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Mechanism of antibacterial activity of copper nanoparticles

This content has been downloaded from IOPscience. Please scroll down to see the full text. 2014 Nanotechnology 25 135101 (http://iopscience.iop.org/0957-4484/25/13/135101) View the table of contents for this issue, or go to the journal homepage for more

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Nanotechnology Nanotechnology 25 (2014) 135101 (12pp)

doi:10.1088/0957-4484/25/13/135101

Mechanism of antibacterial activity of copper nanoparticles Arijit Kumar Chatterjee, Ruchira Chakraborty and Tarakdas Basu Department of Biochemistry and Biophysics, University of Kalyani, Kalyani-741 235, West Bengal, India E-mail: [email protected] Received 14 August 2013, revised 18 December 2013 Accepted for publication 15 January 2014 Published 28 February 2014

Abstract

In a previous communication, we reported a new method of synthesis of stable metallic copper nanoparticles (Cu-NPs), which had high potency for bacterial cell filamentation and cell killing. The present study deals with the mechanism of filament formation and antibacterial roles of Cu-NPs in E. coli cells. Our results demonstrate that NP-mediated dissipation of cell membrane potential was the probable reason for the formation of cell filaments. On the other hand, Cu-NPs were found to cause multiple toxic effects such as generation of reactive oxygen species, lipid peroxidation, protein oxidation and DNA degradation in E. coli cells. In vitro interaction between plasmid pUC19 DNA and Cu-NPs showed that the degradation of DNA was highly inhibited in the presence of the divalent metal ion chelator EDTA, which indicated a positive role of Cu2+ ions in the degradation process. Moreover, the fast destabilization, i.e. the reduction in size, of NPs in the presence of EDTA led us to propose that the nascent Cu ions liberated from the NP surface were responsible for higher reactivity of the Cu-NPs than the equivalent amount of its precursor CuCl2 ; the nascent ions were generated from the oxidation of metallic NPs when they were in the vicinity of agents, namely cells, biomolecules or medium components, to be reduced simultaneously. Keywords: copper nanoparticle, membrane depolarization, reactive oxygen species, lipid peroxidation, protein oxidation, DNA degradation S Online supplementary data available from stacks.iop.org/Nano/25/135101/mmedia (Some figures may appear in colour only in the online journal)

1. Introduction

widely utilized as cheap and effective materials for sterilizing liquids, textiles and also human tissues for centuries [2]. Therefore, metallic Cu-NPs may be a promising antibacterial agent in future. But the most challenging task in this direction is the synthesis of stable metallic Cu-NPs, since they undergo rapid oxidation to Cu2+ ions in air or aqueous media. We have recently developed a method of synthesis of Cu-NPs, which remain stable in ambient condition for about a month [3], and their bactericidal activity on both Gram-negative and Gram-positive bacteria is much higher than those prepared and reported by others [4–6]. However, to use them as a substitute for antibiotics or as an antibacterial sterilizing agent, investigations on (a) the molecular mechanisms of bacterial cell killing and (b) the mode of NP action in cell killing need

Gradual development of different antibiotic-resistant bacterial strains has nowadays made it imperative to research new drugs or materials with a wide spectrum of effective antimicrobial activities. Recent studies on nanomaterials elicit that different metallic and metal oxide nanoparticles (NPs) may have very promising and potent roles as antimicrobial agents. Such NPs, due to their large surface to volume ratio and crystalline structure, trigger biological responses different from those produced by the traditional ionic form of the metals. Moreover, metallic NPs were found to have (a) 7–50 times less toxic effect to mammalian cells than their corresponding ionic forms and (b) prolonged effect as a source of elements in an organism [1]. Of the different metals, copper and its complexes have been 0957-4484/14/135101+12$33.00

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c 2014 IOP Publishing Ltd

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dye to the membrane made it fluorescent, finally measuring any alteration of cell membrane potential from the change in fluorescence intensity of the bound dye. To carry out the experiments, synchronized cells of E. coli K12 [20] were freshly grown up to the log phase (i.e., OD600 nm = 0.2 or cell number of about 108 cells ml−1 ) in Luria–Bertani (LB) medium [21]. The grown cells were then treated with 3.0 and 7.5 µg ml−1 Cu-NPs (the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC), respectively) for 1 h, centrifuged at 8000 rpm for 5 min, and the cell pellets were re-suspended in starvation buffer (SB) [21]. As negative and positive controls, cells alone (i.e. cells treated with nothing) and cells treated with 7.5 µg ml−1 CuCl2 (the precursor of the NPs) respectively were also taken. The subsequent steps of cell labeling by fluorescent dye and spectrofluorimetric (Perkin Elmer-LS50B) and flow cytometric (Beckton Dickinson FACS Calibur) measurements of intact cell membrane potential were made according to the method described previously [19].

to be done. Although the mechanisms behind the biocidal activity of metallic nanostructures are not yet fully understood, three hypothetical mechanisms are the most accepted and reported in the literature: (1) accumulation and dissolution of NPs in the bacterial membrane changing its permeability, with subsequent release of lipopolysaccharides, membrane proteins and intracellular biomolecules and dissipation of the proton motive force across the plasma membrane [7–9], (2) generation of reactive oxygen species (ROSs) or/and their corresponding ions from NPs, with subsequent oxidative damage to cellular structures [10–13], and (3) uptake of metallic ions derived from NPs or of NPs as whole into cells, followed by depletion of intracellular ATP production and disruption of DNA replication [14–17]. There are few reported studies, particularly on the mechanism of bactericidal activity of Cu-NPs. Raffi et al [4] and Ruparelia et al [5] suggested that Cu ions originating from the NPs may interact with phosphorus and sulfur-containing biomolecules such as DNA and protein to distort their structures and thus disrupt biochemical processes. In another study, Wu et al [18] also reported in favor of the involvement of Cu ions for the antibacterial activity of mesoporous copper-doped silica xerogels, where the increase of the copper concentration from 1 to 5% in the gel was found to cause 99% cell killing in gradually reduced time from 24 h to 1 h, respectively. Although, these studies hypothesize few probable modes of bacterium–Cu-NP interaction, a detailed and systematic study (both in vivo and in vitro) is much needed. The present study deals with the more organized and methodical investigations on the mechanistic pathway behind the Cu-NP-mediated occurrence of bacterial filamentation and cell killing as well as the mechanism of functioning of Cu-NPs as a strong antibacterial agent. Our experimental results reveal that the filamentation is caused by the NP-mediated depolarization of the cell membrane, whereas the cell killing is caused by the NP-mediated ROS generation in cells, which results in cellular lipid peroxidation, protein oxidation, and DNA degradation; for all these effects of Cu-NPs, the prime effector is the nascent Cu2+ ions, originating from the oxidation of the metallic Cu atoms of the NPs.

2.2. Estimation of the production of reactive oxygen species (ROSs) in E. coli cells

The extent of ROS production in bacterial cells was estimated using the chemical 20 ,70 -dichlorodihydrofluorescein diacetate (DCFH-DA) as a visual indicator to look inside the cell, according to the spectrofluorimetric method described previously [22]. The basic principle of the assay was that the DCFH-DA could easily cross the cell membrane and was hydrolyzed into DCFH by cellular esterase; subsequent oxidation of DCFH by intracellular ROSs produced highly fluorescent DCF, which was measured by a spectrofluorimeter. Therefore, the extent of DCF fluorescence intensity gave a quantitative measurement of ROS generation within cells. Log phase grown cells, prepared as described in section 2.1, were treated with 3.0 and 7.5 µg ml−1 Cu-NPs for 1 h, centrifuged and suspended in saline (8.0 gm l−1 NaCl, 0.2 gm l−1 KCl). The negative and positive control experiments, as described in section 2.1, were also done. The probe (1 µl of 10 mM DCFH-DA) was added to each of the four sets and incubated for 2 h at ambient temperature in the dark. The fluorescence of each sample was then measured in the spectrofluorimeter with excitation and emission wavelengths of 485 and 530 nm, respectively.

2. Methods

This study was conducted using the widely studied Gramnegative bacterium E. coli. All the experiments were done with synchronized cells, prepared as described in our previous study [3]. When such cells were allowed to grow in fresh medium, all the cells grew similarly and divided at the same time for a number of generations.

2.3. Assay of lipid peroxidation in E. coli cells

To estimate the extent of lipid peroxidation of the cell membrane, the spectrophotometric technique of thiobarbituric acid (TBA) assay, described previously [23], was employed. The basic principle of the assay was that the lipid peroxidation, or in other words the oxidation of polyunsaturated fatty acids, produced malonaldehyde (MDA), which reacted with TBA, producing a chromophore with absorption maximum at 532 nm. Therefore, the level of chromophoric absorption intensity at 532 nm was a quantitative measurement of lipid peroxidation. Log phase grown cells in saline (prepared as described in the preceding experiment) were divided into four parts:

2.1. Determination of membrane potential of intact E. coli cells

The membrane potential was determined by both spectrofluorimetric and flow cytometric methods, using the fluorescent dye 3,30 -diphenylthiocarbocyanine iodide, as described in detail in our previous study [19]. The basic principle of the methods was that the incorporation of the dye into cell membrane was potential dependent and the binding of the non-fluorescent free 2

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to two parts 3.0 and 7.5 µg of Cu-NPs were added separately, to one part 7.5 µg ml−1 of CuCl2 was added, whereas the fourth part was treated as control and nothing was added. All four cultures were incubated for 1 h at 37 ◦ C with shaking. 1.0 ml of each culture was centrifuged; the cell pellet was suspended in 100 µl of SDBME buffer [24]. 100 µl of cell extract was mixed with 200 µl of TBA–trichloroacetic acid (TCA)–hydrochloric acid (HCl) reagent (15% TCA and 0.375% TBA dissolved in 0.25 N HCl) and heated for 15 min in a boiling water bath. After cooling, the flocculent precipitates were removed by centrifugation at 12 000 rpm for 10 min. The absorbance of the supernatant was measured at 532 nm in a UV–vis spectrophotometer (Shimadzu, UV-1800), to estimate the level of lipid peroxidation according to ml−1

grown cells of E. coli K12 were divided into two parts: to one part 7.5 µg ml−1 of Cu-NPs was added and to the other part 7.5 µg ml−1 of CuCl2 was added. Both the parts were allowed to incubate at 37 ◦ C. From the Cu-NP-treated cells aliquots of 1.5 ml were withdrawn after 30, 60 and 120 min, and from the CuCl2 -treated cells an aliquot of the same volume was withdrawn after 120 min of incubation. From each aliquot, genomic DNA was isolated and subsequently electrophoresed for 1 h in 1% (w/v) agarose gel, using Tris–acetate–EDTA buffer. 2.6. UV-spectrophotometric study on DNA–Cu-NP interaction

The surface plasmon resonance property of Cu-NPs was used for this in vitro study. Here, Cu-NP solution was titrated with DNA and the spectrometric study was carried out at 298 K. Cu-NP solution was taken in a cuvette along with a reference cuvette containing gelatin and CuCl2 (the precursor components of the NPs) in the same amounts as present in the test cuvette. DNA solution from the prepared stock was then added stepwise (0.5 µg in each step) in both the sample (containing Cu-NP) and reference cuvettes and mixed thoroughly by turning the cuvettes upside down several times, and after each step of DNA addition the Cu-NP spectrum was recorded. DNA has negligibly small and constant absorbance in the wavelength region of 500–700 nm, which was due to scattering, but as it was added to both the sample and reference cuvettes the contribution due to the scattering of DNA in the Cu-NP spectra was automatically eliminated. The binding interaction was analyzed from the Hill plot of the absorbance data [29]. For any biomolecule–ligand interaction, the Hill equation was represented as log[θ/ (1 − θ )] = γ logK b + γ logCf , where K b is the binding constant, γ is the Hill coefficient, Cf is the concentration of free ligand and θ (fraction of macromolecule bound to ligand) = (A0 − AI )/(A0 − AR ), where A0 , AI and AR are the (absorbance)575 nm of Cu-NPs in the absence of DNA, at any DNA concentration and at the DNA concentration for which the maximum binding took place, respectively. The plot of log [θ/(1 − θ )] versus log Cf represented the Hill plot. The values of K b and γ were determined from the intercept on the x-axis (where log Cf = −logK b ) and the slope in the region of the intercept, respectively. The importance of γ was that it characterized the nature of cooperativity, that is, γ < 1, γ = 1 and γ > 1 signified a negative cooperative, non-cooperative and positive cooperative nature of the binding, respectively.

LPO activity = {(absorbance × total volume)} × {(time × sample volume × molar extinction coeff. × protein concentration)}−1 nM/mg/unit. (1) The protein concentration in the cell lysate was assayed by the Bradford method [25] and the value of molar extinction coefficient of the chromophore was known to be 1.56 × 105 mol−1 cm−1 . 2.4. Estimation of protein oxidation in E. coli cells

The level of protein oxidation in the bacterial cells was measured according to the method described previously [26]. The basic principle of measurement was that the protein carbonyls, products of protein oxidation, were known to react with dinitrophenylhydrazine to form a stable dinitrophenylhydrazone product, of which the 2,4-dinitrophenol (DNP) absorbed UV light at 370 nm. Thus, the extent of protein oxidation was proportional to the intensity of DNP at 370 nm. Cell lysates were prepared as in the case of the lipid peroxidation study. The lysates were centrifuged at 12 000 rpm for 10 min at 4 ◦ C. To the supernatants, 20% cold TCA was added and kept at −20 ◦ C for 1 h for precipitation. The precipitates were made sticky by centrifuging further at 12 000 rpm for 15 min at 4 ◦ C. To each sticky pellet 0.4 ml of 0.2% 2,4-dinitrophenylhydrazine (DNPH) in 2 N HCl was added and incubated in the dark, with vortexing at intervals of 10 min up to 1 h. The protein hydrazone derivatives were extracted through precipitation by 10% cold TCA. The precipitate was treated with ethanol–ethyl acetate (1:1 v/v) and the hydrazones were re-extracted by 10% cold TCA. The resulting precipitate was dissolved by vortexing in 6 M guanidine hydrochloride (GuHCl), kept at 37 ◦ C for 15 min and again centrifuged at 5000 rpm for 2 min to remove the undissolved fragments. The OD value of the supernatant was measured at 370 nm.

2.7. Determination of the NP size

2.5. Study on DNA degradation in E. coli cells

The average hydrodynamic size of the NPs was determined using a particle size analyzer (Malvern, Nano ZS). With the measurement of size, the instrument also determined the count rate of the NPs, i.e. the number of particles studied (to measure the average size) per second of investigation.

In order to observe the fate of DNA (degraded or not) in E. coli cells, genomic DNA was first isolated as described by Wilson [27] and the isolated DNA was then subjected to electrophoresis in agarose gel according to the method described by Sambrook and Rusell [28]. Here, log phase 3

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Table 1. Membrane potential of Cu-NP-treated E. coli cells measured spectrofluorimetrically.

Cell alone −184.5 (±12) mV

Cell + 7.5 µg ml−1 CuCl2 −159.8 (±13.5) mV

Cell + 3 µg ml−1 Cu-NPs −104.3 (±10.8) mV

3. Results and discussion

Cell + 7.5 µg ml−1 Cu-NPs −76.5 (±9.5) mV

division [38, 39]. It was shown there that the membrane potential modulated distribution of several conserved cell division proteins such as MinD, FtsA and the bacterial cytoskeletal protein MreB. Administration of the well known protonophore carbonyl cyanine m-chlorophenyl hydrazone, which blocked proton motive force across the E. coli cell membrane and thus dissipated the membrane potential, made the cells filamentous [38]. This was further supported by the observation that indole, a bacterial signaling molecule and a protonophore, inhibited cell division in E. coli by disrupting the trans-membrane potential [39]. Depolarization of the cytoplasmic membrane was found to inactivate the ‘Min’ site selection system, preventing localization of the FtsZ ring and thereby inhibiting cell division [39]. We also investigated if any depolarization of the E. coli plasma membrane occurred in Cu-NP-exposed intact cells, by both spectrofluorimetric and flow cytometric methods separately, as described in section 2.1. Table 1 shows that the bacterial (E. coli K12) cell membrane was depolarized by the Cu-NP treatment, and the extent of depolarization was dependent on the concentration of NPs used. The normal E. coli membrane potential was measured to be about (−)185 mV, which decreased to almost −105 and −75 mV when treated with 3.0 and 7.5 µg ml−1 of NPs, respectively; whereas a small drop of potential by about 25 mV took place when the cells were treated with 7.5 µg ml−1 of CuCl2 . The flow cytometric results have been depicted in figure 1. The main three quadrants of the cell-representative dots were LL—lower left, LR—lower right and UR—upper right. The dots in the LL quadrant signified cells having negligible fluorescence (1–10 arbitrary units), i.e. unlabeled cells. On the other hand, dots in the quadrants LR and UR signified cells with considerable fluorescence (10–104 arbitrary units) and their extents of fluorescence and size were understandable from the X - and Y -axis values, respectively. Table 2 represents the percentage distributions of cells in different quadrants, when the cells were kept under different conditions. Nearly 100% of the control unlabeled cells were represented by the dots in the quadrant LL (figure 1(A) and table 2, row 1). When the cells were labeled with the fluorescent dye 3,30 -diphenylthiocarbocyanine iodide for 2 h, about 76.35% of cells acquired considerable fluorescence label and the remaining 23.65% cells did not (table 2, row 2); of the labeled cells, the major population (76%) was represented by the dots in quadrant LR and a negligible minority (0.3%) of large-sized cells were represented by the dots in quadrant UR (figure 1(B)). Table 2, rows 4 and 5, shows that when the cells were treated with 3.0 (MIC) and 7.5 µg ml−1 Cu-NPs (MBC), only about 36.56 and 21.89% of cells were labeled. The decrease in the amount of labeling in the NP-treated cells (by about 40 and 54.5% for the NP concentrations 3.0 and

Our earlier communication [3] indicated that the gelatinstabilized Cu-NPs (synthesized by us) had high potential for cell filament formation and subsequent cell killing. The filament size was found to increase with increasing concentration of Cu-NPs up to 3.0 µg ml−1 (the MIC), above which there was a gradual decrease in size. Above the MIC, the filament size could not increase due to NP-mediated cell death. Filamentation is an anomalous growth of bacteria, when cells continue to elongate with multiple chromosomal copies and do not undergo septum formation, causing no cell division [30]. It is a primary defense mechanism for cells under SOS such as stress response. The SOS response is known to play a role in exposure of E. coli cells to DNA-damaging or DNA replication-interfering agents, by inducing about 30 different proteins for damage tolerance [31, 32]. One of the SOS gene products, SulA protein, encoded by the gene sfiA, is responsible for filament formation. SulA inhibited cell division by blocking the formation of the cell-septum-forming ring, made of FtsZ proteins, and thus led to filamentation. SulA interacted directly with FtsZ monomer, thereby preventing the polymerization and GTPase activity of FtsZ. By sequestering FtsZ, the cell could directly link DNA damage to inhibit cell division [33–35]. In the present study, it was investigated whether the SOS response was induced in Cu-NP-treated E. coli cells, by observing if any SOS repair of the UV-damaged bacteriophage 8 X174 [21, 36] occurred within the NP-treated cells. The result showed that no repair of the UV-inactivated 8 X174 took place in cells treated with 7.5 µg ml−1 of Cu-NPs (MBC) for 3 h, indicating that at least the SOS DNA repair genes were not induced in the E. coli cell by Cu-NP treatment (experimental details and results are shown in supplementary section 1 available at stacks.iop.org/Nano/25/135101/mmedia). Moreover, when the cells of E. coli SulA mutant strain SG13109 [37] were treated with 2.5 µg ml−1 (sub-MIC) and 3 µg ml−1 (MIC) of Cu-NPs in LB medium, for each concentration of the NPs they were found to form filaments within 3 h of treatment, indicating that the SulA protein had no role in the cell filamentation on Cu-NP treatment (experimental details and results are shown in supplementary section 2 available at stacks.iop.org/Nano/25/135101/mmedia). Therefore, the Cu-NP-induced cell filamentation was clearly not a SOS-dependent process. 3.1. Study to determine the membrane potential of Cu-NP-treated E. coli cells

Not much was known about the SOS-independent mode of filamentation, until a few recent studies showed that the cell membrane potential had a determining role in proper cellular 4

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Table 2. Percentage distribution of cells in different quadrants of flow cytometric profiles.

Serial no

Cells under different conditions

Lower left (LL) (%)

Lower right (LR) (%)

Upper right (UR) (%)

1 2 3 4 5

Control (unlabeled) Control (labeled) 7.5 µg ml−1 CuCl2 treated (labeled) 3 µg ml−1 Cu-NP-treated (labeled) 7.5 µg ml−1 Cu-NP-treated (labeled)

99.60 23.64 40.06 63.44 78.11

0.22 76.04 59.82 36.45 21.80

0 0.31 0.12 0.11 0.09

Figure 2. ROS generation in the Cu-NP-treated E. coli cells. The

shown values deviate within 5% from the corresponding average values. The statistical significance of the increase in ROS production in Cu-NP-exposed cells compared to that in untreated control cells was measured according to the Student t-test method, where p-values less than 0.5–0.001 were considered significant. ∗∗∗ = p < 0.001 and ∗∗ = p > 0.01.

of the cell membrane; however, the extent (about 16%) was significantly less than that in the Cu-NP-treated cells. Thus, both the spectrofluorimetric and flow cytometric results clearly signified that the exposure of E. coli cells to CuNPs caused cell membrane depolarization in a concentrationdependent manner, and this dissipation of membrane potential was the most probable basis of cell filamentation. Figure 1. Flow cytometric analysis of membrane potential of intact E. coli cells: (A) unlabeled control cells; (B) labeled control cells; (C) cells treated with 7.5 µg ml−1 CuCl2 ; (D) cells treated with 3.0 µg ml−1 Cu-NPs; (E) cells treated with 7.5 µg ml−1 Cu-NPs.

3.2. Study to estimate the ROS production, if any, in Cu-NP-treated E. coli cells

The hallmark of metallic NP-mediated bacterial killing was the ROS production and the consequent cellular damage. Again, metals such as copper act as catalysts in Fenton-like reactions to generate ROSs such as O2 • , OH• etc [40]. Therefore, an experiment was performed to study the ROS production, if any, and its extent in the Cu-NP-exposed E. coli cells. The experimental result clearly demonstrated the generation of ROSs in Cu-NP-treated cells, and the extent of ROS production was dependent on Cu-NP concentration (figure 2). Compared to the level in the untreated control cell, the ROS level in 3.0 and 7.5 µg ml−1 Cu-NP-treated cells was almost 1.8 and 2.5 times higher, respectively. Although the presence of a small amount of ROS (about 20% over the amount in control cell) was found in the CuCl2 (7.5 µg ml−1 )-treated

7.5 µg respectively) with respect to the amount of labeling (about 76%) in the NP-untreated control cells implied that Cu-NPs caused dissipation of the E. coli plasma membrane potential because the dye binding to the cell membrane and the consequent generation of fluorescence was membrane potential dependent. The cells with depolarized membranes were populated in quadrant LL (figures 1(D) and (E)), finally increasing the cell population there by about 40 and 54.5% compared to that in the case of control labeled cells (about 23.5%). Moreover, it is also found from figure 1(C) and table 2, row 3, that when the cells were treated with 7.5 µg ml−1 CuCl2 (the principal ingredient of the NPs) there was depolarization ml−1 ,

5

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Figure 3. Lipid peroxidation level in the Cu-NP-treated E. coli cells.

Figure 4. Protein oxidation level in the Cu-NP-treated E. coli cells.

The shown values deviate within 4% from the corresponding average values.

3.4. Study to estimate protein oxidation, if any, in Cu-NP-treated cells

cells by the copper ions, the amount was highly insignificant with respect to that in the NP-treated cells. This significant production of ROSs might cause free-radical-mediated cellular damage by Cu-NPs, which motivated us to study the extent of cellular lipid peroxidation, protein oxidation and DNA damage, because these were generally the most obvious effects of ROS production.

At the cellular level, exposure of proteins to ROSs was known to modify amino acid side chains with consequent alteration of the protein structure [42]. These modifications led to functional changes that disturbed cellular metabolism. As Cu-NP treatment generated considerable ROSs in E. coli cells, it was necessary to determine whether the protein oxidation phenomenon occurred in the Cu-NP-treated E. coli cells. Our experimental result demonstrated that treatment of E. coli cells with 3.0 and 7.5 µg ml−1 of Cu-NPs for 1 h made the cellular protein carbonyl level or oxidized protein content about 25 and 50 times respectively that in the untreated control cells (figure 4). This suggested that, besides lipid peroxidation, protein oxidation was also a probable reason for the cell killing by Cu-NPs. Figure 4 also shows that the exposure of the cells to 7.5 µg ml−1 of CuCl2 made the protein oxidation level eight times that of the control cells, implying further that the bactericidal effect was specific to NPs instead of only copper ions. It should be mentioned here that the repair of damage to proteins was limited to the reduction of oxidized derivatives of the sulfur-containing amino acid residues; no other kind of repair of protein oxidation was reported. Oxidized proteins were better substrates for proteolytic digestion, and E. coli has specific proteases that selectively degrade oxidized proteins, finally killing cells [43].

3.3. Study to estimate lipid peroxidation, if any, in Cu-NP-treated cells

The lipid peroxidation phenomenon refers to oxidative degradation of polyunsaturated lipids, causing three in vivo damaging consequences to the cellular plasma membrane, namely decrease in membrane fluidity, increase in membrane leakiness and damage in membrane-bound proteins, ultimately leading to toxicity and cell death [41]. Of the different reactive oxygen species, singlet oxygen and hydroxyl radicals remove a hydrogen atom from a methylene group on a polyunsaturated fatty acid, leaving an unpaired electron on the carbon atom. The carbon atom then rearranges itself to a conjugated diene, which reacts with oxygen to form a peroxy radical [23]. Our experimental result on lipid peroxidation, depicted in figure 3, implied that on exposure of the cells to 3.0 and 7.5 µg ml−1 Cu-NPs for 1 h their lipid peroxidation levels were respectively about 35 and 50 times that in the untreated control cells. On the other hand, bulk copper salt (7.5 µg ml−1 of CuCl2 ) caused a small increase in lipid peroxidation by only eightfold. This result suggested that high lipid peroxidation, i.e. high level of oxidation of unsaturated fatty acids, in E. coli membranes might be one of the initiating events for Cu-NP-mediated bacterial killing. Moreover, very little increase of lipid peroxidation (only eightfold) by ionic copper of bulk CuCl2 signified that the NPs, not the copper ions, had a specific effect on bacterial killing. Unsaturated fatty acids are known to be essential and irreplaceable components in biological membranes [41]. Changes in fatty acid composition altered the physical properties of the lipid bilayer, affecting membrane fluidity and thus indirectly regulating the activity of integral membrane proteins.

3.5. Study to investigate whether DNA damage occurred in Cu-NP-treated cells

Free radicals were known to attack both the base and the sugar moieties of DNA, producing lesions such as single and double-strand breaks, adducts of base and sugar groups, and cross-links to other molecules blocking DNA replication [44]. Therefore, an experiment was performed to investigate the fate of DNA in the Cu-NP-treated bacterial cells. Figure 5 clearly demonstrates the degradation of genomic DNA isolated from the Cu-NP-treated E. coli cells. The intact DNA band in lane 1 signifies that no degradation of genomic DNA occurred in cells 6

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1.0, it confirmed that the binding between DNA and Cu-NPs was positively cooperative in nature. The spectrophotometric study of Cu-NP–DNA interaction was also performed at 310 K. From the Hill plot analysis, the values of K b and γ were found to be 1.36 × 105 M−1 and 2.393, respectively, at 310 K. Since (K b )310 K > (K b )298 K , it signified strengthening of the NP–DNA binding interaction with the rise of temperature. These values of K b at two different temperatures, 298 K (say T1 ) and 310 K (say T2 ), were put in the places of K 1 and K 2 , respectively, in the well known van’t Hoff equation:     −1H 1 1 K2 = − (2) ln K1 R T2 T1 to find out the value of the thermodynamic parameter (1H ), i.e. the value of the enthalpy change in the Cu-NP–DNA interaction. The value was calculated to be −6.5 kcal mol−1 ; the negative value of the enthalpy change clearly implied that the NP–DNA interaction was exothermic in nature. Moreover, the values of 1G (change in free energy) at temperatures 298 and 310 K as calculated from the thermodynamic equation

Figure 5. Agarose gel electrophoretic pattern of the chromosomal DNA isolated from the Cu-NP-treated E. coli cells.

exposed to CuCl2 for 2 h. On the other hand, the smear-like band pattern in lane 2 implies chromosomal degradation in E. coli cells when treated with 7.5 µg ml−1 Cu-NPs for only 30 min. Moreover, incubation of the cells with NPs for 1 h caused such an extensive cleavage that many short DNA fragments went out of the gel during electrophoresis and so the total intensity of the smear in lane 3 was less than those of the bands in lanes 1 and 2. In the case of the cells incubated with NPs for 2 h, DNA was fragmented into such small pieces that most of the pieces left the gel in the running time, as observed from the DNA profile in lane 4. It can therefore be concluded that in vivo chromosomal DNA degradation in E. coli started within 30 min of treatment with Cu-NPs (7.5 µg ml−1 ), and more degradation took place with increase of the NP exposure time.

1G = (−)RT ln K b

(3)

were found to be −5.19 and −6.35 kcal mol−1 , respectively, and the negative value of 1G (at each of the two said temperatures) implied that the DNA–Cu-NP binding reaction was a spontaneous phenomenon. 3.7. Study to investigate the mechanism of DNA degradation by Cu-NPs

Here, an in vitro experiment was performed to investigate whether the degradation of DNA was caused either by the Cu2+ ions leached from the oxidized NPs or by the Cu-NPinduced generation of ROSs (singlet oxygen and hydroxyl radicals) or by the Cu-NPs themselves, by adding different radical scavengers and a Cu-ion chelator to the DNA–CuNP reaction mixture. This study was done with pUC19 DNA. As scavengers of singlet oxygen and hydroxyl radicals tris(hydroxyl methyl) amino methane and DMSO were used, respectively, and as a chelator of copper ions EDTA was used. The DNA band patterns and their intensity distributions under different reaction conditions have been presented in figure 7 and table 3, respectively. It is clear from the DNA band profiles in lanes 1–4 of figure 7 that Cu-NPs caused severe degradation of plasmid DNA when incubated at 37 ◦ C for different times. Measurement of the intensity of the bands in lane 1 demonstrated that about 81.5% of the purified control DNA was in the native, circular, supercoiled, compact form (also called replicative form I or RFI) and moved faster than the remaining 18.5% of the DNA, which was nicked (cleavage of a phosphodiester bond in a single strand of DNA) and so non-supercoiled or large relaxed circular—also called RFII DNA. Lane 2 of figure 6 and row 2 of table 3 show that within 5 min of incubation with Cu-NPs almost 90% of the RFI DNA was converted to singly nicked RFII DNA (about 66%) and doubly nicked (single nick in both the strands) linear RFIII DNA (about 25%). It is known that, although

3.6. Spectrophotometric study on DNA–Cu-NP interaction

In order to investigate whether the Cu-NP-induced DNA degradation in E. coli was due to direct interaction of the NPs with the chromosomal DNA or due to some secondary interaction, a spectrophotometric study was performed. Since the E. coli DNA was circular and supercoiled, this in vitro study was done with circular, supercoiled plasmid pUC19 DNA [28] of size 2.68 kbp. Here 0.5 ml of fivefold diluted Cu-NP solution was placed in a 1.0 ml quartz cuvette and was titrated with purified pUC19 DNA. From the absorbance spectra of Cu-NP solution titrated with different concentrations of DNA at 298 K, it was seen that the plasmon peak of Cu-NPs at 575 nm decreased gradually with increasing addition of DNA (figure 6(A)), which indicated some interaction between DNA and Cu-NPs. When the binding isotherm of DNA–NP interaction was plotted from the data of the spectrophotometric results, the isotherm looked sigmoidal in shape, signifying the cooperative mode of interaction (figure 6(B)). From the Hill plot (figure 6(C)), as discussed in section 2.6, values of the binding constant (K b ) and the Hill coefficient (γ ) were found to be 1.22 × 105 M−1 and 2.704, respectively, at 298 K. As the value of γ was greater than 7

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Figure 6. Spectrophotometric data of Cu-NP–DNA interaction. (A) Change in the absorption spectrum of Cu-NPs (1 mM) with the stepwise

addition of pUC19 plasmid DNA at 298 K; (B) the binding isotherm of the NP–DNA interaction at 298 K; (C) Hill plot of equilibrium binding between Cu-NPs and DNA at 298 K. Table 3. Percentage of intensity distribution of pUC19 DNA in different conformations, as measured by the 1D gel analysis software in

Typhoon 9210 (GE Healthcare). Lane no Experimental conditions

RFI (supercoiled) (%) RFII (nicked circular) (%) RFIII (linear) (%)

1 2 4 3 5 6 7 10

81.5 9.44 9.7 — 16.79 7.3 59.35 68.74

DNA alone DNA + Cu-NPs (5 min) DNA + Cu-NPs (15 min) DNA + Cu-NPs (30 min) DNA + Cu-NPs + 0.2 M Tris (O2 •− scavenger) DNA + Cu-NPs + 0.2 M DMSO (OH• scavenger) DNA + Cu-NPs + 20 mM EDTA (ion chelator) DNA + CuCl2 (30 min)

the molecular weights of all three forms of DNA–RFI, RFII and RFIII, are the same, their gel electrophoretic mobilities are different depending on their compactness: supercoiled RFI moved faster than linear RFIII, which again moved ahead

18.5 65.87 58.09 62.45 61.03 70.4 40.65 31.26

— 24.69 32.21 37.55 22.18 22.3 — —

of relaxed circular RFII [45]. After 15 min of incubation, more DNA degradation took place, converting gradually singly nicked circular RFII DNA to doubly nicked linear RFIII DNA, as observed from lane 4 of figure 6 and row 3 of 8

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table 3. Lane 3 shows that at 30 min of interaction between pUC19 and Cu-NP the RFI band was completely abolished and both the RFII and RFIII bands became less intense; this result together with the DNA profiles in lanes 2 and 4 implied that with increasing the time of interaction RFI converted to RFII, which then degraded to RFIII, and finally further degradation of RFII and RFIII occurred, to such a high extent that most of the smaller DNA fragments went out of the gel during electrophoresis and some comparatively larger fragments produced faint smears, observed in the lanes. Lanes 5 and 6 represent the DNA profiles when the DNA–NP interaction was carried out in the presence of tris(hydroxyl methyl) amino methane—a scavenger of singlet oxygen—and DMSO—a scavenger of hydroxyl radical, respectively; with respect to the percentage of RFI DNA in lane 1, those in lanes 5 and 6 were only 9 and 20% respectively, as observed from column 3 of table 3. Therefore, the band patterns of lanes 5 and 6 clearly signified that the scavengers had no significant inhibitory role in the Cu-NP-induced DNA degradation, i.e. the Cu-NPs did not degrade DNA through the production of ROSs such as singlet oxygen or hydroxyl radicals. Instead of the radical scavengers, when the copper-ion chelator EDTA was added to the DNA–NP reaction mixture (lane 7), degradation of RFI DNA was found to be significantly inhibited by almost 73% (comparing the values of column 3 in rows 1 and 7 of table 3), signifying that the DNA degradation effect of Cu-NPs was mostly Cu2+ ion mediated. Lane 9 shows that when fivefold diluted Cu-NP was used little DNA degradation took place; therefore, comparison of the band intensities in lanes 3 and 9 indicates that Cu-NPs degraded DNA molecules in a concentration-dependent manner. If the ion-mediated effect occurred, the CuCl2 (the main precursor of Cu-NPs) should also have the DNA degradation property. However, rows 1, 3 and 10 of table 3 indicate that 1.0 mM CuCl2 in 30 min caused only about 16% cleavage of RFI, whereas 1.0 mM Cu-NPs in 30 min caused complete abolition of RFI. Therefore, the prime outcome of this experimental result was that, although the copper ions appeared to be the main effector for DNA degradation, the nascent ions released from the NPs with their oxidation were perhaps more effective in DNA cleavage than the copper ions present in already ionized CuCl2 .

Figure 7. In vitro degradation of pUC19 DNA by Cu-NPs in the

absence and presence of free radical scavengers and a copper-ion chelator.

in mind that strong chelation of the Cu2+ by EDTA might cause more and more leaching of the ions from the NPs and thus ultimately reduce the size of the Cu-NPs with time. For this, EDTA at a final concentration of 20 mM was added to fivefold diluted Cu-NPs and the size of the NPs was measured, as described in section 2.7, at different intervals of time. The results represented by figure 8 show that the size and the count rate of Cu-NPs decreased gradually with the time of incubation with EDTA. The decrease of both the parameters reached saturation at nearly 30 min of incubation. The size of the NPs was found to decrease from 62.5 to 23.4 nm, whereas the count rate decreased from 282.5 kcps (kilocounts per second) to 130.5 kcps. These results suggest that the Cu-NPs became highly destabilized in the presence of EDTA due to gradual leaching of Cu ions from the NP surface, with simultaneous chelation of the ions by the EDTA. As a consequence, not only did the size of the core particle decrease fast, but almost half of the initial particles were abolished (as observed from the decrease in particle count rate), when kept in the presence of EDTA for about 30 min only. On the other hand, the NPs alone in suspension were highly stable and their size (about 60 nm) remained unchanged. Therefore, binding of the Cu ions to the chelator EDTA caused more leaching of ions from the NPs. Similarly, when the NPs and DNA were in the same place, Cu2+ binding over DNA resulted in more release of ions from NPs and consequently more degradation of DNA. This was further supplemented from our experimental results on the gradual dissolution of the NPs in the defined growth medium, named MOPS (3-(N -morpholino)propanesulfonic acid) medium [46]. Since the MOPS medium was composed of MOPS buffer supplemented with different amino acids, nucleotides, vitamins and a carbon source, incubation of the NPs in this medium also caused a decrease in size of the particles with time; however, the rate and the extent of decrease were small (figure 8(B)). It is also found from figure 8(B) that, as soon as the NPs were added to the MOPS medium, their size increased from about 60 to 185 nm, indicating formation of some binding complex of the NPs with the

3.8. To study the effect of EDTA on the size and count rate of Cu-NPs

The result of the previous experiment 3.7 suggested that the nascent copper ions released from the NPs were responsible for the heavy DNA degrading effect of the NPs. In the presence of DNA, the oxidation of metallic copper atoms of the NPs to Cu2+ was expected, with the simultaneous reduction of DNA by receiving the electrons liberated from the copper atoms. These Cu ions perhaps came out of the NP assembly in nascent form and acted upon the phosphodiester bonds of DNA, and possibly, with the gradual engagement of the Cu ions due to binding with DNA, more ions were leached out of the NPs. To check this possibility, the physical state, i.e. the size and count rate, of the Cu-NPs in the presence of the divalent metal ion chelator EDTA was investigated, keeping 9

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Figure 8. (A) Decrease in size and count rate of Cu-NPs with time of incubation in presence of EDTA; (B) decrease in size of Cu-NPs with time of incubation in MOPS medium. The red point signifies the size of Cu-NPs in water.

organic macromolecules present in the medium; the size of the large complex particles reduced gradually from 185 to 125 nm when incubated in MOPS medium for 12 h. The high chelating property of EDTA for divalent cations Cu2+ justified our results of lower rate and lower extent of size reduction of NPs in the presence of different organic molecules than in the presence of EDTA.

and a single type of Cu-NP synthesized by us were sufficient enough to draw any generalized conclusion on the pathways of cell killing and NP action. Here, it should be emphasized that the results of our previous study [3] showed that similar antibacterial effects of the Cu-NPs also occurred in Gram-positive bacteria B. subtilis and S. aureus, besides the Gram-negative E. coli. Since E. coli is known to be a widely studied organism, and most of the fundamental molecular mechanisms of DNA replication, gene expression, mutation etc were established using E. coli as a biological tool, the use of E. coli as the single organism in our study, to understand the fundamental basis of bacterial cell killing by Cu-NPs, was justifiable enough. In this study, regarding the use of our synthesized Cu-NPs only, it should be mentioned that, although there are different methods, namely laser irradiation [47], γ -irradiation [48], thermal decomposition [49, 50], thiol-induced reduction [51], reduction in micro-emulsions and reverse micelles [7, 52, 53], vapor deposition [54], the sono-electrochemical process [55], flame spray [56], chemical reduction [12, 57, 58] etc, of synthesizing Cu-NPs of different sizes and stabilized by different agents, our goal of innovating a new method of synthesis of Cu-NPs was to prepare very stable NPs, stabilized by the highly biocompatible gelatin, so that they can be used in future as a substitute for antibacterial agents. In our previous study [3], it was reported that our NPs were encapsulated by the stabilizer gelatin molecules, which was expected to be advantageous for interaction of the particles with cell membranes and their subsequent entry into the cell cytosol. Therefore, in the present study emphasis was given exclusively to our synthesized NPs, to investigate the mechanism of their antibacterial role. However, studies are in progress in our laboratory to understand the following important points: (1) the mode of physical interaction between the NPs and the cell membrane, (2) the mode of interaction between the NPs and cell medium organics, (3) NP-mediated alteration of the different physical and chemical characteristics of the cell membrane, (4) localization of the NPs in cells, (5) whole protein expression behavior of the NP-treated cells and (6) whether the nascent ion specific effect of our Cu-NPs for bacteriotoxic potency was also true for Cu-NPs of different sizes, prepared by other methods etc. Finally, an important point of emphasis is that our unpublished results on cupric oxide NPs indicated that the mechanism of antibacterial action

4. Conclusion

In this study, investigations were carried out to unravel the mechanism of bacterial filament formation and cell killing by the action of Cu-NPs and also the mechanism of the action of Cu-NPs. Bacterial cell filamentation was known to occur by the action of an external agent, when the agent either induced an SOS-like stress response in cells or dissipated the cell membrane potential. Our experimental results revealed that the Cu-NP-mediated bacterial cell filamentation was not due to the induction of the cellular SOS response, but due to the depolarization of the cell membrane. Our results also demonstrated that the treatment of E. coli cells by Cu-NPs at the MBC caused an increase in the cellular ROS level by 2.5-fold, compared to the level in the NP-untreated control cells, and this NP-mediated overproduction of ROSs resulted in considerable lipid peroxidation, protein oxidation and DNA degradation, finally killing the cells. ROS inhibitors such as DMSO and tris(hydroxyl methyl) amino methane were found to have little effect on the Cu-NP-mediated in vitro DNA degradation, justifying the view that the degradation was a ROS-independent phenomenon. However, the degradation was highly inhibited in the presence of divalent metal ion chelator EDTA, indicating a positive role of Cu2+ ions behind the process. The findings on the faster decrease in size as well as the count rate of the NPs in the presence of EDTA signified that the nascent Cu ions liberated from the NPs were more reactive than the already present Cu ions in the precursor CuCl2 solution. The release of nascent ions was facilitated by the oxidation of metallic NPs with the simultaneous reduction of the agents such as cells, biomolecules or medium components in the vicinity of the NPs. On this mechanistic study, questions may be raised of whether the use of a single type of bacterium such as E. coli 10

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of CuO-NPs was not through the direct route of Cu ions liberated from the NPs, but rather through the formation of some reactive complex between NPs and cellular medium organics, and no considerable decrease in size of the NPs occurred in the presence of EDTA; this implied that no single mechanism was valid for the antibacterial role of metallic Cu-NPs and NPs of other Cu-containing compounds.

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Acknowledgments

We are indebted to the Department of Biotechnology, Government of India, for financial assistance (project no BT/PR11477/ NNT/28/416/2008). We also acknowledge the Department of Science and Technology, Government of India, for its ‘FIST Programme—2001–2011’ and ‘PURSE Programme—2012–2015’, and the University Grants Commission, Government of India for its ‘Special Assistance Program—2011–2016’ going on in our department/university, to provide the different instrumental and infrastructural support.

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