Design Parameters for Injectable Biopolymeric Hydrogels with Dynamic Covalent Chemistry Crosslinks

Introduction:: Hydrogels have extensive biomedical applications, including tissue engineering, medical devices, and therapeutic delivery vehicles. One critical benefit of hydrogels is their tunable range of mechanical properties (e.g., stiffness, self-healing, and remodeling) that allow them to replicate essential features of the natural extracellular matrix (ECM). Furthermore, the potential extrudability of hydrogels is beneficial for clinical translation, as an injection is a favorable route for delivering cells and pharmaceuticals, including proteins and gene therapies. However, producing hydrogels that are simultaneously injectable, encompass a broad range of stiffness, and retain their mechanical properties post-extrusion remains challenging in the field. For this reason, we have investigated dynamic covalent chemistry (DCC) as an approach to hydrogel crosslinking. The covalent nature of DCC provides mechanical strength to the hydrogel, while the transient behavior allows the polymer chains to associate and dissociate. This can enable injectability and recovery of the polymer network post-extrusion. However, not all hydrogels with transient crosslinks are easily extrudable and completely self-heal. For this reason, we hypothesized that two additional design parameters must be considered when formulating DCC-crosslinked hydrogels 1) degree of functionalization and 2) polymer molecular weight (MW).


Materials and Methods:: Recombinant elastin-like protein (ELP) was synthesized by bacterial expression and purified by inverse temperature cycling. To modify the ELP with hydrazine (HYD) groups, we performed an amidation of hydrazinoacetic acid onto the 14 lysine residues within the primary ELP amino acid sequence, resulting in ELP-HYD. Recombinant hyaluronic acid (HA) was purchased and bioconjugated with benzaldehyde (BZA) through the amidation of propargyl amine, followed by a copper-mediated click-chemistry reaction to synthesize HA-BZA. To synthesize distinct subfamilies, we utilized HA of varying molecular weights (20, 40, 60, and 100 k-Da), and the degree of functionalization with BZA was tuned to 6, 12, 20, and 30 %. When mixed in a 1:1 ratio (by mass, a final concentration of 1% wt ELP-HYD and 1 wt% HA-BZA) of ELP-HYD to HA-BZA, a gel is rapidly formed through the formation of hydrazone bonds that crosslink the two biopolymers together.


Results, Conclusions, and Discussions::
The resulting hydrogels had a range of stiffnesses, G’ ~ 10-1000 Pa, and extrudability, attributed to the combined effects of DCC crosslinks and polymer entanglements. We observed that increasing HA molecular weight increased the hydrogel stiffness due to chain entanglements and longer percolation paths through the network, creating a more effectively crosslinked gel. For lower HA molecular weights, the percolation paths and entanglements are limited, so defect formation dominates at higher HA functionalities. Concurrent with this effect, gelation time rapidly decreased with an increase in the degree of functionalization within each molecular-weight subfamily. This rapid crosslinking leads to nonideal network formation and increases the presence of defects. Overall, lower MW formulations required lower forces for injectability, regardless of stiffness. The higher degree of functionalization formulations exhibited more rapid self-healing. Gel extrusion through a cannula (2-m length, 0.25-mm diameter) demonstrated the potential for minimally-invasive delivery for future biomedical applications.

In summary, this work highlights how the degree of functionality and polymer molecular influence the injectability and network formation of DCC-crosslinked hydrogels and aims to guide the future design of injectable hydrogels. The tunning of these properties allows optimal chemical crosslinking to occur and limits defect formation during the rapid gelation of high-functionality systems. Our results suggest that future efforts to design injectable DCC gels should focus on formulations with crosslinking reaction stoichiometry close to 1:1 and using medium-sized molecular weight polymers.






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