Hussein et al (2019): ZnO-nanoparticles for antibacterial use December 2019 Vol. 22 (12) Synthesis and Characterizations of Zinc Oxide Nanoparticles-Loaded Chloramphenicol for Antibacterial Applications 1Khalfa EF, 1Nafaee ZH, 1Abdullah FN, 1Bdair GS, 1Niema RM, 1Salman HD, 2*Hussein FH. 1College of Pharmacy/ University of Babylon, Babylon, Iraq, 51002 2Al-Mustaqbal University College, Babylon, Iraq *Corresponding author: Hussein F. H. E-mail: [email protected] Abstract Background: Zinc oxidehas low toxicity, biocompatibility and biodegradability making it a material of interest for biomedicine, widely used in antibacterial materials and in pro-ecological systems. The aim of current study was to synthesize zinc oxide nanoparticle (ZnO) from Zn (NO3)2.4H2O by precipitation method. The study includes adsorption of chloramphenicol drugs on ZnO NPs to enhance the bioactivity of ZnO as topical antibacterial. Methods: Zinc oxide nanoparticles were synthesized via a precipitated chemical method for antibacterial applications by Zn (NO3)2.4H2O as precursor. The synthesized Zinc oxide nanoparticles were also modified by loading biologically active compound which was chloramphenicol. The characterization of Zinc oxide nanoparticles wasinvestigated by Scanning Electron Microscopy (SEM), Fourier Transform Infrared (FTIR). Chloramphenicol was adsorbed on ZnO nanoparticles at room temperature using 10%ethanol as solvent. The synthesized Zinc oxide nanoparticles loaded with chloramphenicol were validated for antibacterial activity against S. aureus, Acinetobacter, P. aeruginosa, E. coli and K. pneumoniae. Results: Generally, Zinc oxide nanoparticles loaded with chloramphenicol (100mg/mL) had better response for S. aureus and Acinetobacter while MIC against P. aeruginosa and E. coli was 300mg/ml. On the contrary, K. pneumoniae was the most resistant bacteria in this test and didn’t exhibit any response to the action of (Zinc oxide nanoparticles at any concentration. Keywords: Biological Applications, Chloramphenicol, Zinc Oxide, Pathogenic bacteria, Zinc Oxide Nanoparticles. How to cite this article: Hussein FH, Khalfa EF, et al (2019): Synthesis and characterization of zinc oxide nanoparticles-loaded chloramphenicol for antibacterial applications , Ann Trop Med & Public Health; 22(IV): S383. DOI: http://doi.org/10.36295/ASRO.2019.221217 Introduction Zinc oxide (ZnO) is an inorganic compound. Itis a white powder and is widely used as an additive substance in numerous materials.Zinc oxide is a semiconductor material with high chemical stability and strong photosensitivity with a band gap of 3.3eV at room temperature (1). In addition, ZnO can be used as a sensor, converter, energy generator and photo catalyst in hydrogen production because of its piezo- and pyro-electric properties (2, 3). Zinc oxide was used as photo catalyst in degradation of textile dyeing waste water. Zinc oxide has photo decolorization activity because activating energy of zinc oxide in photo degradation was 24±1kJmol-1. However, the photo degradation involved a mixture of suspension of zinc oxide with titanium dioxide under ©Annals of Tropical Medicine & Public Health S383 Hussein et al (2019): ZnO-nanoparticles for antibacterial use December 2019 Vol. 22 (12) ultraviolet irradiation condition (4). It has low toxicity, biocompatibility and biodegradability making ZnO a material of interest for biomedicine, widely used in antibacterial materials and in pro-ecological systems (5-7). Various synthetic methods have been reported in the literature for synthesis of ZnO nanoparticles, classified into either chemical or physical methods; for example, physical vapor deposition (PVD), chemical vapor deposition, spray conversion processing, sol-gel process and precipitation method (8, 9). These methods are to grow a variety of ZnO structures such as nanoparticles, nanowires, nanorods, nanotubes, nanobelts and other complex morphologies (10). Precipitation method is simple process for the synthesis of nanopowders of metal oxides, which are highly reactive in low temperature sintering (11). This method includes reducing the temperature of the reaction where homogenous mixtures of the reagents are precipitated. However, ZnO nanoparticles were prepared by ultrasound, microwave-assisted combustion method (12). Metal oxides such as ZnO have been studied extensively to explore their utility as potential pharmaceutical agents(11).There are differences between ordinary ZnO powder and ZnO nanoparticles (ZnO NPs) that have a large specific surface area and small size effect (10,11). The direct precipitated synthesized ZnO NPs were combined with sol-gel synthesized titanium dioxide to make evaluation of UV light photo catalytic activity. The photo catalytic degradation of ZnO NPs and titanium dioxide was carried out different RB5 concentrations at different light intensities (13).Nanoparticle can be involved in drug delivery, as the nanoparticle get entrapment of drugs, are either enhanced delivery to, or uptake by target cells and/or a reduction in the toxicity of the free drug to non-target organs (14). ZnO NPs have been reported to possess anti-microbial activity. These particles can significantly reduce skin infection, bacterial load and inflammation in mice, and also improve infected skin architecture (15). The mechanism of ZnO bioactivity is the electrostatic attraction between negatively charged bacterial cells and positively charged particles. This interaction is not only inhibitingbacterial growth but also induces reactive oxygen species (ROS) generation resulting in cell death (16-18). Similarly, it has been suggested that ZnO NPs have ability to inhibit the growth of bacteria due to disorganization of bacterial membranes, which increases membrane permeability leading to accumulation of nanoparticles in the bacterial membrane and cytoplasmic regions of the cells (19).The activity of ZnO NPs on the surface of bacteria or accumulation of NPs either in the cytoplasm or in the periplasmic region causes disruption of cellular function or disruption and disorganization of membranes (20, 21). It has been suggested that ZnO NPs can protect intestinal cells from bacterial infection by inhibiting adhesion and internalization of bacteria. This leads to prevent the increase of tight junction permeability and modulating cytokine (19). The antimicrobial activity of ZnO NPs has been studied against various microorganisms (bacteria) such as Pseudomonas aeruginosa, campylobacter jejuni, Escherichia coli and Gram-positive bacteria such as Bacillus subtillis and Staphylococcus aureus (22). The aim of current study was to synthesize zinc oxide nanoparticle (ZnO) from Zn(NO3)2.4H2O by precipitation method. The study includes adsorption of chloramphenicol drugs on ZnO NPs to enhance the bioactivity of ZnO as topical antibacterial. The ZnO NPs have tested against various types of bacteria including Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter. Material and Methods Materials All chemicals used were analytical grade and without any further purification. The precursor chemicals are zinc nitrate tetrahydrate (Himedia, India), sodium hydroxide (Alpha chemika, India), Ethanol (VWR chemicals, ©Annals of Tropical Medicine & Public Health S383 Hussein et al (2019): ZnO-nanoparticles for antibacterial use December 2019 Vol. 22 (12) France), chloramphenicol (Jing Lu biotechnology, China). Other solvents included deionized water, and Di- methyl Sulphoxide (Vardaan house, Daryaganj, New Delhi India). Synthesis methods In this study, wet chemical method was used for the preparation of ZnO nanoparticles. About 20mg of Zn (NO3)2.4H2O was dissolved in 300ml of deionized water in a beaker with stirring for 10min using magnetic stirrer. Then, 50mL of 0.25M NaOH solution was added slowly drop by drop into the beaker containing the Zn(NO3)2.4H2O solution under stirring condition and controlled pH above 9.The resulting solution was heated under constant stirring, at atemperature of 70°C for 4hrs. Then, the white suspension was formed; this mixture was left at room temperature for 72hrs. The mixed solution was filtered bycentrifugation at 2500rpm for 10min, for further separation, the precipitate wasdried at 70°C for two days. Finally, the precipitate was calculated at 300°C for 5hrs in a muffle furnace. Preparation solution of the chloramphenicol A stock solution of 500μg/mL chloramphenicol was prepared by dissolving 50mg of chloramphenicol into 100mL of aqueous ethanol (10%). Then, five different diluted chloramphenicol solutions (20, 40, 60, 80, and 100)μg/mL were prepared via dilution from the chloramphenicol stock solution. Loading of ZnO nanoparticles on chloramphenicol The adsorption of chloramphenicol on both types of zinc particles (nano and non-nano)was done by taking twice25mL of each diluted chloramphenicol solutions separately (20, 40, 60, 80, and 100)μg/mL and transferring these solutions into five 100mL flask.Next, 50mg of zinc oxidenanoparticles and zinc oxide non-nanoparticles, respectively,were added for each diluted chloramphenicolsolutions separately.Then, 10 mixtures (chloramphenicol with both types of zinc oxide particles) were shaken at room temperature for two hrs. Finally, the mixtures were centrifuged at 6000rpm for five minutes, and then the 10 filtrates were scanned by UV-Vis spectrophotometer while the precipitates were dried for bacteria culture activities. Antibacterial activity Collection of specimens and bacterial identificationweredone