Synthesis and Characterizations of Zinc Oxide Nanoparticles-Loaded Chloramphenicol for Antibacterial Applications

Hussein et al (2019): ZnO-nanoparticles for antibacterial use December 2019 Vol. 22 (12)

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

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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,

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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 by selecting five samples of pathogenic bacteria including Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia and Acinetobacter. These samples were obtained from Microbiologylab at College of Pharmacy/ University of Babylon. These samples were identified by biochemical tests, culture and tested for antibiotic sensitivity. However, antibacterial activity of Zinc Oxide particles was tested by measurement of minimum inhibitory concentration (MIC) usingserial dilution methods.Loop-full growths from bacterial isolates were inoculated into nutrient broth incubated at 37°C for 18hrs. The bacterial suspensions were diluted with normal saline. Turbidity was compared with standard tube (McFarland number 0.5) to yield a uniform suspension containing 1.5 x 108CFU/mL. ZnO particles powder was suspended in DMSO for achievement the interaction of these nanoparticles particles with the bacteria then different concentrations(100, 200, 300, 400, 500)mg/ml of zinc oxide particles-loaded chloramphenicolwereadded to Mueller–Hinton (MH) mediumbroth. The bacterial culture was incubated at 37°C. Broth micro dilution method was used for measurement of MIC values. The test tubes were incubated at 37°C. The MIC was defined as the lowest concentration required for inhibiting visible growth of bacteria in the test tube after incubation(showed no turbidity) while the minimum bactericidal concentration

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(MBC) determined by sub-culturing 50μl from each test tube showing no apparent growth(clear); if there was no growth, this concentration taken as MBC.

Results and Discussion

Scanning Electron Microscopy (SEM) patterns of ZnO nanoparticles

The morphological feature of the carbon nanotubes were determined by using Scanning Electron Microscopy (SUPRA- 55VP) using electron beam energy of 10KV and 15KV. Figure (1) showedthe SEM images of ZnO

NPs synthesized using Zn (NO3)2.4H2O. SEM investigated the morphology of ZnO nanoparticles. The SEM images of the ZnO NPs have different shapes and sizes. The variation of the shape and size of the ZnO NPs is due to theirprecursors, calcination temperature and preparation processes. Similarly, on the right side of the spherical shape of the ZnO NPs was confirmed. The particles agglomeration was very high compared to the previous methods. The SEM images at higher magnification illustrated that the particles with size less than 100nm and also it gave clear idea about particles separation. ZnO particles agglomeration was marginal in comparison to previous methods in other studiesbecause there was formation of nano particles.

Figure 1: SEM images of synthesized ZnO NPs.

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The morphological feature of the loaded ZnO NPs with chloramphenicol was determined by using Scanning Electron Microscopy (SEM, quanta, USA). The loaded ZnO NPs powder was mounted on a sample holder foil layer. The morphology of ZnO nanoparticles synthesized from Zn(NO3)2.4H2O was grain (Figure 2) and spherical. The SEM images of the NPs have different shapes and sizes (Figure 2), the variation of the shape and size of the loaded ZnO NPs is due to the preparation conditions such as, calcination temperature and preparation processes. In addition, the surface of ZnO NPs was loaded using biologically active compound, chloramphenicol that led to variation of ZnO NPs morphology. The adsorption of negatively charged molecules on to ZnO NPs leads to dispersion of already aggregated nanoparticles. Therefore, disaggregation of ZnO NPs and adsorption of chloramphenicol on the surface alter the typical environment of the nanoparticles. The adsorption of negatively charged chloramphenicol onto ZnO NPs made the surface negatively charged resulting in repulsion between nanoparticles leading to dispersion of already aggregate nanoparticles.

Figure 2: SEM images of synthesized ZnO NPs-loaded chloramphenicol.

The UV-Vis absorption pattern of chloramphenicol loaded onZinc oxide nanoparticles

The UV-Vis absorption peaks of chloramphenicol before and after loading on ZnO particles were recorded with UV-Vis spectroscopy (Sheimazo, Lambda 950 UV/VIS Spectrophotometer) in the wavelength region of 200- 400nm at room temperature. The chloramphenicol was dissolved in ethanol 10% and its absorbance measured

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Table 1: Absorption of chloramphenicol after loading on both types of ZnO

No. Conc(μg/mL) CMF Abs/ adsorb on ZnO CMF Abs./ adsorb on ZnO NPs 1 1 20 0.991 0.537 2 40 1.002 0.972 3 60 2.296 1.803

4 80 2.658 1.917 5 5 100 3.740 2.494

Figure 3: Absorbance of chloramphenicol after its Figure 4: Absorbance of chloramphenicol after its adsorption on zinc oxide nanoparticles. adsorption on zinc oxide particles.

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Figure 5: Absorbance of chloramphenicol after its adsorption on normal and nanoparticles zinc oxide.

Adsorption efficiency of zinc oxide particles

The adsorption efficiency of chloramphenicol(278nm) loaded on both types of ZnO was done using five concentrations of chloramphenicol solution(Figures3&4).The highest adsorption efficiency was obtained when the concentration of chloramphenicol was high(Table 1), along with increasingchloramphenicol concentration, the adsorption efficiency increases as well as the loading quantity increases.The loading chloramphenicol on ZnO nanoparticles was higher in comparison with ZnO non-nanoparticles, because nanoparticles have small particle size and large surface area; therefore, the latter increases adsorption. The absorption line (Figure 5) of chloramphenicol loaded on ZnO non-nanoparticles was higher than absorption line of chloramphenicol loaded on ZnO nanoparticles; therefore chloramphenicol adsorption has reverse proportionwith the size of particles.

Fourier Transform Infrared Spectroscopy

The quality of ZnO NPs was also characterized by Fourier Transform Infrared Spectroscopy (OPUS, Spectrum FTIR) in the wavenumber region of 4000-400cm-1 according to the procedures reported. The FTIR measurements of the ZnO NPs were performed by mixing the powder ZnO with KBr pellet. Figure (6A-6C) showed the FT-IR spectra of ZnO nanoparticles, chloramphenicol, and ZnO NPs-loaded chloramphenicol. The broad band around 3350cm-1 assigned to O-H stretching mode of hydroxyl group which represents the presence of water molecule on the surface of ZnO nanoparticles or start materials such as Zn (OH) 2. The small peak between 2830 and 3000cm-1wasdue to C-H stretching vibration of alkane groups. Similarly, the band observed at 1630 and 1384cm- 1wasdue to asymmetric and symmetric stretching carboxylate attached to the ZnO nanoparticles during synthesis. The carboxylate probably came from the reactive carbon-containing plasma species during synthesis of ZnO NPs. The sharp peak observed in the range of 550 to 600cm-1 was attributed to the vibrational phonon of ZnO NPs (Figure 6A).

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In comparison with other studies, there was sharp peak observed in the range of 433 to 510cm-1 which was attributed to the vibration of ZnO NPs. This result indicatedthe successful production of ZnO nanoparticles -1 -1 (Figure 6A). FT-IR spectrum chloramphenicol (Figure 6B) exhibited the NO2 group from 1520cm to 1563cm , C=O from 1686cm-1 to 1694cm-1, OH and NH groups hadcombined peak ranged from 3300cm-1 to 3500cm-1 while CH hadsharp peak at 3078cm-1. This spectrum mentioned the presence of chloramphenicol. The spectrum of ZnO NPs-loaded chloramphenicol (Figure 6C) exhibited thepresence ofsame spectrum readings of chloramphenicol with slight shifting tofunctional groups as well as to Zn-O group that ranged between 500cm-1 to 600cm-1.

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Figure 6: FT IR spectra of ZnO nanoparticles synthesized using: zinc nitrate tetrahydrate(A), Chloramphenicol (B) and ZnO nanoparticles loaded Chloramphenicol(C).

Antibacterial Activity of Zinc oxideNanoparticles-Loaded Chloramphenicol

Increase in antibiotic resistance, is one of the most important reasonsforhospital-acquired infections and its control requires use of drugs combinations with more side effects; therefore, the researchers focused on using a combination of low-risk and effective control of bacterial resistance (23). Using nano-material and evaluation of their antimicrobial effects is one of the ways to reduce the Hospital Acquired Infections (HAI) (23).ZnO non- nanoparticles have been appeared to be strongly resistance to microorganisms; whereas ZnO NPs exhibit strong antibacterial activities on broad-spectrum pathogenic bacteria such as Staphylococcus aureus, Bacillus subtilis, Escherichia coli, E. coli O157:H7, Salmonella enteritidis, Salmonella typhimurium, Pseudomonas fluorescens, and Listeria monocytogenes(24, 25). In current study, the Minimum inhibitory concentration (MIC) of(ZnO) Nano particles (Table 2) against Acinetobacter isolates was found to be 100mg/mL. In another study on pathogenic Gram-positive and Gram-negative bacteria using ZnO NPs, indicated that all tested bacteria were affected in different manners, but there was no effect or antagonism relationship was observed between ZnO NPs and antibiotics on Gram-positive isolates and for Gram-negative isolates. This meant that the combination of antibiotics with ZnO NPs increased the biological activity of these nanoparticles against bacteria (26). In the present investigation, the antimicrobial activity of Zinc Oxide (ZnO) Nano particles-loaded chloramphenicol was evaluated against pathogenic bacteria isolates using broth dilution technique with different concentrations. The Minimum inhibitory concentration (MIC) of ZnO NPs-loaded Chloramphenicol was performed against various bacteria (Table2) and it was found to be 100mg/ml in general 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 (ZnO) Nano particles at any concentration (Figure 7).

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Table 2: Bioactivity of ZnO Nano particles-loaded chloramphenicol

E.coli P. aeruginosa

S. aureus K. pneumoniae

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Acinetobacter

Figure 7: ZnO NPs-loaded chloramphenicol inhibits bacterial growth by Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC).

Ethical Clearance

The Research Ethical Committee at scientific research by ethical approval of both environmental and health and higher education and scientific research ministries in Iraq

Conflict of Interest

The authors declare that they have no conflict of interest

Funding: Self-funding

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