Abstract
INTRODUCTION: Wound healing involves controlled proliferation of fibroblasts and synthesis of extracellular matrix (ECM). Imbalance in this process leads to excessive fibroblast activity, collagen deposition, and fibrosis. Current pharmacological therapies target the inhibition of profibrotic signaling pathways, and surgical procedures are reserved for advanced pathologic conditions, necessitating research on local therapies. The present study focuses on investigating the antifibrotic potential of Cu-doped ZnO (Cu-ZnO) nanoparticles (NPs) synthesized using a microwave-assisted green synthesis method. METHODS: ZnO and Cu-ZnO NPs were synthesized utilizing microwave energy using O sanctum hydroalcoholic extract. Synthesized NPs were characterized using appropriate characterization techniques to elicit the crystallinity, size, morphology, and optical properties. Using the disk diffusion assay, antimicrobial activity of NPs was determined against S aureus, and hematocompatibility was evaluated using chick blood. Inhibition of fibroblastic activity and mitochondrial membrane potential (MMP) was determined to explore antifibrotic activity. RESULTS: X-ray diffraction analysis confirmed successful Cu²⁺ incorporation into the ZnO lattice, with distinct (200) and enhanced (101) diffraction peaks and a reduction in crystallite size at higher dopant levels. Morphological assessment revealed a transition from irregularly shaped ZnO NPs to well-defined tripod-like Cu-ZnO nanoforms, indicating directional growth. UV-Visible (UV-Vis) spectroscopy showed a red shift, reflecting the narrowing of the band gap upon Cu doping. Antimicrobial evaluation against Staphylococcus aureus demonstrated a dopant- and dose-dependent enhancement in activity, with the bacterial zone of inhibition (ZOI) increasing from 8.5 mm (undoped ZnO) to 14 mm (3 M Cu-ZnO) at the highest tested concentration of 1.0 mg/mL, and measurable inhibition (5 mm) persisting even at 0.25 mg/mL for 3 M Cu-ZnO NPs. Hemolysis remained below 5% for all formulations, confirming hemocompatibility. As per the results of antifibrotic activity assays, 3 M Cu-ZnO NPs showed the strongest suppression of 3T3 fibroblast proliferation, with JC-1 analysis confirming mitochondrial membrane depolarization leading to the apoptosis of fibroblasts due to excessive ROS production by Cu-ZnO NPs. CONCLUSIONS: Cu-ZnO NPs exhibited strong antimicrobial activity, maintained hemocompatibility, and effectively regulated the proliferation of the fibroblasts. The enhanced antifibrotic effect is mainly driven by elevated ROS levels, which induce mitochondrial membrane depolarization, as confirmed by the JC-1 assay, leading to mitochondrial stress and apoptotic elimination of hyperproliferative fibroblasts.