Poly(N-isopropylacrylamide)-based hierarchical microgels: sterical stabilization of the particles and supramolecular hydrogel formation via host-guest int

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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.86, 87, 88 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. 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.
M. Zan et al. have recently described a host-guest nanogels-based drug delivery system with dual pH-responsiveness.92 They used random copolymers of two types: adamantyl (Ada)-benzoic imine-conjugated poly[poly(ethylene glycol) monomethyl ether methacrylate]-co-poly(2-hydroxyethyl methacrylate) (PPEGMA-co-PHEMA-Ada); doxorubicin (DOX)-hydrazone 27 and βcyclodextrin (βCD)-conjugated poly[N-(2-hydroxypropyl) methacrylamide]-co-poly(3-azidopropyl 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: 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 DOX-hydrazone (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.
The reorganized nanoparticles are much smaller (~25 nm) and able to penetrate deeper (>2 mm vs. 200 μm for large precursor NPs) inside the dense matrix of tumor tissue, herein mimicked by collagen hydrogel. Having slightly positive ζ-potential, the reorganized nanogels are internalized by the endolysosomes once inside the tumor cells. The hydrazone bonds used to link the DOX to the polymer backbone are further cleaved at pH 5, found in the endo-lysosomal microenvironment, leading to the anticancer drug targeted sustained release (76.9% after 72h of incubation).
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 (Mw ~ 5000) and a polyaspartamide block (Mw ~ 1500), the latter carrying βCD units on the side chain (PEG-b-PCD).93 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 pseudo-amphiphilic 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.94 The particle size of the assemblies was tunable and could be decreased by dropping 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-N-acroyloxysuccinimide (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.95 The thermoresponsive (LCST = 38°C) NIPAM-co-NAS block was also modified with MPEG grafts (Mn ~ 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).
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.96 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 pseudo-copolymer, where PS-βCD was replaced by thermoresponsive PNIPAM-βCD as a host segment.97 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.e. disassemble under the effects of oxidant (H2O2) 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: 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 al.
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).98 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).99 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.
It has been shown that simultaneous delivery of chemotherapeutic drugs and apoptosis-inducing genetic material in tumor cells might increase the cancer treatment efficacy in a synergetic way. Several approaches to design such co-delivery systems via host-guest mediated “bricks and mortar” strategy were recently reported. The strategy comprises several key steps: 1) synthesis of hydrophobic pro-drugs by conjugation of either doxorubicin100 or paclitaxel (PTX) 101 with adamantyl moiety; 2) inclusion of the pro-drugs into the βCDs linked to the positively 32
charged linear PEI (PEI-CD); 3) hierarchical electrostatic self-assembly of PEI-CD/Ada-DOX and PEI-CD/Ada-PTX with either apoptosis-encoding pDNA100 or survivin shRNA101 respectively (Figure I.19). For instance, in the case of PEI-CD/Ada-DOX/pDNA polyplexes stable sizes of ~230 nm in normal saline were achieved when nitrogen to phosphorus (N/P) ratio reached 25. They showed good in vivo doxorubicin retention ability. The cellular internalization of such co-delivery formulations occurred through endocytosis, followed by efficient endosomal escape due to the proton sponge effect of PEI. Usage of PEI-CD/Ada-DOX/pDNA allowed to decrease significantly the tumor growth rate in mice.
Photothermal therapy for cancer treatment has been attracting a lot of attention over the past decades due to its providing a unique opportunity to spatially localize the cell damage effects to the irradiated region.102, 103 Gold and other noble metal nanostructures as photothermal agents, allow to overcome limitations of organic-dye-based photothermal agents, such as low light absorption and undesired photobleaching.104 However, the searched photophysical profiles are often found for large nanoparticles (> 100 nm), known for their unsatisfactory bioclearance (i.e., accumulation in the liver, spleen, and kidneys).105 To solve this contradiction Wang et al. used aforementioned “bricks and mortar” approach to self-assemble size-controlled Au-supramolecular nanoparticles (Au-SNPs) from 2 nm Au colloids.102 The Au-SNPs comprised three building blocks: Ada-grafted 2 nm Au colloids, CD-PEI, and Ada-PEG. By simply tuning the βCD/Ada mixing ratio between the Au colloids and CD-PEI they managed to obtain a series of Au-SNPs with variable sizes ranging between 40 and 118 nm (Figure I.20). The Au-SNPs exhibited pronounced photothermal activity and selectivity as a result of the collective effects between associated Au colloids. Moreover, it was possible to thermally disassemble the Au-SNPs, thus allowing their in vivo bioclearance.
In another recent example the “bricks and mortar” approach was employed to develop a magnetothermally responsive doxorubicin delivery/release system.106 The SNPs in this case were composed of a fluorescently labeled anti-cancer DOX and the four other building blocks: Ada-grafted polyamidoamine dendrimers (Ada-PAMAM), βCD-grafted branched polyethylenimine (βCD-PEI), Ada-functionalized PEG (Ada-PEG), and 6 nm Ada-grafted Zn0.4Fe2.6O4 superparamagnetic nanoparticles (Ada-MNP). The embedded Ad-MNP were used as built-in transformers converting radiofrequency external alternative magnetic field into heat, allowing for spatio-temporal controllability of in vivo DOX release from the nanoparticles.
Much effort is being made to prepare nanoscale supramolecular carriers where CDs play the role of the barrier, slowing down the release kinetics of loaded hydrophobic compounds. Thus, El Fagui et al. described core-shell nanoparticles composed of hydrophobic biodegradable polylactic acid (PLA) core and physically adsorbed β-cyclodextrin polymer (pβCD) shell.107, 108 The PLA core could accommodate model hydrophobic compounds such as benzophenone107 or triclosan108 and the pβCD shell at the periphery ensured their sustained in vitro release and opened the pathway to additional functionalization of the particles with targeting agents, fluorescent labels etc. through inclusion complexation with free CD-cavities. Moreover, using layer-by-layer (LbL) self-assembly technique, deposition of multiple (up to 6) alternate layers of oppositely charged pβCD on the PLA core was possible. It led to further improvement in the benzophenone release profile and demonstrated the potential of these nanoparticles as long-circulating drug carriers.
In yet another example by Agostoni et al. βCD-based shell formation was exploited to stabilize nanometric MOF (nanoMOF) in biological media and to establish a pathway for their further non-covalent functionalization with furtive PEG chains or targeting agents.110 They prepared non-toxic mesoporous Fe(III) carboxylate nanoMOF built up from Fe(III) octahedra trimers and trimesate linkers (1,3,5-benzene tricartricarboxylate) that self-assemble to yield ~220 nm porous architectures delimited by large (29 Å) and small (24 Å) mesoporous cages. Antiretroviral hydrophilic drugs such as azidothymidine-triphosphate (AZT-TP) could be loaded inside the nanoMOF with high encapsulation efficiency (> 99%) due to their coordinate bonding with Fe(III) sites. Phosphate-bearing βCD (βCD-P) was further used to form a non-covalent bioavailable coating around the drug-loaded nanoMOF through the Fe(III)-phosphate interactions. Unlike their uncoated counterparts, βCD-P/nanoMOF demonstrated no signs of aggregation in water for more than 3 days and the coating proved to be stable under physiological simulated conditions (9.5 mM PBS, pH 7.4, 37°C) for 24 hours. Furthermore, βCD-P/nanoMOF could be endowed with “stealth” properties by the inclusion complexation of βCD-P with Ada-modified 5000 Da PEG chains (Figure I.21). This PEGylation strategy also takes advantage of βCD-P as a “gatekeeper” which prevents MPEG-Ada from entering the 5 and 9 A˚ nanoMOF windows and interfering with the drug loading process.

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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 cross-linked chemical gels and non-covalently constructed physical gels. In addition to their inherent stimuli-responsive nature114, physical hydrogels possess a plethora of other advantageous features, e.g. compositional flexibility, tailorability and self-healing properties.
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 CD-hydrogels 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.

Table of contents :

Chapter 1: Literature review: Hierarchical self-assembly via cyclodextrin-mediated host-guest interactions
Table of contents
1. 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
2. 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
Chapter 2: Host-guest polymers with hydrophilic poly(ethylene glycol) spacer, their inclusion properties and self-assembly in water
Table of contents
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 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-hexynoate
1.3.3 Synthesis of 6-Monodeoxy-6-monoazido-βCD (βCD-N3)
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 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
3.2.1 Individual host- and guest polymers
3.2.2 Host-guest nanoparticles of different types
Experimental section
Materials and reagents
Methods and instrumentation
Synthetic procedures
Chapter 3: Bifunctionalized dextrans for surface PEGylation via multivalent host-guest interactions
Table of contents
a. PEG and PEGylation. General information
b. βCD host-guest chemistry and PEGylation
1. Synthesis of PEGylated guest polymers – D40-XGP-YAda-ZPEG
2. Binding properties of D40-GP-Ada-PEG in solution. ITC studies
3. Binding properties of D40-GP-Ada-PEG on the surface. SPR studies
3.1. Adsorption kinetics by SPR
Experimental section
Materials and reagents
Methods and instrumentation
Synthetic procedures
Chapter 4: Poly(N-isopropylacrylamide)-based hierarchical microgels: sterical stabilization of the particles and supramolecular hydrogel formation via host-guest int
Table of contents
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. Steric stabilization of the particles 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
Experimental section
Materials and reagents
Methods and instrumentation
General conclusions and perspectives


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