Click Hydrogels for Controlled Local Antibody Delivery

This thesis was focused on the development of click hydrogels for the controlled local delivery of therapeutic antibodies. In 2001, the term “click chemistry” was first introduced by Sharpless and coworkers as an umbrella term for “spring-loaded” reactions that are modular, wide in scope, stereospecific, give high yields, proceed at simple conditions, and do not form toxic by-products. Since that time, click reactions have had tremendous influence in many research areas including pharmaceutics and material science. Without a doubt, the Diels-Alder (DA) reaction is one of the click reactions with the greatest potential for the development of biomaterials and drug delivery systems. For example, besides the above-mentioned general advantages of click chemistry, the DA reaction does not require a metal catalyst. Therefore, the DA reaction has already been utilized in various biomedical areas, such as the synthesis of polymers and dendrimers, surface functionalization, bioconjugation, nanotechnology, and hydrogel design. For many of these applications the diene-dienophile pair furan and maleimide was utilized as they are readily available and the functionalization is comparably simple. For example, multi-armed poly(ethylene glycol) (PEG) functionalized with maleimide and furan have been utilized to prepare DA-hydrogels. However, the DA reaction is also associated with a number of disadvantages. For example, the reaction is reversible which may be unfavorable for the preparation of stable conjugates. However, the two most serious drawbacks of the reaction are its low reaction rate and potential side-reactions, e.g., the reaction of maleimide with nucleophilic amino acid side-chains (Chapter 1). The goal of this thesis was to utilize the DA reaction for the development of hydrogels that can be applied in local antibody therapy. In order to exploit its full potential, the two main disadvantages of the DA reaction had to be circumvented while its advantages had to be utilized. To be more specific, DA hydrogels that gel more rapidly and that do not undergo side-reactions with proteins had to be developed. To this end, various approaches including chemical modification, the use of protective additives or combination with other click reactions were employed (Chapter 2). For DA hydrogels, gelation is achieved through covalent cross-linking. Therefore, it was hypothesized that gel formation can be accelerated by facilitating the interaction between the reactive groups. Amphiphilic macromonomers were prepared by introducing hydrophobic 6-aminohexanoic acid (C6) and 12-aminododecanoic acid (C12) spacers between the polymer backbone and the functional end-groups. The general associative nature of the macromonomers was verified by an increase in viscosity and the formation of associates or micelles. As a consequence of the hydrophobic association, the reactive groups were brought into close proximity and gelation occurred significantly faster, e.g., twice as fast using a C12 spacer. Interestingly, gel times did not decrease when a modified and a non-modified component were combined, e.g., unmodified PEG-maleimide with modified PEG-furan. This finding further emphasizes the importance of hydrophobic association for accelerated gel formation. Moreover, hydrogels with hydrophobic modification were characterized by a lower average network mesh size and a higher elastic modulus which suggested a more efficient cross-linking process. Furthermore, through hydrophobic modification an increase of hydrogel stability could be achieved. This could be explained by the combined effects of higher cross-linking density and the increased hydrolytic resistance of maleimide moieties resulting from N-alkylation. All of these effects were influenced by spacer length: C12-modification exhibited stronger effects on gelation, stability, and stiffness than C6-modification. In addition, it was found that hydrophobic modification can be used as a tool to achieve delayed antibody release. While the in vitro release of bevacizumab from the unmodified DA-hydrogel was completed after only 10 days, hydrophobic modification delayed the release for about 30 days using C6 and almost 60 days using C12 spacers (Chapter 3). Although gel times could be significantly decreased using hydrophobic modification instantaneous gelation still could not be achieved. In order to develop DA-hydrogels that provide immediate gelation a dual approach was employed. Instead of eight-armed PEGs, thermoresponsive four-armed poloxamines were utilized for macromonomer synthesis and functionalized with maleimide and furyl moieties. Aqueous solutions of these macromonomers exhibited an immediate gelation at body temperature. Concomitantly, the functional end-groups led to covalent cross-linking of the gels. In this way, the rapid sol-gel transition of physical gelation and the stability of chemically cross-linked gels were combined in a hybrid system. In addition, further branches were introduced to create a more versatile hydrogel platform that allowed for tailoring of the core characteristics, i.e., mechanical properties and stability. Hydrogel stability could be precisely controlled in the range of 14 to 329 days depending on the composition used. Finally, controlled release of the model antibody bevacizumab could be achieved over a period of 7, 21, and 115 days in vitro. The release curves were characterized by a notably low burst and a triphasic shape. Most importantly, almost all of the loaded antibody could be recovered after release and approximately 87% displayed functional binding. In conclusion, DA-Poloxamines are rapidly gelling, mechanically stable, degradable, nontoxic, and provide controlled antibody release. As they can be tailored to match the demands of various applications they present a powerful material for controlled local antibody delivery (Chapter 4). Besides slow gelation, the second major drawback of DA-hydrogels are undesired side-reactions with proteins. As potential approach to solve this issue, antibodies could be incorporated into hydrogel microparticles to safeguard them from detrimental cross-linking reactions. Moreover, such antibody-loaded microgels could find use as a delivery platform for controlled local release. However, the fabrication of antibody-loaded microgels with a narrow size distribution and without impairing protein stability is a challenging task. To achieve this goal, a fabrication method combining microfluidics and thiol-ene photoclick chemistry was employed. Microfluidics is a well-characterized approach for the generation of uniform droplets that does not expose materials to harmful stress conditions. On the other hand, the thiol-ene reaction is known to be compatible with proteins and can be triggered using visible light. To fabricate the microparticles, first aqueous droplets containing antibody, macromonomers and reactants were generated using a microfluidic device. Then, green light was used to covalently cross-link the droplets and encapsulate the antibody. In order to tailor microgel properties a macromonomer library comprising both hydrolytically labile and stable eight-armed PEGs with various molecular masses was synthesized. Then, rheology was used to determine the necessary irradiation time and to study mechanical properties. These microgels had a rod-like shape and a narrow size distribution with an approximate width of 380 μm and lengths of 1400 μm or 2150 μm, depending on the process parameters. Bevacizumab was successfully incorporated into the microgels and a sustained release could be achieved over a period of 28 and 46 days. Moreover, it was confirmed that the process developed does not significantly impair the binding ability of bevacizumab. Therefore, the strategy is suitable for loading antibodies into microgels and presents a promising starting point for further development. In future experiments, the general hypothesis that the incorporation into microgels safeguards proteins needs to be verified. Moreover, for delivery purposes microgels with smaller dimensions should be generated to allow for injection or inhalation. This could be achieved by fine tuning of the flow ratio and by using tubes with smaller diameters (Chapter 5). Although encapsulation into microgels might be an effective approach to overcome protein-polymer conjugation during cross-linking, it is a comparably complicated approach. It would be ideal if Michael-type reactions of maleimide could be avoided by simply adding a protective additive. For this purpose, a number of pharmaceutically relevant polyanions were evaluated, i.e., alginate, dextran sulfate, heparin, hyaluronic acid, and poly(acrylic acid). Electrostatic interactions led to reversible binding of the polyanions to the protein surface. Thereby, the reaction of maleimide with nucleophilic moieties on the protein surface could be prevented. These results were confirmed using the model protein lysozyme and by simulating the reaction conditions with monofunctional mPEG5k-maleimide and mPEG5k-furan. For example, at pH 7.4 and without polyanions about 61% of lysozyme was PEGylated and the activity had decreased to about 20% of the initial activity. In comparison, when dextran sulfate had been added an activity of 98% remained and no PEGylation was detected. Overall, dextran sulfate, heparin, and poly(acrylic acid) were identified as the most effective additives to shield proteins during cross-linking. In addition, it could be confirmed that the “shielding” is solely based on electrostatic interactions as the effect could be reversed by adding high salt concentrations. Furthermore, it could be demonstrated that the protective effect can be utilized at acidic, neutral, and basic pH which makes it a particularly versatile tool for protein formulation and delivery. Nevertheless, in order to optimally protect proteins from undesired reactions, cross-linking should be carried out at acidic pH and with polyanions present. These conditions are optimal because on the one hand, at acidic pH the reactivity of nucleophilic groups (e.g., amines) is decreased and on the other, proteins carry a higher positive charge than at neutral pH which facilitates electrostatic interactions with polyanions (Chapter 6)

The Diels-Alder reaction: A powerful tool for the design of drug delivery systems and biomaterials

Click reactions have the potential to greatly facilitate the development of drug delivery systems and biomaterials. These reactions proceed under mild conditions, give high yields, and form only inoffensive by-products. The Diels–Alder cycloaddition is one of the click reactions that do not require any metal catalyst; it is one of the most useful reactions in synthetic organic chemistry and material design. Herein, we highlight possible applications of the Diels–Alder reaction in pharmaceutics and biomedical engineering. Particular focus is placed on the synthesis of polymers and dendrimers for drug delivery, the preparation of functionalized surfaces, bioconjugation techniques, and applications of the Diels–Alder reaction in nanotechnology. Moreover, applications of the reaction for the preparation of hydrogels for drug delivery and tissue engineering are reviewed. A general introduction to the Diels–Alder reaction is presented, along with a discussion of potential pitfalls and challenges. At the end of the article, we provide a set of tools that may facilitate the application of the Diels–Alder reaction to solve important pharmaceutical or biomedical problems

Ligand Density and Linker Length are Critical Factors for Multivalent Nanoparticle−Receptor Interactions

Although there are a large number of studies available for the evaluation of the therapeutic efficacy of targeted polymeric nanoparticles, little is known about the critical attributes that can further influence their uptake into target cells. In this study, varying cRGD ligand densities (0-100% surface functionalization) were combined with different poly(ethylene glycol) (PEG) spacer lengths (2/3.5/5 kDa), and the specific receptor binding of targeted core-shell structured poly(lactic-co-glycolic acid)/poly(lactic acid)-PEG nanoparticles was evaluated using alpha(v)beta(3) integrin-overexpressing U87MG glioblastoma cells. Nanoparticles with 100% surface functionalization and short PEG2k linkers displayed a high propensity to form colloidal clusters, allowing for the cooperative binding to integrin receptors on the cellular membrane. In contrast, the high flexibility of longer PEG chains enhanced the chance of ligand entanglement and shrouding, decreasing the number of ligand-receptor binding events. As a result, the combination of short PEG2k linkers and a high cRGD surface modification synergistically increased the uptake of nanoparticles into target cells. Even though to date, the nanoparticle size and its degree of functionalization are considered to be the major determinants for controlling the uptake efficiency of targeted colloids, these results strongly suggest that the role of the linker length should be carefully taken into consideration for the design of targeted drug delivery formulations to maximize the therapeutic efficacy and minimize adverse side effects

Polyanions effectively prevent protein conjugation and activity loss during hydrogel cross-linking

In situ encapsulation is a frequently used method to prepare hydrogels loaded with high quantities of therapeutic proteins. However, many cross-linking reactions, such as Michael-type addition or Diels-Alder (DA) reaction are not tolerant toward nucleophiles; therefore, side-reactions with proteins can occur during cross-linking. This may lead to undesired protein conjugation, activity loss and incomplete protein release. In this study, a number of polyanions, namely alginate, dextran sulfate, hyaluronic acid, heparin, and poly(acrylic acid), were screened for their capability to protect proteins during covalent cross-linking. To this end, lysozyme was incubated with furyl-and maleimide-substituted methoxy poly(ethylene glycol); different pH values were tested. The degree of PEGylation and the residual activity of lysozyme were investigated. Without polyanions, 61.1% of the total lysozyme amount was PEGylated at pH 7.4; the residual activity was 20.3% of the initial activity. With the most effective polyanion (dextran sulfate), PEGylation could be completely suppressed; the residual activity was 98.4%. The protective effect of polyanions was attributed to electrostatic interactions with proteins; the "shielding" could be reversed by adding high salt concentrations. Furthermore, the protective effect was dependent on the concentration and molecular mass of the polyanion, but almost independent of the protein concentration. As a proof of concept, hydrogels were loaded with lysozyme and bevacizumab during cross-linking via DA reaction. Without polyanions, a large fraction of the protein was covalently bound to the polymer network resulting in degradation-controlled release; the residual activity of lysozyme was 50.0%. With polyanions, the protein molecules were mobile and their release was diffusion-controlled. The residual activity of lysozyme was 88.9%; the released bevacizumab was structurally intact. Polyanions can, therefore, be used as protective additive to prevent chemical protein modification during hydrogel cross-linking. (C) 2016 Elsevier B.V. All rights reserved

Fabrication of antibody-​loaded microgels using microfluidics and thiol-​ene photoclick chemistry

Reducing burst effects, providing controlled release, and safeguarding biologics against degradation are a few of several highly attractive applications for microgels in the field of controlled release. However, the incorporation of proteins into microgels without impairing stability is highly challenging. In this proof of concept study, the combination of microfluidics and thiol-ene photoclick chemistry was evaluated for the fabrication of antibody-loaded microgels with narrow size distribution. Norbomene-modified eight-armed poly(ethylene glycol) with an average molecular mass of 10,000 Da, 20,000 Da, or 40,000 Da were prepared as macromonomers for microgel formation. For functionalization, either hydrolytically cleavable ester or stable amide bonds were used. A microfluidic system was employed to generate precursor solution droplets containing macromonomers, the crosslinker dithiothreitol and the initiator Eosin-Y. Irradiation with visible light was used to trigger thiol-ene reactions which covalently cross-linked the droplets. For all bond-types, molecular masses, and concentrations gelation was very rapid ( < 20 s) and a plateau for the complex shear modulus was reached after only 5 min. The generated microgels had a rod-like shape and did not show considerable cellular toxicity. Stress conditions during the fabrication process were simulated and it could be shown that fabrication did not impair the activity of the model proteins lysozyme and bevacizumab. It was confirmed that the average hydrogel network mesh size was similar or smaller than the hydrodynamic diameter of bevacizumab which is a crucial factor for restricting diffusion and delaying release. Finally, microgels were loaded with bevacizumab and a sustained release over a period of 30 +/- 4 and 47 +/- 7 days could be achieved in vitro

Gold-Tagged Polymeric Nanoparticles with Spatially Controlled Composition for Enhanced Detectability in Biological Environments

Organic nanoparticles offer the advantage of high biocompatibility for biomedical applications but suffer frequently from poor visibility in biological environments. While fluorescent-labeling is convenient and allows for fast and extensive histological analysis, fluorescence imaging and quantitative analysis are limited by low resolution and significantly hindered by tissue autofluorescence. Labeling of polymeric nanoparticles with an additional gold tag would allow for high resolution imaging via transmission electron microscopy (TEM) and for quantification of particles by inductively coupled plasma optical emission spectrometry (ICP-OES). However, spatially uncontrolled gold-tagging can cause significant fluorescence quenching. To overcome this restraint, 2.2 nm gold nanoparticles were introduced at the interface between the hydrophobic fluorophore-loaded core and the hydrophilic shell of polymeric nanoparticles. Due to the small size of gold labels and the spatially controlled stratified composition of hybrid nanoparticles, fluorescence quenching by gold tags was minimized to 15.1%, allowing for concomitant detection of both labels via optical microscopy after enhancement of the gold tags. Multilayered hybrid nanoparticles exhibited outstanding detectability in TEM, even without additional sample staining. Furthermore, they were capable of producing remarkable image contrast inside cells after gold or silver enhancement. The interfacial gold layer increased the hydrodynamic particle size only marginally from 71.8 to 89.5 nm and had no negative impact on biocompatibility in vitro. The gold content (0.75% m/m) is sufficiently high for future quantification in tissues after systemic administration. With their clean-cut structure and superior detectability, multilayered hybrid nanoparticles constitute an outstanding blueprint and a precious tool for the development of nanomedicines

Hydrogel microspheres evading alveolar macrophages for sustained pulmonary protein delivery

Pulmonary delivery is a highly attractive alternative to injections for biologics such as therapeutic proteins. However, bioavailabilities generally suffer from the presence of phagocytic cells that clear particulate matter entering the lung. In this study, microgel particles were developed using an all-aqueous two-phase system approach and evaluated for their efficacy as an inhalable controlled release system. Norbornene- and thiol-modified four- and eight-armed poly (ethylene glycol) with an average molecular mass of 10,000 Da were prepared as macromonomers for microgel formation. Emulsions of precursor solution droplets containing macromonomers and Irgacure 2959 as photocatalyst were prepared in a dextran solution. Irradiation with UV light was used to covalently crosslink the droplets by triggering the thiol-ene reaction. The resulting microgels were processed to dry powder inhaler formulations, and respirable aerodynamic sizes were assessed in vitro. Microgels were loaded with the model proteins lysozyme and bovine serum albumin, with encapsulation efficiencies of 51.5% and 73.6%, respectively. Depending on the macromonomer type, protein-loaded microgels released their cargo over a 6-14 day period. In an MTT assay, the particles did not show significant cytotoxicity, and their recognition by alveolar macrophages was considerably lower than for polystyrene control particles. This makes the microgels a promising pulmonary delivery system for proteins and other biologics

Interaction of funtionalized nanoparticles with serum proteins and its impact on colloidal stability and cargo leaching

The majority of effort in the area of polymeric nanocarriers is aimed at providing controlled drug delivery in vivo. Therefore, it is essential to understand the delicate interplay of polymeric NPs with serum proteins in order to forecast their performance in a biological system. In this study, the interaction of serum proteins with functionalized polymeric colloids as a function of particle charge and hydrophobicity was investigated. Moreover, impact on NP stability and cargo leaching was assessed. The hard protein corona of polymeric NPs with either uncharged methoxy groups (methoxy-NPs), positively charged amine groups (amine-NPs), negatively charged carboxylic acid groups (carboxyl-NPs) or zwitterionic NPs decorated with amine and carboxylic acid groups (zwitterion-NPs) was quantitatively and qualitatively analyzed and correlated with the respective colloidal stability using fluorescence resonance energy transfer. Positively charged amine-NPs displayed an enhanced interaction with serum proteins via electrostatic interactions resulting in a hard corona consisting of diverse protein components. As revealed by FRET and agarose gel electrophoresis, the enhanced adsorption of proteins onto the colloidal surface significantly altered the NP identity and severely impaired the colloidal integrity as the lipophilic cargo was continuously leached out of the hydrophobic NP core. These results highlight the importance of generating a profound knowledge of the bio-nano interface as adherence of biomolecules can severely compromise the performance of a colloidal drug delivery system by changing its identity and integrity

Controlled Antibody Release from Degradable Thermoresponsive Hydrogels Cross-Linked by Diels−Alder Chemistry

Amine-modified four- and eight-armed poloxamines were prepared and subsequently functionalized with maleimide or fury' groups. Aqueous solutions of these polymers exhibited an immediate gelation at a temperature above 37 C. Concomitantly, Diets Alder reactions gradually cross-linked and cured the gels. Different ratios between four- and eight-armed macromonomers were used to tune hydrogel stability and mechanical properties. In this way, hydrogel stability could be precisely controlled in the range of 14 to 329 days. Controlled release of the model antibody bevacizumab was achieved over a period of 7, 21, and 115 days. Release profiles were triphasic with a low burst; approximately 87% of the released antibody was intact and displayed functional binding. The hydrogels presented in this study are degradable, nontoxic, rapidly gelling, stable, and provide controlled antibody release. They can be tailored to match the demands of various applications and present an attractive platform for antibody delivery

Design of hydrogels for delayed antibody release utilizing hydrophobic association and Diels–Alder chemistry in tandem

Biodegradable hydrogels were prepared from furan-and maleimide-functionalized eight-armed poly-(ethylene glycol) with an average molecular mass of 40000 Da (8armPEG40k-furan and 8armPEG40k-maleimide) using the Diels-Alder (DA) reaction as a cross-linking mechanism. Hydrophobic 6-aminohexanoic acid (C-6) and 12-aminododecanoic acid (C-12) spacers were introduced between the polymer backbone and the functional end-groups; the influence on the gel properties was studied. Modification with C-6 and C-12 spacers induced hydrophobic interactions between the macromonomers leading to association and increased viscosity of the polymer solutions; both effects were influenced by the spacer length. In combination with DA cross-linking, hydrophobic derivatives of 8armPEG40k-furan and 8armPEG40k-maleimide led to hydrogels with improved properties. Upon introduction of C-12 spacers, gelation of 8armPEG40k hydrogels occurred twice as fast. Interestingly, no effect was observed when only one of the two components had been modified. Our experiments suggest that the association of macromonomers by hydrophobic interactions facilitates chemical cross-linking via DA chemistry. This hypothesis is supported by calculations of the network mesh size and the Young's modulus of compression, which showed an increased cross-linking density of hydrophobically modified hydrogels. As a consequence of the increased cross-linking density, the degradation stability of C-12-modified hydrogels increased by a factor of 4. Moreover, hydrophobic modification improved the hydrolytic resistance of maleimides; this also contributes to gel stability. The in vitro release of bevacizumab, which served as a model antibody, could be delayed for almost 60 days using modification with C-12. Similar trends were observed for C-6-modified 8armPEG40k hydrogels; however, the effects were considerably weaker. In summary, utilizing hydrophobic association and chemical cross-linking in tandem is a promising approach to create biodegradable hydrogels for delayed antibody release