ZnO nanoparticles enhanced antibacterial activity of ciprofloxacin against Staphylococcus aureus and Escherichia coli Maryam Banoee, 1 Sepideh Seif, 1 Zeinab E. Nazari, 2 Parisa Jafari-Fesharaki, 2 Hamid R. Shahverdi, 3 Ali Moballegh, 3 Kamyar M. Moghaddam, 1 Ahmad R. Shahverdi 1 1 Department of Pharmaceutical Biotechnology and Nanotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran 2 Biotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran 3 Department of Materials Engineering, Faculty of Engineering, Tarbiat Modares University, Tehran, Iran Received 12 January 2009; revised 15 December 2009; accepted 16 December 2009 Published online 11 March 2010 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.b.31615 Abstract: Nanoparticle metal oxides offer a wide variety of potential applications in medicine due to the unprecedented advances in nanobiotechnology research. In this work, the effect of zinc oxide (ZnO) nanoparticles prepared by mechano–chemical method on the antibacterial activity of dif- ferent antibiotics was evaluated using disk diffusion method against Staphylococcus aureus and Escherichia coli. The av- erage size of ZnO nanoparticles was between 20 nm and 45 nm. Although ZnO nanoparticles (500 lg/disk) decreased the antibacterial activity of amoxicillin, penicillin G, and nitrofur- antoin in S. aureus, the antibacterial activity of ciprofloxacin increased in the presence of ZnO nanoparticles in both test strains. A total of 27% and 22% increase in inhibition zone areas was observed for ciprofloxacin in the presence of ZnO nanoparticles in S. aureus and E. coli, respectively. The enhancing effect of this nanomaterial on the antibacterial ac- tivity of ciprofloxacin was further investigated at three differ- ent contents (500, 1000, and 2000 lg/disk) against various clinical isolates of S. aureus and E. coli The enhancing effect of ZnO nanoparticles on the antibacterial activity of ciproflox- acin was concentration-dependent against all test strains. The most enhancing activities were observed in the contents of the 2000 lg/disk. VC 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 93B: 557–561, 2010. Key Words: bioactive material, nanomedicine, nanotechnology INTRODUCTION Despite prodigious efforts, bacterial resistance is still con- sidered as a major drawback in chemotherapy of many infection diseases. 1 Recently, however, certain natural prod- ucts and synthetic compounds have successfully shown to increase antibacterial activity of antibiotics against different clinically isolated resistant test strains. 2–11 Moreover, there are extensive reports on antibacterial effects of silver 12–15 and copper nanoparticles. 16 Also the combination of silver nanoparticles with antibiotics was investigated in our previ- ous work, where we demonstrated that antibacterial activity of penicillin G, amoxicillin, erythromycin, clindamycin, and vancomycin against Staphylococcus aureus and Escherichia coli significantly increases in the presence of silver nanopar- ticles. 17 However, the effect of other nanomaterial such as zinc oxide (ZnO) nanoparticles in combination with antibiot- ics has not been yet investigated. ZnO is widely used as food additive, food supplement, and pharmaceutical ingredi- ent. 18–21 Among other metal oxide nanomaterials, ZnO nano- particles are famous for their catalytic efficiency, chemical stability, and strong adsorption ability. 22 In this study, for the first time, the antibacterial activity of different antibiotics was evaluated against different clinical strains of S. aureus and E. coli either in presence and absence of sub-inhibitory concentrations of ZnO nanoparticles, using disk diffusion assay. ZnO nanoparticles showed considerable enhancing effects on the antibacterial activity of ciprofloxacin against different clinical strains of S. aureus and E. coli. MATERIALS AND METHODS Synthesis of ZnO nanoparticles ZnO nanoparticles were freshly prepared by a recently described mechanochemical method using anhydrous ZnCl 2 , anhydrous Na 2 CO 3 , and NaCl as starting materials. 23 NaCl was used as dilutive additive to the starting powder. The stoichiometric mixture of the starting powders was milled according to the reaction below: ZnCl 2 þ Na 2 CO 3 þ 8 : 6 NaCl ! ZnCO 3 þ 10 : 6 NaCl The diameter of balls and their w/w ratio to powder mass were 10 mm and 10:1, respectively. Mechanochemical milling process was then carried out with planetary mill for 9 h at 250 rpm. Powder was calcined in air at 300  C for 30 min. 23 ZnCO 3 ! ZnO þ CO 2 ðgÞ Finally, ZnO nanoparticles were obtained when calci- nated sample powders were washed with distilled water for thrice and dried. A colloidal stock solution of ZnO nanopar- ticles in ethanol (10 mg/mL) was prepared. This colloidal Correspondence to: A. R. Shahverdi; e-mail: shahverd@sina.tums.ac.ir Contract grant sponsor: Deputy of Research, Tehran University of Medical Sciences, Tehran, Iran VC 2010 WILEY PERIODICALS, INC. 557