(C-95) Combination therapy of microporous hydrogel scaffolds displaying supramolecular peptide assemblies to enhance wound healing

Chronic wounds are an inflammatory disorder in which the body fails to reinstate proper barrier function of wounded skin, leading to diminished tissue function and structure. Because of the multiple overlapping immunological processes occurring during wound healing, there has been a recent growing interest in the development of immunomodulatory therapies. Recently, microporous annealed particle scaffolds (MAPS) hydrogels composed of poly (ethylene glycol) and crosslinked with matrix metalloproteinase-degradable peptide (MMP) have been shown to integrate with surrounding tissue while serving as a scaffold to promote rapid tissue network formation. Changing the chirality of the crosslinking peptide of these HMPs resulted in a weak activation of the adaptive immune system which led to de novo tissue regeneration. Supramolecular peptide nanofibers are a promising approach for augmenting immune responses because of their ability to raise T-cell and B-cell responses without adjuvants or other immunostimulant factors. Therefore, we aim to develop a combination therapy of microporous hydrogel scaffolds displaying epitope-containing peptide nanofibers to further engage the adaptive immune system during wound healing, which we hypothesize will result in enhanced tissue regeneration.

By means of Solid Phase Peptide Synthesis, the Coil29 (Ac-QARILEADAEILRAYARILEAHAEILRAD-NH2) and Q11 (Ac-QQKFQFQFEQQ-Am) backbones were synthetized displaying L-chiral and D-chiral MMP epitopes on the N-terminus of the peptide and purified through High-performance liquid chromatography (HPLC) and verified through Mass Spectroscopy. For the Q11 vaccine formulation, the self-assembling randomized polypeptide KEYA is needed as an adjuvating agent, whereas the self-adjuvating Coil29 backbone did not require external immunostimulants and no additional sequences were added.  The Coil29 backbone alone was used as positive control. Subsequently, all peptides were assembled into nanofibers under physiological conditions as confirmed with Atomic Force Microscopy and used for immunogenicity studies in vivo settings. IgG quantification and antibody subclass phenotyping were performed in streptavidin-treated ELISA plates against biotinylated epitope peptides. To evaluate epitope-specific T cell responses, splenocytes were collected from vaccinated mice 7 days after their last immunization and 0.25 million splenocytes were plated in ELISpot plates followed by stimulation with the corresponding epitope peptide. A microfluidic device was used to produce hydrogel microparticles of 6.5% 4 arm PEG norbornene (MW 20 kDa) crosslinked with a di-cysteine containing peptide that is labile by matrix metalloproteinase (MMP) and modified by 500uM of RGD and the addition of 10mM LAP as the photoinitiator. Peptide nanofibers were then conjugated to or loaded between and within hydrogel beads and are currently being used in vivo and in vitro studies.

Through ELISA, we found epitope-dependent antibody secretion starting at one week after the initial inoculation (Figure 1a) and a statistically significant increased anti-MMP antibody titer for the groups vaccinated with the Coil29 backbone (Figure 1b) when compared to the Q11 groups. Even though D amino acids are usually poorly immunogenic, we found that vaccination against D-chiral MMP epitopes was able to raise a specific antibody secretion that is not significantly differently than that of the L-chiral epitope. We also found that the self-adjuvanting backbone Coil29 can induce higher immune responses compared to the KEYA-Q11 backbone and that all nanofiber formulations elicit a Th2-biased antibody phenotype, as confirmed with the biased IgG1 antibody subclass elicited by the nanofibers (Figure 1d). Through ELISpot we found that spleenocytes extracted from those mice inoculated with DMMP-Coil29 are highly stimulated with soluble DMMP peptide, suggesting the presence of a T cell epitope on the DMMP sequence (Figure 1e). We then demonstrated that peptide nanofibers can be encapsulated at different concentrations in bulk gel without affecting mechanical properties of the hydrogel drastically (Figure 2a) and that nanofibers can be delivered within individual beads (Figure 2a) and between them (Figure 2b) to form a network of annealed microparticles that can deliver immunogenic fibers as cargo (Figure 2c). At of the time of submission of this abstract, in vitro and in vivo immunogenicity studies are currently evaluating immune activation and polarization (M1/M2, Th1/Th2) when this new combination material is delivered subcutaneously to C57BL/6 mice.