Enhanced Wound Healing With β-Chitosan-Zinc Oxide Nanoparticles: Insights From Zebrafish Models

Abstract

Introduction: Wound healing is a complex physiological process essential for the restoration of tissue integrity and function. Novel therapeutic approaches are urgently needed to enhance wound-healing outcomes. Nanotechnology, particularly zinc oxide nanoparticles, has shown promise due to its antimicrobial, anti-inflammatory, and regenerative properties. β-chitosan, derived from squid pens, possesses superior solubility and bioactivity compared to α-chitosan, making it a valuable biomaterial for biomedical applications. Through the integration of β-chitosan and zinc oxide nanoparticles, this study seeks to use the complementary properties of both substances to overcome present constraints in wound care treatments.

Methods: β-chitosan was extracted from squid pens and characterized for its molecular weight, degree of deacetylation, and solubility properties. Further characterization of the synthesized zinc oxide nanoparticles involved Fourier transform infrared spectroscopy to analyze chemical bonding and functional groups, ultraviolet-visible spectroscopy to determine optional properties such as band gap energy, X-ray diffraction spectroscopy to confirm the crystalline phase and calculate crystallite size, and the size was confirmed with the scanning electron microscope. Each technique provided complementary information, ensuring a comprehensive understanding of the synthesized nanoparticles' properties and their potential applications. Adult zebrafish (six to eight months old) were employed as a model organism due to their genetic similarity to humans and regenerative capabilities. Zebrafish were wounded and divided into treatment and control groups, with β-chitosan and β-chitosan-derived zinc nanoparticles treatments administrated at 50 µg/ml, while control groups received 0.05% phosphate buffer saline. The treatments, conducted in triplicate, enabled a comparative analysis of wound closure activity between β-chitosan-derived zinc nanoparticles' healing effects against standard and baseline treatments. Further, gene expression analysis on Bax, BCl-2, IL-2, IL-6, and tumor necrosis factor-alpha (TNF-a) was done using reverse transcriptase polymerase chain reaction.

Results: Characterization studies confirmed the successful synthesis of β-chitosan-derived zinc oxide nanoparticles and a crystalline structure corresponding to zinc oxide. Treatment with β-chitosan-derived zinc oxide nanoparticles significantly accelerated wound closure compared to controls and other treatment groups. Microscopic analysis demonstrated enhanced epithelialization, reduced inflammatory cell infiltration, increased collagen deposition, and improved tissue organization in wounds treated with β-chitosan-derived zinc oxide nanoparticles. Gene expression analysis revealed downregulation of inflammation-causing genes such as BCl-2, IL-2, IL-6, and TNF-a, hence it showed wound-healing activity. The results were statistically significant (p < 0.05).

Conclusion: β-chitosan-derived zinc oxide nanoparticles show promising potential as a novel therapeutic strategy for enhancing wound healing. The synergistic effects of β-chitosan and zinc oxide nanoparticles address multiple aspects of wound healing, including antimicrobial activity, inflammation modulation, and tissue regeneration. This study highlights the advantages of nanotechnology in wound care and underscores the need for further research to optimize nanoparticle formulations for clinical applications.

Keywords: antimicrobial; nanotechnology; regenerative properties; wound healing; zebrafish; zinc oxide nanoparticles; β-chitosan.

Figure 1. Scanning electron microscopy (SEM) image of β-chitosan-derived zinc oxide nanoparticles.

The SEM image depicted the morphology and the size distribution of the synthesized chitosan-derived zinc oxide nanoparticle.

Figure 2. Ultraviolet-visible (UV-vis) absorption spectra of β-chitosan-derived zinc oxide nanoparticles.

Figure 3. Fourier transform infrared spectroscopy (FTIR) spectra of β-chitosan-derived zinc oxide nanoparticles.

Figure 4. X-ray diffraction (XRD) pattern of as-prepared and annealed (800°C) β-chitosan-derived zinc nanoparticles.

Figure 5. β-chitosan-derived zinc oxide nanoparticles enhance wound healing in zebrafish.

Zebrafish were superficially wounded and treated with respective compounds and observed for 15 days (control - phosphate buffer saline, β-chitosan alone (30 µg/ ml), and β-Ch-ZnO-NPs (30 µg/ ml). β-Ch: β-chitosan; β-Ch-ZnO-NPs: β-chitosan-derived zinc oxide nanoparticles. Image credit: Meenakshi Sundaram.

Figure 6. Evaluation of wound-healing activity in zebrafish was carried out in distinct groups compared to the control group.

The treatment group exhibited significantly reduced wound size on zebrafish in β-chitosan-derived zinc nanoparticles-treated group in comparison to the control group (Group 1 - β-chitosan-derived zinc nanoparticles; Group 2 - β-chitosan alone). The data were represented as mean ± standard deviation, two-way ANOVA, and Bonferroni post-tests. The results were statistically significant (p < 0.05). P-value was indicated as ** p < 0.01 and *** p < 0.001. Control - phosphate buffer saline, β-chitosan alone (30 µg/ml), and β-Ch-ZnO-NPs (30 µg/ml). β-Ch: β-chitosan; β-Ch-ZnO-NPs: β-chitosan-derived zinc oxide nanoparticles. Image credit: Meenakshi Sundaram

Figure 7. β-chitosan-derived zinc oxide nanoparticles increased Bax expression on wound-healing process in a concentration-dependent manner.

The data were represented as mean ± standard deviation, one-way ANOVA, and Dunnett's multiple comparison tests. The results were statistically significant (p < 0.05). P-value was indicated as ** p < 0.01 and *** p < 0.001. Control - phosphate buffer saline, β-chitosan alone (30 µg/ml), and β-Ch-ZnO-NPs (30 µg/ml). β-Ch: β-chitosan; β-Ch-ZnO-NPs: β-chitosan-derived zinc oxide nanoparticles. Image credit: Meenakshi Sundaram

Figure 8. β-chitosan-derived zinc oxide nanoparticles decreased BCl-2 expression on wound-healing process in a concentration-dependent manner.

The data were represented as mean ± standard deviation, one-way ANOVA, and Dunnett's multiple comparison tests. The results were statistically significant (p < 0.05). The p-value was indicated as *** p < 0.001. Control - phosphate buffer saline, β-chitosan alone (30 µg/ml), and β-Ch-ZnO-NPs (30 µg/ml). β-Ch: β-chitosan; β-Ch-ZnO-NPs: β-chitosan-derived zinc oxide nanoparticles. Image credit: Meenakshi Sundaram

Figure 9. β-chitosan-derived zinc oxide nanoparticles increased lL-2 expression on wound-healing process in a concentration-dependent manner.

The data were represented as mean ± standard deviation, one-way ANOVA, and Dunnett's multiple comparison tests The results were statistically significant (p < 0.05). P-value was indicated as ** p < 0.01 and *** p < 0.001. Control - phosphate buffer saline, β-chitosan alone (30 µg/ml), and β-Ch-ZnO-NPs (30 µg/ml). β-Ch: β-chitosan; β-Ch-ZnO-NPs: β-chitosan-derived zinc oxide nanoparticles. Image credit: Meenakshi Sundaram

Figure 10. β-chitosan-derived zinc oxide nanoparticles decreased lL-6 expression on wound-healing process in a concentration-dependent manner.

The data were represented as mean ± standard deviation, one-way ANOVA, and Dunnett's multiple comparison tests. The results were statistically significant (p < 0.05). P-value was indicated as ** p < 0.01 and *** p < 0.001. Control - phosphate buffer saline, β-chitosan alone (30 µg/ml), and β-Ch-ZnO-NPs (30 µg/ml). β-Ch: β-chitosan; β-Ch-ZnO-NPs: β-chitosan-derived zinc oxide nanoparticles. Image credit: Meenakshi Sundaram

Figure 11. β-chitosan-derived zinc oxide nanoparticles decreased TNF-a expression on wound-healing process in a concentration-dependent manner.

The data were represented as mean ± standard deviation, one-way ANOVA, and Dunnett's multiple comparison tests. The results were statistically significant (p < 0.05). P-value was indicated as ** p < 0.01 and *** p < 0.001. Control - phosphate buffer saline, β-chitosan alone (30 µg/ml), and β-Ch-ZnO-NPs (30 µg/ml). β-Ch: β-chitosan; β-Ch-ZnO-NPs: β-chitosan-derived zinc oxide nanoparticles; TNF-a: tumor necrosis factor-alpha. Image credit: Meenakshi Sundaram