Development of a controlled-release hydrogel platform that leverages dynamic covalent bonding to extend the release of small-molecule, peptide, and protein therapeutics

Introduction: Pharmaceutical drugs have improved outcomes for many diseases; however, maintaining drug levels that are safe and effective can be challenging. Controlled-release drug delivery systems can improve patient outcomes by maintaining safe and effective drug concentrations for long periods of time. Unfortunately, current systems are limited in several ways, including in their ability to maintain the bioactivity of therapeutic proteins through release. Herein, we present a versatile drug delivery platform consisting of a supramolecular peptide hydrogel functionalized with chemical motifs that engage in dynamic covalent bonding with boronic acid-containing drugs to greatly extend the release of small-molecule, peptide, and protein drugs in vivo. Instead of releasing from hydrogels rapidly via diffusion, boronic acid-containing drugs form reversible covalent bonds with the gel, effectively pausing their transport until the drug returns to the unbonded state. After describing the synthesis and characterization of these materials, we show that a single injection of these self-assembling boronate ester release (SABER) hydrogels is capable of enhancing the therapeutic efficacy of multiple drugs in vivo, demonstrating the ability to prevent the growth of Mycobacterium tuberculosis for two weeks in infected mice and maintain normoglycemia for six days in diabetic mice.

Materials and

Methods: We first synthesized two multidomain peptides (MDPs), K2 and E2, known to self-assemble and form hydrogels using solid-phase peptide synthesis. We then functionalized these peptides with a catechol, 4-nitrocatechol, or salicylhydroxamic acid (SHA), which we hypothesized would push the equilibrium between the boronate ester (bonded) and free boronic acid state and offer different degrees of resistance to oxidation. We evaluated the bonding constants for each motif and evaluated the modified MDP’s ability to form beta sheets by circular dichroism and FTIR, its nanostructure via cryo-transmission electron microscopy, and its shear-thinning and self-healing behavior using rheology. We then demonstrated the release of four small-molecule drugs (ganfeborole, bortezomib, tavaborole, and ixazomib), a modified small-molecule drug (1V209), a peptide (insulin), two therapeutic antibodies (amivantamab and omodenbamab), and one antibody-drug conjugate (trastuzumab deruxtecan) from functionalized hydrogels. We then performed in vivo pharmacokinetic and/or efficacy studies for bortezomib, ganfeborole, and insulin. Bortezomib release studies were performed using naïve mice while ganfeborole delivery from SABER hydrogels was evaluated in an acute M. tuberculosis infection model and insulin delivery from SABER hydrogels was evaluated in diabetic mice.

Results, Conclusions, and Discussions: The functionalization of MDPs with boronic acid-binding motifs did not substantially impact the peptide’s ability to self-assemble into beta sheets or form shear-thinning, self-healing nanofibrous hydrogels. SHA-modified hydrogels demonstrated the greatest ability to extend drug release while maintaining the bioactivity of the encapsulated drug. This was in stark contrast to catechol-mediated release, which was only marginally extended and was highly prone to oxidation. Pharmacokinetic studies in mice showed that ganfeborole release from SHA-functionalized hydrogels increased the duration of therapeutic activity from < 3 days to 22 days, an 8.5-fold increase over a soluble injection. This resulted in improved therapeutic efficacy in an acute M. tuberculosis infection model at 1 and 2 weeks compared to both a single bolus injection and repeated oral administration of ganfeborole. Releasing bortezomib, a cancer drug, from SHA-functionalized hydrogels resulted in both drug retention at the injection site for >21 days and a 9-fold reduction in systemic drug compared to a soluble injection, suggesting that this formulation has strong potential as an intratumoral injection. We then sought to use this delivery system for basal insulin therapy. After performing site-specific labeling of insulin with phenylboronic acid, we injected SHA-functionalized hydrogels containing the drug subcutaneously and observed the ability to maintain normoglycemia for 144 hours compared to the less than 3 hours of glycemic correction achieved with a soluble dose of insulin. These same interactions extended the release of boronic acid-labeled antibodies from SHA-functionalized hydrogels for up to 8 weeks in vivo, providing exciting opportunities for passive immunization and other immunotherapies that would benefit from sustained or local delivery. Together, these results suggest that these functionalized hydrogels represent a robust and versatile platform for releasing multiple classes and drugs mediated by dynamic covalent bonding.

Acknowledgements (Optional): Funding for this work was provided by the National Institutes of Health (R35GM143101, R01DE021798, R01DE030140, and R61AI161809) and the Cancer Prevention and Research Institute of Texas (RR190056).