Porównywalna skuteczność chitozanu/miR-200b-3p i Lipofectamine/miR-200b-3p dostarczanych przez hydrożel Pluronic F127 w leczeniu ran cukrzycowych

BACKGROUND: The clinical application of miRNA therapeutics in diabetic foot ulcers (DFUs) is limited by the rapid degradation of miRNAs. We previously reported that topical lipofectamine (Lipo)-based miR-200b-3p delivery by Pluronic F127 (PF127) hydrogel has pro-healing properties in diabetic wounds. However, Lipo's high cost and cytotoxicity concerns impede its clinical use. Chitosan (CS) is an inexpensive biopolymer. We investigated whether the topical delivery of PF127+CS+miR-200b-3p can accelerate wound healing in diabetic mice. METHODS: In vitro experiments were conducted, including the CCK-8 assay, gelation time measurement, scanning electron microscopy, Zetasizer Nano ZS analysis, release kinetics, swelling ratio, gel retardation assay, and uptake assay. In vivo experiments were performed on db/db mice with two 8 mm dorsal wounds. Four treatment groups including PF127, PF127+CS+miR-negative control (miR-NC), PF127+CS+miR-200b-3p, and PF127+Lipo+miR-200b-3p were examined. On day 14, skin tissues were harvested for H&E and immunohistochemical staining, as well as real-time PCR of variable genes and miR-200b-3p. RESULTS: The CCK-8 assay revealed the non-toxicity of CS on HaCaT keratinocytes. Furthermore, PF127 concentration-dependently decreased the positive surface charge of the CS/miR-200b-3p nanoparticles. The PF127 kept the diameter of the CS/miR-200b-3p nanoparticles at roughly 200-250 nm. In vivo ZEB-1 gene expression confirmed that CS and Lipo had comparable transfection efficacy. Both the PF127+CS+miR-200b-3p and PF127+Lipo+miR-200b-3p groups accelerated wound healing compared to the PF127+CS+miR-NC group. The superb healing properties of the PF127+CS+miR-200b-3p group were supported by the expression levels of genes (Col1α2, Col3α1, Nox1, Nox4, HO-1, IL-6, IL-1β, TNF-α, PAI-1, p16, p21, and p53) and proteins (CD68, CD31, and γH2AX), which showed no significant differences between the PF127+CS+miR-200b-3p and PF127+Lipo+miR-200b groups. CONCLUSION: Topical delivery of CS/miR-200b-3p nanoparticles using PF127 is a promising treatment for diabetic wound healing. Our findings demonstrate that chitosan serves as a cost-effective, non-toxic alternative to Lipofectamine in diabetic wound management.