Research Article pubs.acs.org/journal/ascecg
Visible Light Sensitive Mesoporous Cu-Substituted ZnO Nanoassembly for Enhanced Photocatalysis, Bacterial Inhibition, and Noninvasive Tumor Regression Jagriti Gupta† and D. Bahadur* Department of Metallurgical Engineering and Materials Science, Indian Institute of Technology Bombay, Mumbai−400076, India S Supporting Information *
ABSTRACT: Visible-light driven photosensitizers have been recognized for their potential utility for various applications especially in nanomedicine. The aim of this study is to investigate a new strategy to use Cu-substituted ZnO nanoassemblies for induction of photodynamic effect under visible light irradiation. Here, we report the synthesis of Cu substituted ZnO nanoassemblies (Cu-ZnO NAs) with optimized Cu concentration, required for enhanced photosensitive performance for sustained antibacterial and anticancer activity under dark as well as visible-light irradiation conditions. It is noted that the substitution of Cu ions in ZnO NAs remarkably improves its absorption properties and charge separation efficiency as well as reactive oxygen species (ROS) level that make it more appropriate for photodynamic therapy under visible light irradiation for killing of bacterial and cancerous cells. The generated ROS causes a significant decrease in cell viability as well as mitochondrial membrane potential. Moreover, Cu-ZnO NAs show ROS induced cellular apoptosis, DNA fragmentation, and depolarization of mitochondrial membrane and cell cycle arrest in G1 phase. These characteristics enable the use of these NAs as a photosensitizer in cancer therapy. KEYWORDS: Nanoassembly, Mesoporous, Photosensitizer, Reactive oxygen species, Theranostic agent, Cancer, E. coli
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INTRODUCTION Increasing bacterial infections and rising cases of cancer have become major challenges in the clinical settings. The continuous genetic mutations both in bacterial and cancer cells are primarily responsible for their increasing resistance toward antibiotics and medicines which pose a hindrance in healthcare applications. In order to overcome these limitations, there have been developments of alternative strategies. One of the most effective ways is the use of an optically active nanomaterial as a photosensitizer which provides a safe, selective, and noninvasive treatment for cancerous cells while avoiding side effects on healthy cells. Semiconductor oxide nanomaterials have been a current choice due to their photocatalytic activity and associated bacterial and cancer treatment possibilities. For example, TiO2, ZnO, and CuO have been widely explored as photocatalysts for degradation of dyes, and for bacterial and cancer treatment.1−9 Zinc oxide (ZnO) has acquired an increasing level of interest due to its unique optical properties. However, ZnO suffers from some specific limitations such as wide band gap, low charge separation efficiency, fast recombination rate, and inefficient utilization of sunlight particularly in the visible region. It is well documented that substitution of transition metal ions or anions forming metal/semiconductor hybrid nanostructures significantly enhances the photocatalytic efficiency of metal oxides like TiO2 and ZnO and makes them more suitable for improving © 2017 American Chemical Society
photocatalytic performance and antiviral activity under visible light.10−18 In an interesting study, N-doped TiO2 has been used in visible-light induced photocatalysis, the photogenerated holes thus produced exhibit low oxidation power and slower mobility.19,20 Visible-light activation considerably expands the utilization of photochemically active agents, potentially endowing their uses under visible-light exposure to inhibit bacterial growth and tumor regression. In a current scenario, it is required to develop efficient ZnO nanostructures, which are sensitive under visible light and could possibly be used for the degradation of dyes and bacterial inhibition as well as tumor regression. Among the transition metal ions, Cu is one of the best substituents which is known to increase visible-light absorption and hence improve photosensitization ability due to creation of energy states within the band gap along with an interfacial charge transfer (IFCT).21 When photosensetizers interact with light, electron−hole pairs are generated which are unable to react with molecular oxygen through a one-electron reduction process. However, in the presence of Cu2+ ions, these photogenerated electrons are easily consumed and Cu+ ions are formed, which considerably reduce the recombination rate Received: May 8, 2017 Revised: July 5, 2017 Published: August 28, 2017 8702
DOI: 10.1021/acssuschemeng.7b01433 ACS Sustainable Chem. Eng. 2017, 5, 8702−8709
Research Article
ACS Sustainable Chemistry & Engineering of charge carriers.17,22 Therefore, Cu+ ions act as an electron pool to initiate a multielectron reduction process (two-electron reduction O2 + 2H+ + 2e− → H2O2, 0.68 V; or four-electron reduction O2 + 2H2O + 4H+ + 4e− → 4H2O, 1.23 V) and faciliate the reduction of oxygen molecules to produce hydrogen peroxide. The resulting holes generated during the IFCT process possess strong oxidative power for the degradation of organic pollutants.17,20 These characteristic features make Cu an appropriate choice among the other metal ions for improving the properties of ZnO under visible light irradaition.23−25 There have been quite a few reports on the cytotoxicity and genotoxicity induced by CuO NPs. For example, Hossein et al. reported that Cu2+ indues hepatotoxicity. They also demostrated that Cu2+ shows a rise in mitochondrial reactive oxygen species (ROS) formation, lipid peroxidation, and mitochondrial membrane potential which start cell death signaling.26 Akhtar et al. demonstrated the cytotoxicity, genotoxicity, and oxidative stress induced by CuO NP.9 In contrast to this, Laha et al. reported in vitro growth inhibition and autophagy in MCF7 cells induced by CuO NPs. They demonstrated that CuO NPs works as autophagy inhibitor which is essential to induce apoptosis in breast cancer cells and could be an effective therapeutic strategy toward chemoresistant cancer cells.8 Furthermore, fungicides with copper (Cu) have been widely used to protect crops from fungal infections for more than one century.27 Here, Cu substitution in ZnO plays multiple roles in enhancing the interfacial charge transfer, defect chemistry, ROS generation, and bacterial inhibition. Therefore, these features make Cu substitution in ZnO more suitable as visible light driven photosensitizer for biological applications such as for baterial and cancer treatment which are hardly explored.24 Recently, Moussa et al. demonstrated that both the production of reactive oxygen species (ROS) from ZnO quantum dots and their toxicity on E. coli are enhanced by chemisorbed Cu2+ ions.28 Similarly, Sahu et al. reported the improved inactivation potential of Cu-doped TiO2 NPs under UV light due to both enhanced photocatalytic reactions and leached copper ions.29 These studies suggest that substitution of Cu ions in other transition metal oxides not only enhances the absorption but also catalyzes the ROS generation which are more responsible for killing both the bacterial and cancer cells. These ROS play a vital role in biological applications, especialy in the bacterial inhibition and cancer regression. The higher level of ROS in the cells generates the oxidative stress that damages cellular components like proteins and lipids and causes DNA fragmentaion and depolarization of mitrochondrial membrane potential. They force the cells to go for a self-assisted death mechanism through apoptosis or necrosis. However, the mechanism for the generation of ROS by ZnO is still in debate. Very few studies have been reported toward the underlying mechanism for the ROS generation both under dark and light conditions.30,31 Based on the existing literature, we can presume that the mesoporous Cu substituted ZnO nanoassemblies are an alternative solution for the visible light driven photosensitizer for photodynamic therapy for killing bacterial as well as cancer cells. In this work, we report a facile soft chemical approach for the synthesis of mesoporous Cu substituted ZnO nanoassemblies. With optimum Cu concentration, an enhanced visible light driven photocatalytic performance is realized. This is further explored as photosensitizer for killing of bacteria and cancer cells under visible light irradiation. These mesoporous
Cu substituted ZnO nanoassemblies show highly efficient sunlight induced photocatalytic degradation of organic dyes in a much shorter time than the reported Cu substituted ZnO nanostructures.22−25 A possible mechanism for the charge separation, and the generation of ROS, is discussed by considering the defect chemistry and conversion of Cu2+ to Cu+ ions. The antibacterial and anticancer activity and the cell death mechanism have been proposed by determining the ROS level both in dark and visible light, seen as a major reason for cell death. Specifically, the aim of this study is to make a visible light sensitive mesoporous nanoassembly of ZnO with a higher surface area and surface reactivity. These nanoassemblies are enriched with the defect chemistry responsible for production of an abundance of ROS. The improved properties, therefore, allow them to work as visible light active photosensitizers for the inhibition of bacteria and regression of tumors under visible light.
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MATERIALS AND METHODS
Synthesis of Cu Substituted ZnO Nanoassemblies (Cu-ZnO NAs). The porous nanostructure of Cu substituted ZnO NAs was prepared by hydrolysis of zinc acetate dehydrate with different amount of copper acetate (3, 5, and 10 wt %) in diethylene glycol (DEG) as the reaction medium. During synthesis, 2 mmol of the acetate precursors were added into 25 mL DEG with 0.5 g polyethylene glycol (PEG) and the reaction mixture was refluxed up to 170−180 °C for 1 h. After the completion of the reaction, the heat source was removed and the reaction vessel cooled down to ambient temperature (25 °C). The resulting precipitate was centrifuged and washed several times with Milli Q water, ethanol, and acetone alternately. The samples were dried at 80 °C for further characterizations. The samples were referred as Cu-ZnO NAs based on the nominal concentration of Cu and named as Cu3-ZnO, Cu5-ZnO, and Cu10-ZnO NAs in the text. The elaborate experimental procedure followed for the evaluation of photocatalysis, bacterial inhibition, and cancer therapy, for a different system is already discussed in one of our recent studies.18 A brief discussion of these is given in the Supporting Information.
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RESULTS AND DISCUSSION Structural Analysis of Cu-ZnO Nanoassembly. The morphology of Cu-ZnO NAs, analyzed by scanning electron microscopy, has been found similar for all the substituted percentages of Cu (Figure S1, Supporting Information). Thus, further detailed microstructural study is limited to Cu5-ZnO NAs. Figure 1a shows TEM micrograph of Cu5-ZnO NAs exhibiting discrete spherical, and porous morphology of diameter ∼150 ± 50 nm. High magnification TEM micrographs suggest that each nanoassembly is a self-aggregation of numerous individual nanoparticles of size ∼10−15 nm. The HR-TEM image (Figure 1b) shows the lattice fringes with a lattice spacing of 2.9 Å corresponding to (002) plane of ZnO. Figure 1c and d (I−IV) shows SEM micrographs, and its corresponding EDX elemental mapping that shows the presence as well as uniform distribution of Zn, Cu, and O (using Zn−K, O−K, and Cu−K energies). Phase analysis of Cu-ZnO NAs have been performed using XRD and Figure 1e shows XRD patterns of pristine ZnO and Cu-ZnO NAs with varying Cu concentrations. The XRD patterns correspond to the hexagonal single-phase wurtzite structure of ZnO. XRD peaks of Cu-ZnO NAs show a slight shift toward higher angle in comparison to the pristine ZnO with an increase in the Cu content. In addition, a decrease in the peak intensity and broadening of the peak width are observed. This is due to the formation of particles with smaller average diameters as a result 8703
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presence of Cu in ZnO nanoassemblies, XPS analysis was carried out on Cu5-ZnO NAs. The XPS spectra (Figure S2, Supporting Information) revealed that all Zn and Cu signals appeared in the Cu5-ZnO NAs. Figure S2a shows the highresolution XPS spectra of Zn 2p. Two symmetric peaks centered at ∼1021.5 and ∼1044.5 eV are observed which may be assigned to the Zn 2p3/2 and Zn 2p1/2 levels of Zn, respectively. These peaks correspond to Zn2+ in a hexagonal wurtzite ZnO structure. The two characteristic peaks of Cu 2p have been observed at 932.7 (Cu 2p3/2) and 953 eV (Cu 2p1/2) with their corresponding satellite peaks around 943.4 and 962.6 eV (Figure S2b, Supporting Information).24 The asymmetric peak of Cu 2p3/2 has been deconvoluted into two peaks by Gaussian fitting. Peaks centered at 932.7 and 934.4 eV correspond to the Cu 2p3/2 peak of Cu1+ and Cu2+ respectively, suggesting the presence of multivalent state of Cu species.33,34 In addition, satellite peaks of Cu 2p3/2 and Cu 2p1/2 are also observed as peaks (S1) and (S2), respectively, which are characteristic of a partially filled d-orbital (3d9 in the case of Cu2+). XPS results strongly support the multivalent state of Cu(I) and (II) in Cu5-ZnO NAs. Photoluminescence Properties of Cu-ZnO NAs. UV− visible absorbance spectra of Cu-ZnO NAs with increasing Cu content has been studied and shown in Figure S3 (Supporting Information) which show widening of the band gap. Further photochemical properties of Cu-ZnO NAs have also been explored by photoluminescence spectroscopy. Figure 2 shows the photoluminescence (PL) spectra of ZnO and Cu-ZnO NAs excited by a He−Cd lamp (λ = 325 nm) at room temperature. It exhibits two distinct emission bands for pristine ZnO and Cu-ZnO NAs. One sharp near band edge emission (NBE) in UV region (∼380 nm) is due to the radiative recombination of excitons and another broad defect emission band (450−750 nm) arises due to the native defects, such as oxygen vacancy (Vo), zinc vacancy (VZn), interstitial zinc (Zni), oxygen interstitial (Oi), and extrinsic defects by Cu substitution in ZnO.3,35,36 PL spectra clearly show that the intensity of NBE is higher in ZnO NAs whereas it decreases with Cu substitution
Figure 1. TEM micrographs Cu5-ZnO NA (a) Normal, (b) high resolution image and (c) SEM micrograph of Cu5-ZnO NAs, (d) EDX- elemental mapping (I)Mix spectral mapping (II) Zn Kα, (III) O Kα (IV) Cu Kα and (e) XRD patterns of Cu-ZnO NAs.
of increase in lattice disorder upon Cu2+ ions substitution. Zhang et al. also observed a slight shift toward higher angle of ∼0.04° in Cu doped ZnO as compared to the pure ZnO nanowires.32 The calculated crystallize size of Cu-ZnO (Cu3ZnO 15 nm, Cu5-ZnO 10 nm, and Cu10-ZnO 9.8 nm) is smaller than pristine ZnO (17 nm) which could be attributed to the lattice strain. Also, a noticeable decrease in the lattice constants has been seen with an increase in the Cu concentration. For example, the lattice constants of Cu3-ZnO NAs (a = 3.2381 Å, c = 5.1922 Å) is marginally lower than pristine ZnO (a = 3.2300 Å, c = 5.1782 Å). This observation is consistent with the fact that the ionic radius of Cu2+ is 0.57 Å whereas that of Zn2+ is 0.74 Å. The shifting and broadening in XRD peaks and decrease in lattice parameter, with increasing Cu concentration, strongly suggest that Cu is successfully substituted into ZnO lattice at Zn2+ site. Further to confirm the
Figure 2. PL spectra of ZnO and Cu-ZnO NAs at room temperature (circles show change in the intensity of UV region of ZnO and Cu-ZnO NAs). 8704
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Figure 3. Growth inhibition of E. coli in the presence of different concentrations of (a) ZnO, (b) Cu3-ZnO, (c) Cu5-ZnO, and (d) Cu10-ZnO NAs.
degradation activity over that of ZnO NAs.38 Furthermore, the enhancement in the visible light activity of Cu-ZnO NAs can also be explained on the basis of its electronic configuration. Cu2+ ion is a 3d9configuration which has a tendency to capture the photogenerated electrons while Cu+ ions have the tendency to give electrons to the surface absorbed oxygen and thus amplify the interfacial electron transfer and hence play an important role for enhancement in photocatalytic activity. Antibacterial Activity of Cu-ZnO NAs on the Inhibition of E. coli. While considering wastewater remediation, it is also very important to assess the antibacterial behavior of CuZnO NAs to make them more useful for practical applications. Besides the photocatalytic activity, Cu-ZnO NAs show appreciable bacterial inhibition in dark as well as under visible light irradiation. The antibacterial activity of Cu-ZnO NAs has been evaluated by bacterial growth curve using E. coli as a model bacterium. Figure 3 shows the growth curve of E. coli in the absence (control) and presence of different concentrations of (a) ZnO, (b) Cu3-ZnO, (c) Cu5-ZnO, and (d) Cu10-ZnO NAs. Bacterial growth curves show that ZnO and Cu3-ZnO NAs have analogous effects on bacterial inhibition. On the other hand, Cu5-ZnO and Cu10-ZnO NAs show significant inhibition of the bacteria even at low concentration (12.5 μg/ mL). A complete inhibition in the bacterial growth has been observed at higher concentrations of Cu5-ZnO and Cu10-ZnO NAs (200 μg/mL). The antibacterial activity of Cu5-ZnO NAs has also been demonstrated by live dead assay by staining with SYTO9 and PI dye (Figure S5, Supporting Information). This result shows that more death phase has been observed by the increase in the fluorescence intensity ratio of green to red with increasing Cu5-ZnO NA concentrations. The antibacterial activity of ZnO nanostructures strongly depends on the interaction, internalization, and thereafter ROS generation. The strong interaction between particles and bacterial cells starts damaging the cell membrane and provides a pathway for internalization. This, therefore, prompted ROS generation that oxidizes the lipids present in the cell membrane.
in ZnO NAs, indicating that the recombination between electron−hole pair is stronger in ZnO as compared to Cu-ZnO. Besides, defect emission intensity increases with Cu substitution in ZnO due to the replacement of Zn by Cu ions, demonstrating that the Cu is able to donate two electrons in the band formation presented as Cu2+ state (3d9), thereby enhancing the defects’ concentration in Cu-ZnO NAs.37 Therefore, higher defect concentration and efficient charge separation improve the photocatalytic activity of Cu-ZnO NAs. The photosensitization activity of the Cu-ZnO NAs, under visible light, was evaluated for the best optical performance. The samples showed appreciable photocatalytic activity when used toward photocatalytic degradation of Methylene Blue (MB), Rhodamine B (RhB), and Methyl Orange (MO). Figure S4a−c (Supporting Information) shows the photocatalytic degradation of (a) MB, (b) RhB, and (c) MO in the presence of ZnO and Cu5-ZnO NAs. The UV−visible absorbance spectra, the kinetics of photocatalytic degradation of dyes, and rate constants are given in Figure S4d−i and Table S1 (Supporting Information). The solution of MB, RhB, and MO undergoes complete degradation within 20, 45, and 45 min (photocatalytic efficiency = ∼100%), respectively, in the presence of Cu5-ZnO NAs under solar light irradiation. This is done by monitoring the characteristic absorption peak of MB, RhB, and MO at 664, 554, and 464 nm, respectively. It has been seen that Cu5-ZnO NAs exhibits the highest photocatalytic activity as compared to ZnO and Cu3-ZnO and Cu10ZnO NAs. The photocatalytic results indicate that an optimum concentration of Cu is required for making more efficient charge separation between electron−hole pairs that is well supported by PL spectra (Figure 2). It has also been noticed that the intensity of NBE in UV region decreases with increase in Cu substitution up to 5 wt % (Cu3-ZnO and Cu5-ZnO NAs) and again increases with further increase in Cu substitution (10 wt % Cu, Cu10-ZnO NAs). This suggests that the existence of multivalent Cu species (Cu1+ and Cu2+) and higher defect concentration in Cu-ZnO NAs significantly improve the charge separation and, therefore, exhibit enhanced photocatalytic 8705
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Figure 4. Photoinactivation of E. coli under solar light (SL) in the presence of Cu5-ZnO NAs (a) OD at 600 nm and (b) CFU/mL with respect to time.
explored as a photodynamic agent for cancer due to its higher photosensitivity in visible light and efficient charge separation, discussed in earlier sections, while dealing with photocatalysis and photodynamic study of bacteria. Therefore, the evaluation of the photodynamic effect of Cu5-ZnO NAs under visible light was seen using the oral carcinoma (KB) cells (SRB and LDH assay). Figure 5a shows the percent cell proliferation of KB cells
These ROS cause oxidative stress, responsible for killing of bacterial cells. Therefore, losses in the membrane integrity, oxidation of membrane lipids, and ROS generation by Cu5ZnO NAs have been studied using 3′-diethyloxacarbocyanine iodide dye (DiOC2), TBARS assay, and DCFDA dye. These results show that the Cu5-ZnO NAs strongly interacts with the cell membrane and depolarizes it (Figure S6a, Supporting Information) and, hence, oxidizes the membrane lipids, triggered by ROS (Figure S6b and c). In the present study, the antibacterial property of Cu-ZnO NAs is associated with Cu/Zn ions, their higher surface reactivity, and high level of ROS. It is observed by PL spectra that Cu substitution creates higher surface defects and oxygen vacancies in ZnO NAs that are responsible for the ROS generation. The surface of Cu-ZnO NAs is rich in electrons as well as holes. These available electrons on the surface of NAs are captured by Cu2+ ions which get converted into Cu+ while the holes react with water molecules in the microenvironment and enhance the generation of hydroxyl radicals leading to high levels of oxidative stress and bacterial cell death. Sahu and Wu et al. reported the microbial inactivation by Cu doped TiO 2 nanoparticles. They proposed that Cu2+ ions electrostatically interact with the negatively charged cell membrane and form a concentrated ionic zone of copper enhancing the antimicrobial effects of TiO2.29,39 From the observation of the photocatalytic performance of Cu-ZnO NAs, among the three compositions studied, Cu5ZnO NAs exhibit high charge separation efficiency, visible light sensation, and higher ROS level during light interaction. Therefore, Cu5-ZnO NAs have been further used for the photodynamic study on E. coli under solar light irradiation. Figure 4 shows the photodynamic effect of Cu5-ZnO NAs on E. coli under solar light, catalyzed with 200 μg/mL of Cu5-ZnO NAs, and evaluated by (a) OD measurement and (b) colony counting method in nutrient broth. The photodynamic effect (photolysis) on E. coli is negligible without catalyst under solar light irradiation while there is 100% cell death within 30 min when subjected with Cu5-ZnO NAs under solar light irradiation. This could possibly due to a very high photosensitivity with significant level of ROS generation in the presence of light. Cytotoxicity of Cu-ZnO NAs under Visible Light. Unique optical properties of oxide nanomaterials make them potential candidates for biomedical applications, especially for photodynamic therapy (PDT). The nanomaterial dependent PDT is a fast, simple, biologically safe, and noninvasive with the least permeability in normal cells. It has great potential for cancer therapeutics, as an alternative to chemo-, radio-, and surgical therapy. In this direction, Cu5-ZnO NAs has been
Figure 5. (a) Cytotoxicity study, (b) LDH release study, (c) ROS generation in KB cells incubated with different concentration of Cu5ZnO NAs in the absence and presence of visible light after 1 h irradiation, and (d) confocal image of KB cells on treatment with 50 μg/mL of Cu5-ZnO NAs after exposure of light for 1 h.
with and without visible light irradiation for 1 h with different concentrations of Cu5-ZnO NAs. KB cells do not show any cytotoxic effect in the cell proliferation in the absence and presence of light irradiation. However, 92% decrease in the cells proliferation of KB cells is evident when cells have been treated with 100 μg/mL of Cu5-ZnO NAs with exposure of visible light. Cu5-ZnO NAs also show a remarkable decrease in cell proliferation under dark condition. The cytotoxicity effect of Cu5-ZnO NAs is further confirmed by lactate dehydrogenase (LDH) release (Figure 5b) indicating the physical damage to the cells. The cytotoxic effect by Cu-ZnO NAs is also seen by morphological changes and loss in membrane integrity. The stressed and rounded morphology of KB cells is observed by optical micrographs (Figure S7, Supporting Information). A similar photokilling effect of Cu5-ZnO NAs is also observed on 8706
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Figure 6. Representative flow cytometric dot plot and cell cycle analysis of KB cells (a and d) untreated cells and (b and e) cells exposed to 50 μg/ mL of Cu5-ZnO NAs and (c and f) cells exposed to 50 μg/mL of Cu5-ZnO NAs for 1 h prior to visible light irradiation, respectively.
state (vigorous cell proliferation). If tests conducted in the dark with Cu5-ZnO NAs show 42.3% apoptotic cells, this number significantly increased to 61.7% under light irradiation. Therefore, these results strongly indicate that the percentage of apoptosis is increased under light exposure and can be explained based on the generation of more ROS. The effect of ROS generation on the cell cycle progression of KB cells is also analyzed simultaneously, by flow cytometry, after PI staining by detecting cell cycle arrest and DNA damage. Figure 6 shows the cell cycle of (d) controlled KB cells without any treatment, (e) without light, and (f) with light irradiation for 1 h in the presence of 50 μg/mL of Cu5-ZnO NAs. It was reported that nanoparticles showed cell cycle arrest during the different stages of the cell cycle. Sasidharan et al. reported the cell cycle arrest at the S/G2 phase on treatment with ZnO NCs.40 Earlier, we reported the S phase arrest in HeLa cells on treatment with FITC-ZnO nanocomposite.41 Wang et al. reported a G1/G0 cell cycle arrest in tumor cells induced by cuprous oxide nanoparticles.42 Here, the cell cycle analyses show that Cu5ZnO NAs (50 μg/mL without or with light) induced a marked increase in the number of cells in the G0/G1 phase (G1: 47.51% for control, 54.06% without light, and 70.05% with light) and simultaneous decrease in both S phase (S: 36.07% for control, 36.90% without light, and 25.59% with light) and G2/M phase (G2/M: 16.42% for control, 9.04% without light, and 4.36% with light). Thus, as compared to control, the treated KB cells show cell cycle arrest in G1 phase, observed as an increase in cell population in G1 phase. For the controls, the major cell population is observed in G1 phase, whereas for Cu5ZnO NAs treated cells, an increase in G1 population is accompanied by decrease in S and G2/M phase population. Therefore, these results suggest that the Cu5-ZnO NAs may be applied for PDT for cancer therapy of different cancers as a photosensitizer. Scheme 1 shows the possible mechanisms for the enhanced photocatalytic, antibacterial, and anticancer activity of Cu-ZnO photocatalyst in the absence and presence of visible light irradiation. During visible-light irradiation, electrons present in the valence band of ZnO react with Cu(II) through the IFCT, resulting in the conversion of Cu(II) into Cu(I), while holes remain in the VB. This generated Cu(I) ions efficiently reduce
cervical cancer (HeLa) and hepato-carcinoma (Hep G2) cells under similar conditions. Thus, the cytotoxicity study shows that Cu5-ZnO NAs can substantially obstruct cancer cell proliferation. The photocatalysis and antibacterial studies show that Cu5ZnO NAs are very effective and efficient to generate ROS when interacts with light. The ROS generation in KB cells with Cu5ZnO NAs under light irradiation was studied using nonfluorescent DCF-DA dye and the change in the mitochondrial membrane potential (MMP) using JC-10 dye. These were analyzed by the fluorescence spectroscopy and confocal microscopy after treatment with Cu5-ZnO NAs under visible light irradiation for 1 h. Figure 5c shows the ROS generation in KB cells with different concentrations of Cu5-ZnO NAs with or without light, and Figure 5d shows the confocal image of KB cells after treatment with 50 μg/mL of Cu5-ZnO NAs and exposure to light for 1 h. These results confirm that ROS level is higher under visible light irradiation as compared to dark condition. The change in mitochondrial membrane potential (MMP) has been observed after treatment with Cu5-ZnO NAs (Figure S8a, Supporting Information). The MMP results show significant decrease in ratio of red to green fluorescence intensity after treatment with Cu5-ZnO NAs (66.7% cells in a depolarized state as compared to the control cells on treatment with 25 μg/mL of Cu5-ZnO NAs). Similar changes are also observed by confocal microscopic images (Figure S7b and c, Supporting Information). The treated cells with Cu5-ZnO NAs exhibit an increase in bright green fluorescence with a remarkable decrease in red fluorescence, clearly indicating the loss of MMP. Therefore, it may be inferred that Cu5-ZnO NAs destabilize the mitochondrial membrane potential in cancer cells by generating ROS and release of Cu2+ and Zn2+ ions in the cytoplasm.8,29 The mechanism of cell death is further illustrated by cellular apoptotic study by double staining using annexin-V and PI under dark and visible light irradiation. Figure 6 shows the representative flow cytometric dot plots of (a) control KB cells, (b) treated KB cells with 50 μg/mL Cu5-ZnO NAs under dark conditions, and (c) treated KB cells 50 μg/mL Cu5-ZnO NAs under visible light irradiation for 1 h. These results depicted that controlled KB cells show only 5.3% cells in the apoptotic 8707
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generated by reactive oxygen species. To summarize, among these three compositions, Cu5-ZnO NAs seem to be the best for the degradation of organic dyes and inactivation of bacterial pathogens and very effective toward cancer cells.
Scheme 1. Proposed Mechanism for ROS Generation and Bacterial Inhibition under Dark and Light Conditions Using Cu-ZnO NAs
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.7b01433. Further experimental details, Figures S1−S9, and associated references (PDF)
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AUTHOR INFORMATION
Corresponding Author
*Tel.: +91 22 2576 7632. Fax: +91 22 2572 3480. E-mail address:
[email protected]. ORCID
D. Bahadur: 0000-0002-5092-6624 Present Address †
Chemistry Division, Bhabha Atomic Research Centre, Mumbai 400085, India.
oxygen molecules through a multielectron reduction process that again regenerates Cu(II). The holes react with the surface hydroxyl groups and water molecules and form highly reactive hydroxyl (OH•) radicals. These reactive species (OH•) react with the dye molecules adsorbed on the photocatalyst surface and enhance the photocatalytic degradation of dyes. The generation of hydroxyl radicals is further supported by fluorescence spectroscopy, wherein, increasing fluorescence intensity of terephthalic acid is seen under dark and visible light as shown in Figure S9 (Supporting Information). ROS results indicate that Cu-ZnO NAs specifically generate hydroxyl radicals. Additionally, Cu moieties make Cu-ZnO NAs very effective for the antibacterial and anticancer activity under dark conditions as well as under visible light irradiation. During dark and visible light exposure, both hydroxyl radicals and Cu moieties damage the membrane of cells and degrade proteins, and DNA of cells, resulting in cell death. Therefore, Cu-ZnO NAs exhibit antibacteria and anticancer activity under dark as well as visible light irradiation.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS J.G. acknowledges SERB, India, for the award of a National Post-Doctoral Fellowship. The financial support by the Nanomission of DST, Government of India, is gratefully acknowledged. The authors are thankful to the Centre for Research in Nanotechnology & Science (CRNTS) and central facility IIT Bombay for TEM and XPS analysis.
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REFERENCES
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CONCLUSION A facile soft-chemical approach for the synthesis of Cu substituted ZnO NAs and studies relating to its structural, photocatalytic, antibacterial and anticancer properties have been reported. With different Cu concentrations, these nanoassemblies exhibit fascinating alterations in structural, optical, and photocatalytic properties. The porous morphology of Cu-ZnO NAs provides efficient interaction, diffusion, and transportation of organic dyes with ZnO during the photocatalysis. The formation of different energy states, change in absorption properties, existence of Cu in two valence states along with an interfacial charge transfer (IFCT) in Cu substituted ZnO NAs significantly diminish the recombination rate of photogenerated charge carriers. The higher concentration of ROS and the presence of Cu substantially improve the antibacterial activity of the nanoassembly. The unique properties of Cu-ZnO NAs enable it as a visible light sensitive photosensitizer, which exhibits remarkable photodynamic effect causing significant reduction in viability of the cancer cells. This could be attributed to a higher level of oxidative stress 8708
DOI: 10.1021/acssuschemeng.7b01433 ACS Sustainable Chem. Eng. 2017, 5, 8702−8709
Research Article
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