Anti-bacterial and wound healing-promoting effects of zinc ferrite nanoparticles

Abstract

Background: Increasing antibiotic resistance continues to focus on research into the discovery of novel antimicrobial agents. Due to its antimicrobial and wound healing-promoting activity, metal nanoparticles have attracted attention for dermatological applications. This study is designed to investigate the scope and bactericidal potential of zinc ferrite nanoparticles (ZnFe₂O₄ NPs), and the mechanism of anti-bacterial action along with cytocompatibility, hemocompatibility, and wound healing properties.

Results: ZnFe₂O₄ NPs were synthesized via a modified co-precipitation method. Structure, size, morphology, and elemental compositions of ZnFe₂O₄ NPs were analyzed using X-ray diffraction pattern, Fourier transform infrared spectroscopy, and field emission scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy. In PrestoBlue and live/dead assays, ZnFe₂O₄ NPs exhibited dose-dependent cytotoxic effects on human dermal fibroblasts. In addition, the hemocompatibility assay revealed that the NPs do not significantly rupture red blood cells up to a dose of 1000 µg/mL. Bacterial live/dead imaging and zone of inhibition analysis demonstrated that ZnFe₂O₄ NPs showed dose-dependent bactericidal activities in various strains of Gram-negative and Gram-positive bacteria. Interestingly, NPs showed antimicrobial activity through multiple mechanisms, such as cell membrane damage, protein leakage, and reactive oxygen species generation, and were more effective against gram-positive bacteria. Furthermore, in vitro scratch assay revealed that ZnFe₂O₄ NPs improved cell migration and proliferation of cells, with noticeable shrinkage of the artificial wound model.

Conclusions: This study indicated that ZnFe₂O₄ NPs have the potential to be used as a future antimicrobial and wound healing drug.

Keywords: Antibiotics; Antimicrobial activity; Biocompatibility; Hemocompatibility; Nanoparticles; Spinel ferrites; Wound healing; Zinc ferrites.

Fig. 1

a X-ray diffraction (XRD) patterns, b Fourier-transform infrared spectroscopy (FTIR) spectrum of the synthesized zinc ferrite (ZnFe₂O₄) NPs

Fig. 2

a Field-emission scanning electron microscopy (FE-SEM) image of zinc ferrite nanoparticles (ZnFe₂O₄ NPs) exhibits spherical particles with an average size of 47.9 ± 2.5 nm. b Energy-dispersive X-ray spectroscopy (EDX) spectrum of ZnFe₂O₄ NPs shows the elemental compositions reflecting with the determined weight percentage and atomic percentage of all elements (i.e., zinc, Zn; iron, Fe; and oxygen, O) according to stoichiometry

Fig. 3

In vitro biocompatibility assay. Representative fluorescence microscopy images of human dermal fibroblast cells (HDF) stained with ethidium homodimer-1 (red color; dead cells) and calcein-AM (green color; live cells) after 5 days incubation with ZnFe₂O₄ NPs at concentrations of a 0 µg/mL (control), b 62 µg/mL, c 125 µg/mL, d 250 µg/mL, e 500 µg/mL, and f 1000 µg/mL. Scale bars show 500 µm. g The percent (%) metabolic activity of HDF cells on day 1 and 5 after subjecting to different concentrations of ZnFe₂O₄ NPs. Error bars denote the standard deviations of 4 replicates, and * represents the significance of the reduction in % metabolic activities of treated cells as compared to control. The data were analyzed by two-way ANOVA, and *p ≤ 0.05, ****p ≤ 0.001 were considered statistically significant

Fig. 4

Hemocompatibility of zinc ferrite nanoparticles (ZnFe₂O₄ NPs). a Photographs of hemolysis assay to detect the presence of hemoglobin in the supernatant of ZnFe₂O₄ NPs treated samples. b Hemolysis percentage of ZnFe₂O₄ NPs treated samples versus positive and negative control. PEG and Triton X-100 lysed blood cells served as a negative control (NC) and positive control (PC), respectively. The values presented in the graph are mean ± SD of triplicate and *p ≤ 0.05, ****p ≤ 0.0001 were considered statistically significant

Fig. 5

Bacterial growth inhibition of zinc ferrite nanoparticles (ZnFe₂O₄ NPs). Representative zone of inhibition images after treatment of S. aureus bacteria with different concentrations of ZnFe₂O₄ NPs (12.5–100 µg/mL) compared to the vehicle control (DMSO) and positive control (1 % silver sulfadiazine cream)

Fig. 6

Bacterial live/dead assay. Fluorescence micrographs of E. coli and S. aureus after treatment with 100 µg/mL of ZnFe₂O₄ NPs. a Negative control E. coli cells without any treatment. b S. aureus negative control without treatment. c E. coli treated with tetracycline (positive control). d S. aureus treated with tetracycline (positive control). e E. coli treated with ZnFe₂O₄ NPs (100 µg/mL). f S. aureus treated with ZnFe₂O₄ NPs (100 µg/mL). g Percent (%) dead cells of E. coli and S. aureus after ZnFe₂O₄ NPs treatment as compared to the negative control (NC) and positive control (PC). Experiments were performed in triplicate. The percent dead cells of E. coli and S. aureus were significantly higher in nanoparticle-treated groups and positive control as compared to the negative control. *p ≤ 0.05 and ****p ≤ 0.0001 are statistically significant. Scale bars show 200 µm

Fig. 7

Membrane permeability assay. Fluorescence images of E. coli and S. aureus showing the influx of FITC after treatment with zinc ferrite (ZnFe₂O₄) NPs and tetracycline (positive control). a Untreated E. coli (negative control). b Untreated S. aureus (negative control). c E. coli treated with tetracycline (positive control). d S. aureus treated with tetracycline (positive control). e E. coli treated with 100 µg/mL of ZnFe₂O₄ NPs. f S. aureus treated with 100 µg/mL of ZnFe₂O₄ NPs. Tetracycline and ZnFe₂O₄ NPs induced membrane damage to both Gram-positive and Gram-negative bacteria, resulted in the membrane permeability to the green fluorescent FITC dye. Scale bars show 100 µm

Fig. 8

Protein leakage assay. The concentration of protein detected in ZnFe₂O₄ NPs (100 µg/mL) treated E. coli and S. aureus after 8 h of treatment versus untreated bacteria as a negative control (NC). All experiments were done in triplicate, and the data are presented as mean ± SD. ****p ≤ 0.0001 was considered statistically significant

Fig. 9

Reactive oxygen species (ROS) assay. Intracellular ROS production after 8 h of treatment with ZnFe₂O₄ NPs (100 µg/mL) in E. coli and S. aureus. Hydrogen peroxide (H₂O₂) treated cells were taken as a positive control (PC), and bacteria without treatment served as a negative control (NC). All experiments were performed in triplicates, and data are presented as mean ± SD. **p ≤ 0.01 and****p ≤ 0.0001 were considered as statistically significant

Fig. 10

Scratch assay of NIH-3T3 fibroblast cells in the presence of ZnFe₂O₄ NPs (100 µg/mL). a Bright-field images of the negative control (untreated cells) and ZnFe₂O₄ NPs treated cells after 0, 18, and 36 h of incubation. b Percent (%) scratch shrinkage of ZnFe₂O₄ NPs treated cells versus the untreated control after 18 h. The experiments were performed in triplicates, and data are presented as mean ± SD. The data were analyzed using t-test, where **p ≤ 0.01 was considered statistically significant