A.A. San Agustin1, K.S. Fischer2, B.D. Lyttle3, A. Apte1, E.H. Bolouvi1, A.S. Hauger1, S. Skopp1, J.B. Canchis Angulo1, K. Chen2, G.C. Gurtner2, K.W. Liechty1,4 1University of Arizona Health Sciences Center, Banner Children’s at Diamond Children’s Medical Center, Laboratory For Fetal And Regenerative Biology, Department Of Surgery, Tucson, AZ, USA 2University of Arizona Health Sciences Center, Department Of Surgery, Tucson, AZ, USA 3Laboratory for Fetal and Regenerative Biology, University Of Colorado School Of Medicine And Children’s Hospital Colorado, Aurora, CO, USA 4Ceria Therapeutics, Inc., Aurora, CO, USA
Introduction: The wound healing impairment in diabetic patients is multifactorial, with chronic inflammation and an oxidant/antioxidant imbalance leading to increased oxidative stress as central features. We developed a novel therapeutic strategy targeting both the dysregulated inflammatory response and elevated reactive oxygen species in diabetic wounds. We used cerium nanoparticles (CNP) for their antioxidant properties, conjugating with a miR-146a mimetic (CNP-miR146a) to target dysregulated inflammatory signaling. However, the exact mechanisms by which oxidative stress and inflammatory signaling affect wound healing at the cellular level remain unclear. We hypothesize that CNP-miR146a improves healing in diabetic wounds by modulating the various cell types within the wound microenvironment.
Methods: Single-cell RNA sequencing was employed to interrogate gene expression across more than 5,000 individual genes. The study included three murine treatment groups: non-diabetic wounds (D3 -DB, D7 -DB), untreated diabetic wounds (D3 +DB -T, D7 +DB -T), and diabetic wounds treated with CNP-miR146a (D3 +DB +T, D7 +DB +T). Wounds were collected on days 3 and 7 post-wounding. Cell-specific markers were utilized to quantify the distribution of various cell populations in each group.
Results: On day 3, non-diabetic wounds exhibit a dominant presence of pro-inflammatory macrophages and neutrophils (Pro I Mps 60%; Pro I Neu 38%), and minimal populations of monocyte, dendritic cells (DCs), and other macrophage types (<1%). In contrast, untreated diabetic wounds show a reduction in pro-inflammatory macrophages (Pro I Mps 26%) and an increase in pro-inflammatory neutrophils (Pro I Neu 72%). CNP-miR146a treatment led to an increased presence of pro-inflammatory neutrophils (Pro I Neu 80%) and a decrease in pro-inflammatory macrophages (Pro I Mps 19%). By day 7, non-diabetic wounds show a high population of pro-regenerative macrophages (44%) and monocytes (40%). Untreated diabetic wounds exhibited upregulated in DCs (14%) and pro-inflammatory neutrophils (9%), along with pro-regenerative macrophages (23%), neuronal signaling cells (28%), and lower expression of monocytes compared to non-diabetic wounds (26%). CNP-miR146a-treated diabetic wounds maintain elevated levels of pro-regenerative macrophages (42%) similar to non-diabetic wounds, while monocyte distribution was similar to that observed in untreated diabetic wounds (28%).
Conclusion: Overall, our data highlights the adverse impact of diabetes on wound healing and suggests that targeting inflammation and oxidative stress with CNP-miR146a treatment can modulate and restore immune cell populations to enhance wound healing.