Person:

Desai, Rajiv M.

ECM-mimicking biomaterial research has driven numerous advances in tissue engineering in recent years, as new developments in polymer chemistry and cell-material interactions have allowed bioengineers to create complex rationally designed yet physiologically relevant biomaterials. These new 3D biomaterial platforms can provide new insights for bioengineering as we study cell fate, drug delivery, or the ability for material platforms to guide regenerative medicine. Despite these recent advances, there is still a need for a ubiquitous material crosslinking system that is capable of addressing multiple scientific questions, rather than specific materials tailored to specific problems in tissue engineering. Thus, the guiding hypothesis of this thesis is that we can functionalize naturally occurring biopolymers to crosslink via a biocompatible click chemical reaction to create a tunable biomaterial platform for tissue engineering or 3D bioprinting applications. This thesis will explore the ability to crosslink different alginate or gelatin polymer systems with a bioorthogonal, cytocompatible, and facile click chemical reaction between tetrazine and norbornene. Polymer chemical characterization, hydrogel mechanical characterization, and biological cell characterization studies are shown to demonstrate the utility of these novel biomaterial systems. A 3D bioprinting application of the click gelatin materials is also presented to highlight the amenability of this crosslinking chemistry to create novel tissue constructs. Together, the results presented in this thesis showcase a novel polymer platform that improves the functionality of commonly used biopolymers to allow bioengineers to answer complex research questions in the field of tissue engineering.

Alginate hydrogels are well-characterized, biologically inert materials that are used in many biomedical applications for the delivery of drugs, proteins, and cells. Unfortunately, canonical covalently crosslinked alginate hydrogels are formed using chemical strategies that can be biologically harmful due to their lack of chemoselectivity. In this work we introduce tetrazine and norbornene groups to alginate polymer chains and subsequently form covalently crosslinked click alginate hydrogels capable of encapsulating cells without damaging them. The rapid, bioorthogonal, and specific click reaction is irreversible and allows for easy incorporation of cells with high post-encapsulation viability. The swelling and mechanical properties of the click alginate hydrogel can be tuned via the total polymer concentration and the stoichiometric ratio of the complementary click functional groups. The click alginate hydrogel can be modified after gelation to display cell adhesion peptides for 2D cell culture using thiol-ene chemistry. Furthermore, click alginate hydrogels are minimally inflammatory, maintain structural integrity over several months, and reject cell infiltration when injected subcutaneously in mice. Click alginate hydrogels combine the numerous benefits of alginate hydrogels with powerful bioorthogonal click chemistry for use in tissue engineering applications involving the stable encapsulation or delivery of cells or bioactive molecules.

Injectable gelatin hydrogels formed with bioorthogonal click chemistry (ClickGel) are cell-responsive ECM mimics for in vitro and in vivo biomaterials applications. Gelatin polymers with pendant norbornene (GelN) or tetrazine (GelT) groups can quickly and spontaneously crosslink upon mixing, allowing for high viability of encapsulated cells, establishment of 3D elongated cell morphologies, and biodegradation when injected in vivo.

Targeting small molecules to diseased tissues as therapy or diagnosis is a significant challenge in drug delivery. Drug-eluting devices implanted during invasive surgery allow the controlled presentation of drugs at the disease site, but cannot be modified once the surgery is complete. We demonstrate that bioorthogonal click chemistry can be used to target circulating small molecules to hydrogels resident intramuscularly in diseased tissues. We also demonstrate that small molecules can be repeatedly targeted to the diseased area over the course of at least one month. Finally, two bioorthogonal reactions were used to segregate two small molecules injected as a mixture to two separate locations in a mouse disease model. These results demonstrate that click chemistry can be used for pharmacological drug delivery, and this concept is expected to have applications in refilling drug depots in cancer therapy, wound healing, and drug-eluting vascular grafts and stents.