Resveratrol-Ampicillin Dual-Drug Loaded Polyvinylpyrrolidone/Polyvinyl Alcohol Biomimic Electrospun Nanofiber Enriched with Collagen for Efficient Burn Wound Repair

Abstract

Background: The healing of burn wounds is a complicated physiological process that involves several stages, including haemostasis, inflammation, proliferation, and remodelling to rebuild the skin and subcutaneous tissue integrity. Recent advancements in nanomaterials, especially nanofibers, have opened a new way for efficient healing of wounds due to burning or other injuries.

Methods: This study aims to develop and characterize collagen-decorated, bilayered electrospun nanofibrous mats composed of PVP and PVA loaded with Resveratrol (RSV) and Ampicillin (AMP) to accelerate burn wound healing and tissue repair.

Results: Nanofibers with smooth surfaces and web-like structures with diameters ranging from 200 to 400 nm were successfully produced by electrospinning. These fibres exhibited excellent in vitro properties, including the ability to absorb wound exudates and undergo biodegradation over a two-week period. Additionally, these nanofibers demonstrated sustained and controlled release of encapsulated Resveratrol (RSV) and Ampicillin (AMP) through in vitro release studies. The zone of inhibition (ZOI) of PVP-PVA-RSV-AMP nanofibers against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) was found 31±0.09 mm and 12±0.03, respectively, which was significantly higher as compared to positive control. Similarly, the biofilm study confirmed the significant reduction in the formation of biofilms in nanofiber-treated group against both S. aureus and E. coli. X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) analysis proved the encapsulation of RSV and AMP successfully into nanofibers and their compatibility. Haemolysis assay (%) showed no significant haemolysis (less than 5%) in nanofiber-treated groups, confirmed their cytocompatibility with red blood cells (RBCs). Cell viability assay and cell adhesion on HaCaT cells showed increased cell proliferation, indicating its biocompatibility as well as non-toxic properties. Results of the in-vivo experiments on a burn wound model demonstrated potential burn wound healing in rats confirmed by H&E-stained images and also improved the collagen synthesis in nanofibers-treated groups evidenced by Masson-trichrome staining. The ELISA assay clearly indicated the efficient downregulation of TNF-alpha and IL-6 inflammatory biomarkers after treatment with nanofibers on day 10.

Conclusion: The RSV and AMP-loaded nanofiber mats, developed in this study, expedite burn wound healing through their multifaceted approach.

Keywords: ampicillin; burn wound healing; collagen; electrospinning; nanofiber; resveratrol.

Graphical abstract

Scheme 1

Schematic diagram for the preparation of the collagen decorated and crosslinking of PVA-PVP nanofiber scaffolds encapsulated with RSV and AMP.

Figure 1

SEM images and their corresponding average diameter of Placebo (A and B), PVP-RSV nanofibers (C and D), PVA-AMP nanofibers (E and F) and PVP-PVA-RSV-AMP nanofibers (G and H).

Figure 2

(A) Plot representing FTIR spectra of AMP, RSV, PVA, PVP and drug-loaded nanofiber (PVP-PVA-RSV-AMP) and (B) XRD graphs representing the 2θ values of the AMP, RSV and PVP-PVA-RSV-AMP nanofibers.

Figure 3

Graph demonstrating (A) swelling index of nanofibers (PVP-RSV, PVA-AMP and PVP-PVA-RSV AMP nanofibers), (B) Biodegradability (%) of PVP-RSV, PVA-AMP and PVP-RSV-PVA-AMP nanofibers and (C) In-vitro drug release plots of RSV and AMP from PVP-RSV-PVA-AMP nanofiber. All the data represented as mean±SD (n=3).

Figure 4

Plate representing the zone of inhibition against (A) S. aureus and (B) E.Coli of positive control, negative control, F1 (PVP-RSV), F2 (PVA-AMP) and F3 (PVP-PVA-RSV-AMP). (C and D) shows the bar graph of A and B, respectively. *p <0.05, ***p <0.001, one-way ANOVA followed by Bonferroni multiple comparison test. ns denotes non-significant difference between groups. All the data represented as mean±SD (n=3).

Figure 5

(A) Microbial penetration assay shows Cotton plugged vial as a negative control, Open vial as a positive control, F1 (PVP-RSV), F2 (PVA-AMP) and F3 (PVP-PVA-RSV-AMP) are covered with the drug-loaded nanofibrous mat on 0 day, 3 days, and 7 days. (B) Bar diagram representing the optical density of negative control, positive control and the nanofiber samples F1 (PVP-RSV), F2 (PVA-AMP) and F3 (PVP-PVA-RSV-AMP). (C) Bar diagram representing the reduction in CFU after-treatment of S. aureus with Positive control (VCM), PVP-RSV, PVA-AMP, and PVP-PVA- RSV-AMP nanofiber formulations. *p <0.05, ***p <0.001, one-way ANOVA followed by Bonferroni multiple comparison test. All the data represented as mean±SD (n=3).

Figure 6

Effect of various test samples, including drug-loaded nanofibers on the growth of biofilm; (A) photograph representing biofilm growth in 96-well plate and (B) plot representing a comparative reduction in biofilm after treating with nanofiber formulations. (C) SEM images depicting biofilm growth and reduction upon nanofiber treatment of Bacterial film (positive control) (i), Reduction in biofilm after treatment with nanofiber of PVP-RSV (ii), PVA-AMP (iii), and PVP-PVA-RSV-AMP (iv). All the data given as mean±S.D. **p<0.01, one-way ANOVA followed by Bonferroni multiple comparison test. All the data represented as mean±SD (n=3).

Figure 7

(A) Photograph showing haemolysis of control and nanofiber groups after incubation with RBCs. (B) Graph representing their corresponding haemolysis rate (%) of +Ve control, -Ve control, PVP-RSV, PVA-AMP, and PVP-PVA-RSV-AMP nanofibers. (C) Microscopic images of RBCs after 30 min of incubation with control and nanofiber groups. ****Represents p<0.0001. All the data represented as mean±SD (n=3).

Figure 8

MTT assay of nanofibrous scaffolds of control, PVP-RSV, PVA- AMP, and PVP-PVA-RSV-AMP nanofibers on HaCaT cell line on day 1, 3 and 5. *p <0.05, one-way ANOVA followed by Bonferroni multiple comparison test. All the data represented as mean±SD (n=3).

Figure 9

Cell adhesion of nanofibrous scaffolds of (A–D) PVP-RSV, B–E) PVA- AMP, and C–F) PVP-PVA-RSV-AMP nanofibers on HaCaT cell line on day 1 and 3.

Figure 10

(A) Photographic images representing the burn wound healing of all the groups (Control, PVP-RSV, PVA-AMP, and PVP-PVA-RSV-AMP nanofiber-treated groups) on days 0, 5, 10, 15, and 21, (B) Representing the traces of wound area calculated by ImageJ software on different days 0, 1, 5, 10, 15 and 21 and (C) Bar diagram representing the % wound area (mm²) after treating the burn wound with drug-loaded nanofibrous mats (PVP-RSV, PVA-AMP, and PVP-PVA-RSV-AMP) and control group. *p <0.05, **p<0.01, and ***p <0.001, one-way ANOVA followed by Bonferroni multiple comparison test. All the data represented as mean±SD (n=3).

Figure 11

Histopathology images of (A) H&E-stained tissue samples of different groups (Control, PVP-RSV, PVA-AMP, and PVP-PVA-RSV-AMP nanofiber-treated groups) at day 5, 10, 15 and 21, scale bar (50µm). The re-epithelization and granulation in nanofiber-treated groups improved with enhanced fibroblast proliferation and granulation tissue infiltration (E: Epidermis; (G) Granulation tissue; Ede: Edema) and (B) Photographs represents the mason-stained histological sections of (Control, PVP-RSV, PVA-AMP, and PVP-PVA-RSV-AMP nanofiber-treated groups) at day 10 and 15 showing the improved collagen formation and maturation (M: Muscle growth in red color; (C) Collagen formation in blue color).

Figure 12

The level of expression of (A) IL-6 and (B) TNF-α in control, PVP-RSV, PVA-AMP, and PVP-PVA-RSV-AMP nanofiber-treated groups after day 1 and 10 post treatment. *p <0.05, ***p <0.001, one-way ANOVA followed by Bonferroni multiple comparison test. All the data given as mean±S.D (n=6).