discussion essay Microbiology |Biology
Shape-dependent bactericidal activity of copper oxide nanoparticle mediated by DNA and membrane damage
Dipranjan Laha a, Arindam Pramanik a, Aparna Laskar c, Madhurya Jana a, Panchanan Pramanik b, Parimal Karmakar a,* aDepartment of Life Science and Biotechnology, Jadavpur University, 188, Raja S C Mallick Road, Kolkata 700032, India bDepartment of Chemistry, Indian Institute of Technology, Kharagpur 721302, India cCSIR-Indian Institute of Chemical Biology, Kolkata 700032, India
A R T I C L E I N F O
Article history: Received 7 May 2014 Received in revised form 14 June 2014 Accepted 22 June 2014 Available online 10 July 2014
Keywords: B. Chemical synthesis A. Metals C. Atomic force microscopy
A B S T R A C T
In this work, we synthesized spherical and sheet shaped copper oxide nanoparticles and their physical characterizations were done by the X-ray diffraction, fourier transform infrared spectroscopy, transmission electron microscopy and dynamic light scattering. The antibacterial activity of these nanoparticles was determined on both gram positive and gram negative bacterial. Spherical shaped copper oxide nanoparticles showed more antibacterial property on gram positive bacteria where as sheet shaped copper oxide nanoparticles are more active on gram negative bacteria. We also demonstrated that copper oxide nanoparticles produced reactive oxygen species in both gram negative and gram positive bacteria. Furthermore, they induced membrane damage as determined by atomic force microscopy and scanning electron microscopy. Thus production of and membrane damage are major mechanisms of the bactericidal activity of these copper oxide nanoparticles. Finally it was concluded that antibacterial activity of nanoparticles depend on physicochemical properties of copper oxide nanoparticles and bacterial strain.
ã 2014 Elsevier Ltd. All rights reserved.
1. Introduction
Nanostructured materials offer promising opportunities for improved applications in different area of modern life due to their unique physicochemical properties, caused by their nanosized dimensions and large surface/volume ratios [1]. More recently, several natural and engineered nanomaterials have been shown to possess strong antimicrobial properties including silver nano- particles [2], TiO2 [3], ZnO [4] and SiO2 [5]. Some nanocomposite consisting of different materials are possess bactericidal activity. For example, microfibril bundles of cellulose substance with titania/chitosan/silver-nanoparticle composite films and hierar- chical nanofibrous titania–carbon composite material deposited with silver nanoparticles are lethal to various bacterial strains [6,7].
Application of antibacterial agents in the textile industry, water disinfection, medicine, food packaging etc. are well known. Unlike conventional chemical disinfectants, the antimicrobial nanomate- rials are not expected to produce harmful disinfection by products
(DBPs). Among these several metal based nanoparticles (e.g., copper based nanoparticle) are increasingly recognized as a suitable alternative due to its high redox potential property and relatively lowercostofproduction[8]. Previously, ithas beenreportedthatCuO NPs exibit strong antimicrobial activity against broad spectrum of gram positive and gram negative bacteria [9]. Though, the constit- uents of cell wall in gram-positive and gram-negative bacteria are mainly responsible for their sensitivity to CuO NPs but other factors can also influence the sensitivity. For instance, gram negative E (!) is highlysensitive,but S.aureus(+)andB.subtilis (+)arelesssensitiveto CuO NPs [8]. On the other hand bactericidal activity of such nanoparticles in part depends on size, stability, shape and concentration in the growth medium [10,11].
The mechanisms by which such metal oxide nanoparticles induce bactericidal activities is not fully known but amount of ion release and subsequent production of ROS is supposed to be the main cause [12]. The rate of dissolution of such nanoparticles depends on their morphology as well as their nature [13]. Additionally, by electrostatic interaction nanoparticles are able to attach to the membrane of bacteria and interfere with bacterial membrane [14]. Depending on these two factors many metal oxide nanoparticles act differentially on different strain. As the way by which bacteria is killed by such nanoparticles is different from the
* Corresponding author. Tel.: +91 3324146710; fax: +91 3324137121. E-mail address: pkarmakar_28@yahoo.co.in (P. Karmakar).
http://dx.doi.org/10.1016/j.materresbull.2014.06.024 0025-5408/ã 2014 Elsevier Ltd. All rights reserved.
Materials Research Bulletin 59 (2014) 185–191
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antibiotic their proper evaluation is necessary. Thus a comprehen- sive knowledge about their size and morphology depended anti bacterial activity must be evaluated. In light of these, we undertook the effort to assess the morphology dependent activity of CuO NPs on different bacterial strain. We have synthesized two different shapes of CuO NPs and characterized them for their antimicrobial activity. The antibacterial activity was examined on a broad range of bacterial species including E.scherichia coli wild type, Micrococ- cus luteus, Bacillus subtilis and Proteus vulgaris. While sheet shape CuO NPs are potentially active against gram positive bacteria and spherical shaped CuO NPs are more effective on gram negative bacteria. Both membrane damage and ROS mediated DNA damage are responsible for their antimicrobial activity.
2. Materials and methods
2.1. Materials
In this study all chemicals of analytical grade were used. Copper acetate [Cu(CH3COO)2], glacial acetic acid [CH3COOH], sodium hydroxide [NaOH], copper nitrate trihydrate [Cu(NO3)2″3H2O] was obtained from SRL, India, ethanol (99%), sodium acetate [CH3COONa] from Qualigen, India. Alizarin red S (ARS), Hanks balanced salt solution (HBSS), nitroblue tetrazolium (NBT) were obtained from Sigma–Aldrich, USA. Hydrochloric acid (35%), dimethyl sulfoxide (DMSO), Muller–Hinton agar (MHA) medium and Muller–Hinton broth (MHB) medium were obtained from Hi-media, India.
2.2. Synthesis of CuO NPs (nanospherical, nanosheet)
Different shaped CuO NPs were prepared using co-precipitation method where either copper acetate or copper nitrate is used to form CuO NPs and NaOH acts as stabilizing compound [15,16].
2.2.1. Synthesis of CuO nanospherical 300 ml of 0.02 M copper acetate was taken in a conical flask.
1 ml of glacial acetic acid was added to it. The solution is heated at 80–90 #C on a hot plate with vigorous stirring for 10 min by a magnetic stirrer. 0.8 g NaOH was added rapidly to maintain the pH 6–7. The mixer was kept for 1 h in stirring condition. The resultant solution was centrifuged at 8000 rpm for 10 min. Pellet was dried at 37 #C for 3 days. After that it was homogenized by pestle–mortar and stored.
2.2.2. Synthesis of CuO nanosheet 80 ml of 0.02 M copper nitrate was slowly added to 5 M NaOH
solution in a conical flask at 82 #C. Additional 80 ml of same copper nitrate solution was added to above solution, a total of 32 g of NaOH pellet was added to the flask reactor to maintain the constant concentration of NaOH. The resultant solution was centrifuged at 8000 rpm for 10 min. The pellet was collected and washed with water. Pellet was dried at 37 #C for 3 days. After that it was homogenized by pestle–mortar and stored.
2.3. Particle characterization
Thephaseformationandcrystallographicstateofdifferentshaped CuO NPs were determined by XRD with an Expert Pro (Phillips) X-ray diffractometer using CoKa radiation (a = 0.178897 nm). Samples were scanned from 20# to 80# of 2u increment of 0.04# with 2 s counting time. Presence of surface functional groups was investi- gated by FTIR spectroscopy (Thermo 132 Nicolet Nexus FTIR, model 870). The particle size and nanostructure were studied by high- resolution transmission electron microscopy in a JEOL 3010 (HRTEM), Japan operating at 200 KeV. Dry powder of particles was suspended in de-ionized water at a concentration of 1 mg/mL and
then sonicated at room temperature for 10 min at 40 W to form a homogeneous suspension. After sonication and stabilization, the samples were prepared by coating on carbon-coated copper grids and air dried before TEM analysis. The hydrodynamic size of dispersed CuO NPs in aqueous phase was measured in a Brookhaven 90 Plus particle size analyzer. Copper based nanoparticles were dispersed in water to form diluted suspension of 0.5 mg/ml using sonicator for 30 min. The particles were analyzed by DLS after they were completely dispersed in water.
2.4. Bacterial strains and culture conditions
Well characterized cells of B. subtilis (ATCC 6633), M. luteous (ATCC 9341), E. coli (ATCC 10,536), P. vulgaris (ATCC 13,387), DH5a (k12) were maintained on MHA. Prior to incubation with NPs, the bacteria were cultured overnight in 4 ml of MHB in shaker at 37 #C until the optical density (OD) of the culture reached 1.0 at 600 nm, which indicates 109 CFU ml!1. The overnight cultures were diluted to 107 CFU ml!1 with sterile broth.
2.5. Antibacterial assay
Antibacterial activity of different shaped CuO NPs was affirmed through determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentrations (MBC) [17,18]. MIC is defined as the lowest concentration of antimicrobial agent at which no growth is observedin broth medium. Test tubescontaining 4 ml of broth was inoculated with overnight cultures of the bacteria and then various concentrations of different shaped CuO NPs (0 mg/ml– 0.4 mg/ml) were added in each tube. The tubes were left for shaking at 37 #C for 24 h. Then optical density of each tube was measured at 600 nm for the determination of bacterial growth. To substantiate antibacterial activity further, MBC was determined by inoculating a loop of NPs treated bacterial culture on MHA plates and left at 37 #C for 24 h. MBC is defined as the lowest concentration of NPs where no growth of bacteria is noted on agar plates. Growth curve was studies for both gram positive and gram negative bacteria with and without LD50 dose of these nanoparticles for 8 h.
2.6. Reactive oxygen species (ROS) assay
The production of intracellular reactive oxygen species (ROS) was measured using the same protocol mentioned in our earlier publication [19].
2.7. In vitro copper ion release study
Release of copper ion from the adsorbed NPs in nutrient broth was studied by the metallochromic dye ARS. To each test tube, 4 mg of different shape CuO NPs (nanospherical, nanosheet) were added in 1 ml of MHB. Then the test tubes were kept under shaking condition at 37 #C. Supernatant from each test tube was collected after 2, 4, 6, 12 and 24 h by centrifugation at 10,000 rpm for 10 min. Next, to each collected supernatant a 100 ml of ARS was added from stock (10!5M) along with sodium acetate buffer to maintain acidic pH. The solution was kept for 10 min and then optical density (OD) was measured at 510 nm by UV–vis spectrophotometer. The intensity of absorption depends on the amount of Cu–ARS complex which in turn depends on the concentration of Cu2+. The experiment was carried out three times and reproducible data were obtained [20].
2.8. DNA damage assay
The effect of different shaped CuO NPs on DNA was observed inside bacterial cell. Reporter (b-galactosidase) gene expression
186 D. Laha et al. / Materials Research Bulletin 59 (2014) 185–191
assay was performed. They were inoculated on agar plates containing X-gal and IPTG in the medium and incubated for 12 h at 37 #C for blue color forming colonies [20].
2.9. Cell morphology study by AFM
The effect of different shaped CuO NPs on bacterial cell morphology was studies using atomic force microscopy (AFM, Vecco, USA). Fresh E. coli bacterial culture (OD 0.2) were treated with LD50 dose of NPs for 3 h and then washed with phosphate buffered saline (pH 7) for three times and the cells were fixed with 2.5% glutaraldehyde. A drop of diluted cell suspension was placed on a cover slip and allowed to dry before AFM study [21].
2.10. Cell morphology study by SEM
The effect of different shaped CuO NPs on bacterial cell morphology was studies using scanning electron microscopy (SEM, Vecco, USA). Fresh bacterial culture (OD 0.2) were treated with LD50 dose of different shaped CuO NPs for 3 h and then washed with phosphate buffered saline (pH 7) for three times and the cells
were fixed with 2.5% glutaraldehyde. A drop of diluted cell suspension was placed on a cover slip and allowed to vacuum dry before SEM study [22].
2.11. Data analysis
A Student’s t-test was used to calculate the statistical significance of changes. In all cases, differences are significant for p < 0.05. Data analysis was performed using the Origin Pro v.8 software(Origin Lab).
3. Results and discussions
3.1. DLS and TEM analysis
The hydrodynamic size of different shaped CuO NPs was measured by DLS. Table 1 summarize their physical characteriza- tion. The TEM micrograph of different shaped copper oxide is shown Fig. 1A. From the Fig. 1A, the size of spherical and sheet shaped CuO NPs were 35 $ 5.6, 257.12 $ 13.6 % 42 $ 5.10, respec- tively. As seen in the table, the hydrodynamic sizes of the
Table 1 Characterization of the different shaped CuO NPs used in this study morphology primary size hydrodynamic diameter zeta potential pDia (TEM) TEM (nm) DLS (nm).
Morphology Primary size Hydrodynamic diameter Zeta potential pDia
(TEM) TEM (nm) DLS (nm) CuO spherical 33.20 $ 6.18 235 !27.6 0.305 CuO sheet 257.12 $ 13.6 % 42 $ 5.10 372 !23.1 0.346
a Polydispersity index.
Fig. 1. Physical characterization of different shaped CuO NPs (A) X-ray diffraction patterns of CuO nanosheet and CuO nanospherical; (B) FTIR spectra of of CuO nanosheet and CuO nanospherical; (C) transmission electron microscopic (TEM) image and dynamic light scattering CuO nanosheet and CuO nanospherical.
D. Laha et al. / Materials Research Bulletin 59 (2014) 185–191 187
synthesized NPs were significantly larger than those indicated by their TEM images. This is possibly due to the fact that TEM measures size in the dried state of the sample, where as the DLS measures the size of the hydrated state of particle.
3.2. X-ray diffraction pattern
We first characterized the purity of CuO NPs by XRD. The XRD pattern of CuO NPs was compared and interpreted with standard data of the JCPDS file (JCPDS International Center for Diffraction Data, 1991). Fig. 1B shows the XRD pattern of two different shaped CuO NPs, the characteristic peaks at 2u = 32.25#, 33.12#, 35.28#, 48.62#, 53.42#, 58.09#, 65.95#,67.90# and 72.24# which are in agreement with JCPDS card no. 44-0706.
3.3. Compositional and optical analysis of synthesized different shaped copper oxide nanoparticles (CuO NPs)
The functional or composition quality of the synthesized product was analyzed by the FTIR spectroscopy. Fig. 1C shows the FTIR spectrum in the range of 500–4,000 cm!1. The pure CuO NPs exhibited strong band at 1640 cm!1, characteristic of the CO stretch and the broad band around 3440 cm!1, indicates the presence of !!OH groups (Fig. 1C) for both CuO NPs. Table 1 summarized the physical characteristic of CuO NPs.
3.4. Evaluation of antibacterial properties
The antibacterial activities of these two different shaped CuO NPs against gram positive and negative bacteria were investi- gated using E. coli, P vulgaris, B.subtilis and M. luteus as model organisms. Shape dependent activity of CuO NPs was measured by determining minimum inhibitory concentration (MIC) and mini- mal bactericidal concentration (MBC) as shown in Tables 2 and 3, respectively. The growth of gram negative bacteria P. vulgaris and E.coli was completely inhibited by spherical CuO NPs at a concentration of 0.16 mg/ml and 0.20 mg/ml, respectively where as CuO nanosheet was more active on gram positive bacteria B. subtilis and M. luteous (0.22 mg/ml and 0.20 mg/ml, respectively). Significance of each MIC value is also determined. Difference in dose required for both types of nanoparticles to inhibit the growth of same bacterial strain is also shown on the last column of Table 2. From the Table 2, it is seen that for nanosheet the MIC value is 120–140 ug/ml less than nanoshperical for gram positive bacteria where as for gram negative bacteria, spherical CuO NPs is 120–80 ug/ml less than nanosheet indicating nanosheet CuO NP are more effective in gram positive bacteria and spherical CuO NP is effective in gram negative bacteria. We also determined the MBC of all bacterial strains after treating them with different shaped CuO NPs. A comprehensive table, showing MIC and MBC of different bacterial strain and the ratios of MIC and MBC are
Table 2 MIC value of different shaped CuO NPs on different strain.
Bacterial strain (106 CFU/ml)
Nanospherical (mg/ml) Nanaospherical (mg/ml) p value Difference doses between nanospherical and nanosheet
B. subtilis (+) 0.22 $ 0.028 0.36 $ 0 Nanosheet > nanospherical (p < 0.05)
140 mg/ml
M. luteous (+) 0.20 $ 0.010 0.32 $ 0 Nanosheet > nanospherical (p < 0.01)
120 mg/ml
E. coli (!) 0.28 $ 0.024 0.20 $ 0.05 Nanospherical > nanosheet (p < 0.01)
80 mg/ml
P. vulgaris (!) 0.28 $ 0.0 0.16 $ 0 Nanospherical > nanosheet (p < 0.05)
120 mg/ml
Table 3 MBC and MBC/MIC value of different shaped CuO NPs on different strain.
B. subtilis (+ve) M. luteus (+ve) P. vulgaris (!ve) E. coli (!ve)
Sph Sheet Sph Sheet Sph Sheet Sph Sheet MBC(mg/ml) 0.36 0.24 0.32 0.24 0.36 0.36 0.36 0.32 MBC/MIC 1 1.12 1 1.5 1.28 1.28 1.5 1
Fig. 2. (A,B) Growth curve (optical density) of E. coli, P. vulgaris, B. subtilis, M. luteous treated with respective LD50 dose of CuO nanosheet and CuO nanospherical respectively.
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presented in Table 3, For all the cases the ratio of MBC to MIC is 1 or greater than 1 indicating the potential bactericidal activity. LD50 value of different shaped CuO nanoparticles on different strain was also determine (data not shown). Fig. 2 represents growth kinetics of different strain bacteria in the presence of sheet (Fig 2A) and spherical (Fig. 2B). As seen in the Fig 2 the growth of CuO nanosheet treated bacteria was inhibited after 10 h whereas CuO NPs spherical treated bacteria reached a stationary phase after 10 h of growth. In case of all the four microbial strains, it was observed that with the increase in time of incubation beyond 10 h, with different shaped CuO NPs, OD value was decreased.
To determine the possible mechanism of different shaped CuO NPs on bacterial strains, we assayed in vitro copper ion release by ARS. As shown in Fig. 3A, copper ion release from spherical shaped CuO NPs was less than sheet shaped CuO NPs
at early time point but with increasing time the ion release became same for both the nanoparticles. One step further, we assayed ROS for bacterial strain E.coli and B. subtilis after the treatment of CuO NPs at LD50 dose. In E. coli spherical NPs produced more ROS compared to sheet but for B. subtillis ROS production was almost same for both the NPs. To check the DNA damage induced by NPs we used plasmid based reporter gene assay. In Fig. 3C, reporter gene b-galactosidase was assayed by transforming DH5a with the plasmid and followed by NPs treatment. The amount of blue colonies (due to the hydrolysis of X-gal by b-galactosidase enzyme) reduced significantly for the bacterial cells treated with NPs. We also used atomic force microscope to determine the effect of CuO NPs on E. coli. As seen in Fig 4, both spherical and sheet CuO NPs attached to bacterial cell membrane. Finally, we used SEM to visualize any membrane damage of bacteria. From the SEM image it was observed that
Fig. 3. (A) In vitro copper ion release of these two different shaped CuO NPs. (B) Determination of reactive oxygen species (ROS) of E. coli and B. subtilis in presence of these of different shaped CuO NPs. (C) Reporter gene (b-galactosidase) assay on nanoparticle treated and mock treated pUC 19 transformed DH5a.
Fig. 4. Atomic force microscopy (AFM) images of different shaped copper oxide nanoparticles treated or mock-treated gram negative E. coli bacterial cells.
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spherical shaped produced more membrane damage on E. coli compared to sheet and sheet shaped induced more membrane damage on B. subtilis (Fig. 5).
4. Discussion
In this study, we have reported the antibacterial activity of spherical and sheet shaped CuO NPs. Our results showed that the antibacterial effect of CuO NPs not only depends on size, but also on specific morphology and nature of the bacterial strain. Being transition metal, copper plays an important role in cellular redox cycling and antibacterial activity of copper based NPs are reported earlier [23,24]. Here we showed that apart from its size, CuO NPs morphology is also important for antibacterial activity. Previously Marsili et al. reported morphology dependent antibacterial activity of zinc oxide nanoparticles [25]. In our case we observed differential antibacterial activity of rod and spherical shaped CuO NPs. However, the mechanism of bactericidal actions of these nanoparticles are still not well understood, but it was proposed that surface charge of free metal surface is responsible for the interaction with the bacterial membrane [26]. As a matter of fact nanoparticles may associate with bacteria through several types of interaction such as hydrophobic, electrostatic or van der Waals interaction which may help to damage the cell membrane [27]. In a previous report it was shown that the interaction between silver nanoparticles and constituents of the bacterial membrane caused structural changes in membranes and finally leading to cell death [28]. Similarly surface modification of gold nanoparticles with BSA has been shown to determine its biological effects [29].
We observed that gram positive bacteria are more sensitive to nanosheet CuO NPs where as gram negative are more sensitive to spherical CuO NPs. It may be due to the fact that large sheet shaped CuO NPs can not penetrate the outer membrane of gram negative bacteria, where as small spherical shaped CuO NPs easily penetrate inside the bacterial cell. On the other hand having more surface charge, sheet shaped CuO NPs induced more damage to gram positive bacteria. Such large surface area of diethylaminoethyl
dextran chloride (DEAE-D) functionalized gold nanoparticles also shown to induce hemolysis in RBC [29].
Previously, several studies reported that two possible mecha- nisms are involved in the toxicity of nanoparticles on bacterial cell (1) production of increased level of ROS mostly hydroxyl radicals and singlet oxygen (2) deposition of nanoparticle on the surface of bacteria, resulting accumulation of nanoparticles either in the cytoplasm or in the periplasmic region causing disruption of cellular function. We have also seen the accumulation of CuO NPs on bacterial cell surface by AFM. The differential activity of these two shaped nanoparticles may be due to their difference in ROS generation inside the cells. In vitro Cu ion release is almost same at the higher time for both shaped CuO NPs and the amount of ROS generation is also same by two CuO NPs in B. subtilis strain. It is likely that sheet shaped NPs have less access inside the cells but their accumulation in the membrane or periplasmic region perturb the structure of membrane of such bacteria. This is also observed in our SEM studies where more membrane damage are observed in B. subtilis by nanosheet CuONPs. The thick shield of peptidoglycan layer or its constituents may thus be the target of sheet shaped CuONPs where as small size spherical CuO NPs easily permeable to thin peptidoglycan layer of gram negative bacteria and produce more ROS inside the cell. As a matter of fact these NPs can locally change microenvironments near the bacteria and produce ROS or increase the NPs solubility, which can induce bacterial damage. Thus both spherical and sheet shaped CuO NPs produce membrane damage to gram negative or gram positive bacteria, as observed by SEM. The exact mechanisms of action is not known but it seems likely that constituent of bacterial cell surface may contribute largely by interacting with specific nanoparticles. Additionally we found both the nanoparticles produce DNA damage. Large amounts of ROS could be generated even when only small amounts of CuO NPs are incorporated into cells. Nanoparticles can induce ROS directly, once they are exposed to the acidic environment of lysosomes or interact with oxidative organelles, such as mito- chondria. Thus, antibacterial activity of these two CuO NPs may depend on several factors including physiochemical properties of
Fig. 5. Scanning electronic microscopic image (SEM) of different shaped copper oxide nanoparticles treated or mock-treated gram negative and gram positive E. coli bacterial cells.
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nanoparticles and nature of bacterial surface. Thus the nature of bacterial strain and the surface properties of CuO NPs (e.g., size, shape, zeta potential etc.) are responsible for the antibacterial activity.
5. Conclusion
In this study, we presented the antibacterial activity of two different shaped CuO NPs on different strain. The particles size and morphology were characterized by DLS and TEM. Chemical characterization was done by XRD, FTIR. The studies of antibacte- rial activity of different shaped CuO NPs showed that the NPs were effective on variety of gram positive and gram negative bacteria as well as sheet shaped CuO NPs is more active on gram positive where as spherical shaped CuO NPs is more active gram negative bacteria. ROS induced DNA damage and membrane ruptures are the possible mechanisms of antibacterial activity of both shaped CuO NPs.
Acknowledgements
The authors would like to acknowledge for financial support for this research work the Department of Biotechnology, Government of India (No. BT/PR14661/NNT/28/494/2010). We also express sincere thanks to Indian Institute of Chemical Biology (IICB), Kolkata, India for providing the facilities to transmission electron microscopy and atomic force microscopy.
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