CuO and CuO@SiO2 AS A POTENTIAL ANTIMICROBIAL AND ANTICANCER DRUG
AUTHORSPreetha Bhadra, Biplab Dutta, Debopriyo Bhattyacharya, Sampad Mukherjee
Copper has found to be one in every of the main trace components among of all found within the human body and additionally co-factor of quite three hundred class enzymes, plays a very important role in maintaining cellular processes which are crucial as well as stress, desoxyribonucleic acid replication, repairing of DNA, progression of cell cycle and programmed cell death. Thus, it has found to be very evident that Associate in Nursing alteration in Copper levels in willcer cells can cause a harmful result. analysis has shown that low concentration of Copper in cells ends up in the initiation and progression of cancer and high concentration of Copper shows harmful effects. Copper-mediated super molecular activity situation and aerophilous stress through reactive oxygen species (ROS) might also be the probable cause of this cytotoxic result. CuO contains a neutral hydroxyl radical connected to its surface, that plays a very important role in the changes of behaviour of the surface . Our aim is to indicate that the result of Copper oxide and silicon coated copper oxide on totally different microbes and cancer cells. Characterization of Cu nanoparticles have been done by using different analyzing techniques i.e. UV-Vis Spectroscopy, DLS (Dynamic Light Scattering) and SEM (Scanning Electron Microscope). The effect of the Cu nanoparticles on microbes has been measured by the cup disk method where as the effect on cancer cell line (HeLa) (Human Cervical Cancer Cell Line) has been measured by Fluorescence Anisotropy, MTT assay, Reactive Oxygen Species (ROS). The effect on different enzymatic action has also been measured.
KEYWORDSCopper Oxide, Fluorescence Anisotropy, MTT assay, Reactive Oxygen Species, UV-Vis Spectroscopy
There are numerous concoction and natural mixes with prescription movement like, penicillins (β-lactams gathering) and regular item that execute microorganism or subside their development (Wright et.al,2014) Among them, nanoparticles (metallic and semiconductor) have as of late gotten a great deal of consideration (Li, 2010) Reactive substance component species (ROS) like superoxide anion (O2-), hydrogen peroxide (H2O2), hydroxyl radicals (HO-) and natural hydroperoxides (OHP), nanoparticles statement on the outside of microorganisms and nanoparticles collection inside the cytoplasm/periplasmic district of microorganism, might be result in microorganisms demise (Li, 2013). Bio-nano atoms are those, whose estimate is practically identical nanoparticles, assume an ineluctable indispensable job in charge changed cell cycles of the body and keeping up urgent cell homeostasis (Gunjan and Sagar, 2016). With right bioengineering, Nanoparticles might be sent in an exceedingly limited condition in any arrangement of the body thus it will join the movement of organic parts, so imitating the natural arrangement of the body reliable with the necessity for human benefit. Nanoparticles are very solvent gratitude to their little size and in this manner their dissolvability might be any duplicated by right surface alteration and the high degree to volume quantitative connection of these particles, fabricate them having adequate degree to epitomize drug and elective materials, so giving the higher restorative payload. Another property of those nanoparticles might be spoken to in light of the fact that the particular focusing on nature, so Nanoparticles will explicitly release a helpful payload onto the objective, lessening the feature impacts on customary cells (McNeil et.al, 2009, Wang et.al, 2013). investigation and improvement inside the field of designing ar developing hack cleave all through the planet (Vidya et al., 2013). a genuine commitment of this field is that the advancement of most recent materials inside the micromillimetre scale (nanoparticles). These ar now and then particulate materials with at least one element of however a hundred nanometers (nm), even the particles likely could be zero measurement inside the instance of quantum dabs (Vidya et al., 2013). Metal nanoparticles are of decent intrigue on account of their particular alternatives like compound activity, optical, attractive and electrical properties (Garima, et al., 2011). Nanoparticles display completely new or improved properties with bigger particles of the dominant part materials, and these novel properties ar inferred gratitude to the variety in explicit attributes like size, dissemination, and morphology of the particles (Ravindra, et al., 2011). essentially, nanoparticles (NP) made of metal oxides with sizes yet a hundred nm display antimicrobial exercises because of their unique attributes (for example little molecule measure, goliath surface territory), that smaller scale or full scale estimated particles don’t have. Copper oxide is generally used in the division of substance activity, superconductors, and earthenware production as a kind of crucial inorganic materials. It might be utilized as an impetus and impetus support, yet as conductor dynamic materials like corruption of chuckling gas with alkali and substance response of CO, natural compound and phenol in basic water (Motogoshi et.al, 2010). Metal substance compound nanoparticles, similar to oxide (CuO), have pulled in consideration mainly inferable from their antimicrobial and biocide properties and that they could likewise be used in a few medication applications (Perreault et.al, 2012, Nations et.al, 2015). oxide might be a semiconductor metal with particular optical, electrical and attractive properties and it’s been utilized for fluctuated applications, similar to the occasion of supercapacitors, close infrared channels, in attractive capacity medium media, sensors, catalysis, semiconductors, and so on. (Dagher et.al, 2014, Devi et.al, 2014, Zhang et.al, 2014). For this situation, one in everything about fundamental connected materials inside the exchange is an oxide (CuO) and its combination in nanometer scale (Huang et.al 2010, Longano et.al. 2012, Mudunkotuwa et.al, 2012, Taran et.al., 2016). Additionally, these aluminous nanoparticles might be utilized as an other for silver and gold nanoparticles (Athanassiou et.al, 2006, Rubilar et.al, 2013). Metal concoction compound nanoparticles like CuO might be utilized as antimicrobial operators attributable to their adequacy on safe strains of microbial pathogens, low poisonous quality, and warmth opposition (Emami et.al,2011). CuO nanoparticles are successful in murdering a spread of microorganism. Be that as it may, CuO nanoparticles with high focus are expected to get the disinfectant effect. The disinfectant property of such nanoparticles relies upon their size, steadiness, and focus extra to the extension medium, that gives bigger maintenance time to microorganism NP association (Ravishankar et.al, 2011, Azam et.al, 2012). Regardless of antimicrobial medicinal consideration, dismalness and mortality identified with these microorganism contaminations remain high, somewhat because of the adaptability of those life forms to create protection from almost all anti-infection agents. New techniques are therefore required to spot and build up the resulting age of prescription or operators to manage microorganism contaminations
MATERIAL AND METHODS
Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% heat inactivated fetal calf serum (Gibco™, Thermo Fisher Scientific), penicillin (100 U/mL) and streptomycin (100 μg/mL) (Merck, India), TMA-DPH (1-(4-Trimethylammoniumphenyl)-6-Phenyl-1,3,5-Hexatriene p-Toluenesulfonate) ( Thermo Fisher Scientific), Dry N,N,dimethylformamide (DMF; Merck, India), nickel chloride hexahydrate (NiCl2, 6H2O, Mw = 237.69 g mol−1; Merck, India), sodium hydroxide (NaOH; Merck, India), ethyl alcohol (C2H6O; Merck, India), tetraethyl orthosilicate (TEOS; Merck, India), and ammonia solution 25% (NH4OH; Merck, India) were used in this work. All the materials were used in the experiments without further purification.
PREPARATION OF CuO@SiO2 NANOPARTICLE
The aqueous procedure has been acclimated with blend the oxide nanoparticles (Dutta et.al., 2017). Subtleties of that aqueous technique for the amalgamation of metal-synthetic compound nanomaterials are alluded from (Dutta et.al., 2015). A changed Stöber (Stober et.al., 1968) system, a generally utilized procedure for the blend of oxide nanoparticles has been acclimated orchestrate the silica-covered CuO. Amid this ordinary amalgamation technique, hydrothermally combined CuO nanoparticles (Dutta et.al., 2017) were added to the arrangement of water and alkyl liquor (in volume apportion rough 4:1). to understand an all-around scattered blend, the arrangement was sonicated for ten min. From that point onward, smelling salts were added to the blend (in volume quantitative connection one.4:50) visit drop to change express the CuO nanoparticles in alcoholic media. The blend was yet again sonicated for forty min when the option of smelling salts, and finally, TEOS was extra visit drop to the blend (in volume quantitative connection pretty much zero.4:50). a definitive blend was solid beneath vigorous attractive mixing (500 rpm) for eighteen h. The very much blended colloid was centrifuged at 4000 rpm and washed by grain liquor to dispose of the residuals from the stock. The gathered item was dried at 80°C and used for extra portrayal.
Figure 1 and Figure 2 are demonstrating the FTIR investigation and XRD examination report.
Figure 1 FTIR analysis of CuO nanoparticles
FTIR spectra were recorded in solid part using the KBr pellet technique within the regions of 3500–400 cm−1. FTIR spectra of CuO nanoparticles square measure shown in Figure 1. FTIR spectra of CuO, nanoparticles exhibited vibrations within the region 400–600 cm−1, which might be attributed to the vibrations of M−O (M = Cu) that confirms the formation of CuO nanoparticles. A weak band at around 2300 cm−1 is also attributed to the vibrations of part carbon dioxide. The current findings agree well with the values reported within the accessible literature (Ansari et.al, 2011, Arshad et.al, 2011).
Figure 2 XRD analysis of CuO nanoparticles
The regular XRD examples of the CuO nano¬particles toughened at 400°C are appeared in Figureure 2. The peak positions of samples display the hexagonal, monoclinic, and rhombohedral structures of CuO. Besides, no debasement tops were seen in the XRD designs, as the majority of the three metal oxides indicated single-stage test development. The crystallite estimate was determined to utilize the Scherrer equation,
D = 0 9λ/cos β θ
where λ is the wavelength of X-beam radiation and β is the full width at half limit of the tops at the diffract¬ing edge θ. Crystallite sizes were determined to be 18 nm, 22 nm, and 26.1 nm for ZnO, CuO, and Fe2O3 nanoparticles, individually.
Figure 3 Uv-Vis spectroscopy analysis of different concentration of CuO nanoparticles
ANTI MICROBIAL ACTIVITY: CUP-DISC METHOD
The number of the zone of inhibition has been deduced from three parallel studies and those are taken as the mean value of those. These studies were compared with the known drugs available in the market. The CuO and CuO@SiO2 showed an average value of the zone of inhibition where the combination of these two nano-particles showed a maximum zone of inhibition. The lower concentration of the mixed drug has an effect on the bacterial and the fungal growth which has been measured by calculating the zone of inhibition and the values are (+/-) SD of three parallel measurements. The bacterial culture method has been shown in Figure 4. (A,B, C) are the different concentration of CuO nanoparticle solutions.
Figure 4 Schematic diagram of cup disk method
Human cervical malignant cancer tissue growth cell lines (HeLa) were kept up in Dulbecco’s Modified Eagle’s Medium (DMEM) enhanced with 10% warmth inactivated Fetal brovine serum (FBS) (Gibco™, Thermo Fisher Scientific), penicillin (100 U/mL) and Streptomycin sedate (100 μg/mL) at 37°C in a very humidified air containing 5% CO2gas. Hela cells at a measure of 1.5×105 cells/mL were experienced childhood in an exceptionally 25 cm2 flagon of complete culture medium. At 90% intersection, Hela cells were then trypsinized and seeded on a 96 well tissue culture plate for medium-term with the selection of examinations.
Approximately 1 × 105 mL−1 Hel cells in their exponential growth section were seeded in an exceedingly flat-bottomed 96-well Tissue culture plate for 24hr at 37°C in in a 5% CO2 incubator. Different concentrations (5, 25, 50, 100, and 250 μg/mL) of CuO and CuO@SiO2 nanoparticles were added within the medium to the plate in an exceedingly triplicate manner. Cytoxicity analysis of CuO and CuO@SiO2 nanoparticle was performed using MTT assay and MTT was added to every well and also the plates incubated for 3hr in an exceedingly dark chamber. 100 μl of DMSO was else to dissolve the formazan crystals and also the absorbance browse at 540 nm victimization enzyme-linked-immunosorbent serologic assay reader (EPOCH, BIOTEK) (Zhu et al., 2001). The viable cells were calculated with respect to untreated cells as 100 %.
% of Viable Cells = (A) Control – (B) test
Control X 100
Where (A) test is the absorbance of the test sample and (B) control is the absorbance of the control sample. Non-treated cells were used as the control, and the samples were imaged using an inverted photomicroscope. The Values of MTT assay correspond to mean and standard deviations of three independent experiments.
The fluorescence anisotropy of HeLa was assessed by the determination of TMA-DPH steady-state fluorescence polarization after the cell membrane exterior phospholipid layer permeation of the probe (Dowell, 2002; Pearson, T.,1996; Pearson, T. et.al,2001, Shrivastava, et.al. 2007; Katona,2004; Lakowicz,2004; Hollan, 1996).
For the measuring of the changes within the TMA-DPH fluorescent properties following the membrane permeation, we added 2.5µM TMA-DPH to a 2 ml of cell in the measuring cuvette. The cell suspension with the fluorescent probe was incubated for 30 min at 37°C. The measurement has been done between excitation and emission state, 360 nm and 430 nm respectively.
Membrane fluidity of cancer cells was shown to possess a decisive role within the direct cell to cell contact and also the modulation of the activity of membrane enzymes area unit to be stricken by the enhancement of the reactive oxygen species (ROS) (Garden, 2001). For the measuring of the animate thing ROS, DCF-DA was more to a two milliliter of Hela suspensions. The cell suspension with DCF-DA was incubated for sixty min at 370 C in a very dark condition. Cells not treated with Nanoparticles were used as management. Fluorescence intensity was measured in a Fluorescence radiation photometer (model Hitachi, USA) at excitation and emission wavelengths of 504 and 529 nm respectively.
ANTIOXIDANT ENZYMES ACTIVITIES
The antioxidant activity of the cells has been measured by the Catalase (CAT) and Superoxide dismutase (SOD) test with commercially available kits. The 7×105 cells were plated into 12- well plates and according to supplier’s recommendations, all experimental measurements were performed.
RESULT AND DISCUSSION
CUP DISK METHOD
The Copper Oxide Nano Particles have shown better effect than the Silica coated nano Particles. In some cases the Copper Oxide nanoparticles have shown good results in comparison to the known drugs which is a positive indication of using these nanoparticles as a potential antimicrobial drugs. We also checked their activity on the fungal growth and these particles have also shown a great effect on reducing the fungal growth. These nanoparticles can also be used to reduce the fungal growth and the contamination occurs from it. The results of the zone of inhibition in bacteria and the antibiotics have shown in table 1 and table 2 and the zone of inhibitions for fungi and available anti-fungal have shown in table 3 and table 4.
Table 1 Antibacterial Activity
zone of inhibition of bacteria: (values are mean of three parallel measurements = number of zones of inhibition.) (Concentration in µg/ml and Zone of inhibition in mm)
|Drug Name||Drug Concentration
|E. coli||S. typhi||S aureus||S.pyrogenes|
Table 2 Known drugs used for bacteria
|Drug Name||Drug Concentration
|E. coli||S. typhi||S aureus||S.pyrogenes|
|50 26 24 23 20|
|100 28 27 25 22|
|250 30 29 28 25|
Table 3 Anti Fungal Activity
Zone of inhibition of fungi: (values are mean of three parallel measurements = number of zones of inhibition). (Concentration in µg/ml and Zone of inhibition in mm)
|Drug Name||Drug Concentration
|A. nigar||C. clavus||C. albicans|
Table 4 Known Drug Concentration
|Drug Name||Drug Concentration
|A. nigar||C. clavus||C. albicans|
Figure 5 have shown the graph for the fluorescence anisotropy. The decreasing value of the graph has clearly shown the minimal interaction of CuO particle with the cell membrane, which can be a positive result for the use of metal oxide nanoparticle. These results have shown that the nanoparticles were interacted with the cellular membrane and dissociated the membrane proteins to enter into the cell and to interact with the cytoplasmic organelles. Thus these nanoparticles have interacted with the mitochondria and reduce the growth of the cancer cells. These findings have also been supported by the ROS analysis.
Figure 5 Fluorescence Anisotropy of HeLa cells treated with both the nanoparticles with different concentration. Data represented are mean ± SD of five independent different experiments
MTT assay was performed to check the cell viability in those cells which are stressed by Copper Oxide nanoparticles and Silica coated Copper Oxide nanoparticles. First we evaluated the effects of CuO Nanoparticles on HeLa cells viability. Incubation with 5µg/ml, 25µg/ml, 50µg/ml, 100µg/ml, 250µg/ml for 3, 6, 12 and 24 hr which has been found in a decrement of concentration dependent cell viability, the LC50 was 79.83 ± 0.856 μg/ml. Based on these results Copper Oxide nanoparticles and Silica coated Copper Oxide nanoparticles at submaximal concentrations after 12 h, 50 and 100 μg/ml were selected in this study. The significant findings of this assay was the LC50 of Copper Oxide nanoparticles and Silica coated Copper Oxide nanoparticles was 79.83 μg/ml and submaximal concentrations of 80 and 100 μg/ml were selected for the particular study. There are similarities in the results found in the previous findings and with ours dose dependent reduction value of MTT in HeLa cellswhich were treated with CuO nanoparticles and Silica coated CuO nanoparticles, though cells were different (M. Ahamed ,2011, L. Capasso, et.al. 2014).
According to the Natiaonal Cancer Institute (USA), vegetables crude extracts are cytotoxic considered when their IC50 values are less than 30 μg/ml (M. da, et.al. 2013). After a large screening, CuO nanoparticles and Silica coated CuO nanoparticles (60 and 80 μg/ml) concentrations was being selected because of their best actions. The present study agree with the results of Remila et al. (2015) who have demonstrated that the pre treatment with P. lentiscus extracts for 24 hr in THP-1 cells were strongly inhibited H2O2 damage, with maximum protection at 100 μg/ml (S. Remila, et.al. 2015). The triplicate study of the cell culture has shown that the number of cells is decreasing by the increment of time and the concentration of the drugs respectively.
Figure 6 MTT Assay of HeLa cells treated with both the nanoparticles with different concentration. Data represented are mean ± SD of five independent experiments. Where X-axis is showing the Number of cells and Y-axis represents the drug concentration.
To discover the impact of Copper Oxide nanoparticles and Silica-covered Copper Oxide nanoparticles by actuating cytotoxicity in the cells through the creation of ROS, HeLa cells were treated with the diverse chose groupings of the Copper Oxide nanoparticles and Silica-covered Copper Oxide Nanoparticles. We have recognized a huge decrement of ROS level in cells which were being treated with Copper Oxide nanoparticles and Silica-covered Copper Oxide nanoparticles (Figure: 7). Oxidative pressure has been observed to be an irregularity between the generation ROS and the cancer prevention agent frameworks which were favoring an abundance creation of ROS which has been found as a typical system for harming the phone. Amid the oxidative pressure, the ROS is delivered fundamentally from the electron transport chain of mitochondria. To upgrade the harm prompted by the age of ROS, free radicals from various nanoparticles (which has been utilized for the treatment) can be changed to different less lethal atoms (E. Huerta-García, et.al, 2014). Nanoparticles were having a higher surface zone and have just been exhibited as creating more free radicals and ROS than bigger particles (C. Sioutas, et.al, 2005). It has been discovered that the NiO Nanoparticles can decrease cell feasibility and can incite oxidative worry by consumption of glutathione and enlistment of responsive oxygen species in HEp-2 and MCF-7 cells (M.A. Siddiqui, et.al, 2013), can likewise cause cell passing by various portion subordinate action so as to guarantee the apoptotic pathway and ROS age in HepG2 (M. Ahamed, et.al, 2012), CuO nanoparticles has also been found to increase the production of intracellular ROS, apoptosis (the natural cell death) and necrosis (induced cell death) in BEAS-2B and A549 cells (L. Capasso, et.al, 2014), Our results confirmed that Copper Oxide nanoparticles and Silica-coated Copper Oxide nanoparticles are toxic to HeLa cells. In Figure 6, ROS analysis has been shown in triplicate studies. These analyses showed that the requirement of the oxygen got low with the increase of time and concentration of the drug. These need for oxygen lead the cells to the apoptosis and thus the cell dies due to the treatment of the drugs. These have also coincided with the result of the MTT assay. With the increment of the nanoparticles concentration, the number of viable cells decreased. The nanoparticles showed the better result as the variation of the valance electron was increased as a result those reacted with the protein particles of the cells and dissociated it which leads the cells to destroy.
Figure 7 ROS of HeLa cells treated with both the nanoparticles with different concentration. Data represented are mean ± SD of five independent experiments. Where X-axis is showing the Number of cells and Y-axis represents the drug concentration.
ANTIOXIDANT ENZYMES ACTIVITIES
The HeLa cells were pre-incubated with concentrations of 50 and 100 μg/ml of Copper Oxide nanoparticles and Silica-covered Copper Oxide nanoparticles which is directed to expanding the cancer prevention agent enzymes, Superoxide dismutase and Catalase, exercises appeared in Figures. 5 and 6. Essentially, the Copper Oxide nanoparticles and Silica-covered Copper Oxide nanoparticles likewise found to prompt huge weariness of cancer prevention agents. The aggregation of ROS, for example, superoxide radicals (O2%) and hydroxyl free radicals (%OH) has observed to be diminished which likewise decline the guarded impacts of cancer prevention agent catalysts of cells, for example, e.g. SOD, CAT (A. Li, et.al. 2012). Introduction of HeLa cells to CuO nanoparticles brought about a diminishing in the movement of SOD and the other detoxification proteins which has been found in comparative as hippocampal cells (K. Niska, et.al. 2015).
Figure 8 Effects of both the nanoparticles on the Superoxide dismutase activity in HeLa cells. These HeLa cells were treated with different concentrations for 3, 6, 12 and 24 hrs. Data are mean ± SD of five independent experiments.
Figure 9 Effects of both the nanoparticles on the activity Catalyse in HeLa cells. Cells were treated with different concentrations for 3, 6, 12 and 24 hrs. Data are mean ± SD of five independent experiments.
In drug, nanoparticles are observed to be the most up to date and the rising point of intrigue. The promising qualities of the nanoparticles in the in vivo use of nanoparticles are as yet uncommon. Thus, an expansive possibility of working together among clinicians, scholars and material researchers square measure required for the comprehension of malignant growth science and shrewd style of nanoparticles for his or her higher clinical use. A dynamic coordinated effort could lead on to the occasion of good nanoparticles that show unrivaled exactness of property and harmfulness towards malignancy cells though causing no hurt to customary cells. This is, actually, partner degree accomplishable point, considering the incredibly encouraging qualities of CuO nanoparticles and their inborn nature of property and lethality towards malignancy cells, making them without uncertainty a key instrument for cutting edge disease treatment.
CuO nanoparticles gave noteworthy antibacterial properties against various sustenance borne pathogens just as organisms and the inhibitory impacts of the nanoparticles expanded concerning the expanded centralizations of CuO nanoparticles. CuO nanoparticles could misshape bacterial cell layer, promoting loss of intracellular parts, and at last the passing of cells. These outcomes exhibit that CuO nanoparticles could be possibly considered as a powerful antibacterial specialist for securing rural and sustenance wellbeing.
Consequently, we have discovered that the CuO can be a potential anticancerous and antimicrobial medication for the up and coming age of treatment.
We like to thank Department of Biotechnology (Government Of India) and Centurion University of Technology and Management, Bhubaneswar for funding our work.
Acknowledgement: We want to acknowledge the Department of Biotechnology (Govt. of India) for the financial support. We would also want to thank Dr. Gautam Bose from J.C. Bose Institute and Department of Biotechnology of Jadavpur University for running of the different experiments.
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