Synergistic effect of zinc oxide-cinnamic acid nanoparticles for wound healing management: in vitro and zebrafish model studies

Abstract

Wound infections resulting from pathogen infiltration pose a significant challenge in healthcare settings and everyday life. When the skin barrier is compromised due to injuries, surgeries, or chronic conditions, pathogens such as bacteria, fungi, and viruses can enter the body, leading to infections. These infections can range from mild to severe, causing discomfort, delayed healing, and, in some cases, life-threatening complications. Zinc oxide (ZnO) nanoparticles (NPs) have been widely recognized for their antimicrobial and wound healing properties, while cinnamic acid is known for its antioxidant and anti-inflammatory activities. Based on these properties, the combination of ZnO NPs with cinnamic acid (CA) was hypothesized to have enhanced efficacy in addressing wound infections and promoting healing. This study aimed to synthesize and evaluate the potential of ZnO-CN NPs as a multifunctional agent for wound treatment. ZnO-CN NPs were synthesized and characterized using key techniques to confirm their structure and composition. The antioxidant and anti-inflammatory potential of ZnO-CN NPs was evaluated through standard in vitro assays, demonstrating strong free radical scavenging and inhibition of protein denaturation. The antimicrobial activity of the nanoparticles was tested against common wound pathogens, revealing effective inhibition at a minimal concentration. A zebrafish wound healing model was employed to assess both the safety and therapeutic efficacy of the nanoparticles, showing no toxicity at tested concentrations and facilitating faster wound closure. Additionally, pro-inflammatory cytokine gene expression was analyzed to understand the role of ZnO-CN NPs in wound healing mechanisms. In conclusion, ZnO-CN NPs demonstrate potent antioxidant, anti-inflammatory, and antimicrobial properties, making them promising candidates for wound treatment. Given their multifunctional properties and non-toxicity at tested concentrations, ZnO-CN NPs hold significant potential as a therapeutic agent for clinical wound management, warranting further investigation in human models.

Keywords: Cinnamic acid; Nanomedicine; Wound healing; Wound infection; Zebrafish model; Zinc oxide nanoparticle.

Fig. 1

(A) SEM analysis of Zn-CN NPs revealing a rod-shaped morphology, (B) FTIR spectrum represent specific vibrational modes corresponding to functional groups, (C) XRD analysis of Zn-CN NPs illustrating the presence of a crystalline phase and amorphous phase, and (D) UV-Vis spectrum of synthesized Zn-CN NPs

Fig. 2

(A) Albumin denaturation Inhibition and (B) Hydroxyl radical scavenging activity of Zn-CN NPs at different concnetrations (10 µg/mL, 20 µg/mL, 40 µg/mL, and 80 µg/mL). Diclofenac and Trolox was used as a positive control. The * represented the level of significance (p < 0.05) when the results were compared to the control. Data were presented as mean ± SD of three independent experiments

Fig. 3

MIC of Zn-CN NPs at different concentration against dental pathogens of S. aureus, P. aeruginosa, E. coli, and V. vulnificus. Amoxicillin was used as positive control. The * represented the level of significance (p < 0.05) when the results were compared to the control. Data were presented as mean ± SD of three independent experiments

Fig. 4

Zone of inhibition of different dental pathogen by Zn-CN NPs. (A). A – Control, B – Amoxicillin (50 µg/mL), C - Zn-CN NPs at 40 µg/mL, and D - Zn-CN NPs at 80 µg/mL. (B) Zone of inhibition measured at mm

Fig. 5

3D and 2D interaction images of CN and wound-infecting bacterial pathogens. (A) Sortase A (B) LasR, (C) Outer membrane porin, (D) EpsD. The interaction is observed with amino acids such as PRO (Proline), PHE (Phenylalanine), TYR (Tyrosin), LYS (Lysine), ILE (Isoleucine), VAL (Valine), SER (Serine), TRP (Tryptophan), LEU (Leucine), HIS (Histidine), ARG (Arginine), ASN (Asparagine), ALA (Alanine) and GLU (Glutamic acid)

Fig. 6

The survival rate analysis of zebrafish with different groups of control, wounded zebrafish, and exposure to ZnO-CN NPs (40 µg/mL and 80 µg/mL). The independent three experimental data were expressed as mean ± SD (n = 6/group). The significant difference at p < 0.05 was expressed as *

Fig. 7

(A-D): Representative images of zebrafish wound healing under different treatment conditions. (A) Healthy control, (B) Wounded control, (C) Wounded zebrafish treated with ZnO-CN NPs at 40 µg/mL, and (D) Wounded zebrafish treated with ZnO-CN NPs at 80 µg/mL. (E) Graphical representation of wound closure measured using ImageJ software. Data are presented as mean ± SD from three independent experiments (n = 6/group). Statistical significance was determined with p < 0.05, indicated by *

Fig. 8

Histological analysis of wounded zebrafish tissue. (A) Healthy control, (B) Wound control (C) ZnO-CN NPs treatment at 40 µg/mL, and (D) ZnO-CN NPs treatment at 80 µg/mL. The tissue layers include skeletal muscle, dermal layer, granulation tissue, and epithelial layer with immune cell infiltration

Fig. 9

Effect of ZnO-CN NPs treatment group on the mRNA expression level of mmp13, tnf-α, il-1β. Data were expressed as mean + SD of three independent experiments. * p < 0.05 as compared to the control