Effect of the Graphene- Ni/NiFe2O4 Composite on Bacterial Inhibition Mediated by Protein Degradation.

Recent investigations have demonstrated that nickel ferrite nanoparticles and their derivatives have toxicity effects on bacterial cells. In this study, we have prepared nickel ferrite nanoparticles (Ni/NiFe2O4) and nickel/nickel ferrite graphene oxide (Ni/NiFe2O4-GO) nanocomposite and evaluated their toxic effects on E. coli cells ATCC 25922. The prepared nanomaterials were characterized using X-ray diffraction, Raman spectroscopy, scanning electron microscopy, transmission electron microscopy, and vibrating sample magnetometry techniques. The toxicity was evaluated using variations in cell viability, cell morphology, protein degradation, and oxidative stress. Ni/NiFe2O4-GO nanocomposites likewise prompt oxidative stress proved by the age of reactive oxygen species (ROS) and exhaustion of antioxidant glutathione. This is the first report indicating that Ni/NiFe2O4-GO nanocomposite-initiated cell death in E. coli through ROS age and oxidative stress.

Introduction

Magnetic nanoparticles have indicated promising outcomes in organic applications. Among various iron oxide nanoparticles, spinel ferrite nanoparticles have incredible potential that has been investigated for organic applications and unclean water treatment.

The general formula for spinel ferrite nanoparticles is MFe2O4 (where M is +2 cation of Ni, Mn, Zn, or Co). They have intriguing magnetic and electrical properties with great chemical and thermal strong qualities.[1] These nanocrystalline materials are utilized in numerous applications including magnetic extraction, magnetic resonance imaging, cell naming, drug delivery conveyance, and hyperthermia.[2−5] Nickel ferrite (NiFe2O4) is one of the main spinel ferrites.[6−8] Regardless of the widespread utilization of nickel ferrite nanoparticles, there is a genuine absence of data concerning the poisonousness of these nanoparticles at the cell and atomic levels.

Concurrent reduction of graphene oxide and development of nanocomposite would mean low cytotoxicity. A couple of examinations detailed the likely cytotoxicity of spinel ferrite nanoparticles including nickel ferrite nanoparticles.[9−11] Nickel ferrite nanoparticles demonstrated insignificant changes in HeLa cell expansion at 10 g/mL and low practicality at concentrations of 100 g/mL.[12]

Because of the wide range utilization of magnetic nanoparticles, it is reasonable to assess their possible dangers to the environment and human wellbeing. Results available in the scientific literature concerning the poisonous capability of iron-based nanoparticles are clashing. These nanoparticles have been reported to be poisonous.[31−33,18] However, others report that they do not just show great biocompatibility yet, in addition, apply exceptionally low harmfulness.[34,35] One of our past investigations has demonstrated that the nickel (Ni) nanoparticles are toxic to human lung epithelial cells.[36] These previous results attract further examination on the harmfulness of Ni/NiFe2O4–GO to other bacterial cells (e.g., E. coli (ATCC 25922)).

Oxidative stress is proposed as a traditional mechanism of cell harm prompted by numerous kinds of nanoparticles.[14,15,17] For instance, iron-based nanoparticles created poisonous to natural frameworks by producing reactive oxygen species (ROS).[31,32,37] Cells have various systems of security against oxidative harm, incorporating direct communication with enemies of oxidants. ROS has been proposed to flag particles for the commencement and execution of the apoptotic passing system.[38,39] The creation of ROS, specifically, has likewise been related to modified cell demise in numerous conditions—for example, stroke, lung edema, ischemia, aggravation, and neuro-degeneration.[40,41]

The studies of the molecular harmfulness of nanoparticles are in progress. One component in the debate is the enlistment of oxidative harm to cell constituents, either because of the age of ROS or through the inactivation of the antioxidant defense framework.[13,14] Trial proofs suggest that metal oxide nanoparticles actuated nucleic acid harm and apoptosis via ROS age and oxidative stress.[15−18] Similar investigations demonstrate that silver, copper oxide, and silica nanoparticles initiate cytotoxicity through lipid peroxidation, ROS age, and oxidative stress.[19−21] Protein degradation of E. coli could clarify the activity by Ni/NiFe2O4–GO nanocomposite. The present research reports the preliminary results of the toxic effects of the as-synthesized Ni/NiFe2O4–GO nanocomposite on the bacterial cell of E. coli ATCC 25922. It presents a detailed study regarding protein denaturation and oxidative stress conducted on E. coli cells.[22−25] Moreover, similar to our previous reports on different ferrites for antibacterial activity,[26−30] this material also exhibits anomalous magnetism behavior.[6]

Materials and Methods

Materials

All the chemicals were of analytical grade. Hydrogen peroxide (30 wt %), graphite powder, sodium nitrate, potassium permanganate, sodium acetate (NaAc) sulphuric acid (98 wt %), CoCl2·6H2O, FeCl3·6H2O, and polyethylene glycol (PEG) were purchased from SD-Fine and used as-received without further purification.

Methods

Synthesis of the Ni/NiFe2O4–GO Nanocomposite

GO was prepared from normal graphite by using a modified Hummer’s technique. Nickel ferrite nanocomposite was integrated by a solvothermal process. In an ordinary system, 300 mg of GO, 1 mmol of NiCl2·6H2O, and 6 mmol of FeCl3·6H2O were blended and broken down in a proper amount of ethylene glycol followed by sonication for nearly 2 h. At that point, the blend was exposed to mixing by adding sodium acetate and PEG for some time. The blend was fixed in a Teflon-cut autoclave, kept on a hot-air stove at 200 °C for 10 h, and then saved for cooling at room temperature. The product mixture was isolated by centrifuging a few times with water and ethanol, and afterward, the item was dried at 60 °C.

Characterization

X-ray diffraction (XRD) patterns were recorded on a Rigaku Miniflex using the Cu Kα radiation (λ = 1.5406 Å) over a 2θ range from 10–80 °C. Raman measurements were done in the backscattered geometry using a laser excitation source (He–Ne laser) emitting at 633 nm with 20 mW power coupled with an ARAMIS (Horiba Jobin Yvon, France) micro-Raman spectrometer. The microscopic details of the samples were analyzed by field-emission scanning electron microscopy (Hitachi S-4800) and high-resolution transmission electron microscopy (HR-TEM) with an energy dispersive spectrometer (JEOL-2000EX, JEOL, Tokyo, Japan) operated at 120 kV. X-ray photoelectron spectroscopy (XPS) measurements were also carried out.

Toxic Effect of the As-Prepared Ni/NiFe2O4–GO Nanocomposite

All synthetic substances were bought from Sigma-Aldrich and utilized minus any additional purging. Standard E. coli culture was secured from Haffkine Institute, Mumbai. An overnight developed culture of E. coli was vaccinated in an isotonic saline arrangement. The suspensions of bacterial cells were weakened to acquire cell tests containing 106 to 107 CFU/mL. A 100 μg of GO, NiFe2O4, and Ni/NiFe2O4–GO nanocomposite was separately scattered in a 1 mL isotonic saline arrangement (0.9 w/v % of NaCl) and sonicated for 30 min. E. coli cells were independently brooded with GO, Ni/NiFe 2O4, and Ni/NiFe2O4–GO nanocomposite, scattering in isotonic saline arrangements at 37 °C for 2 h under stirring at 200 rpm. The misfortune % of the practicality of E. coli cells (ATCC 25922) was assessed by the colony counting strategy. Momentarily, an arrangement of 10-overlay cell weakening (100 μL each) was spread onto supplement agar plates and left to become for the time being at 37 °C. States were checked and contrasted with those on control/reference plates to ascertain variations in the cell development restraint. The E. coli in the isotonic saline arrangement was utilized as a control. All treatments were set up in sets of three and rehashed at any rate on three separate events. Further, focus and time subordinate examinations were likewise performed. A 0–200 ppm of Ni/NiFe2O4–GO was read for concentration subordinate examinations and 0–90 min for time subordinate investigations.

Cell Morphology Observation

ATCC 25922 E. coli cells were treated with GO and Ni/NiFe2O4–GO for 1 h. The resultant samples were washed with ethanol followed by centrifugation at 2000 rpm in a microcentrifuge to eliminate debris. The acquired pellet was washed two times and scattered in sterile distilled water. It was then covered with a coverslip, dried at room temperature, and coated with a conductive layer on the pellet for SEM imaging.

Estimation of Total Protein Degradation in E-coli by the Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) Method

E. coli (ATCC 25922) treated with GO, Ni/NiFe2O4, and Ni/NiFe2O4–GO dispersions were mixed independently with 5 mL of phosphate buffer followed by centrifugation at 8000 rpm for 20 min. The supernatant was gathered and rehashed multiple times. The acquired supernatant was weakened with 50 mL phosphate buffer. 1 mL 20% Tri chloro-acetic acid was then added and kept for 30 min. Centrifugation of 20 min at 8000 rpm is then carried out. It was washed with acetone aliquot followed by centrifugation, and 5 mL of 0.1 N NaOH was added to extract protein from E. coli cells. Further electrophoresis was led utilizing the HTP001 kit containing the Himedia Protein Molecular Weight Marker in the range of 29–205 kDa, mol. wt. It was utilized as a protein marker. Eventually, the protein removed from E. coli cells was denatured in 2 mL of gel test buffer containing 1.25 mL of 0.5 M Tris–HCl (pH 6.8), 1 mL β-mercapto-ethanol, 2 mL glycerol, and 0.4 mL 1% bromophenol blue to 0.4 g SDS, and the last volume was made to 10 mL with water. 25 μL of these protein tests were stacked in each well of the electrophoretic gel. At first, 10–15 mA current was given for 10–15 min until the samples began passing through the stacking gel. At that point, the current inventory was expanded to 30 mA until the bromophenol blue color came to approach the lower part of the gel piece. The gel chunk was kept in a box containing the staining arrangement (200 mL methanol, 1.25 g Coomassie Brilliant Blue R-250, and 35 mL frosty acidic corrosive made to 500 mL by adding water) until clear bands were noticed. Excess stain was taken out by keeping the gel in a de-staining arrangement (75 mL of icy acidic corrosive and 50 mL of methanol, the last volume made to 1 L with water).

Glutathione Oxidation Test

Ellman’s test is utilized to assess the amount of thiols in GSH. Before the treatment, E. coli (ATCC 25922) treated with GO, Ni/NiFe2O4, and Ni/NiFe2O4-G dispersions were mixed independently with 225 μL of GSH (0.8 mM in the bicarbonate buffer) to start oxidation. GSH arrangement without graphene-based materials was utilized as a negative control. Oxidization using 1 mM H2O2 and 0.4 mM GSH was utilized as a positive control after 2 h incubation at 27 °C. Then the GSH–GO, GSH–Ni/NiFe2O4–GO, and GSH–Ni/NiFe2O4–GO scatterings were separately moved into a 24-well plate sealed with alumina foil to forestall light exposure. This 14-well plate was then positioned on a shaker at 150 rpm and hatched at room temperature for 2 h. Afterward, 785 μL 0.05 M Tris–HCl and 15 μL of DNTB were added to the 24-well plate to yield a yellow product. The item yield was sifted through a 0.45 μm polyethersulfone channel. 250 μL aliquant of separated arrangements from each example was then moved in a 96-well plate, and using a Varian microplate spectrophotometer, absorbance at wavelength 412 nm was estimated. All tests were prepared in triplicate.

Results and Discussion

XRD Analysis

The XRD for RGO-Ni/NF nanocomposite is shown in Figure , with all peaks matching three phases. The 2θ values 18.19, 30.02, 35.40, 43.13, 53.70, 57.12, 62.67, 71.12, 74.16, and 76.31° correspond to the (111), (220), (311), (400), (422), (511), (440), (620), (533), and (622) crystal planes, respectively, for Ni/NiFe2O4–GO. The obtained peaks match well with the standard JCPDS card no. 86-2267. The peaks with the symbol (Δ) denote a normal NF spinel structure with the Fd3m space group, while peaks with the symbol ($) denote Nickel (Ni) nanoparticles with the Fm3m space group. The presence of RGO is established by the peak (about 2θ = 23°), indicated by the symbol (*). Using the Scherrer equation and the main peak, the crystallite size of Ni nanoparticles is predicted to be 7 nm (111)where D = crystallite size, λ = wavelength of X-rays used, and θ = Bragg’s angle

Figure 1

XRD pattern of the Ni/NiFe2O4–GO nanocomposite. Inset: Williamson–Hall plot. “Reprinted XRD graph with permission from [Applied Physics Letters, https://doi.org/10.1063/1.4892476]. Copyright [2014] [AIP Publishing].”

The nano range size of the product obtained is indicated by the broad nature of the XRD peaks. However, there is a small hump at 25.54°, which corresponds to the (002) plane of graphene. The RGO layers crumple (as illustrated in Figure ) around the magnetic nanoparticles of (Ni/NF) due to intense adsorption. This could result in some structural instability at the surface of magnetic nanoparticles, as well as some strain. The crystallite size and strain (inset of Figure ) formed in the NF phase of the nanocomposite are estimated using the Williamson–Hall plot (W–H plot) (β cos θ = sin θ +0.9λ/D), and the values are determined to be 7.7 nm and 18 × 103, respectively. The XPS findings of graphene oxide solely demonstrate the existence of carbon and oxygen, with no additional peaks attributable to magnetic impurities (see graph in Supporting Information S1.

Figure 3

HR-TEM images of Ni/NiFe2O4–GO with various magnifications.

Morphological characterization

Morphology of the surface and size of the particles of Ni/NiFe2O4–GO nanocomposite were additionally inspected by FE-SEM and HR-TEM techniques. From the FE-SEM pictures, as appeared in Figure , it was seen that the Ni/NiFe2O4 nanoparticles are decorated with homogeneous spherical particles onto sheets of graphene, and the assessed bunch size is 140–160 nm. HR-TEM was additionally used to analyze the structure of the Ni/NiFe2O4–GO nanocomposite. It demonstrates that the NiFe2O4 spheres with an average diameter of 150 nm were anchored on graphene nanosheets (Figure ).

Figure 2

FE-SEM images of Ni/NiFe2O4–GO at various magnifications.

Therefore, the abovementioned results demonstrate very well that the solvothermal course is a homogeneous technique for the preparation of the Ni/NiFe2O4–GO nanocomposite.

Raman Spectroscopy Analysis

Figure shows the Raman spectra Of Ni/NiFe2O4–GO. It has a dominant d-band and g-band peaks at 1347 and 1591 cm–1, respectively. The peaks in the range of 200–700 cm–1 correspond to the NiFe2O4 nanoparticles.

Figure 4

Raman Spectra of Ni/NiFe2O4–GO nanocomposite.

VSM Analysis

Magnetization of the nanocomposite with varying applied fields (−10,000 ≤ H ≤ 10,000 Oe) was estimated at 300 K. Super paramagnetic nature of the Ni/NiFe2O4–GO and exposed NiFe2O4 is evident in their hysteresis loop as shown in Figure S2. A saturation magnetization of 24.282 and 36.107 emu/g was noticed for Ni/NiFe2O4–GO and exposed NiFe2O4, respectively. When contrasted with bare NiFe2O4, the saturation magnetization diminishes because of the commitment of graphene layers.[30]

Antibacterial Activity Effect of the Ni/NiFe2O4–GO Nanocomposite on E. coli cells

Figure shows that only 4.25% of cells were practically viable under treatment with Ni/NiFe2O4–GO and about 17.25% of cells were reasonably viable when treated with NiFe2O4, in contrast with 53.78% viable cells when treated with GO. These exercises were additionally analyzed for model sample concentration and time subordinate conduct of Ni/NiFe2O4–GO on E. coli ATCC 25922 cells. Distinctive grouping of Ni/NiFe2O4–GO (0 to 200 ppm) dispersions were incubated with E. coli cells for about 60 min and, afterward, plated for looking at cell viability.

Figure 5

Cell viability of E. coli when treated with GO, NiFe2O4, and Ni/NiFe2O4–GO. (a) concentration-dependent study of 0–200 ppm reduced graphene oxide-Ni/NiFe2O4 with E. coli cells. (b) time-dependent (0–90 min) study of reduced graphene oxide-Ni/NiFe2O4 treated with E. coli cells.

The control was nontreated E. coli cells. It was seen that 25 ppm Ni/NiFe2O4–GO could restrain 57% of cells, as seen in Figure a. Likewise, 100 ppm was sufficient to hinder bacterial organisms (E. coli) as the cell viability is 26.84%. Then, again in time subordinate examination, it was seen that when 100 ppm of Ni/NiFe2O4–GO scattering was treated with cells of E. coli for different time stretches from 0 to 90 min, at that point 60 min was enough to eliminate bacteria, indicating cell viability of 23.74% (Figure b). The viability loss of E. coli dynamically diminishes with the increments of Ni/NiFe2O4–GO concentrations.

Figure 6

FEG-SEM images of (a) living E. coli and (b) its destruction after treatment with Ni/NiFe2O4–GO.

E. coli reasonability has nearly diminished from 57 to 21% by increasing the amount of Ni/NiFe2O4–GO nanocomposite from 25 to 100 ppm. A larger part of E. coli cells was executed after brooding with Ni/NiFe2O4–GO at the convergence of 100 ppm. Along these lines, the cell viability diminished from 64.66 to 19.66% by varying the treated time periods from 15 to 90 min.

Destruction of the Bacterial Membrane

The SEM measurements were utilized to outline the interaction between Ni/NiFe2O4–GO and E. coli cells. In Figure a, E. coli cells as control show the whole cell divider with no crack, while Figure b has the total breaking down of the cell divider. Spikes made on E. coli cells are an evident sign that Ni/NiFe2O4–GO has associated with the cell divider. This sort of cell wall break is irreversible harm initiated after direct contact with Ni/NiFe2O4–GO. Therefore irreversible harm can be prompted to bacterial cells after direct contact with Ni/NiFe2O4–GO.

Estimation of Total Protein Degradation in E. coli by the SDS-PAGE Method

Figure displays the results of protein degradation in nanocomposite-treated E. coli cells. It is obvious from the protein degradation that, stress-intervened protein debasement of the cell layer left just a single higher density protein unblemished, though the rest of the proteins were denatured, which is not normal for proteins under treatment with GO or control.

Figure 7

Protein degradation by graphene oxide (GO) and Ni/NiFe2O4–GO.

Glutathione Oxidation due to the Ni/NiFe2O4–GO Treatment

Irregularity of the H2O2 pathway in the cell layer creates oxidative stress. To inspect oxidative pressure in vitro GSH, oxidation was intervened. GSH is a tripeptide with thiol gatherings. It acts as an antioxidant in bacteria in the range of 0.1 and 10 mM concentrations. GSH can forestall harm to cell segments brought about by oxidative stress. Thiol gatherings (−SH) in GSH are oxidized into disulfide bonds (−S–S−). The disulfide bond containing GSH is called glutathione disulfide. GSH is used as an oxidative pressure marker in cells. Ellman’s assay can quantitatively evaluate the centralization of thiol bunches in GSH. When 0.4 mM GSH is treated with 100 ppm Ni/NiFe2O4–GO, the GSH oxidation progressively advances with a broadening response time up to 60 min. Figure shows the portion of GSH oxidized by Ni/NiFe2O4–GO increments in contrast with graphene. Similarly, Ni/NiFe2O4–GO has fundamentally higher oxidation reactivity, up to 89.32%, than graphene at a similar response time and concentration of 60 min in 125 μg/mL of Ni/NiFe2O4–GO. The GSH oxidation in a roundabout way confirms that Ni/NiFe2O4–GO is able to intercede ROS-autonomous oxidative pressure toward bacterial cells.

Figure 8

Oxidative stress release by E. coli because of the effect of the reduced graphene oxide- Ni/NiFe2O4 composite.

Conclusions

Graphene–nickel ferrite composites were successfully obtained and tested on E. coli cells. The nickel ferrite nanocomposite prompted a harmful reaction in E. coli cells through ROS age and GHS consumption. Taking everything into account, we have demonstrated that graphene-based Ni ferrite nanoparticles cause critical cytotoxicity to E. coli cells in a portion subordinate way in the concentration range. This nanocomposite was additionally found to prompt oxidative stress on E. coli cells by the enlistment of the ROS level and exhaustion of the GSH level via oxidation. This in vitro study indicated the acceptance of apoptosis by graphene-based Ni ferrite nanoparticles and warrants further examination to decide whether in vivo openness results may exist for the graphene-based application of Ni ferrite nanoparticles.