Probing the Effects of Chirality on Self-Assembling Peptides: Hydrogel Formation, Degradation, Antigen Release, and Adjuvancy.

INTRODUCTION: Multidomain peptides (MDPs) are amino acid sequences that self-assemble to form supramolecular hydrogels under physiological conditions that have shown promise for a number of biomedical applications. K(2)(SL)(6)K(2) ("K(2)"), a widely studied MDP, has demonstrated the ability to enhance the humoral immune response to co-delivered antigen. Herein, we sought to explore the in vitro and in vivo properties of a peptide with the same sequence but opposite chirality (D-K(2)) since peptides composed of D-amino acids are resistant to protease degradation and potentially more immunostimulatory than their canonical counterparts. METHODS: K(2) and D-K(2) hydrogels were characterized and evaluated in vitro using circular dichroism, rheology, cryo-electron microscopy, and fluorescence recovery after photobleaching studies. In vivo experiments in SKH-1 mice were conducted to evaluate both ovalbumin release from the hydrogels and hydrogel degradation. The injection site of the hydrogels was analyzed using histology and humoral immunity was assessed by ELISA. RESULTS: In vitro, the enantiomeric hydrogels exhibited similar rheological properties, and fluorescence recovery after photobleaching experiments demonstrated that the diffusion of ovalbumin (OVA), a model antigen, was similar within both hydrogels. In vivo, K(2) and D-K(2) peptide hydrogels had similar OVA release rates, both releasing 89% of the antigen within 8 days. Both hydrogels elicited a similar antigen-specific humoral immune response. However, the in vivo degradation of the D-K(2) hydrogel progressed significantly slower than K(2). After 4 weeks in vivo, only 23 ± 7% of the K(2) hydrogel remained at the injection site compared to 94 ± 7% of the D-K(2) hydrogel, likely due to their different protease susceptibilities. CONCLUSION: Taken together, these data suggest that peptide chirality can be a useful tool for increasing hydrogel residence time for biomedical applications that would benefit from long persistence times and that, if an antigen releases over a sufficiently short period, release can be largely independent of degradation rate, though slower-diffusing payloads may exhibit degradation rate dependence. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12195-024-00806-1.