Poster O1 - Early wound phase modulation using self-assembled nanoparticles for pressure wound healing after spinal cord injury

Introduction: Spinal cord injury patients face prolonged periods of bed rest as rehabilitation can take weeks to years depending upon the type of injury. As a result, these patients are susceptible to chronic secondary complications, such as pressure wounds. About 40% of injured patients develop pressure wounds during their initial hospitalization while >80% are readmitted due to the presence of pressure wounds. These wounds can take months to heal or not heal completely as pressure wounds developed following spinal cord injury result in delayed wound healing due to a decrease in immune cell response at the wound site. We hypothesize that modulating the immune cells such as monocytes and macrophages around the wound site via exogenous application of self-assembled chemokine and neuropeptide fused with elastin-like polypeptide nanoparticles promotes wound healing after spinal cord injury.

Materials and

Methods: To achieve the objective, recombinant monocyte chemotactic protein-1 (MCP-1) and alpha-melanocyte-stimulating hormone (ɑ-MSH) were fused with elastin-like polypeptides (ELP). Briefly, recombinant MCP-1 and ɑ-MSH were cloned in front of elastin cassette V40C2 in the cloning vector pET25b+ using the protocol developed by members of our lab. As per the published protocol, plasmids were transformed into Escherichia coli, cultured overnight at 37°C, purified using centrifugation, and characterized using SDS-PAGE and Western Blot. Purified nanoparticles were tested for their size, in an in vitro system of migration for their biological activity, and later using an animal model of pressure wounds after complete spinal cord injury. 10-week-old Balb/C male mice were used to induce complete spinal cord injury and full-thickness wounds below the injury site. Mice were divided into 6 treatment groups: Control, ELP, MCP-1, MCP-ELP, α-MSH, and MSH-ELP. 100 ng of each nanoparticle treatment was administered topically on each wound every day for the first 7 days. On days 3 and 7, mice from each group were sacrificed and wound site tissue samples were excised to characterize the acute phase of wound healing. The rest of the mice were sacrificed on day 26 after complete wound healing to identify long-term wound healing properties. Tissue samples were stained for H&E, Trichrome, and F40/80. Images were captured using an ECHO Revolution microscope and analyzed using ImageJ (NIH) software.

Results, Conclusions, and Discussions: We developed pure, low-cost MCP-ELP, and MSH-ELP self-assembled nanoparticles which induced comparable biological activity as compared to native proteins. A transwell migration assay was conducted to evaluate the biological activity of these nanoparticles using THP-1 monocytes (Figure 1). We found that THP-1 cell migration as a chemotactic response was significantly better in the case of native (MCP and α-MSH) and nanoparticles (MCP-ELP and MSH-ELP) compared to the ELP and Control groups. However, MSH-ELP induced better migration as compared to all other groups and its native form (p < 0.001). Immunohistological studies at different time points (days 3, 7, and 26) showed successful migratory effects of macrophages to the wound sites in a time-dependent manner. Wound healing was enhanced overall compared to the control group by continuously applying up to 7 days of these nanoparticles (MCP-ELP and MSH-ELP). In conclusion, we successfully developed and purified the fusion nanoparticles (MCP-ELP and MSH-ELP) using inverse transition cycling which is an easy and cost-effective process. In vitro, studies suggested enhanced chemotactic response. Animal studies suggested successful recruitment of macrophages to wound sites and thereby enhanced wound healing after SCI.

Acknowledgements (Optional): This work was funded by Rutgers Busch Biomedical Grant 2021. The authors thank Rick Cohen from Biomedical Engineering, Rutgers University for his technical support.