gene delivery was introduced by Kissel and coworkers. They synthesized a new type of ternary copolymers by grafting PEG-b-poly(ε-caprolactone) (PCL) diblock copolymers onto branched PEI. The DNA polyplexes of such ternary copolymers demonstrated neutral surface ζ-potential and

  complexes with both PEG and PCL, α-CD end up threaded preferentially along the PCL block, making it more hydrophilic and preventing its association with PEI (Figure I.14). The resulting hierarchically structured polyplexes showed excellent stability, bioavailability and DNA transfection efficiency.

Figure I. 14 :

 14 Figure I.14: Formulation of supramolecular gene vectors: a) Rotaxanation of the PCL block of the brPEI-PCL-PEG ternary copolymer by inclusion complexation with α-cyclodextrin; b) Formation of polyplexes via electrostatic association with DNA. Adapted from Shuai et al.[START_REF] Shuai | Supramolecular gene delivery vectors showing enhanced transgene expression and good biocompatibility[END_REF]

  benzoic imine-conjugated poly[poly(ethylene glycol) monomethyl ether methacrylate]-copoly(2-hydroxyethyl methacrylate) (PPEGMA-co-PHEMA-Ada); doxorubicin (DOX)-hydrazone and βcyclodextrin (βCD)-conjugated poly[N-(2-hydroxypropyl) methacrylamide]-co-poly(3azidopropyl methacrylate) (PHPMAco-PPMA-DOX-CD) (Figure I.15, a). The 220 nm host-guest nanogels were obtained by dialysis of the mixture of PPEGMA-co-PHEMA-Ada and PHPMAco-PPMA-DOX-CD solutions in DMSO against phosphate-buffered saline (PBS) at pH 7.4. The benzoic imine linker between Ada and PPEGMA-co-PHEMA backbone can be cleaved at pH 6.5. As a result, nanogels undergo size reorganization when placed in a slightly acidic tumor cells microenvironment (Figure I.15, b).

Figure I. 15

 15 Figure I.15: a) Chemical structures of used guest (PPEGMA-co-PHEMA-AD) and host (PHPMAco-PPMA-DOX-CD) polymers with pH-sensitive AD-benzoic imine (cleaved at pH 6.5) and DOXhydrazone (cleaved at pH 5) links respectively; b) Schematic illustration of the formation of host-guest self-assemblies and their dual pH-responsive rearrangement endo-lysosomal internalization and drug release. Adapted from Zan et al.[START_REF] Zan | Dual pH-triggered multistage drug delivery systems based on host-guest interaction-associated polymeric nanogels[END_REF]

  off the temperature below the lower critical solution temperature (LCST) of PNIPAM or increased, by increasing the PNIPAM/PEG-b-PCD weight ratio. The subsequent release of a model hydrophobic drug study showed that the payload could be released in a sustained manner, and the release rate could be modulated via adjusting the temperature. Zhuo et al. described simultaneously thermo-and pH-responsive doxorubicin delivery system. They used α-β cyclodextrin dimer to prepare amphiphilic supramolecular polymers via selective host-guest complexation of α-CD with phenyl (attached to the hydrophilic NIPAM-co-Nacroyloxysuccinimide (NAS) block) and of βCD with adamantane (linked to the hydrophobic PCL block) (Figure I.16). The resulting polymers showed well defined CMC lying between 0.15-0.30 mg/mL above which they hierarchically self-assemble into spherical core shell micelles.[START_REF] Quan | Core-shell nanosized assemblies mediated by the α-β cyclodextrin dimer with a tumor-triggered targeting property[END_REF] The thermoresponsive (LCST = 38°C) NIPAM-co-NAS block was also modified with MPEG grafts (M n ~ 2000) and targeting peptide sequences in a bottle-brush manner. Under normal physiological conditions (pH ~ 7.4, T = 36.6 °C) the hierarchical micelles are stable and protected against immunogenic interactions. Upon reaching the solid tumor tissue with pH < 6.8 the PEG grafts are removed by the cleavage of benzoic imine bonds connecting them to the main chain and the micelles are internalized by the malignant cells. Furthermore, once the PEG grafts are gone the LCST of the hydrophilic block drops down to 35.5 °C which along with higher than normal (38 °C) temperature of cancer cells leads to the collapse of the micelles and release of the payload (doxorubicin).

Figure I. 16 :

 16 Figure I.16: Formation of hierarchical core-shell nanoparticles for delivery of doxorubicin with switchable tumor cell triggered targeting and release. Reproduced from Quan et al.[START_REF] Quan | Core-shell nanosized assemblies mediated by the α-β cyclodextrin dimer with a tumor-triggered targeting property[END_REF]

  e. disassemble under the effects of oxidant (H 2 O 2 ) or temperature via different mechanisms: (1) the oxidant induces dissociation of βCD-Fc, thus removing the PEO-Fc + protective shell from PNIPAM-βCD cores; (2) at temperatures below the LCST, the hydrophobic PNIPAM becomes hydrophilic, hence the hydrophobic core dissociates. The micelles were successfully loaded with doxorubicin and its release could be triggered by either of two stimuli.

Figure I. 17 :

 17 Figure I.17: Schematic representation of the formation and redox-triggered disassembly of the TPPC6-SS-Ada/PEG-βCD hierarchical micelles for photodynamic therapy. Adapted from Liu et

  Akin to the previous two examples, the TPPC6-SS-Ada/PEG-β-CD pseudo-copolymer further self-assembles into hierarchical micelles in water (Figure I.17). The micelles proved to be efficiently up-taken by the MCF-7 malignant cells due to the Enhanced Permeability and Retention (EPR) effect. Moreover, given the presence of redox-sensitive S-S bonds the nanostructures demonstrate the aforementioned biological responsiveness for gene transfection vectors, i.e. disassembly and the porphyrin photosensitizer payload release occur in a more reductive intracellular microenvironment. To proceed with the topic of stimuli-responsive hierarchical nanostructures based on cyclodextrins, Ma et al. recently reported a simple way to supramolecular vesicles constructed via hydrophobic self-assembly of the amphiphilic βCD/tyrosine inclusion complex (Figure I.18).

Figure I. 18 :Figure I. 19 :

 1819 Figure I.18: Schematic illustration of the proposed mechanism of the hydrophobic self-assembly and competitor-induced disassembly of the βCD/tyrosine-based hierarchical vesicles.Reproduced from Ma et al.[START_REF] Ma | A multistimuli-responsive supramolecular vesicle constructed by cyclodextrins and tyrosine[END_REF]

Figure I. 20 :Figure I. 21 :

 2021 Figure I.20:Host-guest mediated "bricks and mortar" approach for the preparation of sizecontrolled gold supramolecular nanoparticles (Au-SNPs). Reproduced from Wang et al.102

  consist of two steps: 1) formation of CD-PPRTX as such, by threading multiple CDs along an appropriate, typically PEO, polymer chain via inclusion complexation; 2) aggregation of CD-PPRTX due to the intermolecular hydrogen bonding between the threaded CDs and microcrystalline domains formation, the latter being able to hold the resulting 3D-network together (Figure I.22). Overwhelming majority of so far described CD-PPRTX gels are based on αCD/PEG inclusion complexation due to the perfect matching in size between the internal cavity of unmodified αCD and chain cross-section area of PEG.122

Figure I. 22 :

 22 Figure I.22: Schematic illustration of the 2-step hierarchical CD-PPRTX gel formation mechanism: a) hydrophobically-driven inclusion of high molecular weight PEG chains into αCD; b) hydrogen bonding between the threaded αCD microcrystalline domains leading to gelation. Adapted from Liu et al. 122

(Figure I. 23 :

 23 Figure I.23: Schematic illustration of the inclusion-driven formation of αCD/PEG-PHB-PEG polypseudorotaxane (a) and αCD/PEG-PHB-PEG-based doubly cross-linked hydrogel (b).

Figure I. 24 :

 24 Figure I.24: Schematic illustration of the preparation of αCD/PEG PPRTX hierarchical hydrogels containing pDNA polyplexes for gene transfection and sustained release. Reproduced from Li et al. 131

Figure I. 26 :

 26 Figure I.26: Schematic illustration of the formation and temperature-or competitor triggered dissociation of QD-containing hierarchical hydrogels made of hybrid inclusion complexes (HIC).Reproduced from Liu et al.114

  of polymerization of PDMA to PNIPAM blocks (Figure I.26). In addition to temperature change, competitor binding114, 141, 142 or Fc chemical oxidation to Fc + in the case of Fc-PDMA-b-PNIPAM 142 could be used to disrupt the 3D network and trigger the sol-gel transition. Moreover, the presence of inorganic nanomaterials in the hydrogels endowed them with improved rheological properties. Thus, for Fc-containing HIC at a concentration of 12.5 wt% the sol-gel crossover occurs at 40.5°C with G' reaching ~ 2000 Pa, indicating a strong gel formation.142 The innate fluorescence of QD in such systems might be used as a visualization tool in biosensors elaboration.

(

  HA-Azo) led to a hydrogel (Figure I.27).

Figure I. 27 :

 27 Figure I.27: Schematic illustration of the UV/Vis irradiation and competitor binding responsiveness of the bacteriophage M13-βCD/HA-Azo hierarchical hydrogel. Reproduced from Chen et al. 143

Figure I. 28 :

 28 Figure I.28: Schematic illustration of the formation of hierarchical chemically-responsive hydrogels containing amphiphilic βCD supramolecular vesicles as cross-linking points. Adapted from Himmelein et al. 116

Phenomena and Macrocyclic Chemistry in 2014. 20 1. 1 . 1 Figure II. 1 :

 20111 Figure II.1: Strategy of modular synthesis of dextran-based polymers with hydrophilic PEG spacer.

Scheme II. 1 :

 1 Scheme II.1: Reaction scheme representing the 2-step synthesis of thiolated dextran (DT40-SH).21

Figure II. 2 :

 2 Figure II.2:1 H-NMR spectrum (DMSO, 400 MHz) of DT40-4NC (DS 4NC = 24.5 mol.%). Traces of DMF are complicated to remove completely. However, it doesn't interfere with accuracy of DS determination and is used as a reaction medium in the next step.

Figure II. 3 :

 3 Figure II.3: Influence of the 4-NC/AHG ratio on the degree of substitution of 4-NC activated dextrans.

Figure II. 4 :

 4 Figure II.4: 1 H-NMR spectrum (D 2 O, 400 MHz) of thiolated dextran (DT40-SH, DS= 17.5 mol.%).

23 Scheme II. 2 :

 232 Scheme II.2: Synthesis of PEG-acryloyl-5-hexynoate by Steglich esterification.

Figure II. 5 :

 5 Figure II.5: 1 H-NMR spectrum (CDCl 3 , 400 MHz) of PEG-acryloyl-5-hexynoate. The signal of -CH 2of 5-hexynoic acid in the α-position to the carbonyl is shifted from 2.38 to 2.47 ppm once it's coupled to PEG. The signal at 2.38 ppm on the spectrum corresponds to the excess of 5hexynoic acid.

3 :

 3 Scheme II.3: General thiol-ene couplings via: a) free radical and b) Michael addition processes.

  Scheme II.4: Synthesis of dextran grafted with alkyne-terminated 375 Da PEG chains -DT40-g-PEG-hex-5-ynoate via nucleophile-mediated thiol-Michael addition to the acrylate C=C bonds.

68 being

 68 Scheme II.5: Assumed mechanism of the nucleophile-mediated hydrothiolation of an activated C=C bond (EWG stands for electron withdrawing group). Adapted from Hoyle et al.16

Figure II. 6 :

 6 Figure II.6: 1 H-NMR spectrum (DMSO-d 6 , 400 MHz) of DT40-g-PEG-hex-5-ynoate (DS= 16.2 mol.%). The signals at 4.20-4.30 ppm correspond to -CH 2 -protons of PEG adjacent to -O-COgroups of acrylate and 5-hexynoate.

  H-NMR in DMSO-d 6 by comparing the integrals of βCH 2 -of 5-hexynoic acid moiety at 1.69 ppm with the massive of the anomeric and 3 hydroxyl protons of dextran (4H) at 4.4-5.3 ppm (Figure II.6). The conversion was as high as 96%.

  H-NMR (Figure II.7) and FTIR (Figure II.8).

Figure II. 7 :

 7 Figure II.7: 1 H-NMR spectrum (DMSOd 6 , 400 MHz) of 6-monodeoxy-6-monoazido-βCD (βCD-N 3 ).

Figure II. 8 :

 8 Figure II.8: IR-spectrum of mono-(O-6-deoxy-6-azido)-βcyclodextrin (βCD-N 3 ) showing the single peak at 2105 cm -1 corresponding to the grafted azides.

7 :

 7 Scheme II.7: Synthesis of host polymer (DT40-g-PEG-βCD) by CuAAC with CuSO 4 /Na-Asc+TBTA catalytic system.

Figure II. 9 :

 9 Figure II.9: 1 H-NMR spectrum (DMSO-d 6 , 400 MHz) of DT40-g-PEG-βCD (DS CD = 3.7mol.%) prepared by CuAAC in pure water without TBTA (P0). Redoubling of β-CH 2 -protons of 5hexynoate at 1.6-1.9 ppm is clearly observed.

  2 section of this chapter. Assuming the DS CD determined by NMR correct, one should calculate the binding stoichiometry, n, between the grafted βCD and the titrant, 2-(1-adamantyl)ethyl trimethylammonium bromide (AdaTMA). Ideally the latter should equal to unity and DS CD (ITC) = DS CD (NMR). However, due to adsorbed water, inevitably present in freeze-dried polymer samples (~10-15 wt.%), the experimental n value, and consequently the resulting DS CD are slightly lower than those obtained by NMR. As one can see, the ITC experiment confirmed the incompleteness of alkynes conversion for P1 click assay (Table II.1).

Figure II. 10 :

 10 Figure II.10: 1 H-NMR spectrum (DMSO-d 6 , 400 MHz) of DT40-g-PEG-βCD (DS CD = 16.5 mol.%, P3 in Table II.1).

  ) while both P0 and P1, which contain residual terminal alkynes, have shown limited solubility in water (gel formation at concentrations higher than 10 g/L).

  glucopyranose units undergo a spontaneous 360° rotation in water (Figure II.11). As a result, part of the spacers end up being deeply included inside one of the βCD cavities. Thereby, the latter become no longer available for inclusion complexation.

Figure II. 11 :

 11 Figure II.11: Schematic illustration of the substituted glucopyranose inversion process for the case of para-isomer. Reprinted from Menuel et al.29

Figure II. 12 :

 12 Figure II.12: 1 H-NMR spectrum (D 2 O, 400 MHz) of DT40-g-PEG-βCD (P2). A single triazole proton signal is observed at 7.70 ppm.

1. 4 . 1

 41 Scheme II.8: Synthesis of PEG-acrylate-Ada: a) by DBTDL-catalyzed isothiocyanate-alcohol reaction; (b) DMAP-catalyzed esterification with 1-adamantanecarbonyl chloride.

Figure II. 13 :

 13 Figure II.13: 1 H-NMR spectra (CDCl 3 , 400 MHz) of the products of PEG-acrylate modification via: a) DBTDL-catalyzed coupling with Ada-NCS; b) DMAP-catalyzed esterification with 1adamantanecarbonyl chloride (3h reflux in CHCl 3 ); c) DMAP-catalyzed esterification with 1adamantanecarbonyl chloride (2h at 0°C + 3h reflux in CHCl 3 ).

  Scheme II.9: Synthesis of DT40-PEG-Ada by nucleophile-mediated thiol-Michael addition.

Figure II. 14 :

 14 Figure II.14: 1 H-NMR spectrum (D 2 O, 400 MHz) of DT40-g-PEG-Ada (DS CD = 10.5 mol. %).

2. 1

 1 Figure II.15: ITC enthalpograms characterizing the interactions of βCD as monomeric hosts with DT40-g-PEG-Ada1 (a, 5.1 mol.% Ada) and DT40-g-PEG-Ada2 (b, 10.5 mol.% Ada) as hosts. Upper panel: amounts of heat released upon successive injection of 15 μL aliquots of βCD (5 mM) into solutions of DT40-g-PEG-Ada (0.5 mM in Ada). Bottom panel: integrated heat data, giving a differential binding curve which was fitted to a standard single-site binding model.

Figure II. 16 :

 16 Figure II.16: Structural comparison of the investigated host (a) and guest (b) polymers.

Figure II. 17 :

 17 Figure II.17: Schematic representation (a) and chemical structure (b) of pβCDN+.

Figure II. 18 :

 18 Figure II.18: Schematic representation of an SPR experiment for evaluation of D40Ada and DT40-gPEG-Ada binding properties on the surface (HG stands for host-guest interactions).

Figure II. 19 :

 19 Figure II.19: SPR sensograms of the host-guest driven adsorption of D40Ada9 and DT40-gPEG-Ada2 on top of gold/ pβCDN+ bilayers.

2. 2

 2 Figure II.20: ITC enthalpograms characterizing the interactions between AdaTMA as monomeric guest and different DT40-g-PEG-βCD as hosts: a) P1; b) P2; c) P3. Upper panel: amounts of heat released upon successive injection of 15 μL aliquots of AdaTMA (5 mM) into solutions of DT40g-PEG-βCD (0.5 mM in βCD), both dissolved in 0.1 mol•L -1 NaCl. Bottom panel: integrated heat data, giving a differential binding curve which was fit to a standard single-site binding model.

  Comparison of the structural formulas of the investigated polymers is shown in Figure II.16, b. This finding was surprising given the nature of flexible hydrophilic spacers which were supposed to increase the availability of βCD moieties.

  ) was used as the 1 st layer to saturate the gold surface of the sensor. Its deposition resulted in an increase of the measured refractive index ΔRI 1 . A layer of the guest polymer with PEG spacer (DT40-g-PEG-Ada, DS = 10.5 mol.%) was then adsorbed on top of pβCDN+ layer via host-guest interactions, resulting in a 2 nd increase in refractive index (ΔRI 2 in Figure II.21).

Figure II. 21 :

 21 Figure II.21: Schematic representation of an SPR experiment for evaluation of DT70GPβCD8 and DT40-gPEG-βCD binding properties on the surface.

Figure II. 22 :

 22 Figure II.22: SPR sensograms of the host-guest driven adsorption of DT70GPβCD8 and DT40-gPEG-βCD on top of gold/ pβCDN+/ DT40-g-PEG-Ada multilayers.

  growing aggregates was observed (Figure II.23, a). This resulted in a fast drop of scattering intensity, caused by associative phase separation and precipitation (Figure II.23, b).

3. 1 . 1 Figure II. 23 :

 1123 Figure II.23: Hydrodynamic diameters (a) and scattered intensity count rates (b) of the freshly prepared HG nanoassemblies prepared from DT40-gPEG-Ada2 (Ada) and DT40-gPEG-βCD (CD) solutions stored for different periods of time. Patterned area corresponds to precipitation region.

Figure

  II.23, b).

Figure II. 23 ,

 23 a). In this case the most efficient nanoassemblies formation was observed, once again, at equimolar βCD/Ada ratio as evidenced by the maximum position in the scattered light intensity (green curve in Figure II.23, b). However, the absolute scattering intensity at maximum is slightly lower comparing to the experiment where non-aged host polymer solution was used. It might be due to a partial degradation of the dextran polymers during the extended periods of storage.

3. 1 . 2 Figure II. 24 : 3 . 1 . 3

 1224313 Figure II.24: Hydrodynamic diameters (a) and scattered intensity count rates (b) of the HG nanoassemblies stored for 5 days, prepared from DT40-gPEG-Ada2 (Ada) and DT40-gPEG-βCD (CD) solutions stored for different periods of time.

Figure II. 25 : 3 . 1 . 4

 25314 Figure II.25: Hydrodynamic diameters of the HG nanoassemblies stored for 5 days, prepared from fresh host polymer and continuously aged guest polymer stock solutions at C tot of the polymers: a) 0.68 g/L, b) 1.36 g/L (βCD/Ada = 1:1). The experimental data points fitted to the 1 st order exponential decay function.

Figure II. 26 :

 26 Figure II.26: Hydrodynamic diameters and scattered intensity count rates of the HG nanoassemblies prepared from DT40-gPEG-Ada2 preliminary incubated for 24h with various amounts of monomeric βCD (βCD mon ). C tot of the polymers 1.36 g/L, βCD/Ada = 1:1.

  SAXS measurements of the individual HG polymers of different types and nanoassemblies prepared from them were done using the laboratory instrument at Aarhus University (Denmark) in collaboration with our colleagues Beatrice Plazzotta and Prof. Jan Skov Pedersen. The SAXS data were complemented by SLS experiments performed at University of Maine with the kind support from Dr. Céline Charbonneau and Dr. Taco Nicolai.

Figure II. 27 :

 27 Figure II.27: Determination of the particles morphology by combining of the SAXS and DLS data.

  results of SLS experiments for the individual host-guest polymers with various structures are represented in Figure II.28a. The weight concentrations were chosen so that to keep a constant C CD(Ada) value (5•10 -4 M) and lie in the range of 1.2-1.9 g/L.

Figure II. 28 :

 28 Figure II.28: a) Rayleigh ratios angular dependency (𝑅(𝑞)) by SLS and b) SAXS scattering curves for the individual host and guest polymers without PEG spacer and with hydrophilic PEG spacer (samples with the same concentrations and times of storage were used for both kinds of experiments).

99 An

 99 analogous trend was found in the SAXS data for the individual polymers (Figure II.28b). The first thing to notice while analyzing the low-q range of the curves is a considerably higher scattering intensity for the host polymer with PEG spacer comparing to the other polymers. Two ways to obtain their 𝑅 𝑔 were used: a) the construction of Guinier plots; b) analysis of the pairdistance distribution functions (PDDF) obtained via inverse Fourier-transformation of the oscillating part of the form factor in the middle-𝑞 range (described in detail in the Experimental section). Both ways of calculation lead to twice as high 𝑅 𝑔 values for DT40-gPEG-βCD compared to its PEG-containing guest counterpart (DT40-gPEG-Ada2) and host polymer without PEG spacer (DT70GPβCD8), as reported in

9 )Figure II. 29 :

 929 Figure II.29: SAXS scattering curves of the DT40-g-PEG-Ada2 (DS CD = 10.5 mol.%) solutions as a function of: a) time of storage (both at 1.2 g/L); b) concentration (both solutions 1 day after preparation).

Figure II. 30 :

 30 Figure II.30: SAXS scattering curves of the DT40-g-PEG-βCD (DS CD = 16.5 mol.%) solutions as a function of: a) time of storage (both at 1.5 g/L); b) concentration (both solutions 1 day after preparation). The scaling factor between the two dataset on the right is coherent with the concentration difference.

Figure II. 31 :Figure II. 32 :

 3132 Figure II.31: Schematic representations of a) a comb-like polymer chain; b) a star-like structure with 4 comb-like arms (DT40-gPEG-βCD); c) the basic subunit (form factor𝑃 𝑠𝑢𝑏 (𝑞)) of a comblike polymer chain (form factor𝑃 𝑎𝑟𝑚 (𝑞)). Derivation of the mathematical expressions for 𝑃 𝑠𝑢𝑏 (𝑞) and 𝑃 𝑎𝑟𝑚 (𝑞) can be found in the Experimental section.

  -guest nanoassemblies of various compositions were prepared by mixing the solutions of individual polymers at equimolar βCD/Ada ratio and C CD/Ada = 5•10 -4 M (aged for 30 days in the case of the polymers with PEG-spacer). At the first stage we collected the scattering data for lower 𝑞 range by performing SLS experiments (Figure II.33).

Figure II. 33 :

 33 Figure II.33: Rayleigh ratios angular dependency (𝑅(𝑞)) for the HG-nanoassemblies of different compositions (TableII.12).

Figure II. 34 :

 34 Figure II.34: SAXS scattering patterns representing the formation of HG-nanoassemblies between the polymers without (a) and with (b) hydrophilic PEG spacer (C CD/Ada = 5•10 -4 M).

Figure II. 35 :

 35 Figure II.35: SAXS scattering patterns of the HG-nanoassemblies between the polymers without (a) and with (b) hydrophilic PEG spacer prepared in the different ways, i.e. either by diffusiondriven mixing of the polymers in the SAXS measuring capillary (red curves) or by preliminary vigorous stirring of the polymer solutions at 800 rpm (black curves).

  nanoassemblies are thus modelled by a polydisperse spherical form factor and a Lorentzian term was introduced to account for the fluctuations of the gel network. Due to the relative low concentrations of the samples the structure factor was neglected while fitting the data. The mathematical description of the model used is provided in the Experimental section. The parameters deduced from the fits using this model are thus: 𝑅 𝑔 , radius of gyration of the nanoassemblies; σ poly , their polydispersity index and  l -correlation length of the network inside the nanoassemblies. Despite a certain shift towards higher 𝑅 𝑔 values, most of the nanoparticle features derived from the modeling were consistent with those obtained by the Guinier approximation and PDDF analysis (Table II.14). Namely, the nanoparticles composed of DT70GPβCD8 and DT40Ada9 are bigger than those produced from their PEG-containing counterparts (Figure II.36a). When host polymer with PEG spacer (DT40-gPEG-βCD) and guest polymer without PEG spacer (DT40Ada9) are used, the 𝑅 𝑔 of the resulting mixed nanoparticles (P6) is roughly an average of those of P2 and P4 (TableII.14). At the same time, the "inversed" mixed nanoparticles self-assembled from DT70GPβCD8 and DT40-gPEG-Ada2 proved to be the smallest ones with 𝑅 𝑔 ~ 28 nm (P5). No difference was found between the nanoparticles prepared by stirring and by diffusion.

Figure II. 36 :

 36 Figure II.36: Experimental SAXS data (symbols) and best fits to a polydisperse spherical form factor model (lines): a) nanoparticles made of the polymers with and without the PEG spacer; b) mixed nanoparticles of two types. The corresponding 𝑅 𝑔 and other parameters are given in

:

  ITC measurements were performed using a MicroCal VPITC microcalorimeter. In each titration, injections of 15 μL of concentrated AdaTMA or βCD solutions (both at 5•10 -3 mol •L -1 in 0.1 mol •L -1 NaCl and in pure water respectively) were added from the computer controlled 295 μL microsyringe at an interval of 180 s into the cell (V= 1.4569 mL) containing the investigated polymer solution (5 •10 -4 mol •L -1 of βCD or Ada groups) while stirring at 450 rpm. The temperature was fixed at 25°C. The raw experimental data are obtained as the amount of heat produced per second following each injection of titrant as a function of time. Integration of the heat flow peaks by the instrument software (after taking into account heat of dilution) provides the amount of heat produced per injection. The experimental data were fitted with a theoretical titration curve using the instrument software with a model assuming a 1:1 stoichiometry for the adamantyl/βCD complex. The enthalpy change, |𝛥𝐻|, the association constant, K a , and the overall stoichiometry, n, were the adjustable parameters.Dynamic light scattering:The mean hydrodynamic diameter and the polydispersity index (PdI) of the nanoassemblies were determined by dynamic light scattering (DLS) using a Zetasizer Nano ZS (Model ZEN3500) Malvern Instrument equipped with a He-Ne laser (λ = 633 nm, scattering angle 173°). Individual polymer solutions in water were filtered using 0.40 μm regenerated cellulose syringe filters prior to measurements and/or nanoassemblies preparation.Each investigated sample was measured fifteen times for ten seconds at 25°C.In DLS, a sample containing a suspension of colloidal particles in solution is irradiated by a laser beam. The laser light which hits particles is scattered in all directions. The scattering intensity shows time-dependent fluctuations as a result of the particles undergoing Brownian motions in the irradiated volume. The fluctuation rate is related to the size of the particles: the smaller the particles, the quicker they will move causing higher fluctuation rates. By the means of special equipment, digital autocorrelators, quantitative information can be derived from the intensity fluctuations. It is represented by so-called intensity autocorrelation function: 𝐺(𝜏) = 𝐵 + 𝐴 ∑ exp (-2𝑞 2 𝐷𝜏) (II.3)where 𝐵 -baseline at infinite time, 𝐴 -amplitude (or intercept), 𝐷-diffusion coefficient, 𝜏correlator delay time, 𝑞 -scattering vector,

  )

  81) was added to the solution followed by 4-NC (3.47 g, 17.2 mmol). The reaction mixture was stirred at 0°C for 2h, precipitated twice in cold EtOH, washed with 1:1 mixture of EtOH/Et 2 O and dried in vacuum overnight at 56°C yielding a white solid (m = 4.06g). DS (from 1 H-NMR = 17%). 1 H-NMR (DMSOd 6 ) δ = 3.0-4.0 (m, dextran glucosidic protons), 4.45, 4.78 and 4.90 (s, dextran hydroxyl protons), 4.67 (s, anomeric proton), 5.29 and 5.53 (s, glucosidic protons at positions bearing nitrophenyl substituents), 7.55 and 8.33 (dd, 2H, aromatic protons). 4-NC activated dextran (4.0 g, 21 mmol of AHG, 3.6 mmol of 4-NC) was dissolved in 35 ml of DMF containing 2 w/v% of LiCl at 55°C. Cysteamine (2.2 g, 28.5 mmol) was dissolved in 50 ml of DMF by warming the solution to 40°C and continuously blowing Argon through it. The solution of cysteamine was added drop wise to the solution of 4-NC activated dextran previously cooled to room temperature. The reaction mixture was stirred at room temperature under argon for 24h, precipitated in cold EtOH and washed with ethanol. The resulting white solid was redissolved in 250 ml of water. Dithioerythritol (0.26 g, 1.7 mmol) was added in order to reduce the disulfide bonds, possibly formed during the reaction. The solution was purified by ultrafiltration (MWCO 10000) against degassed distilled water under nitrogen and finally the product was recovered by freeze drying (m = 3.08 g, 82%). DS (from 1 H-NMR = 17%). 1 H-NMR (D 2 O) δ = 2.66 (t, 2H, NHCH 2 ), 3.32 (d, 2H, CH 2 SH), 3.4-4.1 (m, dextran glucosidic protons), 4.97

  -3.42 (m, overlapped with HDO), 3.40-3.76 (m, 28H), 4.15-4.58 (m, 6H), 4.75-4.92 (m, 7H), 5.56-5.93 (m, 14H), 7.43 (d, 2H), 7.75 (d, 2H). Mono-(O-6-deoxy-6-azido)-βcyclodextrin: βCD-OTs (3.5 g, 2.71mmol) was dissolved in 30 ml of water by warming it to 85°C under vigorous stirring. Excess of sodium azide (2.65 g, 40.7 mmol) was added to the solution and the reaction was stirred at 80°C for 60h. The mixture was precipitated in 320 ml of acetone, filtrated and the product was isolated by drying at 65°C in vacuum. To obtain the pure mono-(O-6-deoxy-6-azido)-βcyclodextrin (βCD-N 3 ) precipitations in acetone were proceeded until the IR spectrum showed no peak from free sodium azide at 2138 cm -1 (Figure II.8). Yield: 2.64 g (84.1%). 1 H-NMR (DMSO-d 6 ) δ = 3.21-3.45 (m, overlapped with HDO), 3.49-3.84 (m, 28H), 4.38-4.60 (m, 6H), 4.75-4.93 (m, 7H), 5.56-5.93 (m, 14H).DT40-g-PEG-βCD: CuSO 4 /Na-Asc path. DT40-g-PEG-hex-5-ynoate (0.2 g, 0.13 mmol of terminal alkynes), βCD-N 3 (0.264 g, 0.23 mmol) and TBTA (0.014 g, 0.026 mmol) were dissolved in 30 ml of preliminary degassed DMSO/water 2:1. The mixture was heated to 50°C while continuously flushing argon through it. After the sodium ascorbate (400 μl of 20 g/L solution, 0.04 mmol) and CuSO 4 •5H 2 O (320 μl of 10 g/L solution, 0.013 mmol) were added, the solution was degassed by bubbling argon under sonication. After stirring at 50°C for 30h the reaction mixture was precipitated in acetone. The precipitate was then washed twice with acetone, dissolved in water and dialyzed against water for 3 days. The βCD terminated Dextran-PEG grafted copolymers (DT40-g-PEG-βCD) were isolated by freeze drying (m = 0.255 g, P2).

Scheme 10 :

 10 Scheme 10: Two-step synthesis of TBTA -Cu(I)-stabilizing ligand.

Figure II. 37 :

 37 Figure II.37: 1 H-NMR spectrum (CDCl 3 , 400 MHz) of tris-(benzyltriazolylmethyl)amine (TBTA)prepared according to the original procedure.28

Scheme III. 1 :

 1 Scheme III.1: Reaction scheme for the synthesis of 1-adamantaneethylazide (a) and MeOPEG-N 3 (b).

  The 1 H-NMR spectra of the resulting polymers (D40-GP) in D 2 O show a well-separated singlet of terminal alkyne protons at 2.90 ppm (Figure III.1).

Figure III. 1 :Scheme III. 3 :

 13 Figure III.1: 1 H-NMR spectrum of alkyne-modified dextran D40-28GP in D 2 O.

  irradiation, the reaction times reduced dramatically. Full conversion of the residual alkynes by the reaction with MeOPEG-N 3 was typically achieved within 40 min at 85 °C.

Figure III. 2 :

 2 Figure III.2: 1 H-NMR spectra in DMSO-d 6 of: a) D40-18GP-18PEG, modified exclusively with 18 mol.% of 5000 g•mole -1 PEG chains. A single triazole proton is found at 8.06 ppm; b) D40-28GP-10Ada-18PEG, modified with 18 mol.% of PEG and 10 mol.% of adamantyl groups. Two partially overlapping triazole protons are observed at 8.06 and 8.10 ppm.

  the next stage we applied ITC to study the interpolymer interactions. The concentration of Ada groups in the solutions of D40-GP-Ada-PEGs was fixed at 5•10 -4 mole/L. The solution of D40-18GP-18PEG, bearing no adamantyl groups, was used at the mass concentration equal to that of D40-28GP-10Ada-18PEG with C(Ada) = 5•10 -4 mole/L. The enthalpograms, shown in Figure III.3b,c are plotted as a function of the βCD/Ada molar ratio. Except for the case of D40-18GP-18PEG (free of Ada groups) where no interaction could be detected (black circles in Figure III.3b,c), the guest polymers display highly exothermic interactions with both neutral pβCD and positively charged pβCDN+.

Figure III. 3 :

 3 Figure III.3: Enthalpograms of the interaction of D40-GP-Ada-PEG polymers with monomeric βCD (a); pβCD (b) and pβCDN+ (c). The solid lines represent the fits of the data by One Set of Sites model (Origin 7.0 ITC custom version).

Figure III. 4 .

 4 Figure III.4. Three injections of 2 g/L solutions were enough to reach the plateau in RI, indicating the surface saturation, as shown in the first part of the sensograms for neutral pβCD (Figure III.5). The resulting 1 st layer obtained after equilibration for 20 min gives a quite stable ΔRI in both cases (8.5 • 10 -4 ± 0.8 and 12.1 • 10 -4 ± 0.7 for pβCDN+ and pβCD respectively).

Figure III. 4 :

 4 Figure III.4: Schematic representation of an SPR experiment for evaluation of D40-GP-Ada-PEG binding properties on the surface.

  0.5M NaCl solution in the present case, electrostatic repulsions are not screened at all and only negligible amounts are adsorbed.

Figure III. 5 :

 5 Figure III.5: Adsorption of D40-GP-Ada-PEG on neutral pβCD layer. DS(Ada): 0 (a), 5 (b), 10 (c) mol.%

Figure III. 6 :

 6 Figure III.6: Adsorption of D40-GP-Ada-PEG on pβCDN+ with 1.6 N + per CD. DS(Ada): 0 (a), 5 (b), 10 (c) mol.%. Consecutive injections are indicated by arrows.

8 .

 8 18PEG was studied next, given that the latter demonstrated the most interesting SPR adsorption profile (Figure III.6c, III.7). A modified version of SPR experiment was used. First, gold surface was saturated with pβCDN+, and then diluted D40-28GP-10Ada-18PEG solutions at various concentrations (0.01 -0.20 g/L) were passed over the pβCDN+ layer. The results are represented in Figure III.At low concentrations (i.e. 0.01 g/L), the RI increases quasi-linearly with time, indicating that every polymer molecule reaching the surface should be adsorbed in this case. The adsorbed amount corresponding to the plateau (0.59 mg/m 2 ) is less than 30% of the saturation amount, obtained from the SPR adsorption experiment. The sensogram recorded at 0.025 g/L changes its character drastically. One observes a steep initial growth and 1.65 mg/m 2 of the deposited polymer at the plateau, which is already 78% of the saturation value.When increasing the concentration up to 0.1 g/L and higher, the initial exponential growth becomes even steeper and the amount of adsorbed polymer at the plateau gradually approaches the saturation level from the experiment with multiple injections of the 1 g/L solution (Figure III.7). The above is an indication of strong affinity between the polymers. Indeed, at such low concentrations as 0.025 g/L one already observes a fast exponential saturation of the surface sites of pβCDN+.

Figure III. 8 :

 8 Figure III.8: The evolution of RI versus time for various D40-28GP-10Ada-18PEG concentrations flowed over a pβCDN+ layer.

9 .Figure III. 9 :

 99 Figure III.9: Experimental response of RI versus time for the adsorption of D40-28GP-10Ada-18PEG on pβCDN+ layer (circles) with the corresponding first order exponential decay fits performed according to the Equation III.3 (solid lines); C(D40-28GP-10Ada-18PEG) = 0.08 (a), 0.16 (b) g/L.

  The polymer was purified by ultrafiltration on regenerated cellulose membrane with the MWCO of 30 000 Da. The final pβCDN+ product was obtained by freeze-drying (m = 3.85 g). The N+ content was determined by 1 H-NMR in D 2 O by comparing the integral of three methyl groups of grafted quaternary ammonium groups (9H, 3.22 ppm) with the integral of anomeric protons of βCD (7H, 4.91-5.53 ppm). The result was 1.62N+ per CD cavity which corresponds to 58.2 wt% of βCD in the final product.

2

 2 Due to the thermoresponsive character of pNIPAm the microgels show VPT triggered by increase of temperature, typically occurring at ~33°C (Figure IV.1). 2

Figure IV. 1 :

 1 Figure IV.1: Schematic illustration showing the VPT process of a responsive cross-linked microgel particle triggered by an external stimulus, herein temperature.

Figure IV. 2 )

 2 often show better mechanical properties and the biocompatibility of microgels/nanoparticles is increased by "hiding" the latter in the biocompatible hydrogel network.

Figure IV. 2 :

 2 Figure IV.2: Illustration of a "plum pudding" hydrogel containing nanoscale delivery vehicle loaded with a drug. Such system is characterized by two diffusion coefficients: 𝐷 1 representing

  microgels (DX microgels) (Figure IV.3). All these types of hydrogels exhibit structural hierarchy with two or more distinct length scales between the adjacent crosslinks represented by the correlation lengths . In the first two cases microgels are either mechanically entrapped (Figure IV.3, i) or covalently linked (Figure IV.3, ii) with the bulk hydrogel matrix. In the case of DX microgels, however, the particles are directly interlinked and no additional hydrogel matrix is required (Figure IV.3, iii).

Figure IV. 3 :

 3 Figure IV.3: Schematic illustration of different types of microgel-containing hydrogels. Reprinted from Richtering and Saunders.23

  At high temperatures (> VPT temperature of the microgels) the collapse of filler microgel particles led to a change in their nature from a soft to a hard filler. Incorporation of 25 g•L -1 of microgels in a hydrogel with total polymer concentration of 75 g•L -1 led to storage moduli twice as high at 20°C and ten times as high at 40°C comparing to the pure PAAm hydrogel with the same mass concentration (Figure IV.4).

Figure IV. 4 : 1 . 1 . 1

 4111 Figure IV.4:The storage modulus G' of pNIPAm microgel-filled PAAm hydrogels as a function of microgel concentration at two different temperatures. Reprinted from Meid et al.30

Figure IV. 5 :

 5 Figure IV.5: Schematic illustration of the pNIPAm microgel cores synthesis by precipitation polymerization technique.

Figure IV. 6 :

 6 Figure IV.6: Evolution of hydrodynamic diameters of pNIPAm-g-100%pAAc microgels as a function of temperature at different pH. The pH was set with 10 mM phosphate buffer solutions.

Figure IV. 7 :

 7 Figure IV.7: pNIPAm/βCDN host polymer coated microgels preparation strategy.

  4 mM (if not indicated otherwise, concentrations are given for the final volume after mixing). The hydrodynamic diameters of the particles were gradually decreasing as more of cyclodextrin polymer was added (Figure IV.8) which might be explained by shrinking of the AAc external shell due to partial negative charge compensation. At roughly stoichiometrical +/-ratio electrophoretic mobility of the microgel/oppositely charged polymer complexes drops down to zero (Figure IV.9), leading to their aggregation at +/-= 0.7-1.3 (Figure IV.8). However, when excess of pβCDN+ is introduced (corresponding to +/-= 1.3-1.4) pNIPAm/βCD1.62N host microgels become positively overcharged and regain their colloidal stability. The excess of positive charges induces an increase in the osmotic pressure of the solvent in the shell, leading to a partial reswelling of the microgels (Figure IV.8). Moreover, the obtained overcharged complexes show good stability over time with their D h only negligibly evolved after 8 days of storing in pure water at ambient temperature (Figure IV.8).

2 Figure IV. 8 :

 28 Figure IV.8: Evolution of hydrodynamic diameters (D h ) of pNIPAm/βCD1.62N host polymer coated microgels as a function of introduced (+)-charges concentration; stability of coated microgels after 8 days of storage at room temperature. C(AAc-) = 0.145 mM. The range of C(+) corresponding to colloidaly unstable particles is greyed out.

Figure IV. 9 :Figure IV. 10 :

 910 Figure IV.9: Electrophoretic mobilities (E mob ) of freshly prepared pNIPAm/βCD1.62N host polymer coated microgels as a function of introduced (+)-charges concentration. C(AAc-) = 0.145 mM. The range of C(+) corresponding to colloidaly unstable particles is greyed out.

Figure IV. 11 :

 11 Figure IV.11: TC experiments for determination of non-bound pβCDN+ in the supernatants after binding with pNIPAm-g-pAAc microgels: a) bare pNIPAm-g-pAAc ultracentrifugation efficiency as a function of pH; b) TC corresponding to pβCDN+ in the mixture before ultracentrifugation (filled circles) and to non-bound pβCDN+ in the supernatants of pNIPAm/βCD1.62N complexes (empty circles) as a function of +/-charges ratio, pH = 2.5, C(AAc-) = 0.5 mM. Ultracentrifugation conditions: 62 400 × g, 40°C, 10h.

. 1

 1 for polymer characteristics) on the coating formation efficiency (Figure IV.12). For this purpose poly-β-cyclodextrins with 3.24 and 5.00 quaternary ammonia N+ per CD cavity were used. Electrophoretic mobility curves evolve in a remarkably similar way as functions of C(+) in the mixture for different polymers.

Figure IV. 12 :Figure IV. 14 :

 1214 Figure IV.12: Electrophoretic mobilities (E mob ) of pNIPAm/βCDN coated with host polymers with different N+ charge density per βCD. C(AAc-) = 0.50 mM.

Figure IV. 15 :

 15 Figure IV.15: Comparison of hydrodynamic diameters (a) and electrophoretic mobilities (b) of pNIPAm/βCD1.62N and pNIPAm/βCD1.62N/PEG microgels prepared at equal +/-molar ratios (Ada/CD = 0.3, C(AAc-) = 0.145 mM).

Figure IV. 16 .

 16 Figure IV.16. Thus obtained 𝐷 (1.7 nm) is significantly lower than the 𝑅 𝐹 of individual PEG chains, indicating the brush-like conformation of the latter. It explains partly the origin of the achieved steric stabilization.

Figure IV. 16 : 1 . 3

 1613 Figure IV.16: Schematic illustration of the approach used to estimate the distance (𝐷) between the adjacent PEG chains on the surface of stabilized pNIPAm/βCD1.62N/PEG microgels. Black dots represent the anchor points of PEG grafts; 𝑎 stands for the area of the square cell occupied by a PEG graft.

Figure IV. 18 :

 18 Figure IV.18: Hydrodynamic diameters as a function of temperature for positively overcharged pNIPAm/βCD1.62N (+/-= 2.2) and neutral, sterically stabilized pNIPAm/βCD1.62N/PEG (+/-= 1.10) microgels recorded at pH 7; the pH of the stock pNIPAm-g-pAAc solution was set with 0.1M NaOH. C(AAc-) = 0.145 mM.

  Ada) as multivalent cross-linkers, which are expected to establish interparticle connections between the microgels through the βCD-Ada inclusion complexation (Figure IV.19).

Figure IV. 19 :

 19 Figure IV.19: Schematic illustration of the strategy for host-guest driven cross-linking of pβCDN+-coated microgels into a DX microgel 3D network.

2 (

 2 = 3.6 • 10 16 𝑝𝑎𝑟𝑡𝑖𝑐𝑙𝑒𝑠 • 𝐿 -1 (or 3.6 • 10 19 𝑝𝑎𝑟𝑡𝑖𝑐𝑙𝑒𝑠 • 𝑚 -3 ) (IV.2)where 𝑁 𝐴 is the Avogadro number and 𝐶 𝜇𝑔𝑒𝑙 is the concentration of microgels in g•L -1 . At +/-= molar ratio used in G3 and G4 samples) the 𝑅 ℎ = 150 nm (1.5•10 -7 m) for host polymer-coated microgels as determined by DLS. Hence, effective hydrodynamic volume of one particle of spherical shape is:

  sweep measurements of storage G' and loss G'' moduli are shown in Figure IV.20,a-c. The G' and

  the evidenced by the frequency sweep data viscoelastic character of the G samples (Figure IV.20), rather poor fits to the Maxwell model were obtained. An example of such fits for the G3 sample is illustrated in Figure IV.21. Only the loss modulus G'' measured data for low frequencies (0.1-1.6 rad•s -1 ) is somewhat coherent with corresponding fit (dashed line).

Figure IV. 21 :

 21 Figure IV.21: Storage G' and loss G'' moduli as a function of angular frequency 𝜔 for the G3 hydrogel sample and non-linear fits of G' (solid line) and G'' (dashed line) to the Maxwell model performed based on the equations IV.6 and IV.7 respectively (25°C, γ = 0.1%).

  their mechanical properties are expected to be temperature-dependent. The evolution of storage G' and loss G'' moduli of G3 sample as a function of temperature was studied in the range of 25-60°C (Figure IV.22,a).

Figure IV. 22 :

 22 Figure IV.22: Oscillatory rheological measurements of the pNIPAm/βCD1.62N/DT110Ada3-G3 hydrogel temperature-induced phase transitions: a) G', G'' evolution under temperature ramps (temperature change rate = 2°C/min, 𝑓= 10 Hz, γ = 0.1%); b) dynamics of G', G'' recovery under instantaneous switching of temperature between 25 and 50°C (𝑓= 10 Hz, γ = 0.1%).

  As showed in Figure IV.12 pβCD3.24N+ with doubled number of N+ per CD cavity work as efficiently as pβCD1.62N+ for coating and electrostatic stabilization of pNIPAm-g-pAAc. Thus, in the next step we tried to cross-link pβCD3.24N+-coated microgels (+/-= 2.0) with DT110Ada3. It turned out that pNIPAm/βCD3.24N/DT110Ada3 continue to remain in a viscous liquid state even at C tot as high as 14.8 wt%. Decreased host-guest cross-linking efficiency in this case should be related to a lower concentration of βCDs in the external shell of the overcharged pNIPAm/βCD3.24N particles.

Figure IV. 24 :(IV. 8 )

 248 Figure IV.24: Evolution of storage G' and loss G'' moduli under heating and cooling temperature ramps (temperature change rate = 2°C/min, 𝑓= 10 Hz, γ = 0.1%) for a) E1 and b) E2 hydrogel samples (Table IV.4).

Figure IV. 25 : 4 .

 254 Figure IV.25: Arrhenius plots for the determination of 𝐸 𝑎 of the host-guest supramolecular hydrogels with different compositions (Tables IV.3, IV.5) constructed according to the equation IV.4. The linear fits of the curves are represented by dash lines.

  weaker mechanical properties comparing to E1 (Figure IV.19, c-d). It may be related to aggregation between the relatively densely packed pNIPAm/βCD3.24N microgels in a freezedried state. Further, such aggregates might not be fully disrupted during the interaction with DT500Ada2 solution resulting in the lower strength of the gel network.

  biomedical applications, proved to have poor colloidal stability. Thus, a pathway to remove this limitation relying on the host-guest chemistry of the pβCDN+ coating was proposed. Neutral pNIPAm/βCDN were successfully sterically stabilized with prepared by CuAAC bifunctionalized (PEG, Ada)-grafted dextrans. Both in the case of pNIPAm/βCDN and pNIPAm/βCDN/PEG microgels, a full preservation of temperature-triggered VPT of pNIPAm core was observed. The developed systems attractiveness for potential drug delivery applications is determined by three main factors: a) pNIPAm core for a temperature-induced release of hydrophobic actives upon shrinking; b) βCD-containing shell intended to slow down the initial "burst release" phenomenon; c) hydrophilic PEG corona known for providing the nanoscale drug carriers with "stealth" properties and longer blood circulation times.In the second part of this work we studied another way of exploiting the surface host-guest chemistry of pNIPAm/βCDN. It involves non-covalent crosslinking of individual microgels with Ada-modified dextrans to yield hierarchical 3D networks. Depending on the total weight concentrations and molar ratios of the components, we observed phase separation, formation of viscous liquids or formation of hydrogels. The elastic storage moduli of the hydrogels with C tot of 10-11 wt% (G' = 1-3•10 3 Pa) are comparable with those reported for cyclodextrin-based supramolecular hydrogels with various architectures. Regarding the poor colloidal stability of pNIPAm/βCDN at high concentrations, the hydrogels can be formulated in two indirect ways: a) by concentrating the mixtures of starting components at lower than gelation point concentrations under vacuum at 55°C; b) by freeze-drying the pNIPAm/βCDN and their subsequent rehydration with a guest polymer solution.

  , due to the sensibility of supramolecular CD/guest links to the presence of low molecular

	weight hosts or guests, in most cases CD-based hierarchical hydrogels are inherently chemically-
	responsive to competitor binding. To illustrate this concept, Ogoshi et al. prepared chemically-
	responsive hydrogel containing single-wall carbon nanotubes (SWNT). SWNT were first covered
	with pyrene-modified βCDs via π-π interaction. Then, the gel network was formed through host-
	guest interactions with poly(acrylic acid) modified with dodecyl groups and could be converted
	back to sol by simple addition of competitors such as 1-adamantane carboxylic acid Na-salt or
	αCD. 144 The same scientific group reported chemically-responsive supramolecular hydrogel
	made from a guest-modified monomeric βCDs. 115 In the first step, inclusion complexation
	between the βCD bearing hydrophobic 2,4,6-trinitrophenyl-6-amino-transcinnamoyl moieties
	yielded supramolecular fibrils, which were further self-assembled into physical hydrogel via
	hydrogen bonding. The gel-to-sol transition could be easily induced either by host-guest
	competitors or by urea, the latter acting as a denaturating agent, able to disrupt hydrogen
	bonds.

Table of contents

 of Introduction ................................................................................................................................... Steglich esterification of poly(ethylene glycol) acrylate with 5-hexynoic acid ............. 1.3.2 Nucleophile-mediated thiol-Michael click of DT40-SH and PEG-acryloyl-5-hexynoate66 1.3.3 Synthesis of 6-Monodeoxy-6-monoazido-βCD (βCD-N3) ............................................. Control experiment with βCD aiming to disrupt hydrophobic Ada-conglomerates in fresh DT40-gPEG-Ada2 solutions ........................................................................................... 3.2 SAXS and SLS investigation of the individual polymers and HG-nanoassemblies of different types ........................................................................................................................................... Experimental section ................................................................................................................... Materials and reagents ............................................................................................................

1. Synthesis of the host-guest polymers with hydrophilic PEG spacer ....................................... 1.1 General synthetic strategy ................................................................................................... 1.2 Synthesis of thiolated dextrans (DT40-SH) .......................................................................... 1.3 Synthesis of host polymers with hydrophilic PEG spacer (DT40-g-PEG-βCD) ...................... 1.3.1 1.3.4 Synthesis of DT40-g-PEG-βCD by copper(I)-catalyzed azide-alkyne cycloaddition ...... 1.4 Synthesis of guest polymers with hydrophilic PEG spacer (DT40-g-PEG-Ada) .................... 1.4.1 Synthesis of hetero-bifunctionalized PEG-acrylate-Ada ............................................... 1.4.2 Synthesis of DT40-g-PEG-Ada guest polymers .............................................................. 2. Binding properties of host-guest polymers with hydrophilic PEG spacers ............................. 2.1 Guest polymers -DT40-gPEG-Ada ........................................................................................ 2.1.1 Isothermal titration microcalorimetry studies .............................................................. 2.1.2 Surface plasmon resonance studies .............................................................................. 2.2 Host polymers -DT40-gPEG-βCD ......................................................................................... 2.2.1 Isothermal titration microcalorimetry studies .............................................................. 2.2.2 Surface plasmon resonance studies .............................................................................. 3. Nanoassemblies prepared from host-guest polymers with hydrophilic PEG spacers ............ 3.1 Size and stability of HG-nanoassemblies by DLS .................................................................. 3.1.1 Effect of the stock solutions aging ................................................................................ 3.1.2 Nanoassemblies stability by DLS ................................................................................... 3.1.3 Minimal required DT40-gPEG-Ada2 aging time ............................................................

3.1.4 3.2.1 Individual host-and guest polymers ............................................................................. 3.2.2 Host-guest nanoparticles of different types ............................................................... Conclusions .................................................................................................................................. Methods and instrumentation ................................................................................................. Synthetic procedures ............................................................................................................... References ...................................................................................................................................

Table II . 1 .

 II1 Characteristics of DT40-g-PEG-βCD polymers prepared by CuAAC with different catalytic systems

	Entry	Catalytic system	DS alkyne (NMR), mol.%	DS CD (NMR), mol.%	DS CD (ITC), mol.%	Alkyne to CD conversion, %	Yield, %
	P0	CuSO 4 /Na-Asc, water	14.5	3.7	-	25.5	61
	P1	Cu(MeCN) 4 PF 6 /Na-Asc+TBTA, DMSO	15.5	11.2	9.5	72.2	63
	P2	CuSO 4 /Na-Asc+TBTA, DMSO/water = 2:1	15.5	13.4	11.3	86.4	85
	P3	CuSO 4 /Na-Asc+TBTA, DMSO/water = 2:1	19.5	16.5	14.3	84.6	81

  Some controversial data on isothiocyanate-hydroxyl group reactivity profile are found in literature. Thus, de Belder et al. described successful modification of dextran by fluoresceinisothiocyanate in the presence of DBTDL and pyridine in DMSO 31 ; at the same time in a more recent example Wiberg et al. have reported much lower reactivity of phenyl isothiocyanate (PhNCS) comparing to phenyl isocyanate (PhNCO) in the reaction with cyclohexanol catalyzed by

Table II . 2 .

 II2 Degree of substitution and yields of DT40-g-PEG-Ada polymers

	Compound	DS (-SH), mol.%	DS (Ada), mol.%	-SH to Ada % conversion,	Yield, %
	DT40-g-PEG-Ada1	6.5	5.1	78.5	81
	DT40-g-PEG-Ada2	12.5	10.5	84.0	84

Table II . 3 .

 II3 Thermodynamic parameters of inclusion complexes formation of βCD with DT40-g-PEG-Ada and with the Ada-substituted dextrans with short spacers (DT40Ada)19

	Polymer	DS NMR (Ada), mol.%	K a , L•mole -1	ΔH, kJ•mole -1	TΔS, kJ•mole -1	ΔG, kJ•mole -1
	DT40-g-PEG-Ada1	5.1	5.22•10 4	-41.2	-14.2	-27.0
	DT40-g-PEG-Ada2	10.5	6.08•10 4	-42.1	-14.7	-27.4
	DT40Ada6	6.6	1.16•10 4	-39.4	-16.2	-23.2
	DT40Ada9	8.8	1.14•10 4	-41.1	-17.9	-23.1

Table II . 4 .

 II4 Comparison of the binding efficiency of D40Ada9 and DT40-gPEG-Ada2 with respect to a positively charged cross-linked βCD-polymer (pβCDN+)

	Experiment	Adsorbed layer	ΔRI, 10 3 •a.u.	Q ads , mg/m 2	N(CD, Ada)/m 2	Ada/CD ratio
	no PEG spacer	pβCDN+ D40Ada9	1.02 1.85	1.09 1.98	3.76•10 17 5.94•10 17	1.58
		pβCDN+	0.86	0.92	3.17•10 17
	PEG spacer	DT40-gPEG-Ada2	3.08	3.30	9.05•10 17	2.85

Table II

 II

	Polymer	DS NMR (CD), mol.%	n	L•	K a ,

.5. Thermodynamic parameters of inclusion complexes formation of AdaTMA with DT40g-PEG-βCD and with the host polymers with short hydrophobic spacers (DT70GPβCD)

12

mole -1 ΔH, kJ•mole -1 TΔS, kJ•mole -1 ΔG, kJ•mole -1 DT40-g-PEG-βCD (P1)

		11.2	0.85	5.24•10 4	-22.4	4.6	-27
	DT40-g-PEG-βCD (P2)	13.4	0.85	5.04•10 4	-23.2	3.6	-26.8
	DT40-g-PEG-βCD (P3)	16.5	0.87	3.45•10 4	-17.7	8.2	-25.9
	DT70GPβCD5	12.7	0.85	2.05•10 5	-31.9	-1.3	-30.6
	DT70GPβCD6	16.8	0.82	1.90•10 5	-31.9	-1.4	-30.5

  8 nm and dn/dc equal to 0.145 mL/g for all investigated polymers. Based on 𝑄 𝑎𝑑𝑠 , the numbers of βCDand Adamoieties the in the 3 rd and the 2 nd adsorbed layers respectively (Figure II.21) were calculated.The results are reported in Table II.6. The CD/Ada molar ratio drops from 4.13 to 0.75 for DT70GPβCD8 and DT40-gPEG-βCD respectively, indicating higher binding efficiency for the host polymer with no PEG spacer. As in the case of the guest polymers, this result is coherent with the ITC data.

	Table II.6. Comparison of the binding efficiency of DT70GPβCD8 and DT40-gPEG-βCD with
	respect to guest polymer (DT40-g-PEG-Ada) surface layer by SPR
	Experiment	Adsorbed layer	ΔRI, 10 3 •a.u.	Q ads , mg/m 2	N(Ada or CD)/m 2	CD/Ada molar ratio
	no PEG spacer	DT40-g-PEG-Ada DT70GPβCD8	3.12 10.12	3.28 10.86	0.90•10 18 3.70•10 18	4.13
	PEG spacer	DT40-g-PEG-Ada DT40-gPEG-βCD	5.11 5.14	5.50 5.46	1.51•10 18 1.13•10 18	0.75

However, in the case of DT70GPβCD8, highly substituted (26 mol.%) by bulky βCD groups, one should also consider the sterical hindrance factor. Due to the local rigidity of DT70GPβCD8, after formation of the initial interpolymer host-guest links, the βCD in their vicinity can be restricted from approaching the surface. Saturation of the adamantyl surface sites should thus result in thicker adsorbed layers. In this case, the same number of free Ada binding sites on the surface is occupied by a higher number of DT70GPβCD8 molecules, leading to higher polymer coverage (𝑄 𝑎𝑑𝑠 ) and CD/Ada ratios (4:1). On the other hand, for DT40-gPEG-βCD with lower DS

(16.5

mol.%) and with βCDs being more loosely attached to the backbone via PEG spacer, the host polymer local rigidity should be much lower. There should be much less hindrance toward linking neighboring βCDs on surface sites. As a matter of fact, in this case association tends to occur close to the stoichiometrical 1:1 CD/Ada ratio. These remarks show that the discussion of the adsorption mechanism of the host and guest polymers is quite complex, given that they involve multivalent interactions with several opposing effects. For instance, high substitution degree should favor cooperative interactions with a zip fastener mechanism leading to stoichiometric host/guest association. However, high substitution ratio can at the same time lead to increased local rigidity and anticooperativity.

Table II . 7 .

 II7 Hydrodynamic diameters of the nanoassemblies obtained from the freshly prepared DT40-gPEG-βCD (16.5 mol.%) and DT40-gPEG-Ada2 (10.5 mol.%) stock solutions

	βCD/ Ada	C(host), g/L	C(guest), g/L	C tot , g/L	5 min	D h , nm 5d	11d 5 min	PDI 5d	CR ** (10 -3 , kpcs) 11d 5 min 5d 11d
	1:1	0.38	0.30	0.68	249	503	pr.	0.05	0.01	pr.	52	16 pr.
	1:2	0.23	0.36	0.59	1000	pr. *	pr.	1	pr.	pr.	17	pr. pr.
	2:1	0.46	0.18	0.64	146	305 401 0.05	0.09	0.10	111 30 15
	3:1	0.57	0.15	0.72	139	295 330 0.06	0.04	0.01	92	29 23
	4:1	0.61	0.12	0.73	128	266 334 0.05	0.11	0.06	59	28 20

* pr. -precipitation; ** -count rate.

Table II .

 II

	βCD/ Ada	C(host), g/L	C(guest), g/L	C tot , g/L	D h , nm 5min 5d 11d 5min	PDI 5d	CR (10 -3 , kpcs) 11d 5min 5d 11d
	1:1	0.38	0.30	0.68	162 251 313 0.02	0.01 0.11 153	56	28
	1:2	0.23	0.36	0.59	217 708 pr. *	0.02 0.21 ** pr.	64	22	pr.
	2:1	0.46	0.18	0.64	124 236 275 0.07	0.03 0.02	81	73	29
	3:1	0.57	0.15	0.72	112 223 234 0.10	0.05 0.01	54	55	35
	4:1	0.61	0.12	0.73	112 211 224 0.14	0.07 0.06	38	51	32

8. Hydrodynamic diameters of the nanoassemblies obtained by mixing of freshly prepared DT40-gPEG-βCD (16.5 mol.%) and aged for 30 days DT40-gPEG-Ada2 (10.5 mol.%) stock solutions * -precipitated, ** -2 nd generation of large aggregates is emerged

Table II .

 II

	Polymer	C, g/L	Time of storage, days	𝑹 𝒈, nm (SLS)	M w, 10 -3 g•mole -1 (SLS)	𝑹 𝑯, nm 1 peak 2 peak 1 peak 2 peak Volume, %
	DT40Ada9	1.9	1	41.0	78 (44)	5.3	81.8	99.9	0.1
	DT40-gPEG-Ada2	1.2 1.2 5.0	30 1 1	45.3	221 (62)	9.9 9.6 13.7	106.3 103.3 >1000	99.3 99.7 99.9	0.7 0.3 0.1
	DT70GPβCD8	1.4	1	26.4	244 (130)	12.0	128.1	99.7	0.3
	DT40-gPEG-βCD	1.5 1.5 0.75	30 1 30	52.2 *	4907 (116)	8.8 9.2 9.1	81.4 80.6 81.1	94.6 94.9 94.7	5.4 5.1 5.3
	DT40-g-PEG-hex-5-ynoate	1.5	2	-		13.6	103.9	99.5	0.5

9.

Characteristics of different types of host-guest polymers determined by light scattering (SLS + DLS); the theoretical 𝑀 𝑤 are given in brackets; 𝑅 𝐻 were measured at a single angle (173°) using a Zetasizer Nano-ZS and SLS measurements were performed at university of Maine with a multi-angle light scattering apparatus. * -experimental data were treated so as to eliminate the contribution from bigger scatterers

Table II .

 II Table II.10. Comparably high 𝑅 𝑔 obtained for the guest polymer with no PEG spacer (DT40Ada9) might be explained by the partial aggregation occurring in the solution of this hydrophobically modified polymer. 10. Radii of gyration 𝑅 𝑔 of different types of host-guest polymers determined from the SAXS data by two methods, Guinier plots construction and PDDF analysis.

	Polymer	C, g/L	Time of storage, days	𝑹 𝒈, nm (Guinier)	𝑹 𝒈, nm (PDDF)
	DT40Ada9	1.9	1	17.8	24.0
		1.2	30	6.9	10.1
	DT40-gPEG-Ada2	1.2	1	15.2	13.3
		5.0	1	21.9	23.7
	DT70GPβCD8	1.4	1	6.8	9.3
		1.5	30	21.5	18.8
	DT40-gPEG-βCD	1.5	1	20.5	19.4
		0.75	30	21.0	19.8
	Interestingly, for the guest polymers with PEG spacer, differences in the SAXS scattering

Table II . 11 .

 II11 Information on the models used to fit the experimental SAXS data and the size and structure parameters obtained for the individual host-guest polymers.

	Polymer	 2	Structure	Number of Branches	𝑹 𝒈 , nm (Model)	𝑹 𝒈 , nm (Guinier)
	DT40-gPEG-βCD 1.5 g/L aged	2.9	Star of comb-like chains	4	19.1±0.7	21.5
	DT40-gPEG-βCD 0.75 g/L aged	1.6	Star of comb-like chains	4	19.0±0.1	21.0
	DT40-gPEG-βCD 1.5 g/L fresh	1.8	Star of comb-like chains	4	18.7±0.4	20.5
	DT40-gPEG-Ada2 1.2 g/L aged	1.9	Comb-like	-	10 ± 2	6.9
	DT40-gPEG-Ada2 1.2 g/L fresh	2.5	Comb-like	-	13.3±0.2	15.2
	DT70GPβCD8 1.4 g/L fresh	1.1	Comb-like	-	9.3±0.5	6.8

However, in the case of DT40-gPEG-βCD, which shows clear signs of cross-linking, the data could be successfully modelled as star-like structure with 4 comb-like arms (Figure II.31b, 32b). This might possibly display only the local structure and the polymer can form a more extended network on a larger scale.

Table II .

 II 12. Characteristics of different types of host-guest nanoassemblies determined by light scattering (SLS + DLS). This might be a proof of a higher chain packing density in P2, as the result of stronger interpolymer interactions. On the other hand, the "mixed" particles (P5) show drastically lower both 𝑅 𝑔 and 𝑀 𝑤 values. Assuming dense packing in the case of P2, (without PEG spacer), 𝑀 𝑤 should vary as 𝑅 𝑔3 , meaning that a decrease of 𝑅 𝑔 by a factor of 2.06 should

	Code	Components	C tot , g/L	Way of mixing	𝑹 𝒈, nm (SLS)	M w, (SLS) 10 -6 g•mole -1	𝑹 𝑯, nm 𝑹 𝒈 /𝑹 𝑯
	P2	DT70GPβCD8; DT40Ada9	1.65	stirring	63.2	283	116.4	0.54
	P4	DT40-gPEG-βCD; DT40-gPEG-Ada2	1.35	stirring	60.7	128	107.1	0.57
	P5	DT70GPβCD8; DT40-gPEG-Ada2	1.30	stirring	30.7	15	52.0	0.59
	P6	DT40-gPEG-βCD; DT40Ada9	1.70	stirring	-*		97.5	-

* -negative slope in Guinier plot, 𝑅 𝑔 determination is not possible.

Interestingly, despite a little difference in 𝑅 𝑔 , nanoassemblies prepared from the polymers without PEG spacers (P2) have significantly higher molecular weight than PEG-spacer containing ones (P4, Table

II

.12). result in 8.74 times lower 𝑀 𝑤 . Comparing P2 and P5, the observed 𝑀 𝑤 decrease is more than twice larger

Table II .

 II As in the case of individual polymers, the 𝑅 𝑔 estimations by two approaches (i.e. Guinier plots and PDDF) were generally in good agreement (TableII.13). The nanoassemblies prepared from the polymers with PEG spacer (P3, P4 in TableII.13) appear to be slightly smaller both in terms of 𝑅 𝑔 and hydrodynamic radius 𝑅 𝐻 compared to their PEG-free counterparts (P1, P2).

	Code	Components	C tot , g/L	Way of mixing	𝑹 𝒈, nm (Guinier)	𝑹 𝒈, nm (PDDF)	𝑹 𝑯, nm	𝐆𝐮𝐢𝐧 𝑹 𝒈 /𝑹 𝑯
	P1	DT70GPβCD8; D40Ada9	1.65 diffusion	36.1	31.4	116.0	0.31
	P2	DT70GPβCD8; D40Ada9	1.65	stirring	36.1	32.0	116.4	0.31
	P3	DT40-gPEG-βCD; DT40-gPEG-Ada2	1.35 diffusion	24.9	24.9	105.5	0.24
	P4	DT40-gPEG-βCD; DT40-gPEG-Ada2	1.35	stirring	27.8	26.1	107.1	0.26
	P5	DT70GPβCD8; DT40-gPEG-Ada2	1.30	stirring	23.1	21.4	52.0	0.44
	P6	DT40-gPEG-βCD; D40Ada9	1.70	stirring	32.7	31.0	97.5	0.34

13. Radii of gyration 𝑅 𝑔 of different types of host-guest nanoassemblies determined by SAXS.

Table II .14. Table

 II

	Code	Components	Way of mixing	𝑹 𝒈 , nm (Model)	𝑹 𝒈 , nm (Guinier)	 poly 	 l , nm	 2
	P1	DT70GPβCD8+ DT40Ada9	Diffusion 45.4 ± 0.04	36.1	0.188 ±0.003	2.4±0.3	5.8
	P2	DT70GPβCD8+ DT40Ada9	Stirring	44.9 ± 0.4	36.1	0.187 ±0.003	2.1±0.3	5.7
	P3	DT40-gPEG-βCD + DT40-gPEG-Ada2	Diffusion	35.1 ± 0.1	24.9	0.207 ±0.004	16.7±0.3	4.0
	P4	DT40-gPEG-βCD + DT40-gPEG-Ada2	Stirring	35.3 ± 0.5	27.8	0.182 ±0.003	16.9±0.9	3.9
	P5	DT70GPβCD8 + DT40-gPEG-Ada2	Stirring	28.4 ± 0.2	23.1	0.182 ±0.002	4.6±0.2	4.6
	P6	DT40-gPEG-βCD + DT40Ada9	Stirring	42.6 ± 0.4	32.7	0.187 ±0.005	10.5±0.2	3.0

II.14. Modelling of the SAXS data for various HG-nanoparticles to a polydisperse spherical form factor model. Contemporary theories of semidilute/concentrated polymer solutions and swollen polymer gel networks define correlation length ( l in Table II.14) as the radius of a volume inside which intramolecular effects are dominant and beyond which intermolecular effects prevail.

  12, 27 NMR analyses were performed in D 2 O or DMSO-d 6 , unless other is indicated, on a Bruker Avance II Ultrashield Plus 400 MHz NMR spectrometer.1 H-NMR spectra were calibrated using the residual signals of D 2 O, DMSO-d 6 or CDCl 3 as the reference signals.

	Methods and instrumentation
	NMR spectroscopy: Thin layer chromatography (TLC): TLC experiments were carried out using Silica gel 60 F 254 TLC
	plates with 0.2 mm silica gel and 2-PrOH/H 2 O/EtOAc/NH 4 OH (c) as a mobile phase and visualized
	with 5% H 2 SO 4 in ethanol.

  ), 1.94 (t, 1H, C ≡CH), 2.23 (td, 2H, hexynoic-CH 2 ), 2.45 (t, 2H, hexynoic-CH 2 ), 3.56-3.64 (m, -CH 2 -PEG), 3.66 (t, 2H, CH 2 CH 2 O-hexynoate), 3.71 (t, 2H, CH 2 CH 2 O-acrylate), 4.20 (t, 2H, CH 2 CH 2 O-hexynoate), 4.28 (t, 2H, CH 2 CH 2 O-acrylate), 5.82 (m, 1H, CH=CH 2 ), 6.12 and 6.42 (m, 2H, CH=CH 2 ).

	DT40-g-PEG-hex-5-ynoate: Thiolated DT40-SH (1.4 g, 1.33 mmol of -SH groups) and lithium
	chloride (0.45 g, 10.6 mmol) were dissolved in 22 ml of DMF at 50°C while bubbling the solution
	with argon. The temperature was lowered to 35°C and the 0.4M solution of PEG-acryloyl-5-
	hexynoate in DMF (6.6 ml, 2.26 mmol) was added to the reaction mixture dropwise followed by
	hexylamine (60 μl, 0.65 mmol). The reaction was stirred at 35°C for 35h under atmosphere of
	argon. Finally DMF was evaporated under reduced pressure; the obtained product was
	redissolved in approximately 50 ml of water and dialyzed against pure water for 3 days. The
	alkyne terminated grafted copolymer (DT40-g-PEG-hex-5-ynoate) was isolated by freeze drying
	(m = 1.49 g). DS (from 1 H-NMR = 16%).

1

H-NMR (CDCl 3 ) δ = 1.

82

(quin, 2H, hexynoic-123 CH 2 1 H-NMR (DMSO-d 6 ) δ = 1.69 (quin, 2H, hexynoic-CH 2 ), 2.20 (td, 2H, hexynoic-CH 2 ), 2.40 (t, 2H, hexynoic-CH 2 ), 2.52-2.66 (m, 4H, CCH 2 CH 2 S-clicked acrylate), 2.71 (t, 2H, NHCH 2 ), 2.79 (s, 1H, C ≡CH), 2.98-3.96 (m, CH 2 SH with dextran glucosidic and PEG main chain protons, overlapped with HDO), 4.06-4.28 (m, 4H, CH 2 CH 2 O-hexynoate and

CH 2 CH 2 O-acrylate), 4.67 (s, anomeric proton), 4.50, 4.85 and 4.91 (m, dextran hydroxyl protons), 5.07 and 5.14 (s, glucosidic protons at substituted positions). 6A-O-p-toluenesulfonyl-βcyclodextrin: βCD (25.9 g, 22.5 mmol) was dissolved in 60 ml of water by warming under vigorous stirring. The temperature was lowered to 25°C and finely ground 1-(p-toluenesulfonyl)imidazole (20 g, 90.1 mmol) was added to the solution. The reaction mixture was stirred for 2h hours at room temperature. Sodium hydroxide (11.7 g, 293 mmol) in 32 ml of water was added over 15 min. The reaction mixture was left under stirring for another 15 min and filtered through a sintered-glass funnel in a flask charged with NH 4 Cl (31.3 g, 585 mmol).

Table III . 1 .

 III1 Characteristics of D40-XGP-YAda-ZPEG (guest) and pβCD (host) polymers

	Guest polymers	GP, mol%	Ada, mol%	PEG, mol%
	D40-18GP-18PEG	18	0	18
	D40-28GP-5Ada-23PEG	28	5	23
	D40-28GP-10Ada-18PEG	28	10	18
	Host Polymers	CD, wt%	M w , kg•mole -1	N+/CD
	pβCD	67.0	208	0
	pβCDN+ a	58.2	-	1.62

.1. a -prepared by modification of pβCD

a •10 -3 , L/mole ΔH, kJ/mole TΔS, kJ/mole ΔG, kJ/mole

	βCD	5 10	60.7 40.3	-44.4 -41.6	-16.9 -15.2	-27.4 -26.3
	pβCD	5 10	3.34 21.9	-40.0 -21.7	-19.8 3.0	-20.2 -24.8
	pβCDN+	5 10	3.02 35.4	-35.6 -18.1	-15.7 7.8	-19.8 -26.0

.2).

Table

III

.2. Thermodynamic parameters of interaction of the D40-GP-Ada-PEG polymers with monomeric βCD and βCD-polymers at 25°C Titrant Ada, mol.% K

Table III .

 III

	3. Association (k a ) and dissociation (k d ) rates between D40-28GP-10Ada-18PEG and
	pβCDN+ layer on gold
	C, g/l	k a , mol -1 s -1	k d , s -1
	0.08	8.6•10 2	0.6•10 -3
	0.10	7.2•10 2	0.8•10 -3
	0.13	8.6•10 2	1.4•10 -3
	0.16	8.3•10 2	2.0•10 -3
	0.20	2.5•10 2	0.6•10 -3
	Average	(7.0±2.6)•10 2	(1.1±0.6)•10 -3

  The reaction mixture was allowed to warm and stirred overnight at room temperature. The solvent was evaporated under reduced pressure and the product was redissolved in 100 mL of EtOAc. The organic layer was washed once with saturated NaHCO 3 and twice with brine, dried over Na 2 SO 4 , filtered and evaporated under reduced pressure. The subsequent filtration through a silica pad (40 g, EtOAc/n-heptane = 1/25) yielded the final product as a white solid. Thin layer chromatography (TLC) was carried out using Silica gel 60 F 254 SOCl 2 ; the reaction mixture was heated to 85 °C and refluxed for 40 h. Excess SOCl 2 was removed by bubbling argon at 65°C. Then 100 mL of DCM were added and the obtained solution was precipitated in 1 L of Et 2 O, filtered and washed with Et 2 O. The subsequent drying in vacuum at 40°C yielded the product as a white solid. Yield: 94.6 % (9.51 g). MeOPEG-Cl (9.40 g, 1.87 mmol) was dissolved in 100 mL of anhydrous DMF and treated with NaN 3 (3.30 g, 50 mmol). The reaction mixture was stirred at 80 °C for 60 h, precipitated in 1 L of Et 2 O and filtered. The product was subsequently purified by dissolving it in 100 mL dichloromethane, washing with 200 mL of water, concentrating the organic layer to 50 mL under reduced pressure and its precipitation in 600 mL of 2-propanol. After filtration and drying in vacuum at 55 °C, the final product was obtained as white flakes. Yield: 70.9 % (6.68 g). Alkyne-substituted dextrans with ether bonds were synthesized according to the procedure described by Nielsen et al.42 Dextran (1.5 g, 9.25 mmol AHG) was dissolved in 10 mL of 0.1M NaOH. It was followed by addition of glycidyl propargyl ether (0.55-0.85 mL, 5.09-7.86 mmol), increase of the reaction temperature to 35 °C and stirring for 16h. The product was precipitated in 220 mL of 2-PrOH, filtered on a sintered-glass funnel and washed with 100 mL of 2-PrOH. The product was then dissolved in ~50 mL of water, dialyzed against pure water for 48h mL of a solution at 0.17 mol/L in DMSO) were dissolved in 17 mL of DMSO. TBTA (0.79 mL of a solution at 0.10 mole/L in DMSO) and Na-Asc (0.20 mL of a solution at 0.28 mole/L in water) were added, and the obtained solution was bubbled with argon for 10 min. Then, Cu(MeCN) 4 PF 6

	D40-28GP-(5,10)Ada-(23,18)PEG: D40-28GP (0.160 g, 0.225 mmol alkyne) and Ada-N 3 (0.23-
	1-Adamantaneethyltosylate: 1-Adamantaneethanol (4.80 g, 26.6 mmol) was dissolved in 25 mL of pyridine, cooled to 0 °C on ice bath and p-toluenesulfonyl chloride (8.37 g, 43.9 mmol) was 0.46 (50 mg, 0,132 mmol) was added, the reaction mixture was bubbled with argon for 5 min under sonication, followed by CuAAC, performed under microwave irradiation (70 °C, 20 min). Further, MeOPEG-N 3 (1.58-1.25 g, 0.32-0.25 mmol) was added to the reaction mixture and microwave irradiation was proceeded (85 °C, 40 min). The reaction mixture was dialyzed against DMSO for 20 h, diluted with 250 mL of water and ultrafiltrated (MWCO 30,000). The product was obtained MeOPEG-N 3 : The IR of the product confirmed the absence of residual NaN 3 , showing a single -N 3 peak at 2117 by freeze-drying as white solid (0.69 g). 1 H-NMR (DMSO-d 6 ) δ = 1.50 (s, 6H, Ada), 1.57-1.72 (m, cm -1 . 1 H-NMR (D 2 O) δ = 3.38 (s, 3H, -OCH 3 ), 3.45-3.95 (m, PEG chain protons) 6H, Ada), 1.92 (s, 3H, Ada), 2.90-3.90 (m, dextran glucosidic and PEG protons, overlapped with added to the solution. TLC plates with 0.2 mm silica gel (EtOAc/heptane = 1/25). Yield: 89.4% (7.96 g). 1 H-NMR (DMSO-d 6 ) δ = 1.30-1.40 (m, 6H+2H, Ada overlapped with Ada-CH 2 ), 1.45-1.70 (m, 6H, Ada), 1.90 (s, 3H, HDO), 4.40-5.20 (m, 4H, dextran hydroxyl + anomeric), 8.06 (s, 1H, triazole-PEG), 8.10 (s, 1H, D40-18,28GP: and freeze-dried, yielding a white solid (1.27-1.56 g). 1 H-NMR (D 2 O) δ = 2.93 (s, 1H, C ≡CH), 3.35-triazole-Ada).
	Ada), 2.42 (s, 3H, -CH 3 ), 4.03 (t, 2H, CH 2 -OTs), 7.49 (d, 2H, Ts), 7.78 (d, 2H, Ts). 4.10 (m, 5H, dextran glucosidic protons), 4.23 (s, 2H, CH 2 -C ≡C), 4.90-5.08 (s, 1H, dextran
	1-Adamantaneethylazide (Ada-N 3 ): 1-Adamantaneethyltosylate (7.96 g, 23.8 mmol) was anomeric proton).
	dissolved in 0.5 mol/L NaN 3 solution in DMSO (167 mL, 83.3 mmol) and stirred at 80°C for 50 h. D40-18GP-18PEG: D40-18GP (0.150 g, 0.155 mmol alkynes) and MeOPEG-N 3 (1.32 g, 0.265
	250 mL of water were added to quench the reaction, and after cooling down 800 mL of EtOAc mmol) were dissolved in 16 mL of DMSO by warming at 50 °C. Subsequently TBTA (0.54 mL, 0.10
	were added. The organic phase was washed 3 times with water and one time with brine, dried M in DMSO) and Na-Asc (0.17 mL, 0.28 M in water) were added to the solution, followed by
	over Na 2 SO 4 and filtered. Concentration under reduced pressure and drying under vacuum gave bubbling with argon for 10 min. Finally, Cu(MeCN) 4 PF 6 (23 mg, 0,062 mmol) was added to the
	yellowish oil. Yield: 74.6 % (3.64 g). The IR spectrum of the product showed a single -N 3 peak at reaction mixture, it was bubbled with argon for another 5 min and azide-alkyne cycloaddition
	2094 cm -1 . 1 H-NMR (CDCl 3 ) δ = 1.38 (t, 2H, Ada-CH 2 ), 1.48 (s, 6H, Ada), 1.57-1.72 (m, 6H, Ada), (CuAAC) was performed under microwave irradiation (85 °C, 40 min). The reaction mixture was
	1.94 (s, 3H, Ada), 3.24 (t, 2H, CH 2 N 3 ). dialyzed against DMSO for 20 h, diluted with 250 mL of water and ultrafiltrated (MWCO 30,000).
	The product was obtained by freeze-drying as white solid (0.58 g). 1 H-NMR (DMSO-d 6 ) δ = 2.90-MeOPEG-Cl: MeOPEG-OH (10 g, 2 mmol) of 5000 g/mol was dissolved in 15 mL (206.5 mmol) of 3.90 (m, dextran glucosidic and PEG protons, overlapped with HDO), 4.40-5.20 (m, 4H, dextran
	hydroxyl + anomeric), 8.06 (s, 1H, triazole).

Table of contents

 of Introduction ................................................................................................................................. a. Sterically stabilized pNIPAm-g-pAAc/pβCDN+ hierarchical microgels ................................. b. Supramolecular hydrogels based on pNIPAm/βCD1.62N hierarchical microgels ...............

	1. Sterically

stabilized hierarchical pNIPAm-based microgels via host-guest interactions - pNIPAm/βCDN/PEG

  ..................................................................................................................... 1.1 Preparation of host polymer coated pNIPAm microgels -pNIPAm/βCDN ....................... 1.1.1 pNIPAm-g-pAAc microgels .......................................................................................... 1.1.2 Host polymer coated pNIPAm/βCDN microgels ......................................................... 1.1.3 Evidence of βCDN+ coating ......................................................................................... 1.1.4 TC analysis of unbound βCDN+ ................................................................................... 1.1.5 Influence of βCDN+ charge density ............................................................................. 1.2 Stabilization of the neutral pNIPAm/βCDN microgels with DT40-GP-Ada-PEG................. 1.2.1 Strategy of steric stabilization ..................................................................................... 1.2.2 Estimation of surface PEG chains conformation ......................................................... 1.3 Thermoresponsive properties of pNIPAm/βCD1.62N and pNIPAm/βCD1.62N/PEG hierarchical microgels ..............................................................................................................

2. Hierarchical hydrogels based on pNIPAm/βCDN through host-guest interactions

  .............. 2.1 Hydrogels based on host-guest interactions between pβCD1.62N+ and DT110Ada ........ 2.1.1 Rheological properties ................................................................................................ 2.2 Hydrogels based on host-guest interactions between pβCD3.24N+ and DT500Ada ........ 2.2.1 Rheological properties ................................................................................................ 2.2.1. Freeze-drying pathway for the preparation of pNIPAm/βCD3.24N/DT500Ada2 hydrogels .............................................................................................................................. Conclusions .................................................................................................................................. Experimental section ...................................................................................................................

Table IV . 1 .

 IV1 Characteristics of the polymers and microgels used

	Microgels	C(AAc-), mM → C, g•L -1	D h , nm (pH 7)
	pNIPAm-g-100%pAAc	1 mM → 0.40 g•L -1	450
	Host polymers	C(+), mM → C, g•L -1	M w, 10 -3 g•mole -1 CD, wt%	N+/CD
	pβCD1.62N+	1 mM → 1.20 g•L -1	238	58.2	1.62
	pβCD3.24N+	1 mM → 0.68 g•L -1	268	51.8	3.24
	pβCD5.00N+	1 mM → 0.49 g•L -1	299	46.1	5.00

Table IV . 2 .

 IV2 Percentage of non-bound pβCDN+ at different C(+)/C(-) ratios determined by TC analysis of the supernatants of the ultracentrifuged samples. Ultracentrifugation conditions: 62

	400 × g, 40°C, 10h.
	C(+), mM C(+)/C(-) pβCDN+ non-bound / pβCDN+ total , %
	0.34	0.67	40.6
	0.48	0.97	47.1
	0.62	1.24	37.4	40 ± 4
	0.76	1.52	37.6
	0.96	1.93	37.2
	1.10	2.2	48.0
	1.38	2.76	54.3

Table IV . 3 .

 IV3 Molar ratios and weight concentrations of the components in supramolecular gel samples prepared from pβCD1.62N+ and DT110Ada guest polymers

	Components -sample code	+/-Ada/CD	C(microgel), wt%	C(pβCDN+), wt%	C(DT110Ada), wt%	C tot , wt%	Observation
	pNIPAm/βCD1.62N/ DT110Ada3 -G1	0.8	1.3	0.6	1.4	3.4	5.4	phase separation
	pNIPAm/βCD1.62N/ DT110Ada3 -G2	1.5	1.3	0.6	2.6	6.3	9.4	viscous liquid
	pNIPAm/βCD1.62N/ DT110Ada3 -G3	2.0	1.3	0.5	3.3	8.1	11.9	gel
	pNIPAm/βCD1.62N/ DT110Ada4 -G4	2.0	1.3	0.6	3.8	7.1	11.6	gel

Table IV . 4 .

 IV4 Characteristics of the control samples containing no DT110Ada guest polymer crosslinker.

	Components -sample code	+/-	C(microgel), wt%	C(pβCDN+), wt%	C(DT110Ada), wt%	C tot , wt%	Observation
	pNIPAm/βCD1.62N -G5	2.0	2.7	16.0	0	11.7	phase separation
	pNIPAm -G6	-	10.1	0	0	10.1 liquid

Table IV . 6 .

 IV6 Activation energies 𝐸 𝑎 of pNIPAm microgel-containing and microgel-free host-guest

	supramolecular hydrogels
	Sample	Composition	C(pβCDN+DT-Ada), wt%	𝑬 𝒂 , kJ•mole -1
	G3	pNIPAm/pβCD1.62N/DT110Ada	11.4	25.5
	E1	pNIPAm/pβCD3.24N/DT500Ada	9.7	26.8
	E2	pβCD3.24N/DT500Ada	9.5	18.1

As mentioned before, amounts of the reagents taken, as well as exact details on the synthetic procedure cannot be unveiled herein for confidentiality reasons (Unpublished data by Kardosh et al.)

Acknowledgements

funded by the European Union. Apart from working in the ICMPE I've had the opportunity to travel a lot, to pass a great deal of top-notch courses and Grant agreement no.: 290251.

Materials and reagents ............................................................................................................ Methods and instrumentation ................................................................................................. References ...................................................................................................................................

at the same conditions. The initial data treatment was performed using the SUPERSAXS program package (C. L. P. Oliveira and J. S. Pedersen, unpublished) using Milli-Q water as a standard. 58 The scattering intensity patterns of the investigated polymer and nanoassemblies in aqueous solutions were recorded as functions of "length of the scattering vector" q. As the resolution range for typical SAXS set-ups lies in the range from 1 to 100 nm, the usage of modified low-q set-up with increased sample-to-detector distance was required to match the experimental parameters with the large sizes of the HG nanoassemblies (> 100 nm in terms of D h ). In this way the q min of 0.0038 Å -1 was achieved. According to the Fourier transformation sampling theorem, information is lost for sizes larger than π/q min 48 , thus indicating that the R max in the experiments discussed below was of 82.6 nm. According to the SAXS theory, the scattering intensity of an ensemble of monodisperse particles is: 𝛥𝐼(𝑞) = 𝑁 • 𝐼 0 (𝛥𝜌) 2 • 𝑉 1 2 • 𝑃(𝑞) • 𝑆(𝑞) (II.8)

where N -number of the particles, 𝛥𝜌 -the difference in electron density between the particles and the matrix (water), 𝐼 0 -primary X-ray beam intensity, 𝑉 1 -volume of the particle, 𝑃(𝑞)form factor and 𝑆(𝑞) -structure factor of the particle. The latter carries information about particle-particle interactions and in cases of dilute systems might be treated as 𝑆(𝑞) = 1. 59 All the components of the equation (3) except the 𝑃(𝑞) and 𝑆(𝑞) are independent of scattering angle and might be treated as a constant, which leads to a simplified version of the equation:

The form factor 𝑃(𝑞) bears information on the shape and the internal density distribution of the particles. At small angles any form factor can be approximated by a Gaussian curve and the curvature of the latter is defined by the overall size of the scatterers, according to Guinier 45,60 :

where 𝐼(0) is the extrapolated zero-angle intensity and 𝑅 𝑔 is the so-called radius of gyration.

𝑃(𝑞) is equal to 𝛥𝐼(𝑞)/𝐼(0). Unlike 𝑃(𝑞), the radius of gyration is model-independent and contains no information on the shape or structure of the nanoparticles. However, if these parameters could be assumed, like it is the case with the host-guest nanoparticles (spherical 0.8 mmol) solution in 15 mL of DMF was added to the reaction mixture followed by hexylamine (152 μl, 1.02 mmol). After stirring at 65°C under argon for 16h, the reaction mixture was precipitated in 420 mL of Et 2 O, washed twice with Et 2 O on a sintered-glass funnel, redissolved and dialyzed against pure water for 5 days. The adamantyl-grafted guest copolymer (here DT40g-PEG-Ada2) was isolated by freeze drying (m = 0.42 g). DS (from 1 H-NMR = 10.5 mol.%). 1

Experimental section

Materials and reagents

Pyridine (99.9%, anhydrous), N,N′-dimethylformamide (DMF, 99.8%, anhydrous), tetrakis(acetonitrile)copper(I)tetrafluoroborate (Cu(MeCN) 4 PF 6 , 97%), thionyl chloride (SOCl 2 , 99+%), 1-adamantaneethanol (98%) and glycidyl propargyl ether (GPE, 90+%) were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France) and used as received. p-Toluenesulfonyl chloride (98%), sodium azide (99%), L-ascorbic acid sodium salt (Na-Asc, 99%) and dimethyl sulfoxide (DMSO, 99.8%, anhydrous) were purchased from Alfa Aesar and used as received.

Sodium hydroxide (98+%, anhydrous pellets) were purchased from Acros Organics and used as received.

Dextran (DT40, M w = 4.3•10 4 g mol -1 , PDI = 1.50, Amersham Pharmacia Sweden), lithium chloride (Alfa Aesar) and poly(ethylene glycol) methyl ether of 5000 Da (MeOPEG-OH, Sigma-Aldrich) were dried overnight in vacuum at 90 °C prior to use.

Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA) 52 was synthesized according to the literature data. Positively charged pβCDN+, modified with quaternary ammonia, was prepared from neutral epichlorohydrin-branched pβCD polymer available in the lab (208 kg•mole -1 , PDI = 1.82), according to the previously described procedures. 47,48 Methods and instrumentation NMR spectroscopy: NMR measurements were performed in D 2 O or DMSO-d 6 , unless other is indicated, on a Bruker Avance II Ultrashield Plus 400 MHz NMR spectrometer. 1 H-NMR spectra were calibrated using the chemical shifts of the residual solvents signals according to the literature data 53 . SPR: SPR measurements were carried out with a Spreeta SPR-EVM-BT from Texas Instruments.

Fourier

The light of an infrared light-emitting diode (LED; λ = 840 nm) reaches the sensor surface at a range of angles above the critical angle. A reflectivity-versus-angle spectrum is obtained, and an apparent refractive index (RI) is derived from it by the Spreeta software. The sensor surface is a gold layer (ca. 50 nm). In the case of adsorption on the golden surface the RI changes provide information on the adsorbed layer parameters. The sensor is integrated in a flow cell and the measurements were carried out at 23°C using a continuous water flow (3 mL/h) which was delivered by a syringe pump (Kd Scientific), and a Rheodyne injection valve to switch to the sample solution.

The sensor surface was cleaned with a 4 % CrO 3 solution in water and rinsed with MeOH before each experiment. After settling the sensor into the flow cell, an in-situ cleaning was performed with one injection (0.1 mL) of a solution of 0.1 M NaOH and 1 % Triton X-100 in water followed by 3 injections (0.1 mL) of 10 % EtOH in water. The sensor was calibrated in pure water, assuming a RI value of 1.33300, and the RI variations after the different polymer injections were monitored as a function of time. the microgel particles in a swollen state, provided that their surface groups and dangling polymer chains are close enough to establish a link. 23 DX microgels were first described in 2000 by Hu et al. 31 Their system was composed of epichlorhydrin crosslinked pNIPAm-g-pAAc microgels. In this case and in many later works, secondary crosslinking of the microgels was done covalently, by functionalizing them with vinyl groups and using free-radical chemistry. 32,33 Such DX microgels were used as injectable formulations to reinforce degenerated intervertebral disc tissue. 33 The microgel cross-linking was performed directly in vivo and the resulting hydrogels had remarkably high toughness (G' = 0.7-1.3•10 5 Pa). However, because of the permanent character of secondary cross-linking such DX microgels lack reversibility and selfhealing capacity.

The introduction of cyclodextrins as structural units of both chemical and physical hydrogels proved to be beneficial given the ease and versatility of CD functionalization (via multiple primary and secondary hydroxyls), their high biocompatibility and the opportunity to exploit the cyclodextrin-hydrophobic guest inclusion chemistry. 34 Over the last decade a large variety of complex supramolecular hydrogels containing CDs have been developed. 18 In some of recent examples βCD-Ada host-guest chemistry was used as a means to cross-link soft nanoscale objects into non-covalent 3D hydrogel networks. For instance, Himmelein et al. used hydrophobically self-assembled cyclodextrin vesicles (CDV) as multivalent 3D joints which were converted to a gel by addition of adamantane-modified hydroxyethylcellulose (HEC-Ad). 22 Due to its reversible nature their system showed shear-thinning/self-healing properties and was injectable through a syringe.

Given the above, in the second part of this work we had a goal to use pNIPAm/βCDN core-shell microgels as a building block for hierarchical DX microgels non-covalently cross-linked with Adasubstituted dextrans through host-guest interactions.

TC analysis of unbound βCDN+

To figure out the actual percentage of bound pβCDN+ we determined the total carbon (TC) amount in the supernatants of ultracentrifuged pNIPAm/βCD1.62N samples for all the investigated C(+) concentration range. The amount of TC is obtained through total combustion of the samples onto a platinum catalyst at 680°C. It leads to the catalytic oxidation of any carbon in the supernatant into carbon dioxide (CO 2 ). The concentration of the latter is then determined using a nondispersive infrared (NDIR) gas analyzer compared with the calibration curve recorded for a series of solutions with known TC concentrations. The procedure is described in more detail in the Experimental section.

Since we aimed to determine the TC amount corresponding to non-bound pβCDN+, it was important to minimize the signal from non-sedimented microgels. Acidifying of the samples to As it was shown, positive overcharging of pNIPAm-pAAc with pβCDN+ is needed to make the resulting pNIPAm/βCD1.62N colloidally stable. At the same time, for the majority of eventual biomedical applications microgels are required to have neutral or moderately negative overall charge. To overcome this drawback we used bifunctional (PEG, adamantane)-grafted dextrans (DT40-GP-Ada-PEG) for sterical stabilization of pNIPAm/βCD1.62N. The applied strategy consisted of 2 steps, namely: pre-mixing of required amount of pβCDN+ with DT40-28GP-10Ada-18PEG polymer bearing 10 mol % of adamantyls and 18 mol.% of 5000 Da PEG grafts 37 (described in detail in Chapter 3), followed by addition of microgel solution (Figure IV.13).

A pNIPAm/βCD1.62N sample from the middle of precipitation range with C(+) = 0.16mM and C(AAc-) = 0.145 mM was chosen to investigate its behavior as a function of Ada/CD molar ratio in the pβCDN+/DT40-28GP-10Ada-18PEG precursor complex. It turned out that already at Ada/CD = 0.3 precipitation was suppressed leading to stable microgels of around 200 nm (Figure

IV.14).

To prove the host-guest driven origin of sterical stabilization we conducted a control experiment with DT40-18GP-18PEG, bearing exclusively 18 mol.% of PEG grafts (Table IV.1) and unable to form inclusion complexes with βCD. No considerable effect on the system behavior was weakness and reversible character of the host-guest links, which are being broken and reformed under applied stress. 44,46,47 The finite lifetimes of the interpolymer cross-links are due to the fluctuations in the relative kinetic energy of the interacting Ada-and βCD-groups. These lifetimes (𝜏 𝑐𝑜 ), also known as relaxation times, may be obtained as the inverse of the angular frequency

) of the cross-over point of G' and G'' in frequency sweeps: Interestingly, the G4 hydrogel differs from G3 only by higher DS(Ada) of the used DT110Ada macromolecular cross-linker (6.2 vs. 4.7 mol.% respectively), whereas the +/-ratio, Ada/CD ratio and total weight concentration of the components are equal. However, G4 has significantly lower compared to G3 gel network strength as evidenced from 𝑓 𝑐𝑜 and G'(10 Hz) values (Figure

IV.20,b-d).

One might expect the opposite effect for more highly substituted DT110Ada4, which is intended to provide higher cross-linking density between the microgels. At the same time, Wintgens et al. found that higher DS(alkyl) leads to associative phase separation instead of homogeneous 3D-network formation in the interaction between alkyl-substituted dextrans and neutral pβCD. 45 Increased number of hydrophobic groups per chain is thought to induce larger cooperativity of the interpolymer interaction. In the case of pNIPAm/βCD1.62N/DT110Ada4 system (G4) the latter might result in DT110Ada4 being tightly attached to the pβCD1.62N+- In order to confirm that the 3D-network is actually formed through the host-guest interactions two control samples containing no guest polymer cross-linker were prepared (Table IV.4). The concentration of the G5 sample composed of pβCDN+ -coated microgels to 11.7 wt% expectedly resulted in a loss of colloidal stability and phase separation.

Uniform pNIPAm microgel dispersions with concentrations corresponding to close-packing (~ 5 wt%) were reported to undergo sol-gel transition at temperatures below LCST. 48 Uncoated 𝜏 𝑐𝑜 = 0.145 s) due to the increased cooperativity of the interactions. Furthermore, comparing to the G3 hydrogel, the contribution from microgels to the C tot is increased more than two times for E1 -from 4.2 to 11.8%.

The gelation process was confirmed to be driven by the βCD-Ada inclusion complexation (Table IV.4). In order to understand the impact of microgels on the mechanical properties of the hydrogels a blank sample E2, containing only pβCD3.24N+ and DT500Ada2, was prepared, so that its C tot was equal to the sum of C(pβCD3.24N+) and C(DT500Ada) in the E1 hydrogel (Table IV ,d)., which can be in part related to the higher total concentration of the latter. On the other hand, the extent of the linear viscoelastic region is significantly higher for microgel-free E2, i.e. the hydrogel maintains its structure up to 250% strain, while in the case of E1 a breakdown is observed already at 100% strain. These results indicate that despite having a high volume fraction and being in close contact pNIPAm-g-pAAc microgels do not mechanically reinforce the resulting hydrogels network. Being highly swollen in water below the VPT temperature, they should behave rather as soft disruptive regions, i.e. the volume of hydrogel occupied by microgels is depleted of host-guest cross-links constituting the 3D-network. In a typical polymerization procedure, calculated amounts of NIPAm together with BA crosslinker and SDS were dissolved in distilled water, introduced in a double-wall Pirex glass reactor and put under vigorous stirring. 1 To keep the temperature of the reaction mixture constant at 80°C, the outer shell of the reactor was connected to a thermostat and controlled temperature water was circulated in it. The reaction mixture was degassed by purging it with nitrogen for 60 min. Then, the reaction was initiated by adding an aqueous solution of APS to the reactor followed by 4h under vigorous stirring at 80°C. After reaching 95% conversion of the NIPAm monomer, AAc monomer was fed into the reaction mixture to form the shell consisting of 100% grafted pAAc chains. The final product, pNIPAm-g-100%pAAc microgels, was purified from unreacted monomers and polymeric byproducts by centrifugation (Hettich 220R centrifuge, 16 000 rpm), decantation, and redispersion. The centrifuged microgels were redispersed in Milli-Q water and the cycle was repeated up to 5 times. Purified microgels were characterized by DLS, UV/Vis spectroscopy and surface tension measurements.

Synthesis of "host" polymers, neutral epichlorhydrin-interconnected pβCD and positively charged pβCDN+, modified with quaternary ammonia, is described in the Experimental section of Chapter 3. 53,54 Synthesis and binding properties of bifunctional (PEG, adamantane)-grafted dextrans (DT40-GP-Ada-PEG) are described in detail in Chapter 3. 37

Methods and instrumentation

Coated microgels preparation: 1. pNIPAm/βCDN core-shell microgels: In a typical procedure 2 mL of pNIPAm-g-pAAc microgels solution at 0.145 or 0.29 mM of (-) charges were mixed with pbCDN+ solutions with increasing concentrations of (+) charges (V = 2mL). The mixing was performed for 2 minutes at 600 rpm and at room temperature (pH = 7.0). Sizes and E mob of the resulting pNIPAm-g-pAAc/pβCDN+ complexes were monitored by DLS and electrophoretic mobility measurements respectively. to protect the thin ice film from any irradiation before imaging and to reduce the irradiation during the image capture. The images were recorded at 93K. The samples were prepared as follows. A drop of the microgel complexes solution at pH 2 was deposited on a Quantifoil grid (Micro Tools GmbH, Germany). The grids were previously treated by glow-discharge in the presence of argon to increase their surface hydrophilicity. The excess of solution was then blotted out with a filter paper, and before evaporation the grid was quench-frozen in liquid ethane to form a thin vitreous ice film. The grid was mounted in a Gatan 626 cryo-holder cooled with liquid nitrogen and transferred in the microscope.

pNIPAm/βCDN

Ultracentrifugation: An ultracentrifuge from Beckman Coulter (Optima Max-XP, type TLA 110 rotor) was used to separate the microgel particles from the liquid phase. The centrifugations were performed at a relative centrifugal force (RCF) 62 400 × g for at least 10h, while heating the solutions to 40°C.

Total Carbon measurements: TC measurements were performed on a Shimadzu TOC-L CSN instrument. A potassium hydrogen phthalate solution in ultrapure water (2.125 g•dm -3 corresponding to 1000 mgC•dm -3 ) was used to establish a calibration curve in the 10-200 mgC•dm -3 range. The samples under investigation were transferred inside TC combustion tubes filled with platinum catalyst at 680°C in oxygen-rich atmosphere. The CO 2 generated due to the total combustion of the samples (required sample V = 8 mL) was then measured using an integrated NDIR gas analyzer and compared with the calibration curve to determine the TC concentration. Rheological measurements: Rheological characterization of the hydrogels was performed with a DHR-2 Rheometer (TA Instruments), equipped with a 1° cone geometry of 40 mm diameter.

Supramolecular hydrogel

An insulated ring was placed around the geometry to prevent water evaporation. The hydrogel samples under investigation (𝑉= 400-500 μm) were placed between the geometry and the Peltier plate with a spatula or with a pipette (in the case of weak gels or viscous liquids).

Oscillatory frequency sweep measurements at 20 °C and 0.1% strain were performed in order to

General conclusions and perspectives

The goal of this PhD project was to elaborate new polysaccharide-based host (β-cyclodextrin modified) and guest (adamantyl modified) polymer architectures with improved binding characteristics, as well as to integrate such polymers as structural elements of complex hierarchical systems with stimuli-responsiveness properties, e.g. sterically stabilized pNIPAmbased microgels and supramolecular hydrogels composed of pNIPAm-based microgels interconnected via host-guest interactions. In view of their potential biomedical applications, the presence of β-cyclodextrin (βCD)-polymers in such systems was aimed to endow them with bioavailability, reversibility and controlled kinetics of drug release. The binding properties of the polymers in solution and on solid surface were studied by ITC and SPR respectively and compared to those of previously reported βCDand Ada-substituted dextrans with short hydrophobic spacers. The presence of flexible PEG spacer proved to enhance the availability of Ada groups in DT40-gPEG-Ada, which resulted in a 5-fold increase of its affinity constant (K a ) to monomeric βCD in solution and improved adsorption on the surficial pβCD layers. However, the presence of PEG spacer proved to be less beneficial in the case of host polymers, where DT40-gPEG-βCD showed in average 4 times lower K a than those of the host polymer with a sort hydrophobic spacer (DT70GPβCD).

In the next step we used a combination of SAXS and light scattering to study the host-guest driven formation of the nanoassemblies between the aforementioned polymers with various architectures. Such nanoassemblies were formed spontaneously upon mixing the host and guest polymer aqueous solutions at βCD/Ada =1 molar ratio. The nature of individual polymers was found to have a significant influence on the size and internal organization of the nanoassemblies. In general, particles containing DT40-gPEG-βCD are smaller and have less compact structure than those containing DT70GPβCD. The same effect, though to a lesser extent, was observed for the particles composed of DT40-gPEG-Ada as a guest polymer. The obtained results contribute to design of host-guest polymers with improved properties, i.e.

polymers capable of self-assembling into more stable, biodegradable nanoparticles with more attractive drug delivery profiles.

In the second part of this work, a series of new (PEG, Ada)-grafted dextrans (denoted as D40-GP-Ada-PEG) were prepared by CuAAC click reaction, using alkyne modified dextrans and azide derivatives of adamantane and 5000 g•mole -1 PEG. The degree of substitution (DS) by PEG grafts was kept similar (18-23 mol.%) while the level of substitution by hydrophobic Ada groups was varied from 0 to 10 mol.%. It was shown that due to the relatively long triazole-ether spacer between the Ada-groups and dextran backbone, D40-GP-Ada-PEG have higher K a with monomeric βCD in solution as compared to previously described Ada-modified dextrans and pNIPAm with Ada-groups rigidly attached to the polymers through short ester and amide bonds respectively.

Furthermore, in the case of interaction with neutral or positively charged βCD-polymers, both in solution and on the surface, the interpolymer affinity proved to be strongly dependent on the DS(Ada), i.e. the K a for more densely substituted D40-28GP-10Ada-18PEG (10 mol.% Ada) is one order of magnitude higher than that for D40-28GP-5Ada-23PEG (5 mol.% Ada). Such non-linear increase in K a is an indication of strong cooperativity effects between the Ada-groups in synthesized polymers. Given the above, (PEG, Ada)-grafted dextrans should be auspicious candidates for applications, where fast and reversible PEGylation of surfaces or nanoparticulate carriers is required.

In the last park of the presented work our goal was to develop an approach to the synthesis and non-covalent sterical stabilization of poly(N-isopropylacrylamide)-g-100%poly(acrylic acid) (pNIPAm-g-pAAc) core-shell microgels coated with positively charged β-cyclodextrin polymer (denoted as pNIPAm/βCDN). Furthermore, we showed that pNIPAm/βCDN can be used as building blocks of thermo-reversible hierarchical hydrogels non-covalently crosslinked via hostguest interactions.

First, we used electrostatic self-assembly to attach the β-cyclodextrin polymer bearing quaternary ammonia groups (pβCDN+) to the surface of negatively charged at pH 7 microgels.

This approach provided an easy way to control the degree of coating and the overall charge of the resulting pNIPAm/βCDN complexes by simply adjusting the +/-molar ratio during the mixing stage, while the βCD content in the shell at a fixed +/-ratio can be varied by using pβCDN+ with different quaternary ammonia N+ substitution levels. The pNIPAm/βCDN aqueous suspensions proved colloidally stable when positively overcharged with pβCDN+ but aggregated at neutral overall charge, which would be a serious limitation for any potential biomedical use. This problem could be solved by exploiting the host-guest chemistry of pβCDN+. Neutral pNIPAm/βCDN were sterically stabilized through a simple 2-step procedure involving: 1) preparation of supramolecular precursor between pβCDN+ and D40-GP-Ada-PEG described above; 2) electrostatic self-assembly of the precursor with pNIPAm-g-pAAc yielding pNIPAm/βCDN/PEG hierarchical microgels. Both positively overcharged pNIPAm/βCDN and neutral pNIPAm/βCDN/PEG microgels show a full preservation of temperature-induced volumephase transition (VPT) of the pNIPAm core.

We also elaborated hierarchically structured hydrogels based on positively overcharged pNIPAm/βCDN by non-covalently crosslinking them with Ada-modified dextrans (DT-Ada).

Rheological measurements and calculations of the effective volume fraction 𝜙 𝑒𝑓 of the microgels in such hydrogels (at 10-11 wt%) suggest that the individual microgels are at a close contact, thus making their architecture similar to that of so-called doubly-crosslinked microgels (DX microgels). To the best of our knowledge, this is the first example of DX microgels noncovalently crosslinked via host-guest interactions. Furthermore, the hydrogels exhibit fully reversible temperature-induced gel-sol transition due to the synergetic effect between shrinking of the microgels and dissociation of βCD-Ada crosslinks at higher temperatures. Due to the presence of microgels the gel-sol transition temperature of pNIPAm/pβCDN/DT-Ada is significantly shifted down to the physiological temperature range (37-41°C) as compared to uniform pβCDN/DT-Ada host-guest hydrogels (51°C).

The obtained results may be extended over a multitude of promising directions.

With regard to the first part our work, the DT40-gPEG-βCD synthesis procedure needs to be optimized in order to avoid the alkyne crosslinking issues at the CuAAC stage. It would also be interesting to replace the PEG spacer with a one responsive to external stimuli, e.g. pNIPAm. It would allow temperature-triggered assembly and dissociation of the resulting host-guest nanoparticles. In collaboration with colleagues from Bristol University (Kathrin Lange, Wuge Briscoe) we began the study of the surface properties of the host-guest nanoassemblies, and the initial surface-force apparatus (SFA) and atomic force microscopy (AFM) results show their high potential as lubrication agents. Finally, we plan to study the entrapment and release of model hydrophobic drugs into the nanoassemblies prepared from the DT40-gPEG-βCD and DT40-gPEG-Ada.

Besides the sterical stabilization of pβCDN+ -coated thermoresponsive microgels, (PEG, Ada)grafted dextrans described in Chapter 3 of this manuscript can find a number of interesting applications. Namely, we envisage using D40-GP-Ada-PEG for the PEGylation of hydrophilic drug loaded metal-organic framework (MOF) nanoparticles coated with phosphated βCD. In this case the bulkiness and superior binding properties of D40-GP-Ada-PEG would allow outperforming simple Ada-substituted linear PEGs which tend to penetrate into the crystalline structure of nano-MOF and hamper their drug-loading capacity.

There is a high potential to go towards the usage of hierarchical pNIPAm/βCDN/PEG microgels as thermoresponsive smart drug delivery vectors. The presence of the host polymer external shell and PEG corona in their structure are expected to provide better control over the loaded drugs release kinetics and to address the issue of pNIPAm low biocompatibility respectively.

Last but not least, the pNIPAm/pβCDN/DT-Ada hierarchical hydrogels might be used as a medium for the differentiation of pluripotent stem cells. Indeed, after completion of the differentiation process the differentiated cells would be collected by increasing the temperature to 37-41°C and inducing the gel-sol transition of the network. However, such applications would require some modifications in the design of the system. First of all, as it was shown by total carbon (TC) measurements the N+ -overcharged pNIPAm/pβCDN contain significant amounts of non-bound pβCDN+. Polymeric materials containing permanent positive charges are known to be less biocompatible than their neutral or negatively charged analogues. Given this we envisage replacing pβCDN+ coating with the one based on neutral βCDs as a future extension of this work. This might be achieved by copolymerizing NIPAm with NH 2 -or alkyne-containing monomers in the external shell. The latter would allow modifying the surface of the microgels with either βCD tosylate (βCD-OTs) βCD azide (βCD-N 3 ) derivatives using nucleophilic substitutions or CuAAC reactions respectively. Hierarchical hydrogels built of such βCD-grafted microgels are expected to be less toxic and less sensible to variations of pH and ionic force in the cell culture media.