Design, production and investigation of the effects of silver/zeolite-based wound dressings In Vitro/In Vivo conditions for the healing of acute CBRN wounds

The aim of CBRN defense is to provide appropriate equipment and materials capable of mitigating threats posed by CBRN agents. In this context, the treatment methods applied in injuries caused by exposure to chemical, biological, radiological, and nuclear (CBRN) agents represent a crucial step in individual protection. With advancements in the field of nanomaterials, it has become possible to design and produce biocompatible, biodegradable, and antimicrobial dressings that accelerate the wound healing process. In this thesis study, it is aimed to investigate the wound healing potential of nanofiber mats produced using polyvinyl alcohol (PVA), silver nanoparticles, and zeolites. Silver nanoparticles (AgNPs) were synthesized using the chemical reduction method. Characterization of the particles was carried out using UV-Vis spectroscopy, DLS, and SEM. Electrospinning technique was used for the fabrication of nanofibers. To obtain effective wound dressings, optimization studies were performed by varying parameters such as concentration, the distance between the needle tip and collector, and solution flow rate. The nanofibers were characterized using SEM and FT-IR. The antibacterial activity of the nanofibers (AgZeo) was determined using the disk diffusion method against Escherichia coli and Staphylococcus aureus. In vitro cytotoxicity was assessed using the Alamar Blue assay, where the healthy keratinocyte cell line HaCaT served as a control, and IC50 values were calculated. The in vitro wound migration assay of the nanofibers was evaluated using the scratch method on HaCaT cells. To determine the in vivo wound healing potential of the nanofiber wound dressings, 24 Wistar albino rats aged 8–12 weeks were used. The rats were randomly divided into 4 groups, with 6 rats in each group, and full-thickness excisional wound models were created on their dorsal areas. In Group 1 (control), no treatment was applied to the wound models. In Group 2 (Sham), 0.9% isotonic sodium chloride was applied to the wound models. In Group 3 (positive control), commercial wound dressing were applied to the wound models. In Group 4 (AgZeo), nanofiber wound dressing produced by the electrospinning method were applied to the wound models and wound contraction percentages were calculated. To enable a detailed understanding of the wound healing process in the study, wound tissue samples were stained with hematoxylin and eosin (H&E) and examined under a light microscope. The research results revealed that AgNPs exhibited a peak absorption at 400 nm, confirming the presence of silver at the nanoscale. Optimal electrospinning conditions were established with a solution-to-collector distance of 15 cm, a voltage of 17 kV, and a solution flow rate of 0.5 mL/h. In the in vitro cytotoxicity study, each layer of the nanofibers was evaluated individually. The IC50 values were found to be 118.2 µg/mL for PVA nanofibers, 100.7 µg/mL for PVAZeo nanofibers, and 119.7 µg/mL for AgZeo nanofibers. The nanofibers showed no cytotoxic effects on human skin keratinocyte cells. In the in vitro wound migration assay, it was observed that by the end of 48 hours, the wound area in the AgZeo nanofiber group had completely closed. As a result of the antibacterial activity test, AgZeo nanofibers formed inhibition zone areas of 163 mm² against E. coli and 189 mm² against S. aureus. In the in vivo wound healing experiment, the wound area in the group treated with AgZeo nanofiber wound dressings was completely closed by day 14. At the end of day 14, wound closure was 8% in the negative control group, 43% in the positive control group, 60% in the sham group, and 96% in the AgZeo group. As a result of the histopathological evaluation, the AgZeo group was observed to exhibit a more pronounced and advanced regenerative response in terms of epidermal thickening, increased collagen fibers, and cellular density. In conclusion, this research suggests that nanofiber wound dressings formulated with nanoscale materials could serve as an effective alternative to traditional wound dressings for therapeutic applications in injuries caused by CBRN (Chemical, Biological, Radiological, and Nuclear) incidents. It is also considered to make a significant contribution to the national CBRN defense field. Key words: Antibacterial nanofibers; electro-spinning; silver nanoparticle; wound healing; Zeolites