ANTIBACTERIAL VERMICULITE-CHLORHEXIDINE / POLYETHYLENE NANOCOMPOSITES
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1 ANTIBACTERIAL VERMICULITE-CHLORHEXIDINE / POLYETHYLENE NANOCOMPOSITES Magda SAMLÍKOVÁ a,b, Sylva HOLEŠOVÁ a,b, Marta VALÁŠKOVÁ a,b, Marianna HUNDÁKOVÁ a, b, Erich PAZDZIORA c, Dušan KIMMER d a Nanotechnology Centre, VŠB Technical University of Ostrava, 17. listopadu 15/2172, CZ Ostrava Poruba, Czech Republic b IT4Innovations Centre of Excellence, VŠB Technical University of Ostrava, 17.listopadu 15/2172, CZ Ostrava Poruba, Czech Republic c Institute of Public Health Ostrava, Centre of Clinical Laboratories, Partyzánské náměstí 7, CZ Ostrava, Czech Republic d SPUR a.s., T.Bati 299, CZ Zlín, Czech Republic magda.samlikova@vsb.cz Abstract Organically modified vermiculite (NaVER/CA) was used as nanofiller for preparation of vermiculitechlorhexidine/polyethylene nanocomposites (NaVER/CA-PE). Organovermiculte was prepared from sodium vermiculite (NaVER) and chlorhexidine diacetate (CA) through the process of intercalation. The set of vermiculite-chlorhexidine/polyethylene nanocomposites was prepared from mixtures containing polyethylene (PE) and clay nanofiller NaVER/CA (weight ratios of clay nanofiller : PE were 3:37, 6:34, 10:30). The mixtures were melt-compounded using a Brabender twin-screw extruder and the plate samples of NaVER/CA-PE nanocomposites were pressed for antimicrobial tests. The structural characteristics of the nanocomposites were studied by X-ray diffraction, infrared spectroscopy and optical microscopy. The antimicrobial activity was studied against Gram negative Escherichia coli and Gram positive Staphylococcus aureus using microbial fingerprints technique with blood agar. The first fingerprints of samples were made after 24 h, next after 48, 72 and 96 h. The results of antibacterial tests show good antimicrobial effect depending upon time of contact of colony forming units of bacteria with the surface of plates. The most intensive degradation of number of bacteria was between 48 and 72 h. Keywords: Nanocomposite, polyethylene, vermiculite, chlorhexidine, antibacterial activity 1. INTRODUCTION Nanocomposites represent broad spectrum of materials in recent technologies. This rapidly expanding field is generating many exciting new materials with novel properties. New unique material can be obtained by combining properties from the parent constituents into a single material [1]. Addition of a clay mineral filler, which has at least one dimension at the nanoscale, to a polymer leads to a clay polymer nanocomposite [2]. A few studies about vermiculite as a nanofiller in some polymers were reported [3-5]. During the last few years, the spread of emerging infectious diseases, especially those caused by drug-resistant pathogens, has become a growing global concern. The development of biocidal polymers, that is, polymers that can inactivate infectious pathogens upon contact, has attracted research interest. Namely, microorganisms often have strong abilities to survive on ordinary polymeric materials. Preparation of antibacterial nanocomposite is one of the possibilities to reduce a risk of contamination of surface of polymer materials [6-11]. Chlorhexidine is considered as one of the most popular agents to reduce bacterial growth. It is substituted biguanidine with strong basic character that forms stable salts with several acids, e.g. Chlorhexidine diacetate (CA). Chlorhexidine has a very wide range of antibacterial activity against a variety of gram-
2 negative and gram-positive organisms. Thus, chlorhexidine is used as topical medication, a bactericide, and in dental applications as an active ingredient in mouthwash to reduce dental plaque and oral bacteria [12, 13]. The term polyethylene (PE) describes a huge family of resins obtained by polymerizing ethylene gas and it is by far the largest volume commercial polymer. It is a thermoplastic polymer consisting of long hydrocarbon chains with chemical formula (C2H4)nH2. This thermoplastic is available in a range of flexibilities and other properties depending on the production process, with high density materials being the most rigid. Polyethylene can be formed by a wide variety of thermoplastic processing methods and is particularly useful where moisture resistance and low cost are required. PE has excellent chemical resistance, meaning that it is not attacked by strong acids or strong bases. It is also resistant to gentle oxidants and reducing agents. This work reports preparation and antibacterial activity of vermiculite chlorhexidine/polyethylene nanocomposites. Vermiculite modified by chlorhexidine was used as a nanofiller for preparation of organoclay/polyethylene nanocomposites. The structural changes of prepared plate samples were characterized by X-ray diffraction (XRD) and infrared spectroscopy (IR). The antibacterial activity was evaluated using microbial fingerprints technique in various time intervals. 2. EXPERIMENTAL 2.1 Materials Vermiculite (VER) from Brazil was selected as the starting clay mineral material. This natural powder material was grounded using planetary ball mill Retsch under constant speed 300 rpm for 20min. After sieving, fraction < 40µm was obtained and utilized for experiment. The crystallochemical formula of vermiculite sample (Si6.16Al1.84)(Al0.08Fe Fe Mg4.88Ti0.12)O20(OH)4(Ca0.30K0.36Na0.16) per formula units was calculated from the results of elemental chemical analysis. The cation exchange capacity (CEC) of VER was 106 meq/100g. Further used chemicals were in analytical quality: Chlorhexidine diacetate (C22H30N10Cl2.2C2H4O2, Sigma-Aldrich), sodium chloride (NaCl, Vitrum) and ethanol (C2H5OH, Vitrum) as a solvent. 2.2 Preparation of organovermiculites The natural VER was converted into Na + form using cation exchange procedure with 1.0 M aqueous solutions of sodium chloride (70 C, 6 h). After cooling, the suspension was washed with demineralized water until free of chloride ions. The sample was dried overnight at 70 C and sign as NaVER. Ethanolic solution of chlorhexidine diacetate (CA) with concentration of CA was prepared accordant with the 1xCEC of vermiculite. This ethanolic solution of CA was mixed with aqueous solution of NaVER, stirred and heated (75 C, 6 h). After centrifugation the organovermiculites was dried overnight at 80 C and labeled as NaVER/CA. 2.3 Preparation of vermiculite-chlorhexidine/pe nanocomposites Powdered and granulated mixture of the industrial low density polyethylene (LDPE), Bralen FB 2-17 (Slovnaft corp., Slovak Republic) without additives was used as a starting material for preparation of composite matrix. The first sample contained 1.2 g (3 wt. %) of nanofiller NaVER/CA and 38.8 g (97 wt. %) of polyethylene Bralen FB Another samples contained 6 and 10 wt. % of NaVER/CA and appropriate amount of polyethylene. These mixtures were blended in the Brabender kneading chamber (BRABENDER GmbH & Co. KG /815604) at 150 C for 10 min in two velocity intervals (10 rpm for 2 min and subsequently 50 rpm for 8 min). Then the composite was pressed by hand pressing for 3 min at 160 C. Prepared 1 mm thin desks with size 100 x 100 mm were cooled down in other water cooled press for 10 min and signed as 3%NaVER/CA, 6%NaVER/CA and 10%NaVER/CA.
3 2.4 Analytical methods X-ray diffraction (XRD) patterns of powder organovermiculite nanofiller were recorded using a Rigaku Ultima IV difractometer with Cu K radiation and scintillation counter. It was measured in 0.1 mm diameter glass capillary under constant conditions (2-60 2θ, scan speed 2 /min, 40kV, 40kA). The nanocomposite desks were measured on the X-ray diffractometer from INEL equipped with a curved position-sensitive detector CPSD 120 (reflection mode, Ge-monochromator, Cu Kα1 radiation) in an ambient atmosphere under constant conditions (2000 s, 35 kv, 20 ma). The IR spectra were obtained by the KBr method using a NEXUS 470 Fourier transform (FTIR) spectrometer (ThermoNicolet, USA). The spectrometer was equipped with Globar IR source, KBr beam splitter, and DTGS detector. For each spectrum, 128 scans were obtained with resolution of 4 cm -1. Range of measurements was cm Antibacterial test Antibacterial activity of prepared nanocomposites was studied using microbial fingerprints technique. This method of direct fingerprints is based on the assumption that the bacteria under the same conditions gradually die. Each sample plate was cut to the three platelets (50x50 mm). Bacterial suspension of Escherichia coli and Staphylococcus aureus were spread on the plates and were left to dry in the laminar box at 21 C. Then the dried bacterial suspension on the surface of plates was stamping using microbial fingerprints technique on the three discs with blood agar in 24, 48, 72 and 96 h time intervals. The bacteria cultivation took place in thermostat at 35 C for 24 h. The number of colony-forming units of bacteria (CFU) at all three fingerprints were counted and averaged. 3. RESULTS AND DISCUSSION 3.1 X-ray powder diffraction XRD patterns in Fig. 1 show intensive reflections of PE and basal reflections of NaVER/CA and NaVER/CA-PE nanocomposites. Fig. 1 XRD patterns of the PE, nanofiller NaVER/CA and NaVER/CA-PE nanocomposites.
4 Basal reflection with value d = nm identified in NaVER/CA (Fig. 1) corresponds to the dehydrated vermiculite phase [14]. Intercalation CA to the interlayered space of NaVER caused expansion of interlayer space from 1.2 nm to nm in NaVER/CA. The semicrystalline PE containing both crystalline and amorphous regions showed two main reflections with the values d(110) = 0.42 nm and d(200) = 0.38 nm corresponding to the orthorhombic crystal structure of PE [4]. Based on the XRD patterns can be presumed that the structure of PE in the NaVER/CA-PE nanocomposites did not change. A new peaks with d = nm may correspond to the further intercalation after exfoliation of NaVER/CA nanofiller to the PE matrix. 3.2 FTIR spectroscopy The spectrum of PE (Fig. 2) shows main bands at 2914, 2846, 1463 and 720 cm -1, which belong to CH stretching asymmetric and symmetric vibrations, CH deformation and rocking vibrations, respectively. The additional band at 1375 cm -1 is also visible and this band is usually observed in the spectra of low density polyethylene, LDPE [15]. The IR spectra of prepared NaVER/CA-PE nanocomposites are shown in Fig. 2. Typical intensive band that occurred in the cm -1 interval was assigned to Si O stretching vibration together with Si O bending vibration at 444 cm -1. Increase of intensity of Si O stretching vibration with increasing amount of NaVER/CA can be also well observed. Fig. 2 IR spectra of prepared NaVER/CA-PE nanocomposites and pure PE. The band that occurred around 1532 cm -1 in all NaVER/CA-PE nanocomposites is due to the NH bending vibration of imine group of chlorhexidine diacetate. Since C=N stretching vibration of imine group appears near 1650 cm -1 it is difficult to distinguish it because this band is overlapped with bending vibration of water. The characteristic band of acetate group is C=O stretching which appears at 1721 cm -1. The band at 1264 cm -1 corresponds to C-O stretching vibration of acetate.
5 3.3 Antibacterial assessment The antibacterial activity of NaVER/CA-PE nanocomposites was tested against Escherichia coli and Staphylococcus aureus by microbial fingerprints technique. Results of antibacterial tests are in Table 1 and Fig. 3. Number of survive bacterial colony-forming units is decreasing with time of exposure. The best antibacterial effect has a sample 10% NaVER/CA which contains the highest amount of CA. The antibacterial tests revealed that Staphylococcus aureus is a less resistant bacteria against prepared nanocomposites: there were only 9 survived colony-forming units of Staphylococcus aureus for 96 h. Table 1 Average number of bacterial colony-forming units (CFU) of Escherichia coli and Staphylococcus aureus at the various time intervals. Escheichia coli Staphylococcus aureus Time of contact of bacteria with the surface of nanocomposite (h) Decline of number of CFU 3% NaVER/CA CN CN % NaVER/CA CN % NaVER/CA The countless number (CN) in Table 1 was in case of more than 500 colonies per composite plate. There is a decline of CFUs of Escherichia coli and Staphylococcus aureus for all samples (Fig. 3) while a rapid decline was observed between 48 and 72 h of acting time. Based on these findings we can presume a long lasting effect of these NaVER/CA-PE nanocomposites Fig. 3 Decreasing number of CFUs of Escherichia coli and Staphylococcus aureus at different times. 4. CONCLUSIONS The vermiculite-chlorhexidine/polyethylene nanocomposites were successfully prepared and tested for antibacterial activity. The XRD analysis signified an exfoliation of NaVER/CA nanofiller in the PE matrix. Homogeneous mixing of NaVER/CA nanofiller in PE was confirmed by IR spectroscopy. Antibacterial test of all prepared samples showed a good antibacterial activity with long lasting effect against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus. A rapid decline of number of survive colonyforming units was in interval from 48 to 72 h. The results of this study are very useful for future study and development of new antibacterial nanocomposites materials.
6 ACKNOWLEDGEMENTS Financial support of the IT4Innovations Centre of Excellence project reg. no.cz.1.05/1.1.00/ ; Ministry of Educations, Youth and Sports of the Czech Republic, project SP2013/78 and Czech Grant Agency, projects GA ČR 210/11/2215 are gratefully acknowledged. REFERENCES [1] Simha Martynková G., Ruemmeli M., Čech Barabaszová K. Introduction to nanocomposite science of layered and tubular materials. Hauppauge, N.Y.: Nova Science Publishers, 2012, p ISBN [2] Ruitz-Hitzky, E., van Meerbeek A. Clay mineral and organoclay-polymer nanocomposites. In: Bergaya F., Theng B.K.G., Lagaly G. (Eds.), Handbook of Clay Science, Developments in Clay Science, vol. 1. Elsevier Ltd., Amsterdam, p [3] Valášková M., Simha Martynková G., Matějka V., Barabaszová K., Plevová E., Měřínská D. Organovermiculite nanofillers in polypropylene. Appl. Clay Sci. 2009, 43, p [4] Valášková M., Tokarský J., Čech Barabaszová K., Matějka V., Hundáková M., Pazdziora E., Kimmer D. New aspects on vermiculite filler in polyethylene. Appl. Clay Sci. 2013, 72, p [5] Wang L., Wang X., Chen Z., Ma P. Effect of doubly organo-modified vermiculite on the properties of vermiculite/polystyrene nanocomposites. Appl. Clay Sci. 2013, 75-76, p [6] Sánchez-Valdes S., Ortega-Ortiz H., Ramos-de Valle L.F., Medellín-Rodríguez F.J., Guedea-Miranda R. Mechanical and antimicrobial properties of multilayer films with a polyethylene/silver nanocomposite layer. J. Appl. Polym. Sci. 2009, 111, p [7] Nien Y., Liao Y., Liao P. Antibacterial activity of poloxamer-modified montmorillonite clay against E. coli. Mater. Lett. 2011, 65, p [8] Nigmatullin R., Gao F., Konovalova V. Polymer-layered silicate nanocomposites in the design of antimicrobial materials. J. Mater. Sci. 2008, 43, p [9] Hundáková M., Valášková M., Pazdziora E., Slobodian P., Kimmer D. Preparation and some properties of polyethylene/vermiculite nanocomposites. In Nanocon 2012 : conference proceedings. Ostrava : Tanger Ltd., p ISBN [10] Fong N., Simmons A., Poole-Warren L.A. Antibacterial polyurethane nanocomposites using chlorhexidine diacetate as an organic modifier. Acta Biomater. 2010, 62, p [11] Bruna J.E., Penaloza A., Guarda A., Rodriguez F., Galotto M.J. Development of MtCu 2+ /LDPE nanocomposites with antimicrobial activity for potential use in food packaging. Appl. Clay Sci. 2012, 58, p [12] Jones Ch.G. Chlorhexidine: is it still the gold standard? Periodontology , 15, p [13] Attin T., Abouassi T., Becker K., Wiegand A., Roos M., Attin R. A new method for clorhexidine (CHX) determination: CHX release after application of differently concentrated HX-containing preparations on artificial fissures. Clin. Oral. Invest 2008, 12, p [14] Calle C. de la, Suquet H. Hydrous phyllosilicates, Vermiculite. Mineral. Geochem. 1988, 19, p [15] Socrates, G. Infrared and Raman Characteristic Group Frequencies: tables and charts. John Wiley Sons, Ltd., Chichester. 2001
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