Poster N4 - Design of a Heat Inducible Self-Assembling Peptide Hydrogel

Introduction: The discovery of self-assembling peptide hydrogels (SAPHs) in the 1990s prompted their assessment throughout various biological systems due to their characteristic biocompatibility and mechanical tunability (e.g., porosity).1 Self-assembly is primarily driven by electrostatic interactions, hydrophobic effects, and hydrogen bonding; all of which can lead to the assembly of α-helices and β-sheets.2,3 The characterization of primary and secondary protein structures can be achieved through circular dichroism (CD), Fourier Transformed Infrared Spectroscopy (FTIR), and nuclear magnetic resonance (NMR). Novelly, the Kumar Lab developed a SAPH with a different backbone design, termed New Candidate 3 (NC3), which served as a basis for the development of the NC3-AA’ sequence. NC3-AA’ is a neutrally charged peptide composed of a single central glycine spacer and flanking palindromic peptide residues. Intriguingly, self-assembly of NC3-AA’ appears to be augmented through heat treatment at 85°C without the addition of a multivalent ion to stabilize the flanking charged peptide residues. The investigation of this novel material may support the discovery of a more effective SAPH that can be applied for research in angiogenesis, neurogenesis, dentinogenesis, and other regenerative processes.

Materials and

Methods: Peptide was synthesized using solid-phase Fmoc peptide chemistry where purity and chemical identity were confirmed by HPLC & MS, respectively. Structural characteristics of the NC3 peptide were determined using FTIR, CD, SEM, & AFM. Modification of the NC3 sequence was performed to yield NC3-AA’, which will be structurally characterized by FTIR, CD, SEM, AFM, & TEM. Heat treatment of NC3-AA’ shows visual improvement in hydrogelation and the mechanism of folding will be validated through micro Differential Scanning Calorimetry (µDSC). In-vitro cytocompatibility will be assessed using a CCK8+ metabolic assay with C2C12s & HUVECs. NC3-AA’ will be incorporated into the conditioned media at varying concentrations in DMEM & MCDB 131 media, respectively, with 1% FBS supplementation, and a no treatment control group. Ultimately, in-vivo efficacy of NC3-AA’ will be assessed within a wound healing model in Wistar rats.

Results, Conclusions, and Discussions: AFM shows NC3 self-assembles into fibrous structures that are capable of recapitulating native tissue architecture. Heat treatment of NC3-AA’ on a heat block is achieved after incubating for 1 hour at 85˚C.

NC3-AA’ demonstrates improved self-assembly following heat treatment at 85˚C for 1 hour. Structural properties, in-vitro cytocompatibility, and in-vivo biocompatibility experiments will be employed to characterize NC3-AA’ as a wound healing scaffold.

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