Decoding Wound Healing Mechanisms of Plasma-Activated Water based Nanoparticles via Raman Spectroscopy

Background Plasma-activated water (PAW) has emerged as a promising therapeutic agent for wound healing due to its ability to modulate inflammatory responses and promote tissue regeneration. Nanoparticles such as zinc oxide (ZnO) and chitosan have also demonstrated strong antibacterial, anti-inflammatory, and pro-healing properties. However, the combination effects of combining PAW with ZnO and chitosan nanoparticles remain unexplored. Materials and methods In this study, pressure ulcer in rats were treated with PAW alone or in combination with ZnO nanoparticles, chitosan nanoparticles, or both. Healing outcomes were assessed through histological analysis, cytokine profiling (IL-6, IL-1β, IL-10, TGF-β), and Raman spectroscopy across key spectral ranges (1000–1200, 1500–1750, and 2700–3000 cm⁻¹). Hierarchical clustering, heatmap similarity analysis, overlay spectra, and principal component analysis (PCA) were employed to evaluate biochemical changes. Results PAW treatment reduced pro-inflammatory cytokines (IL-6, IL-1β, TGF-B) and increased IL-10, consistent with enhanced resolution of inflammation. ZnO nanoparticles promoted neovascularization, collagen deposition, and antibacterial activity, while chitosan nanoparticles contributed to oxidative stress reduction and epithelial regeneration. Raman spectroscopy revealed increased intensities at 1560 cm⁻¹ (amide II) and 1640 cm⁻¹ (amide I), indicating collagen and keratin synthesis. Conclusion The order of healing efficacy was: Control < PAW < PAW-ZnO ≈ PAW-Chitosan < PAW-ZnO-Chitosan. PCA and cluster analyses confirmed that the triple combination (PAW-ZnO-Chitosan) produced the most distinct biochemical profile, correlating with superior wound closure and tissue remodeling. This study demonstrates, for the first time, that PAW enriched with ZnO and chitosan nanoparticles enhances wound healing by modulating cytokine expression, supporting collagen regeneration, and promoting tissue remodeling. Raman spectroscopy provided molecular-level validation of these effects. These findings highlight the potential of nanoparticle-enriched PAW as a novel therapeutic strategy for advanced wound care.