Antibacterial activity and toxity of silver Nanosilver versus ionic silver Libor Kvítek 1 Aleš Panáček 1, Robert Prucek 1, Jana Soukupová 1, Markéta Vaníčková 1, Milan Kolář 2 and Radek Zbořil 1 1 Regional Centre of Advanced Technologies and Materials, Department of Physical Chemistry, Faculty of Science, Palacký University, Olomouc. 2 Department of Microbiology, Faculty of Medicine and Dentistry, Palacký University, Olomouc.
NanoSilver in commercial products Samsung s Silver Nano Technology Libor Kvítek 2
Managed synthesis of silver Modified Tollens process Reduction agent Ag + + 2NH 3 [Ag(NH 3 ) 2 ] + Ag 0 Size managing changing of ammonia concentration and choice of reduction substance AgNO 3-10 -3 mol dm -3 ammonia from 0.005 to 0.2 mol dm -3 Reduction substance - 10-2 mol dm -3 (saccharides) 25 nm 200 nm 400 nm Kvítek L., Prucek R. Panáček A. et al. J Mat Chem 15 (10), 1099-1105, 2005 Libor Kvítek 3
Study of biological properties of silver NPs Silver NPs 25 nm (0,005 mol dm -3 NH 3, reduction by maltose) Antibacterial activity gram-negative and gram-positive bacteria Antifungal activity Candida spp. Cytotoxicity human fibroblasts Toxicity ciliate (P. caudatum), fruit fly (D. melanogaster), algae (S. subspicatus) TEM UV-VIS Libor Kvítek 4
Antibacterial activity of silver NPs First time Size dependent study of antibacterial activity of silver NPs (25 nm 50 nm) Minimum Inhibitory Concentration MIC (mg/l) Size of Ag NPs Bacteria Ionic Ag 50 nm 25 nm 35 nm 44 nm (Maltose) (Lactose) (Glucose) (Galactose ) Enterococcus faecalis CCM 4224 1.69 6.75 54 - - Staphylococcus aureus CCM 3953 1.69 3.38 6.75 6.75 54.0 Escherichia coli CCM 3954 0.84 1.69 27.0 27.0 - Pseudomonas aerugin. CCM 3955 0.84 3.38 13.5 27.0 - Staphylococcus epidermidis 1 0.84 1.69 6.75 13.5 6.75 Staphylococcus epidermidis 2 0.84 1.69 6.75 6.75 54.0 1) (methicillin-susceptible) 2) (methicillin-resistant) Panáček A., Kvítek L., Prucek R., Kolář M.: J Phys Chem B 110 (33), 16248-16253, 2006 Libor Kvítek 5
Antibacterial activity of silver NPs Second time Enhancement of antibacterial activity of silver NPs (25 nm) by their stabilization Bacteria 1) (methicillin-susceptible) 2) (methicillin-resistant) AgNO 3 Nonstabilised silver NPs MIC (mg/l) Stabilised Ag NPs SDS Tween 80 Libor Kvítek 6 PVP 360 Enterococcus faecalis CCM 4224 1.69 6.75 3.38 6.75 6.75 Staphylococcus aureus CCM 3953 1.69 3.38 1.69 3.38 3.38 Escherichia coli CCM 3954 0.84 1.69 1.69 1.69 3.38 Pseudomonas aerugin. CCM 3955 0.84 3.38 1.69 3.38 1.69 Staphylococcus epidermidis1 0.84 1.69 0.84 1.69 1.69 Staphylococcus epidermidis2 0.84 1.69 1.69 1.69 1.69 SDS sodium dodecyl sulfate Tween 80 - polyoxyethylene sorbitan monooleate PVP 360 polyvinylpyrrolidone (M.W.= 360000) Kvítek L., Panáček A., Soukupová J., Kolář M. et al.: J Phys Chem C, 5825-5834, 2008
Antifungal activity of silver NPs Minimum inhibitory concentrations of 25 nm sized silver NPs MIC (mg/l) Candida spp. AgNO 3 Nonstabilised silver NPs Stabilised silver NPs SDS Tween 80 PVP 360 C. albicans I 0.42 0.42 0.052 0.1 0.1 C. albicans II 0.42 0.21 0.1 0.21 0.21 C. parapsilois 1.69 1.69 0.84 0.84 0.84 C. tropicalis 0.84 0.84 0.42 0.42 0.42 Panáček A., Kolář M., Večeřová R. et al.: Biomaterials 30, 6333-6340, 2009 Libor Kvítek 7
Antifungal activity of silver NPs Comparison of antifungal activity of silver NPs with those of common antifungals Amp Amphotericine Itra Itraconazole Flu Fluconazole Vori Voriconazole Casp - Caspofungine non-stabil. SD Tween Amp Itra Flu Vori Casp Ag NPs stabilised Ag NPs Panáček A., Kolář M., Večeřová R. et al.: Biomaterials 30, 6333-6340, 2009 Libor Kvítek 8
Cytotoxicity of silver NPs (human fibroblasts) Silver NPs: LC50(24 hours) = 25 mg/l Ionic silver: LC50(24 hours) = 1 mg/l Fluorescence intensity 80 60 40 Silver NPs Ionic silver Control 20 0 0,06 0,25 0,7 2 5 15 30 60 Concentration of silver (mg/l) Panáček A., Kolář M., Večeřová R. et al.: Biomaterials 30, 6333-6340, 2009 Libor Kvítek 9
Toxicity (ecotoxicity) of silver NPs Paramecium caudatum (ciliate) Silver NPs LC50(1hour) = 39 mg/l Ionic silver 0.4 mg/l (immediate death Kvítek L., Vaníčková M., Panáček A. et al. J Phys Chem C 113, 4296-4300, 2009 chlorophyll a amount (mg/l) 8 7 6 5 4 3 2 1 0 Scenedesmus sub. (algae) Silver NPs LC50 >30 mg/l of silver Ionic silver LC50 = 5 mg/l of silver 0 0,3 1 3 9 30 Ag concentration (mg/l) Ionic Ag Nano Ag Control Libor Kvítek 10
Toxicity (ecotoxicity) of silver NPs Drosophila melanogaster Acute toxicity assay to larval stages - dose dependent study, silver NPs were added into the nutrient at the concentrations from 10 mg/l to 100 mg/l - 10 mg/l of silver no acute toxic effect, slight decreasing in cuticle pigmentation - 20 mg/l of silver - LC50-60 mg/l of silver - LC100 Comparison of flies cultivated on medium without/with silver NPs Libor Kvítek 11
Antimicrobial activity of silver NPs in relation to their toxicity (comparison with ionic silver) NanoAg IonicAg Antibacterial activity (MIC; 24hours) Antifungal activity (MIC; 36hours) Cytotoxicity to human fibroblasts P. caudatum 1-7 mg/l 1-2 mg/l 1 mg/l 1 mg/l 25 mg/l 1 mg/l 25 mg/l 0.4 mg/l Toxicity to S. subspicatus (LC50; 48hours) >30 mg/l 5 mg/l D. melanogaster (LC50) 20 mg/l n.a. Libor Kvítek 12
Acknowledgement - my colleagues from NanoSilver group - Milan Kolář and Renata Večeřová Department of Microbiology, Faculty of Medicine, Palacký University - financial support from Ministry of Education and Czech Science Fondation 1M6198959201 MSM 6198959218 MSM 6198959203 GACR GAP304/10/1316 Thank you for your attention Libor Kvítek 13