Abstract

Background
Diabetic foot ulcers form when wounds fail to progress through the physiological stages of wound healing. They are associated with infection, amputation and high mortality. Small EVs, containing proteins, lipids, RNA, and DNA, may have a role in wound healing and pathological chronic wounds.

Methods
In vitro analysis was performed on diabetic and control human dermal fibroblast cell lines. Each cell line was exposed to a combination of normoxic/hypoxic, normoglycaemia/hyperglycaemic and inflammatory/non-inflammatory conditions. Small EVs were isolated by ultracentrifugation and characterised by transmission electron microscopy (TEM), Nanotracking analysis (NTA), and Western blots. MicroRNA (miRNA) content was analysed.

Small EVs were isolated from patient wound dressings and plasma, from non-diabetic patients, diabetic patients with healing wounds and diabetic patients with non-healing wounds. miRNA was analysed and compared to in vitro samples.

Results
Using in vitro models, fibroblasts exposed to a simulated diabetic wound environment produced sEVs enriched with both known and novel miRNAs. Six known miRNAs (including miR-126-3p, miR-223-3p, and miR-155-5p) and seven novel miRNAs were identified as significantly upregulated. Parallel patient studies identified hsa-let-7a-5p and hsa-miR-486-5p as upregulated in non-healing DFUs, while others hsa-miR-1-3p and hsa-miR-184 were downregulated. Importantly, some novel miRNAs overlapped between patient samples and in vitro models, strengthening their relevance as biomarkers.

Conclusions
sEV-associated miRNAs are key regulators of impaired wound healing and may link local ulcer pathology to systemic inflammation. Future studies will validate these candidates in larger cohorts and assess their translational potential as biomarkers or therapeutic targets.