Toxicity and antibacterial assessment of chitosancoated silver nanoparticles on human pathogens and macrophage cells

Abstract

Background

Pathogenic bacteria are able to develop various strategies to counteract the bactericidal action of antibiotics. Silver nanoparticles (AgNPs) have emerged as a potential alternative to conventional antibiotics because of their potent antimicrobial properties. The purpose of this study was to synthesize chitosan-stabilized AgNPs (CS-AgNPs) and test for their cytotoxic, genotoxic, macrophage cell uptake, antibacterial, and antibiofilm activities.

Methods

AgNPs were synthesized using chitosan as both a stabilizing and a reducing agent. Antibacterial activity was determined by colony-forming unit assay and scanning electron microscopy. Genotoxic and cytotoxic activity were determined by DNA fragmentation, comet, and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assays. Cellular uptake and intracellular antibacterial activity were tested on macrophages.

Results

CS-AgNPs exhibited potent antibacterial activity against different human pathogens and also impeded bacterial biofilm formation. Scanning electron microscopy analysis indicated that CS-AgNPs kill bacteria by disrupting the cell membrane. CS-AgNPs showed no significant cytotoxic or DNA damage effect on macrophages at the bactericidal dose. Propidium iodide staining indicated active endocytosis of CS-AgNPs resulting in reduced intracellular bacterial survival in macrophages.

Conclusion

The present study concludes that at a specific dose, chitosan-based AgNPs kill bacteria without harming the host cells, thus representing a potential template for the design of antibacterial agents to decrease bacterial colonization and to overcome the problem of drug resistance.

Keywords:

• chitosan-silver nanoparticles
• antibiofilm
• cytotoxicity
• genotoxicity

Acknowledgments

This work was supported by a grant (BT/PR13488/ GBD/27/270/2010) from the Department of Biotechnology, Government of India, to AS. The authors thank Dr Sanjeeb Sahoo and Malvern Instruments Ltd for providing dynamic light scattering and zeta potential analysis. The authors also thank Dr Biplab Sarkar for excellent discussions and for his help in the transmission electron microscopy studies. Finally, the authors are grateful to the Sonawane lab members for their fruitful discussions and suggestions.

Disclosure

The authors report no conflicts of interest in this work.

Supplementary figures

Figure S1 Antibacterial activity of silver nanoparticles (AgNPs) by agar diffusion method: chitosan-stabilized AgNPs (CS-AgNPs) at 2 ppm were loaded into the wells formed on plates containing a lawn of Staphylococcus aureus; growth inhibition was determined by measuring the zone of inhibition after 24 hours; chitosan was used as a control.

Figure S2 Cytotoxicity of chitosan-stabilized silver nanoparticles (CS-AgNPs) on mouse macrophage RAW264.7 cells. Cell morphology of RAW264.7 macrophages after treatment with different concentrations (3 and 10 ppm) of CS-AgNPs for 24 hours. Cells grown in medium and chitosan were used as a control.