(B-41) Extracellular Matrix Inspired Biomaterials for Tunable Nucleic Acid Delivery and Expression of Therapeutic Growth Factors in Chronic Wounds

Introduction:: Chronic wounds affect millions of individuals each year, with poor patient outcomes and high treatment costs placing a major burden on the global healthcare system. Underlying pathologies, such as Diabetes Mellitus or poor circulation, are the primary cause of wound chronicity. Developing effective treatments for chronic wounds is challenging for several reasons, including bacterial infection, prolonged inflammation, increased protease activity, and disrupted cellular signaling. To address these challenges, particularly disrupted cellular signaling, our group has designed extracellular matrix inspired hydrogels which utilize the inflammatory wound environment for controlled release and expression of growth factor encoding plasmid DNA. Here, we demonstrate the ability to tune material formulations, control gene expression profiles, and improve wound healing outcomes in acute and diabetic animal models.

Materials and Methods:: The collagen-mimetic peptide (CMP) sequence [(GPP)₃GPRGEKGERGPR(GPP)₃GPCCG] was synthesized using solid phase peptide synthesis and purified with high-performance liquid chromatography. The CMPs were then conjugated to branched polyethylenimine (PEI, M_n = 25,000) using the bifunctional linker sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC). Polyplexes, the nanostructures used for gene delivery, were formed via the electrostatic interactions between positively charged PEI and negatively charged plasmid DNA (pDNA). Hydrogels were then formed by mixing CMP decorated polyplexes with denatured type I collagen containing either hyaluronic acid or fibrin as a secondary matrix component. The secondary structure of CMP was characterized using circular dichroism, and the size, charge, and stability of the polyplexes were characterized using dynamic light scattering and gel electrophoresis. The hydrogels were then seeded with either NIH3T3 mouse fibroblasts or normal human dermal fibroblasts to evaluate gene expression profiles in vitro. Last, the hydrogels were applied to both acute and diabetic animal wound models to evaluate wound healing outcomes.

Results, Conclusions, and Discussions:: The melting temperature of CMP is approximately 45 ºC as determined by circular dichroism, indicating the highest temperature at which the CMP will fold into a triple-helix. Below this temperature, the CMP can hybridize with the native collagen molecules to form a stable tether between the polyplexes and the collagen matrix, enabling direct temporal control over release and expression through the extent of CMP modification on the polyplexes. Dynamic light scattering results for all polyplex formulations demonstrate a suitable size (< 200 nm) and surface charge (~30 mV) for efficient cellular uptake, and complete pDNA condensation is observed through gel electrophoresis. Temporal control of gene expression was confirmed in vitro, with more CMP modification resulting in later peaks in gene expression. In animal models, delayed expression of growth factor pDNA improved wound healing compared to recombinant growth factor delivery and unloaded hydrogels. Taken together, these results show the potential of CMP-based gene delivery from extracellular matrix mimicking hydrogels for treating chronic wounds. Continued work on this project aims to incorporate multiple therapeutic cargo and understand the biological mechanisms of gene carrier release with the goal of optimizing the material for clinical translation.

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