Multifunctional Silver Nanoparticles Based on Chitosan: Antibacterial, Antibiofilm, Antifungal, Antioxidant, and Wound-Healing Activities

Abstract

The purpose of this study is to create chitosan-stabilized silver nanoparticles (Chi/Ag-NPs) and determine whether they were cytotoxic and also to determine their characteristic antibacterial, antibiofilm, and wound healing activities. Recently, the development of an efficient and environmentally friendly method for synthesizing metal nanoparticles based on polysaccharides has attracted a lot of interest in the field of nanotechnology. Colloidal Chi/Ag-NPs are prepared by chemical reduction of silver ions in the presence of Chi, giving Chi/Ag-NPs. Physiochemical properties are determined by Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), transmission electron microscopy (TEM), dynamic light scattering (DLS), and scanning electron microscopy with energy-dispersive X-ray spectroscopy (SEM-EDX) analyses. TEM pictures indicate that the generated Chi/Ag-NPs are nearly spherical in shape with a thin chitosan covering around the Ag core and had sizes in the range of 9-65 nm. In vitro antibacterial activity was evaluated against Staphylococcus aureus and Pseudomonas aeruginosa by a resazurin-mediated microtiter plate assay. The highest activity was observed with the lowest concentration of Chi/Ag-NPs, which was 12.5 µg/mL for both bacterial strains. Additionally, Chi/Ag-NPs showed promising antifungal features against Candida albicans, Aspergillus fumigatus, Aspergillus terreus, and Aspergillus niger, where inhibition zones were 22, 29, 20, and 17 mm, respectively. Likewise, Chi/Ag-NPs revealed potential antioxidant activity is 92, 90, and 75% at concentrations of 4000, 2000, and 1000 µg/mL, where the IC₅₀ of Chi/Ag-NPs was 261 µg/mL. Wound healing results illustrated that fibroblasts advanced toward the opening to close the scratch wound by roughly 50.5% after a 24-h exposure to Chi/Ag-NPs, greatly accelerating the wound healing process. In conclusion, a nanocomposite based on AgNPs and chitosan was successfully prepared and exhibited antibacterial, antibiofilm, antifungal, antioxidant, and wound healing activities that can be used in the medical field.

Keywords: antimicrobial; antioxidant; chitosan; chitosan/silver nanoparticles; wound healing.

Figure 1

FTIR spectrum (A) and XRD pattern (B) of Chi/Ag NPs.

Figure 2

TEM images (A,B), SAED pattern (C), DLS (D),and zeta potential (E) of Chi-Ag-NPs.

Figure 3

(A) SEM image and (B) EDX spectrum of prepared Chi/Ag-NPs.

Figure 4

Resazurin dye test for determining minimum inhibitory concentration against S. aureus ATCC^® 25923™ and P. aeruginosa MTCC1034.

Figure 5

Light-inverted microscopic images of S. aureus (A) and P. aeruginosa (B) biofilms grown with various concentrations of Chi/Ag-NPs. At concentrations above 0.12xMIC bacteria have appeared as aggregated together to perform normal biofilm. P. aeruginosa and S. auerus biofilm inhibition in the presence of Chi/Ag-NPs at Sub. MIC (C). The absorbance of the control was considered to represent 100% of biofilm (results were considered significant when compared to control; * p < 0.05. Data are presented as mean ± SD, n = 4).

Figure 6

Antifungal activity of Chi (1), Ag+ (2), Chi/Ag-NPs (3), and Nystatin (4) toward C. albicans (a), A. fumgatus (b), A. terreus (c), and A. niger (d) using agar-well diffusion method.

Figure 7

Antioxidant activity of Chi, Ag+, and Chi/Ag-NPs.

Figure 8

In vitro cytotoxicity effects on Chi/Ag-NPs and doxorubicin against normal human skin cell line (BJ-1) was assessed by SRB colorimetric assay. Within each column, different letters indicate significant differences among values (p < 0.05) based on one-way ANOVA estimated by GraphPad Instat software, (version 6.01), San Diego, CA, USA.

Figure 9

(A) Effects of different treatments on the wound area contraction (0–96 h). Values are given as mean ± SD (n = 3/group). Different letters indicate significant differences (p < 0.05). (B) Representative phase contrast micrographs of cells treated with 100 μg/mL Chi/Ag-NPs at 0 and 24 h. Wound closure rates are expressed as percentage of scratch closure from after 0 to 96 h compared to initial area. Red and black lines mean center and wide of the wound.