WDS'16 Proceedings of Contributed Papers — Physics, 188–192, 2016. ISBN 978-80-7378-333-4 © MATFYZPRESS

Silver Oxide and Copper Oxide Nanoparticles as Perspective Antibacterial Agents in Plasma Polymer-Based Nanocomposites

D. Nikitin,1,2 M. Vaidulych,1 I. Gordeev,3 J. Hanuš,1 A. Choukourov,1 D. Slavínská,1 H. Biederman1 1Charles University in Prague, Faculty of Mathematics and Physics, Department of Macromolecular Physics, V Holešovičkách 2, 180 00 Prague, Czech Republic. 2G. A. Krestov Institute of Solution Chemistry of the Russian Academy of Sciences, Akademicheskaya 1, 153045 Ivanovo, Russia 3Jan Evangelista Purkyne University in Usti nad Labem, Faculty of Science, Department of Physics, České mládeže 8, Usti nad Labem 400 96, Czech Republic.

Abstract. Silver and copper are known as strong antibacterial agents, and the production of nanocomposite materials based on these elements is of high importance. Recently, it has been argued that silver and copper oxide nanoparticles may have the superior antibacterial efficiency as compared to the metals. This work briefly introduces the field of antibacterial metal oxides and presents a new approach. The first experimental results are also given on the preparation of silver nanoparticles by a Gas Aggregation cluster Source with subsequent oxidation by low-temperature oxygen plasma treatment.

Introduction Through the evolution process, a lot of bacteria mutated and became resistant to the antibiotic action. This motivated scientists for a search of new anti-infective agents. One of the possible decisions is using of metal nanoparticles (NPs). It is well-known that some metals, even at low concentrations, demonstrate strong antibacterial effects. A long time ago, ancient Egyptians, Greeks and Romans used vessels made of silver (Ag) and copper (Cu) for water disinfection and food storage [Paladini et al., 2015]. The first reports about metal-based nanosystems (colloids) appeared in the literature in the 1960s [Le Ouay & Stellacci, 2015]. However, the increase of scientific interest in metal NPs occurred at the turn of the 21th century. First NPs were synthesized by wet chemical methods from solutions of metal salts such as, for example, AgNO3 and CuCl2 [Sondi & Salopek- Sondi, 2004; Anyaogu et al., 2008]. The NP size ranged from several to tens of nm. In the last years, the interest in the development of environmentally friendly methods of NP synthesis remarkably increased: laser ablation, magnetron sputtering, plasma-solution systems and others were successfully utilized. One of the possible routes for the plasma-based NP production is the using of gas aggregation cluster sources (GAS). The GAS takes advantage of magnetron sputtering which transfers metal atoms into the gas phase. Metal atoms subsequently undergo nucleation under elevated pressure of a cooled inert gas. The production of Ag and Cu NPs by means of GAS has already been realized in our group [Polonskyi et al., 2012; Kratochvil et al., 2014]. This method is characterized by the stable deposition rate and small fluctuations in the NP size. The antibacterial character of Ag and Cu NPs was discussed mainly in terms of two mechanisms: the ion release and the generation of reactive oxygen species (ROS) [Le Ouay & Stellacci, 2015; Karlsson et al., 2013]. The reactions corresponding to the ion release are presented below: + + 2Ags + 1/2O2 + 2H → 2Ag + H2O + 2+ Cus + O2 + 2H → Cu + H2O2. Metal ions may destroy membrane of bacteria, react with DNA and stimulate the microorganism’s death. Note that in the case of Cu, the ion release is accompanied by the formation of hydrogen peroxide, and it was supposed that ROS may also contribute to the antibacterial effects. Decoration of surfaces with metal NPs often suffers from their poor adhesion. Embedding of NPs into thin polymeric supports may significantly improve the durability of the antibacterial action. A vast number of papers described the composites of Ag and Cu NPs with polyvinyl alcohol [Eghbalifam et al., 2015], poly(methyl methacrylate) [Siddiqui et al., 2015] poly(ethylene glycol) etc. Generally, it is possible to regulate the ion release from NPs by tuning the properties of the polymer

188 NIKITIN ET AL.: SILVER OXIDE AND COPPER OXIDE NANOPARTICLES IN NANOCOMPOSITES matrix [Vasilev et al., 2011]. Moreover, the polymeric matrix itself may possess an additional and very attractive functionality. For example, poly(ethylene oxide) plasma polymer demonstrates non-fouling properties i.e. the ability to resist the protein adsorption and the accumulation of biofilms [Choukourov et al., 2010]. Therefore, the combination of Ag or Cu NPs with PEO matrix may lead to the development of medical devices with advanced functionalities. The strong antibacterial activity of Ag and Cu NPs was examined on dangerous pathogens such as Escherichia coli and Staphylococcus aureus. Therefore, the application of metal NPs in fabrication of medical devices such as polymeric bandages, catheters, implants and filters has good perspectives. Nevertheless, in spite of all the extensive research already done, deep understanding of the antibacterial mechanisms of Ag and Cu NPs is still lacking. Recently, several works appeared that reported on good antibacterial activity of silver oxide [Lalueza et al., 2011] and copper oxide NPs [Gunawan et al., 2011]. In this case, the kinetic aspects of the ion release can be significantly different and this requires a systematic research. The main experimental aim of this work is to prepare silver oxide (and in perspective copper oxide) NPs by GAS with their potential use as antibacterial agents in metal oxide/plasma polymer nanocomposites. The detaile characterization of Ag oxide NPs is presented below.

Experimental part The experimental set-up used for the production of NPs was identical to the one reported earlier [Vaidulych & Hanus, 2015] and is shown in Fig. 1a. The Ag NPs were produced by means of a Haberland type GAS [Haberland & Karrais, 1992]. The GAS consisted of a cylindrical water cooled aggregation chamber ending in a conical nozzle with a small orifice (diameter 2 mm). A planar magnetron (diameter 81 mm) was equipped with an Ag target (3 mm thick) and the sputtering was performed under direct current of 150 mA. The aggregation length between the target and the orifice was 8.5 cm. Argon was used as a working gas under the pressure of 100 Pa. The GAS was connected to a stainless steel vacuum deposition chamber that was pumped by rotary and diffusion pumps. The pressure in the deposition chamber was 4.5 Pa. A sample holder was placed in the deposition chamber at the distance of 15 cm from the orifice. The deposition rate was measured by quartz crystal microbalance (QCM). Thus-prepared Ag NPs were deliberately oxidized by the oxygen plasma. The schematic image of the oxidation process is presented in Fig. 1b. The oxidation was carried out in a separate deposition chamber that was equipped by a capacitively coupled electrode for plasma generation and by an opposing and grounded substrate holder. The live electrode was powered by an r.f. generator (Dressler Ceasar, 13.56 MHz) through a matching box. The discharge power was 15 W. The oxygen pressure was set at 2.5 Pa. The distance between the live electrode and the sample holder was 5 cm.

(a) (b) Magnetron Capacitively coupled Ar Substrate with Ag NPs electrode Water cooling

Ar plasma RF

Substrate

holder O2 plasma RF generator Deposition Orifice chamber

Figure 1. Schematic images of gas aggregation source (a) and oxidation process (b).

189 NIKITIN ET AL.: SILVER OXIDE AND COPPER OXIDE NANOPARTICLES IN NANOCOMPOSITES

Different types of substrates were chosen including microscopic glass for UV-vis spectroscopy and copper grids with thin carbon foil for transmission electron microscopy (TEM). The UV-Vis spectroscopy measurements were performed by a Hitachi U3300 spectrophotometer. The shape, the size and the structure of the NPs were investigated by means of TEM. The size distribution of NPs was evaluated from the TEM images using the Solarius Particles software. Chemical composition of NPs was studied by means of X-ray photoelectron spectroscopy (Phoibos 100, Specs).

Results and discussion Under the experimental conditions reported, stable beams of the Ag NPs with the flux of 3 µm–2s–1 were produced. The NPs were collected on the substrate for 30 s and they were found to have close to spherical shape (Fig. 2a). After 15 min treatment with oxygen plasma, the number of the NPs increased and part of them joined together to form bigger agglomerates of irregular shape (Fig. 2b).

(a) (b)

N/N , % N/Ntotal, % total 50 70

45 (c) (d) 60 40 35 50

30 40 25 30 20 15 20 10 10 5 0 0 < 5 nm < 15 nm > 30 nm 5 - 10 nm > 20 nm 15 - 20 nm 20 - 25 nm 25 - 30 nm 11 - 15 nm 16 - 20 nm Particle sizes Particle sizes (e) (f)

Figure 2. TEM images (a, b), size distributions (c, d) and high-resolution TEM images (e, f) of Ag (a, c, e) and AgxO (b, d, f) NPs.

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The histograms of the size distribution are presented in Figs. 2c,d where the Y-axis shows the ratio of the amount of the NPs with certain diameter to the general amount of the NPs. It is evident that the mean size of the NPs decreased from 25 nm to less than 10 nm after oxidation. The high-resolution TEM images reveal that the Ag NPs consist of regions with randomly oriented crystalline planes and, hence, they point at their polycrystalline structure Fig. 2e. By contrast, many of the oxidized NPs exhibit close to monocrystalline structure (Fig. 2f). The formation of small particles can be attributed to the result of re-sputtering of Ag NPs upon the oxygen plasma treatment. Sputtered Ag atoms re-deposit on the surface and self-assemble into small nanoclusters. These can further join with the formation of larger agglomerates. Ag NPs are known to produce a distinct absorption band in the visible range due to the particle plasmon resonance [Sondi & Salopek-Sondi, 2004]. The UV-Vis spectra were acquired on the particles before and after the oxygen plasma treatment. It was confirmed that the Ag NPs produced by GAS exhibit the particle plasmon resonance at the wavelength of 375 nm (Fig. 3a). The UV-vis spectrum of the NPs measured after the oxidation shows the degeneration of the plasmon band (Fig. 3b). Such behavior indirectly confirms the success of the oxidation process. The changes in the UV-Vis spectra are supported by the changes in the chemical composition of the NPs were analyzed by XPS (Fig. 4). The high resolution Ag 3d peak demonstrates the negative shift after the oxidation. This fact is usually associated with the formation of silver oxide [Yan et al., 2012]. Moreover, Full-Width-at-Half-Maximum of the peaks also increases after the treatment and this indicates the formation of silver oxides with the different extent of stoichiometry.

(a) (b) 100 a) 100 b)

95 95 T, % T, % 90 90

85 85 400 500 600 700 800 900 400 500 600 700 800 900 λ, nm λ, nm

Figure 3. UV-Vis spectra of Ag (a) and AgxO NPs prepared on microscopic glasses.

Ag 3d5/2

Ag 3d3/2

2

1

362 364 366 368 370 372 374 376 378 380 Binding energy, eV

Figure 4. Comparison of Ag 3d XPS peaks of Ag NPs (1) and AgxO NPs (2).

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Conclusion Ag and Cu NPs and NPs of their oxides were shown to be highly attractive as antibacterial agents. The perspective of their combination with polymer matrices for the production of bactericidal composites was discussed. The advantages of GAS used for NPs deposition were presented. Ag NPs were produced with the mean size of 20 nm. Oxygen plasma treatment of the NPs was demonstrated to produce silver oxide NPs as it was confirmed by UV-Vis and XPS. The treatment results in the decrease of the mean size below 10 nm and in the transformation of their crystalline structure.

Acknowledgments. This work was supported by the Grant Agency of the Czech Republic through the grant GA13-09853S and by the grant SVV-2016-260215.The authors thank Dr. J. Vesely for TEM images.

References Anyaogu, K. C., Fedorov, A. V., & Neckers, D. C. (2008). Synthesis, characterization, and antifouling potential of functionalized copper nanoparticles. Langmuir, 24(8), 4340–4346. Choukourov, A., Gordeev, I., Polonskyi, O., Artemenko, A., Hanyková, L., Krakovský, et al. (2010). Poly(ethylene oxide)-like Plasma Polymers Produced by Plasma-Assisted Vacuum Evaporation. Plasma Processes and Polymers, 7(6), 445–458. Eghbalifam, N., Frounchi, M., & Dadbin, S. (2015). Antibacterial silver nanoparticles in polyvinyl alcohol/sodium alginate blend produced by gamma irradiation. International Journal of Biological Macromolecules, 80, 170–6. http://doi.org/10.1016/j.ijbiomac.2015.06.042 Gunawan, C., Teoh, W. Y., Marquis, C. P., & Amal, R. (2011). Cytotoxic Origin of Copper ( II ) Oxide Nanoparticles : Comparative Studies and Metal Salts, (9), 7214–7225. Haberland, H., & Karrais, M. (1992). Thin films from energetic cluster impact: A feasibility study. Journal of Vacuum Science & Technology A: Vacuum, Surfaces, and Films, 10(5), 3266. http://doi.org/10.1116/1.577853 Karlsson, H. L., Cronholm, P., Hedberg, Y., Tornberg, M., De Battice, L., Svedhem, S., & Wallinder, I. O. (2013). Cell membrane damage and protein interaction induced by copper containing nanoparticles- Importance of the metal release process. Toxicology, 313(1), 59–69. http://doi.org/10.1016/j.tox.2013.07.012 Kratochvil, J., Kuzminova, A., Kylian, O., & Biederman, H. (2014). Comparison of magnetron sputtering and gas aggregation nanoparticle source used for fabrication of silver nanoparticle films. Surface and Coatings Technology, 275, 296–302. http://doi.org/10.1016/j.surfcoat.2015.05.003 Lalueza, P., Monzon, M., Arruebo, M., & Santamaría, J. (2011). Bactericidal effects of different silver- containing materials. Materials Research Bulletin, 46(11), 2070–2076. http://doi.org/10.1016/j.materresbull.2011.06.041 Le Ouay, B., & Stellacci, F. (2015). Antibacterial activity of silver nanoparticles: A surface science insight. Nano Today, 10(3), 339–354. http://doi.org/10.1016/j.nantod.2015.04.002 Paladini, F., Pollini, M., Sannino, A., & Ambrosio, L. (2015). Metal-based antibacterial substrates for biomedical applications. Biomacromolecules, 16(7), 1873–1885. http://doi.org/10.1021/acs.biomac.5b00773 Polonskyi, O., Solař, P., Kylián, O., Drábik, M., Artemenko, A., et al. (2012). Nanocomposite metal/plasma polymer films prepared by means of gas aggregation cluster source. Thin Solid Films, 520(12), 4155–4162. http://doi.org/10.1016/j.tsf.2011.04.100 Siddiqui, M. N., Redhwi, H. H., Vakalopoulou, E., Tsagkalias, I., Ioannidou, M. D., & Achilias, D. S. (2015). Synthesis, characterization and reaction kinetics of PMMA/silver nanocomposites prepared via in situ radical polymerization. European Polymer Journal, 72, 256–269. http://doi.org/10.1016/j.eurpolymj.2015.09.019 Sondi, I., & Salopek-Sondi, B. (2004). Silver nanoparticles as antimicrobial agent: A case study on E. coli as a model for Gram-negative bacteria. Journal of Colloid and Interface Science, 275(1), 177–182. http://doi.org/10.1016/j.jcis.2004.02.012 Vaidulych, M., & Hanus, J. (2015). Preparation of Porous Structures and Their Surface Modification, 77–83. Vasilev, K., Griesser, S. S., & Griesser, H. J. (2011). Antibacterial surfaces and coatings produced by plasma techniques. Plasma Processes and Polymers, 8(11), 1010–1023. http://doi.org/10.1002/ppap.201100097 Yan, Z., Bao, R., & Chrisey, D. B. (2012). Generation of Ag–Ag2O complex nanostructures by excimer laser ablation of Ag in water. Physical Chemistry Chemical Physics, (Angel 2012), 3052–3056. http://doi.org/10.1039/c2cp42668d

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