# In vitro and in vivo evaluation of oleuropein loaded hyalurosomes for diabetic foot ulcer healing

## TL;DR

This study explores a new nanocarrier system loaded with oleuropein to improve healing in diabetic foot ulcers by reducing inflammation and oxidative stress.

## Contribution

The novelty lies in developing and evaluating oleuropein-loaded hyalurosomes for diabetic wound healing using both in vitro and in vivo models.

## Key findings

- OLE-HLs showed a biphasic release profile and enhanced bioactivity compared to free oleuropein.
- OLE-HLs reduced oxidative stress and inflammatory markers while promoting tissue repair in diabetic rats.
- Histopathological and immunohistochemical analyses confirmed improved wound healing with OLE-HLs.

## Abstract

Diabetic foot ulcers (DFUs) are among the most severe and challenging complications associated with diabetes mellitus, primarily due to chronic inflammation, oxidative stress, and impaired tissue regeneration. To develop and evaluate oleuropein-loaded hyalurosomes (OLE-HLs) as a unique topical nanocarrier system for wound healing using an experimental diabetic rat model. OLE-HLs were synthesized using a modified thin-film hydration method, and the nano-formulation was characterized by measuring the size and potential, followed by TEM, encapsulation efficiency, FTIR, in vitro release studies, and cell cytotoxicity assay. The wound-healing efficacy was evaluated in vitro, using scratch assay and in vivo, via studying the wound closure rate, oxidative stress markers, pro-inflammatory cytokines, TGFβ1 gene expression, and histopathological alterations, complemented by immunohistochemical analysis. OLE-HLs exhibited a mean particle size of 254.64 ± 9.84 nm, a zeta potential of − 25.12 ± 2.18 mV, and an encapsulation efficiency of 89.61 ± 1.82%, while TEM revealed spherical, uniformly sized, and well-dispersed vesicles, confirming stability and homogeneity. FTIR analysis verified successful oleuropein encapsulation and compatibility within the hyalurosomal system. The formulation displayed a biphasic release profile, with 26.47 ± 0.86% released in the first hour and 76.72 ± 2.45% within 24 h. Cytotoxicity assays showed enhanced bioactivity (IC50: 269.63 µg/mL) compared with free OLE (644.81 µg/mL). The scratching assay model was validated by the in vivo model, where OLE-HLs significantly promoted wound healing by reducing oxidative stress, as shown by increased GSH and GST levels and decreased MPO activity. It also suppressed inflammatory mediators (IL-6, IL-17, TNF-α, MMP-13, ADAMTS-5) and enhanced TIMP-3 expression. Additionally, regulation of TGFβ1 expression and improved histopathological and immunohistochemical features collectively supported better tissue repair. These data suggest that OLE-HLs notably promote diabetic foot ulcer healing by modulating inflammation, oxidative stress, and cellular regeneration, thereby warranting further clinical investigation.

The online version contains supplementary material available at 10.1038/s41598-026-42804-5.