Hydrogel Based Wound Dressing Material Using Fish Based Collagen And Silver Nanowires as Antimicrobial Agent

Abstract

Chronic wounds fail to heal naturally and do not proceed through the sequential wound healing stages within an expected time frame. These wounds are characterized by extended inflammatory phase, excessive exudate and high alkaline pH (8.9), followed by elevated inflammatory cytokines, high levels of matrix metalloproteinases (MMPs) and obstinate bacterial infections. This disrupts the natural healing process leading to impaired wound healing. The prime objective of this research work was to develop a pH- sensitive smart wound dressing material which can maintain an optimal exudate based on the wound pH, through which MMPs activity can be controlled to achieve better healing and also reduce bacterial infection. In this work, a pH-responsive hydrogel composed of poly(aspartic acid) (PAsp), poly(vinyl alcohol) (PVA) along with collagen and silver nanowires (Ag NWs) was prepared via free radical polymerization method. Collagen used in the wound dressing material was successfully extracted from marine waste i.e., Sole fish skin that can enhance the wound healing process. The extraction process was optimized using Response Surface Methodology (RSM) with Box-Behnken Design (BBD) for achieving maximum yield. The optimal conditions to obtain highest collagen yield was determined to be, an acetic acid concentration of 0.54 M, NaCl concentration of 1.90 M, solvent/solid ratio of 8.97 mL/g and time of 32.32 hrs. The maximum collagen yield of 19.27 ± 0.05 mg/g of fish skin was achieved under the optimal conditions. The analysis of variance (ANOVA) and contour plots exhibited a significant interaction of all the selected variables over collagen extraction process. SDS-PAGE (Sodium dodecyl sulfate- polyacrylamide gel electrophoresis) analysis suggested that the extracted collagen contained three α-chains i.e. (α1)2, α2 (M.W. 118, 116 kDa) and one β chain (M.W. 200 kDa) which was similar to commercially available calf skin type I collagen. Ag NWs used in the wound dressing material act as an antibacterial agent and were synthesized using hydrothermal method by reducing silver nitrate (AgNO3) using fructose in the presence of poly(vinylpyrrolidone) (PVP). Scanning electron microscopy (SEM) analysis showed that ultra-long, uniform and thin silver nanowires were obtained under optimized conditions; 0.02 M AgNO3, 0.016 g/mL of fructose, 0.16 g/mL of PVP at 160 °C within ii 22 hrs. The dynamic light scattering (DLS) analysis revealed that the silver nanowires obtained had an average diameter of 77 nm possessing high level of crystallinity with face centered cubic (FCC) phase that was evident from the X-ray diffraction (XRD) patterns peaked at (111), (200), (220), (311) and (222) planes. A pH-sensitive hydrogel based wound dressing material i.e., PAsp/PVA/Collagen hydrogel loaded with Ag NWs was synthesized and optimized by altering the components namely concentration of PAsp (50mg-200 mg), PVA (4%-10%), collagen (0.5 mg/mL-3 mg/mL), Ag NWs (2.5 mg, 5 mg and 10 mg), ammonium persulfate (APS) (75 mg-150 mg) and ethylene glycol di- methacrylate (EGDMA) (0.25 mM-1 mM) based on the properties like swelling characteristics, physical strength and stability of the hydrogel. A maximum swelling ratio of 1511% have been achieved at the optimised condition in the presence of collagen at pH 10 whereas 1286% was achieved in the absence of collagen. However, the swelling ratio of PAsp/PVA/Collagen hydrogel (1511%) slightly decreased with the addition of Ag NWs and achieved swelling ratio of 1405% at pH 10 when hydrogel loaded with 5 mg Ag NWs. The prepared dressing material had good pH-sensitivity to alkaline environment and exhibited maximum swelling at pH 10 and minimum at pH 3. Through this mechanism, developed wound dressing material can maintain pH by removing excess exudate on wound bed and can retain required moist environment for better healing process. Antibacterial activity of hydrogel loaded with 5 mg Ag NWs exhibited 99.92% reduction in viable E. coli colonies. The hydrogel was assessed for its cytotoxicity on L929 cells by Sulforhodamine B (SRB) assay and it was revealed that hydrogel had not shown any toxicity and promoted cell proliferation. In-vivo wound healing studies showed that PAsp/PVA/Collagen impregnated with 5 mg Ag NWs improves the healing process and exhibited re-epithelization within 12 days with no scar formation, which is also confirmed by assessing histological parameters. Histological evaluation revealed good dermal layer formation with a high healing score. Comprehensively, the results suggested that PAsp/PVA/Collagen hydrogel impregnated with 5 mg silver nanowires can be a novel wound dressing material for chronic wounds.