Chitosan-Stabilized Ag Nanoparticles with Superior Biocompatibility and Their Synergistic Antibacterial Effect in Mixtures with Essential Oils

Abstract

Silver nanoparticles (AgNPs) are considered a promising alternative to the use of antibiotics in fighting multidrug-resistant pathogens. However, their use in medical application is hindered by the public concern regarding the toxicity of metallic nanoparticles. In this study, rationally designed AgNP were produced, in order to balance the antibacterial activity and toxicity. A facile, environmentally friendly synthesis was used for the electrochemical fabrication of AgNPs. Chitosan was employed as the capping agent, both for the stabilization and to improve the biocompatibility. Size, morphology, composition, capping layer, and stability of the synthesized nanoparticles were characterized. The in vitro biocompatibility and antimicrobial activities of AgNPs against common Gram-negative and Gram-positive bacteria were evaluated. The results revealed that chitosan-stabilized AgNPs were nontoxic to normal fibroblasts, even at high concentrations, compared to bare nanoparticles, while significant antibacterial activity was recorded. The silver colloidal dispersion was further mixed with essential oils (EO) to increase the biological activity. Synergistic effects at some AgNP⁻EO ratios were observed, as demonstrated by the fractionary inhibitory concentration values. Our results reveal that the synergistic action of both polymer-stabilized AgNPs and essential oils could provide a significant efficiency against a large variety of microorganisms, with minimal side effects.

Keywords: Ag nanoparticles; antibacterial; essential oil; synergistic effect.

Figure 1

The ultraviolet-visible (UV-vis) spectra of silver nanoparticles (AgNPs) with and without chitosan (CH) stabilizer.

Figure 2

TEM images of AgNPs prepared in distilled water without stabilizer (a), with 0.05% chitosan as stabilizer (b) and (c), and energy dispersive spectroscopy (EDAX) for the AgNP sample with chitosan (d).

Figure 3

(a) The size and size distribution of AgNPs stabilized with high molar weight (HMW) chitosan solution; (b) Zeta potential of the same samples.

Figure 4

The FTIR spectra of AgNPs stabilized with chitosan solution (HMW) (yellow = pure chitosan; blue = AgNPs stabilized in 2% chitosan solution; red = AgNPs stabilized in 0.2% chitosan solution).

Figure 5

The viability of the CCD-1070 Sk cells treated with different concentrations of AgNPs after 24 h. The values are the means of the results for each sample from three separate experiments. Error bars indicate standard errors of the means. The statistical significance is indicated as follows: * p < 0.05.

Figure 6

The lactate dehydrogenase (LDH) released in normal fibroblast cells, CCD-1070 Sk, treated with different concentrations of AgNPs after 24 h. The values are the means of the results for each sample from three separate experiments. Error bars indicate standard errors of the means.

Figure 7

The variation of the fractional inhibitory concentration index of AgNP–chitosan solution mixtures measured for the inhibition of E. coli growth; CH(L) – low molecular weight chitosan; CH(H)—high molecular weight chitosan.

Figure 8

The variation of the fractional inhibitory concentration index (FICI) of bare AgNPs–essential oils (Eos) at various ratios. (a) Thyme oil; (b) Clove oil.

Figure 9

The variation of the fractional inhibitory concentration index (FICI) of mixed systems with AgNPs stabilized with 1% LMW chitosan and essential oils. (a) Thyme oil; (b) Clove oil.