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)
