(719h) Fibroblast Transfection in Diabetic Foot Ulcer Models Using Collagen-Based Gene-Activated Matrices

Diabetic foot ulcers are a significant unmet clinical need that lead to lower extremity amputations and increased five-year mortality rates. Despite substantial research efforts on developing new treatments, progress toward clinically relevant therapies has been limited. The inflammatory nature of these wounds limits the efficacy of growth factor-based approaches, with rapid degradation and clearance requiring repeat applications and high doses. Nucleic acid delivery offers an alternative strategy capable of bypassing these limitations by instructing resident cells to produce therapeutic growth factors. Non-viral delivery offers increased safety and tunability compared to viral approaches, but the primary challenge is low transfection efficiency. To improve transfection rates and increase the likelihood of therapeutic success, our group is exploring the effects of the diabetic wound environment on nucleic acid delivery from gene-activated matrices (GAMs).

To evaluate this research question, we employed a previously developed GAM consisting of a collagen-based matrix loaded with polyethylenimine (PEI) polyplexes. The polyplexes are functionalized with collagen-mimetic peptides (CMPs) that hybridize with native collagen triple helices and physically crosslink the polyplexes to the matrix. Polyplex release from the matrix relies on protease-mediated degradation, and release kinetics can be tuned based on the concentration of the CMP crosslinks. Within the context of this general approach, we hypothesized that the matrix composition can regulate the transfection of a an important dermal cell type, human dermal fibroblasts (HDFs), through integrin-mediated biochemical and biomechanical signaling. Additionally, we hypothesized that the inflammatory diabetic wound environment would have deleterious effects on transfection, necessitating alternative approaches designed to restore transfection in this environment.

To test these hypotheses, we employed 2D and 3D in vitro and excisional in vivo models of the wound environment to monitor cell behavior. Two-dimensional experiments enabled the direct characterization of the effects of integrin binding and inflammatory cytokines on HDF transfection, viability, and phenotype. The more complex 3D experiments allowed us to probe the influence of mechanical properties on transfection, migration, and viability. Moreover, the 3D experiments enabled us to evaluate the interactions between our materials and the inflammatory environment, as well as the development of these interactions and cell behavior over relevant time scales. Designing materials specifically for the diabetic wound environment will be required for successful translation, and elucidating the interactions between materials, cells, and their native environments can inform therapeutic design across numerous diseases.