J Nanopart Res (2012) 14:1063 DOI 10.1007/s11051-012-1063-6
Antibacterial activity of MgO nanoparticles based on lipid peroxidation by oxygen vacancy Karthikeyan Krishnamoorthy • Govindasamy Manivannan Sang Jae Kim • Kadarkaraithangam Jeyasubramanian • Mariappan Premanathan
Received: 9 March 2011 / Accepted: 13 July 2012 / Published online: 8 August 2012 Springer Science+Business Media B.V. 2012
Abstract Antibacterial activity of MgO nanoparticles (NPs) was evaluated against the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa as well as the Gram-positive bacterium Staphylococcus aureus by microtitre plate-based assay incorporating resazurin as an indicator of cell growth. MgO NPs exhibited antibacterial activity with minimal inhibitory concentration of 500 lg/mL against E. coli and 1,000 lg/mL for P. aeruginosa and S. aureus. MgO NPs enhanced ultrasound-induced lipid peroxidation in the liposomal membrane. It was suggested that the mechanism of the antibacterial activity of the MgO NPs relied on the presence of
K. Krishnamoorthy K. Jeyasubramanian M. Premanathan Department of Nanoscience and Technology, Mepco Schlenk Engineering College, Sivakasi, Tamil Nadu, India K. Krishnamoorthy S. J. Kim (&) Nanomaterials and System Laboratory, Department of Mechanical Engineering, Jeju National University, Jeju, South Korea e-mail: [email protected]
G. Manivannan Department of Microbiology, NMSS Vellaichamy Nadar College, Nagamalai, Madurai, Tamil Nadu, India M. Premanathan (&) Department of Biotechnology, Mepco Schlenk Engineering College, Sivakasi, Tamil Nadu, India e-mail: [email protected]
defects or oxygen vacancy at the surface of the nanoparticle which led to the lipid peroxidation and reactive oxygen species generation. Keywords MgO nanoparticles Antibacterial activity Lipid peroxidation ROS generation Oxygen vacancy Resazurin
Introduction Human beings are often infected by microbes such as bacteria, viruses, and yeasts in the living environment. In recent years, the control of microbial population becomes a universal concern due to the ubiquity of microorganisms and their ability to establish themselves. Several bacteria exhibit multi-drug resistance against chemical antibiotic drugs for many years and they continuously emphasize on human health care (Kim et al. 2007). The development of new antibacterial agents are of relevance to a number of industrial sectors including those focused on the environment, food, synthetic textiles, packaging, healthcare, medical care, as well as construction, and home and workplace furnishing (Kumar et al. 2008; Li et al. 2006). In general, antibacterial agents are broadly categorized as organic or inorganic. Both the natural and the synthetic organic antibacterial agents inhibit the growth of a wide variety of bacteria and fungi (Sudarshan et al. 1992; Liu et al. 2001; Okouchi et al.
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1998). However, organic antibacterial agents have some shortcomings, such as low heat resistance, high decomposability, and short life expectancy, which have limited their application. As a result, inorganic antibacterial agents have received more and more recognition in the antibacterial product market (Fang et al. 2006; Jung et al. 2008). Nowadays, the increase in the use of inorganic antibacterial agents comparably to the organic antimicrobial agents is mainly due to their improved safety and stability at higher temperatures (Premanathan et al. 2011). There is always a demand for developing new, effective antibacterial agents with low cost due to their significant impact on the environment and health care. In this regard, nanoparticles are highly recognized as antibacterial agents owing to their ability to inhibit the bacterial growth due to their size, structure, and surface properties (Raghupathi et al. 2011). Nanotechnology offers a way to develop potential antibacterial agents to overcome the multi-drug resistance of bacteria (French 2005). The toxicity of nanoparticles toward microorganism is due to either physical disruption or oxidative stress (Hu et al. 2010; Veerapandian and Yun 2011). Nanostructured materials have been used in textiles and in food industry to limit the growth of bacteria (Ugur et al. 2010). Metal NPs such as silver and copper are traditionally well-known antibacterial materials (Kumar et al. 2008). Metal oxide NPs, such as TiO2, ZnO, and CaO have also been recognized as antibacterial agents (Roselli et al. 2003; Stoimenov et al. 2002; Zhang et al. 2007). Some of the metal oxides, e.g., ZnO and MgO, are essential minerals for human health (Yamamoto 2001). MgO NPs have the advantage of non-toxicity, high thermal stability, biocompatible, low cost, and have considerable potential as an antibacterial agent. Mg plays several vital roles in human biology. Its deficiency has been implicated in the regulation of blood pressure. The institute of Medicine of the National Academy of Sciences has established Recommended Dietary Allowances which defines the average daily intake that is sufficient to meet the requirements is 420 mg (Mureinik and Guy 2003). Mg is ingested as a dietary supplement in the form of MgO and MgOH. These are insoluble in water, but readily soluble in gastric juices, and the Mg ion becomes bioavailable upon dissolution in the stomach. MgO and MgOH serve as pH control agents in dairy
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products and in the manufacture of canned vegetables such as peas. They also serve as flow enhancer and anti-caking agents in dry breakfast cereal, salt production, and powder concentrates such as soft-drink mixes. In medicine, MgO is used for the relief of heartburn, sore stomach, and acid indigestion, as an antacid, detoxifying agent, and for bone regeneration (Bertinetti et al. 2009; Boubeta et al. 2010). Recently, the MgO NPs show promising application in nanocryosurgery for tumor treatment (Di et al. 2012). Hence, the evaluation of antibacterial activity of MgO NPs toward different bacterial strains is of potential interest. In the present investigation, the antibacterial activity of the MgO NPs was explored against Escherichia coli, Pseudomonas aeroginosa, and Staphylococcus aureus by the microtitre plate-based assay with resazurin as an indicator of cell growth (Premanathan et al. 2011) and their minimal inhibitory concentration (MIC) were calculated. The advantage of employing this method is in its use of resazurin, a blue non toxic dye as an oxidation–reduction indicator for the evolution of cell growth (Sarker et al. 2007).
Materials and methods Preparation of MgO NPs The MgO NPs were prepared by wet chemical method using magnesium nitrate and sodium hydroxide (NaOH) as precursors and cellulose as stabilizing agent (Karthikeyan et al. 2009). Briefly, 0.2 M solution of NaOH was slowly added drop wise into a 0.1 M solution of magnesium nitrate in 50 mL of water with vigorous stirring for 2 h. The white precipitate formed containing Mg(OH)2 was washed thoroughly with distilled water and centrifuged at 3,000 rpm for 15 min. The procedure was repeated several times until the precipitate was free from any trace of impurities. Then 1 g of cellulose powder was mixed with the precipitate and dried at 100 C. The dried mixture containing Mg(OH)2 and cellulose was thoroughly mixed by grinding in a pestle and mortar. After that, the powder was calcined at 300 C for 2 h in a muffle furnace. The cellulose powder added here is for the role of capping agent and also to enhance the decomposition of Mg(OH)2 into MgO NPs at 300 C.
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Characterization of MgO NPs Phase purity and grain size were determined by X-ray diffraction (XRD) analysis recorded on a Siefert X-ray diffractometer ((Richard Seifert & Co, Ahrensburg, ˚ ) at Germany)) using CuKa radiation (k = 1.54016 A 60 keV over the range of 2h = 20–80. Fourier transform–infrared (FTIR) spectra of the MgO NPs were recorded using an Alpha-T spectrometer (Bruker Optics GmbH, Ettlingen, Germany). Ultraviolet–visible (UV–vis) spectra of MgO NPs were recorded on a Perkin-Elmer 110 UV-Lambda 25 spectrophotometer (Perkin-Elmer, Norwalk, Connecticut). Photoluminescence (PL) spectra of MgO NPs were measured by Cary Eclipse Fluorescence Spectrophotometer. The zeta potential and particle size measurements were carried out on ZetaSizer (Nano-Z, Malvern Instruments Ltd. UK). Scanning Electron Microscopy (SEM) observations were conducted on FE-SEM (JSM-6700F, JEOL Ltd). Atomic force microscopy (AFM) observation was conducted on a Scanning Probe 115 Microscope XE 70 (Park Systems, Suwan, South Korea). Antibacterial assay and estimation of MIC The microorganisms E. coli (MTCC739), S. aureus (MTCC96), and P. aeruginosa (MTCC1688) were collected from microbial type culture collection and gene bank, IMTECH, Chandigarh, India and maintained in nutrient medium. The antibacterial activity of MgO NPs and commercially available MgO powder was estimated by a microtitre plate-based method (Premanathan et al. 2011). Briefly, a sterile 96-well plate was labeled. A volume of 100 lL of test material (4 mg/mL) in 10 % (v/v) sterile water was pipetted into the first row of the plate. To all other wells, 50 lL of nutrient broth was added and serial dilutions were performed. To each well, 10 lL of resazurin indicator solution was added. Using a pipette 30 lL of 3.39 strength isosensitised broth was added to each well to insure that the final volume was of single strength nutrient broth. Finally, 10 lL of bacterial suspension (5 9 106 cfu/mL) was added to each well to achieve a concentration of 5 9 105 cfu/mL. Each plate had a set of controls: a column with a broad-spectrum antibiotic as positive control (ciprofloxacin in serial dilution), a column with all
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solutions with the exception of the test compound, and a column with all solutions with the exception of the bacterial solution adding 10 lL of nutrient broth instead. The plates were prepared in triplicate, and incubated at 37 C for 48 h. The color change was assessed visually. Any color changes from blue to pink were recorded as positive. The lowest concentration at which color change occurred was taken as the MIC value. The average of the three values was calculated and that was the MIC for the test material and bacterial strain. Lipid peroxidation measurement The free radical modulation activity of MgO NPs was determined by lipid peroxidation assay (Premanathan et al. 2011). Briefly, lipid peroxidation was induced in liposome prepared by ultrasonic irradiation from egg lecithin by adding 5 lL of 400 mM FeCl3 and 5 lL of 200 mM L-ascorbic acid. To this, MgO NPs were added. The control was prepared which contained no compound. The samples were incubated at 37 C for 60 min. The reaction was inhibited by adding 1 mL of stopping solution which contained 0.25 N HCl, 1.5 % trichloroacetic acid, and 0.375 % thiobarbituric acid. These reaction mixtures were kept in a boiling water bath for 15 min, cooled, and centrifuged. The absorbance of the resulting solution was measured at 532 nm. Statistical analysis Data were analyzed by Biostat software (AnalystSoft Inc., Vancouver, British Columbia, Canada) for oneway analysis of variance for statistical significance of the model (P \ 0.05).
Results and discussion This study employed a precipitation method to prepare nanosized MgO. Aqueous solutions of magnesium nitrate and sodium hydroxide were mixed at 25–30 C and a precipitation reaction occurred while stirring for 2 h, resulting in the formation of magnesium hydroxide. Further, the hydroxide powder was calcined at 300 C, which led to the decomposition of Mg(OH)2 to MgO NPs.
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The X-ray diffraction (XRD) pattern of the synthesized MgO NPs as shown in Fig. 1a demonstrated that the MgO was crystalline in nature and the diffraction peaks matched very well with a cubic phase of MgO (Kumar et al. 2009). The peaks at 2h = 36.95, 42.90, 62.27, 74.64, and 78.57 were assigned to the (111), (200), (220), (311), and (222) reflection lines of cubic MgO particles, respectively (Alavi and Morsali 2010). The lattice parameter of the MgO NPs was measured ˚ . The diffraction pattern and interplanar as a = 4.21 A spacing closely matched those in the standard diffraction pattern of MgO. (International Centre for Diffraction Data, Joint Committee on Powder Diffraction Standards 45-0946). No characteristic peaks of any impurities were detected, suggesting that high-quality MgO was obtained. Estimation from the DebyeScherer formula (Feng et al. 2008), D = kk/(bcosh), where D is the average crystal size, k is the Scherer constant, k is the wavelength of the X-ray, b is the peak width of half maximum, and h is the Bragg diffraction angle, the average crystallite size was 25 nm. Figure 1b represents the FTIR spectra of the MgO NPs. The strong band found at 440 cm-1 is due to the stretching vibration of MgO (Rezaei et al. 2011). The other bands in the range of 1,300–1,700 cm-1 are related to the hydroxyl groups of molecular water (Gu et al. 2004; Rezaei et al. 2011). The optical properties of the MgO NPs were studied in detail by means of the UV–vis absorption spectra in the wavelength range of 200–800 nm at room temperature. Figure 2a shows the UV–vis absorption
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spectrum of the MgO NPs depicting the enhanced absorbance in the low UV region. The maximum absorption band of MgO NPs was found at 225 nm, which can be attributed to the electronic excitations of 4-coordinated surface anions at the edges and a shoulder band at 275 nm was attributed to the 3-coordinated surface anions at the corners (Sterrer et al. 2003). The dependence of the absorption coefficient (a) on the photon energy (hm) in the bandedge spectral region for a direct transition is given by the Tauc Model (Zhang et al. 2011): ahm = Const (hm - Eg)1/2 where a is the absorption coefficient, h is the Planck’s constant, m is the frequency of light, and Eg is the band gap of the solid. The inset of Fig. 2a shows plot of (ahm)2 vs. photon energy of the MgO NPs and a direct energy band gap of 5.35 eV (225 nm) was determined from the plot which agrees well with the previous reports (Kumari et al. 2009). In order to study the defect states in MgO NPs, the photoluminescence spectrum were measured which provides significant information about their surface states. It is known that MgO NPs show a broad photoluminescence (PL) spectrum in the blue region due to low coordinated surface ions or defects (Stankic et al. 2005). Figure 2b shows PL spectra of the MgO NPs excited at the wavelength 225 nm. It can be seen that there is a broad peak centered at 390 nm and has been attributed to defects (Kumar et al. 2009). The surface morphology of the MgO NPs was analyzed by means of AFM. The two- and threedimensional topography of the MgO NPs is shown in
Fig. 1 a X-ray diffraction pattern of MgO NPs. b FTIR spectra of MgO NPs
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Fig. 2 a UV–vis spectra of MgO NPs. The inset shows the optical absorption coefficient (ahm)2 of MgO NPs as a function of the photon energy. b Photoluminescence spectrum of MgO NPs
Fig. 3a. Direct observation of the image revealed that the size of many of the MgO NPs was in the size range of 10–30 nm. The particles appeared to be acicular in shape. The size distribution of the MgO NPs along with the line profiles drawn at 3.8 lm horizontally (red line) and 5.5 lm vertically (green line) are shown in Fig. 3b. It clearly indicated that the majority of the MgO NPs fell in the range of 10–30 nm. The histogram analysis also revealed the same. Figure 4 shows the SEM image of the MgO dispersion in water after their deposition on glass substrate by drop casting method. It clearly shows the MgO particles are in the range of nanometer insuring further support for the size of the synthesized MgO. The results obtained from the AFM and SEM matched well in accordance with the XRD results. The important issues in biomedical applications are the stability of nanoparticles in dispersion (Fang et al. 2009). Accordingly, in vitro stability of MgO NPs was evaluated by measuring the particle size analysis and zeta potential in dispersion are shown in Fig. 5. The particle size analysis (Fig. 5a, b) shows 110 nm which is slightly higher when compared with the results of XRD, SEM, and AFM. This increase in size is because DLS measurement methods employed in dispersion in which MgO can easily be hydrated in water. The zeta potential analysis (Fig. 5c) shows that the MgO NPs posses 15.3 mV which reveals the generation of incipient stability in water. Hence, our dispersion of MgO NPs holds good stability.
Antibacterial activity of MgO NPs was evaluated against Gram-negative E. coli, P. aeruginosa, and Gram-positive S. aureus by the resazurin incorporation method as shown in Fig. 6. Resazurin is an oxidation–reduction indicator used for the evaluation of cell growth, particularly in cytotoxicity assays (Krishnamoorthy et al. 2011). It is a blue nonfluorescent and non-toxic dye that becomes pink and fluorescent when reduced to resorufin by oxidoreductases within metabolically viable cells. Resorufin is further reduced to hydroresorufin (uncolored and nonfluorescent). The lowest concentration at which the color changes from blue to pink was taken as the MIC value. There are two types of antibacterials, viz. bacteriostatic, which can prevent the growth of bacteria but not kill, and bacteriocidal which can kill the bacteria. Since the resazurin method is based on enzymatic activity of metabolically active viable cells, MgO is bacteriocidal. Bacteria were incubated with the medium containing serial dilution of MgO NPs, incubated, and observed for the color change. Color change was started after 12 h of incubation and reached maximum by 18 h. There was no difference in color change after 18 h until the end of the incubation time of 48 h. The MICs of MgO NPs against E. coli, P. aeruginosa, and S. aureus were found to be 500, 1,000, and 1,000 lg/mL, respectively. This is in contrast to the previous report that the MIC of MgO against E. coli and S. aureus are found to be at the same concentration
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Fig. 3 a AFM image of MgO NPs. b Line profile and histogram analysis of MgO NPs
(Makhluf et al. 2005). For the comparison, commercially available MgO powder was used for antibacterial assay. No activity was observed with the maximum concentration used in the assay protocol (2,000 lg/mL). This is in agreement with the previous work which demonstrated the smaller particles are more active in the bacteriocidal activity and gradual decrease in the bacteriocidal activity with the increase
in particle size (Makhluf et al. 2005). Sawai and coworkers used very high concentration of MgO powder for antibacterial assay (Sawai et al. 2000). A number of studies indicate that certain nanomaterials, including metal oxide nanoparticles, have the potential to exhibit spontaneous ROS production based on material composition and surface characteristics, while other nanomaterials trigger ROS production only
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Fig. 4 SEM image of MgO NPs
in the presence of selected cell systems (Lovric et al. 2005; Xia et al. 2006; Long et al. 2006). Free-radical modulation activity of MgO NPs was determined by ultrasonic irradiation-induced lipid peroxidation in liposomes from egg lecithin. Ultrasonic radiation caused lipid peroxidation in the liposomal membrane.
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Three reaction products of lipid peroxidation, conjugated dienes, lipid hydroperoxides, and malondialdehydes, have been detected in the ultrasound-exposed liposomal membrane (Jana et al. 1990). The reaction products are produced at different stages of a lipid peroxidation chain reaction mediated by free radicals. Our earlier studies demonstrate that lipid peroxidation is inhibited by free radical scavengers (Kanagalakshmi et al. 2010). This evidence partly proves the involvement of free radicals in the reaction process (Pryor 1977; Pryor 1980). The ultrasound-induced lipid peroxidation thus appears to be the result of a chain reaction, involving oxygen and mediated by free radicals (Chatterjee and Agarwal 1988). MgO NPs enhanced the ultrasound-induced lipid peroxidation. In comparison with the control group, lipid peroxidation was increased by 154 and 129 % after exposure to 100 and 50 lg/mL of MgO (P \ 0.05), respectively. It may be possible that sequential oxidation–reduction reactions may occur at MgO NPs surface to produce reactive oxygen species such as superoxide radical (O2-), hydrogen peroxide (H2O2) and hydroxyl
Fig. 5 a Particle size analysis, b histogram, and c zeta potential of MgO NPs
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may act on the O2- produced by the MgO NPs, resulting in formation of H2O2 that penetrates E. coli and oxidizes their active systems and kill. Whereas, another antioxidant enzyme catalase produced by P. aeruginosa and S. aureus neutralize H2O2 to H2O and O2, hence the less susceptibility. Mechanisms of cellular toxicity as elevated ROS production that exceeds the capacity of the cellular antioxidant defense system causes cells to enter a state of oxidative stress which results in damage of cellular components such as lipids, proteins, and DNA (Xia et al. 2006; Long et al. 2006). The oxidation of fatty acids leads to the generation of lipid peroxides that initiate a chain reaction resulting in disruption of cell membrane and subsequent cell death.
Fig. 6 Antibacterial activity of MgO NPs against E. coli using resazurin; color pink denotes the growth and blue denotes the inhibition of growth. N negative control, P positive control, MgO MgO NPs treated, CF ciprofloxacin treated. (Color figure online)
radical (OH•). The mechanism of the antibacterial activity of the MgO NPs relies on the presence of defects or oxygen vacancy at the surface of the nanoparticle. The presence of defect/oxygen vacancy in the MgO NPs are described with the UV–vis spectra and photoluminescence spectra of MgO NPs (as shown in Fig. 2a, b). Since MgO is readily hydrated and forms a layer of Mg(OH)2 on the surface, it leads to the formation of surface bound electron–hole pair which can decompose into a surface trapped electron and a localized hole state (Berger et al. 2005; Sterrer et al. 2000). These are typical oxide catalysts which will react with the molecular oxygen (O2) and produce superoxide radicals O2-. Proteins in the cell walls of bacteria contain many peptide linkages. The superoxide anion will attack the carbonyl carbon atom in the peptide linkages eventually leading to destruction of the bacteria. It is noteworthy to mention that the mechanism for the differential toxicity is due to the following reason. All the bacteria tested can produce superoxide dismutase a cellular antioxidant defense enzyme that
The key finding of this work supported the view that the toxicity of MgO NPs relies on the presence of defects/oxygen vacancy at the surface of the nanoparticle which leads to lipid peroxidation and ROS generation. This work suggested that the bacterial susceptibility of nanoparticles not only rely on the cell wall structures of Gram-positive and Gram-negative bacteria but also dependent on the cellular enzymes and biochemical events. Acknowledgments The authors are thankful to the Management and the Principal of Mepco Schlenk Engineering College and NMSS Vellaichamy Nadar College for providing the necessary facilities to carry out the work. A part of this work was supported by a National Research Foundation of Korea Grant under contract number 2011-0015829.
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