COMPARISON OF ANTIBACTERIAL AND ANTIPROTOZOAL EFFECTS OF NANOPARTICLES Zn 2+, Cu 2+ a Ag + INTERCALATED ON CLAY MINERALS Erich PAZDZIORA a, Kateřina MATĚJOVÁ a, Marta VALÁŠKOVÁ b, Sylva HOLEŠOVÁ b Marianna HUNDÁKOVÁ b a ZDRAVOTNÍ ÚSTAV SE SÍDLEM V OSTRAVĚ, Partyzánské nám. 7, 702 00 Ostrava, Česká republika, erich.pazdziora@zu.cz b CENTRUM NANOTECHNOLOGIÍ VYSOKÉ ŠKOLY BÁŇSKÉ TECHNICKÉ UNIVERZITY OSTRAVA, 17. listopadu 2172/15, Ostrava-Poruba, Česká republika, valaskova@vsb.cz 1. INTRODUCTION Nanoparticles of metals (e. g. Zn 2+, Cu 2+, Ag + ) have antibacterial properties. Silver was first used clinically to prevent eye infections in newborns. The effectiveness of silver revolves around its low propensity to select for resistance, its broad spectrum of aktivity. Silver is biocidal in the ionic form and, unlike many antibiotics, has at least six mechanisms of action: Ribosome (m-rna) (t-rna)--silver binds disallowing polypeptide initiation, sulfhydryl compounds (Oxidation)-sulfur amino acid is inactivated to the disulfide form, protein denaturation (Ag-salts) -coagulation of enzymatic and other proteins, nucleic acid interactions-interaction with DNA,disrupting mrna synthesis, cytoplasmic membrane-disruption and leaching of metabolites, interference with electron transport in cytochrome system. The multiple targets suggest that at least six mutations are required for organisms to become resistant. Most antibiotics, on the other hand, have one or two mechanisms of action, usually involving binding to a ribosome or inhibiting some aspect of metabolism.(1). At the present time begin use of nanoparticles silver in wound care products, orthopedic and orthodontic (dental implants) products (2). A range of silver-coated or -impregnated dressings are now commercially available against burn-wound pathogens, namely resistant Staphylococcus aureus (MRSA) (3). Biomedical devices colonized by bacteria may cause infection or mortality. To prevent such infections, an effective strategy is to develop novel biomedical devices with antibacterial abilities via various surface modification processes. Biofilm formation is a recurrent complication in implant surgery and may result in loss of implants. Silver nanoparticle may be used as an implantable biomaterial. Nanoparticles Ag +, Cu 2+, Zn 2+, Mn 2+, or Fe 2+ was individually loaded onto chitosan nanoparticles. Their particle sizes and zeta potentials were measured. Antibacterial activity was significantly enhanced by the metal ions loaded, except for Fe 2+ (4). Antimicrobial materials based on copper and zinc-doped hydroxyapatite are attractive in a wide variety of medical applications. (5). Antiprotozoale effects were testing much less. The effect on the viability of Leishmania promastigotes was assessed. The aphidicolin nanosuspension was directly transferred into antiparasitic test systems.(6). The present work studies the antibacterial and antiprotozal effects of metalnanoparticles. For immobilization nanoparticles were using fylosilicates (clay minerals montmorillonites and vermiculites). Clay minerals have continuously attracted attention for the possibility of modifying their layered structure by intercalation (9-12). Cations Na +, K +, Ca 2+, Mg 2+ in the interlayer position of clay minerals are substituted through the use of cations Zn 2+, Cu 2+, Ag + during intercalation.
2. MATERIALS AND METODS 2.1. Materials 2.1.1. The natural Mg-vermiculite and Ca-montmorillonite was ground in a planetary mill, then passed through a 0.045 mm sieve and fraction <40 lm was utilized for experiments. 2.1.2. Bacteria Enterococcus faecalis CCM 4224, Escherichia coli CCM 3988 and Pseudomonas aeruginosa CCM 1960 were obtained from the Czech Collection of Microorganisms (Brno, Czech Republic - CCM). 2.2. Methods 2.2.1. Antibacterial activity test Clay minerals samples with nanoparticles Zn 2+, Cu 2+ a Ag + were proved from 10 % solutions ready for use via dilution 1:3 (suspension micromethod). Bacterial suspensions were after a period 24 hrs for five days transferred to the fresh glucose solutions. The minimum inhibitory concentration (MIC) of prepared montmorillonite and vermiculite was determined by their lowest concentration that completely inhibits bacterial growth. The dilution and cultivation were preceded on the microtitration plate with 96 hollows. The first set of hollows on the plate contained 10% (w/v) montmorillonite and vermiculite. This dispersion was further diluted by a threefold diluting method in glucose stock in such manner, that second to seventh set of hollows contained sample dispersed in concentration of 3.33%, 1.11%, 0.37%, 0.12%, 0.041% and 0.014%. The eight set of hollows contained pure glucose stock as check test (control). A volume of 1µl of glucose suspensions of E. faecalis CCM 4224 (9.3x10 8 cfu ml -1 ), E. coli CCM 3988 (1.1x10 9 cfu ml -1 ) and P. aeruginosa CCM 1960 (6.3x10 8 cfu ml -1 ), was put into hollows. Bacterial suspensions was after the elapse of 30, 60, 90, 120, 180, 240 and 300 min and then during 5 days always in 24 h interval transferred from each hollow to 100 µl of the fresh glucose stock and bacteria were incubated in thermostat at 37 C for 24 and 48 hrs. 2.2.2. Antiprotozoal activity test The tests with nanoparticles in Trichomonas vaginalis 10 6 culture were performed by method tube tests (inoculate potion of protozoa 15 microlitres). Protozoal counts were evaluated in the interval 24 hrs for five days. 3. EXPERIMENTAL RESULTS Labory tests proved antibacterial effect of nanoparticles: Zn 2+ in dilution from 10 % till 3,3 %, Cu 2+ from 3,3 % till 0,041 and Ag + from 0,37 % till 0,12 % in bacterial cultures.
Table 1: : Enterococcus faecalis effective concentrations 3,3 + + - - - - + + + + + + 1,11 + + - + - - + + + + + + 0,37 + + - + - + + + + + + + 0,12 + + - + - + + + + + + + 0,041 + + - + + + + + + + + + Table 2: Escherichia coli effective concentrations 3,3 - - - - - - + + + + + + 1,11 + + + - - - + + + + + + 0,37 + + + + - - + + + + + + 0,12 + + + + - - + + + + + + 0,041 + + + + + + + + + + + + Table 3: Pseudomonas aeruginosa effective concentrations 3,3 + + - - - - + + + + + + 1,11 + + + + - - + + + + + + 0,37 + + + + - - + + + + + + 0,12 + + + + + + + + + + + + 0,041 + + + + + + + + + + + +
Nanoparticles caused a killing of Trichomonas vaginalis in cultures with concentration of solution Zn 2+ from 0,37 % till 0,12 %, with Cu 2+ 0,37 % and Ag + from 0,041 % till 0,014 %. Check samples of clay minerals montmorillonite and vermiculite without of nanoparticles have not neither the antibacterial or antiprotozoal effects. Table 4 Trichomonas vaginalis effective concentrations 3,3 - - - - - - + + + + + + 1,11 - - - - - - + + + + + + 0,37 - - - - - - + + + + + + 0,12 + - + + - - + + + + + + 0,041 + + + + - - + + + + + + 0,014 + + + + - + + + + + + + 0,0046 + + + + + + + + + + + + 0,0015 + + + + + + + + + + + + 4. CONCLUSIONS Montmorillonite (MMT) and vermiculite (VMT) were prepared via intercalation technique. Intercalated nanoparticles Zn (II) Cu (II), Ag (I) in MMT and VMT after 5 days have antibacterial activity. Antiprotozoal activity is higher. The results from this study may be used for future development of new types of biomaterials with antibacterial and antiprotozoal activity. REFERENCES [1.] PRINCE, H., N., PRINCE, D., L. Antimicrobial silver in orthopedic and wound care products. TheFreeLibrary.com, Orthopedic Design & Technology (May 1, 2008). [2.] SUG-JOON A., SHIN-JAE L. et al. Experimental antimicrobial orthodontic adhesives using nanofillers and silver nanoparticles. Dental Materials, 2009, 25, 2: 206-213. [3.] MARGARET I. SAU L. L., VINCENT K. M. P. et al. Antimicrobial activities of silver dressings: an in vitro comparison. J Med Microbiol 55 (2006), 59-63. [4.] WEN L. D., SHAN S. N., et al. Antibacterial activity of chitosan tripolyphosphate nanoparticles loaded with various metal ions. Carbohydrate Polymers 75 (2009) 385 389. [5.] STANIČ V., DIMITRIJEVIČ S. et al. Synthesis, characterization and antimicrobial activity of copper and zinc-doped hydroxyapatite nanopowders. Applied Surface Science, 2010, 256, 20: 6083-6089.
[6.] TEKWANI, B. L., WALKER L. A8-Aminoquinolines: future role as antiprotozoal drugs Current Opinion in Infectious Diseases, 2006, 19, 6: 623-631. [7.] PIKSOVÁ K., WEISEROVÁ M., JEDLIČKOVÁ A., FOJTÍK A. Silver nanoparticles and thein bactericidal effect. Nanocon, 20. - 22. 10. 2009, Rožnov pod Radhoštěm, Česká Republika [8.] KVÍTEK L., PRUCEK R., PANÁCEK A., SOUKUPOVÁ J. Nanočástice stříbra příprava, vlastnosti a aplikace. Nanocon, 20. - 22. 10. 2009, Rožnov pod Radhoštěm, Česká Republika [9.] MALACHOVÁ K., PRAUS P., PAVLÍČKOVÁ Z., TURICOVÁ M. Activity of antibacterial compounds immobilised on montmorillonite Applied Clay Science 43 (2009) 364 368 [10.] HOLEŠOVÁ S., VALÁŠKOVÁ M., PLEVOVÁ E., PAZDZIORA E., MATĚJOVÁ K. Preparation of novel organovermiculites with antibacterial activity using chlorhexidine diacetate. Journal of Colloid and Interface Science 342 (2010) 593 597. [11.] IZQUIRRDO BARBA I., VALET-REGI et al. Incorporation of antimicrobial compounds in mesoporous silica film monolith. Biomaterials 30 (2009) 5729 5736. [12.] HONGPING H., DAN Y. et al. A novel organoclay with antibacterial activity prepared from montmorillonite and Chlorhexidini Acetas. Journal of Colloid and Interface Science 297 (2006) 235 243.