Utilization of surfactant assisted zinc oxide nanodisk against breast cancer cells

Abstract The current work displayed the efficacy of the nanodisk shaped zinc oxide nanostructures (NDs-ZnONSs) processed via hydrothermal solution process and studies the cytotoxicity against human breast cancer cells (MCF-7). The NDs-ZnONSs were analyzed by using instruments such as X-ray diffraction pattern (XRD), Field emission scanning electron microscopy (FESEM), and Fourier transform infra-red spectroscopy (FTIR). The XRD and FESEM shows the crystallinity and morphology of NDs-ZnONSs states the estimated width size of each sheet diameter and length are ∼20-25 nm and 2-3 µm respectively. The cytotoxicity of MCF-7 cells with NDs-ZnONSs at different concentrations (1, 2, 5, 10, 25, 50 and 100 µg/mL) were examined via MTT and NRU assays. The results showed that at initial (1, 2 and 5 µg/ml) concentrations of NDs-ZnONSs didn’t show any cytotoxicity in cancer cells but higher concentrations (25, 50 and 100 µg/mL) of NDs-ZnONSs displayed a remarkable inhibition in cancer cells growth and morphology. Further, the reactive oxygen species (ROS) generation was also examined with NDs-ZnONSs at different concentrations and result showed that it increased with increasing the concentration of NDs-ZnONSs in MCF-7 cells. The expression of apoptotic genes (p53, bax, casp-3) including control via qRT-PCR, reveals that mRNA level of apoptotic genes with NDs-ZnONSs were upregulated.


Introduction
Among a long range of various types of nanomaterials (NMs), the oxide-based nanostructured materials exhibited unique characteristics due to their small size, high surface to volume ratios, enhance thermal stability, and enough electron transportation passages [1,2].Due to these reasons, NMs are largely applied in electronic industry such as LED, solar cells, hydrogen storage, battery, fuel cells, and other related fields [3].Over a long range of oxide NMs, the zinc oxide (ZnO) exhibit a unique characteristic such as a high band-gap ~3.37 eV with 60 meV of binding energy, the wurtzite structure provides the higher chemical and mechanical stability of nanostructures [4][5][6].Also, the ZnO exhibit unique properties such as optical, semiconductor etc. Due to their biocompatible and biocidal properties, it is largely utilize in various directions such as foods, packaging, cosmetics, healthcare formulations, biomedical applications, drug delivery, gene delivery, chemical, and biosensing [7,8] etc.Because of the biocidal as well as anticancer activity of ZnO nanostructures, resulted an increased demand and utilizes as a drug as well as biomedical image contrasting agent in medical and health sectors [9].A number of physical and chemical methods were applied to process the different shaped ZnO nano structures such as high filament chemical vapour deposition (HFCVD), chemical vapour deposition (CVD), plasma enhanced chemical vapour deposition (PECVD), mechanochemical process, precipitation process, sol-gel, solvothermal, microwaves assisted process, emulsion or micro emulsion process, gas phase, spray pyrolysis, sonochemical method, microwaves synthesis, and many others [10][11][12][13][14].Over various adopted methods, the hydrothermal chemical method is highly useful, cheap, and inexpensive method for the large-scale production of nanostructures [15].In this connection, several researches had already been done in the area of ZnO nanoparticles research, but very limited studies are available for different shaped structures especially in the area of biomedical applications [16].Towards to this direction, nanobased structures were applied for the cytotoxic studies such as NRs of ZnO exhibit the biocompatibility against lupus erythematosus (LE) cells and displayed that these rods shaped structures could be safe for the biological utilities [17].Also, NRs does not affect any mortality in zebrafish embryos but it exhibited cytotoxicity of cell-death and their exposure to breast cancer cell line (MCF-7) [18].In another report published by Zanni et al. [2016] [19] shows that the MCF-7 cells exhibited a decrease in proliferation with the surge of apoptosis compared to HaCaT cells once exposed to ZnO nanorods [19].
The present study produces NDs-ZnONSs with using zinc acetate di hydrate, sodium hydroxide, and surfactant sodium dodecyl sulfate (SDS) via hydrothermal solution process.The characterized NDs-ZnONSs were employed for anticancer potential of nanostructured material.These NDs-ZnONSs were studies for the cytotoxicity, genotoxicity, and intracellular ROS in detail, and also establish to authenticate the grown sheets are much effective as an anticancer agent.

Synthesis of NDs-ZnONSs
The synthesis of NDs-ZnONSs were performed via hydrothermal process with using zinc acetate di hydrate (Zn(CH 3 COO) 2 .2H 2 O), sodium hydroxide (NaOH) and surfactant sodium dodecyl sulfate (SDS, CH 3 (CH 2 ) 11 OSO 3 Na) purchased from the Aldrich chemical corporation, U.S.A and utilized as without any further purification.For the synthesis of NDs-ZnONSs, Zn(CH 3 COO) 2 .2H 2 O (0.03 M, ~0.658 g) and NaOH (0.02 M) and SDS (0.005 g) were assorted in 100 mL methanol (MeOH) with constant stirring in a beaker.The pH of the transparent solution was measured and it was touched up to 12.31, thereafter, the solution was transferred to the Teflon beaker and this was fixed in a steel autoclave jacket and heated in an oven at ~100 °C for 8 h.Once the heating was completed, transfer the steel jacket at room temperature and to keep for 24 h.Thereafter, the formed product in Teflon beaker was transferred into beaker, washed well with MeOH repeatedly to remove the ionic impurities.The product was centrifuged (3000 rpm/min for 3 min, Eppendorf, 5430 R, Centrifuge, Germany) and dried at room temperature and was employed for further studies.

Characterizations of the prepared product
The processed product was well characterized with instruments XRD (Rigaku, Japan), FESEM (Hitachi, Tokyo, Japan) and FTIR (Perkin Elmer-FTIR Spectrum-100, U.S.A.).XRD was utilized to know the phase identification, crystallinity of the material and estimated particle diameter of NMs with Cu Kα radiation (λ = 1.54178Å) in the range of 20°-80° with 6°/min scan speed in accelerating voltage of 40 kV and current was 30 mA.The structural evaluation was accessed via FESEM.For the FESEM analysis, the product in the form of powder was sprinkled on carbon tape and pasted on sample holder and thereafter, it was moved to a glass chamber and coated with platinum (~3s) via sputtering process.Once the process was completed, the sample holder was fixed to the clamp of FESEM and analyzed at room temperature.

Reagents and consumables for cell culture
MTT dye, DCF-DA dye, and neutral red dye was procured from Sigma.Cell culture media, FBS, and antibiotics-antimycotic was purchased from Invitrogen, USA.All other plastic wares and consumables were obtained from Nunc, Denmark.

Cell culture
Human breast cancer cell line (MCF-7) was grown in DMEM with FBS (10%) and antibiotic-antimycotic solution (1%).Cell line was maintained in a CO 2 incubator (5% CO 2 and humid atmosphere) at 37 °C.For the experiments, the cells were cultured in 24, 48, and 96 well plates as per the demand of assays.

MTT assay
The cytotoxicity of NDs-ZnONSs was assessed by MTT assay.The assay was done as per the set protocol [20].Briefly, cells were plated in 96-well culture plates and grown in a CO 2 incubator for overnight.These cells were exposed to 1, 2, 5, 10, 25, 50, and 100 µg/ ml of NDs-ZnONSs for 24 h.Then, MTT (10 μL/well of 5 mg/ml stock) was mixed to wells and kept for 4 h in a CO 2 incubator.This solution discarded and then dimethyl sulfoxide (DMSO, 200 µL) was added to wells.The absorbance of the plate was read at 550 nm.The % cell viability was considered using following formula:

NRU assay
NRU assay was also performed to assess the cytotoxicity of NDs-ZnONSs in MCF-7 cells.
The assay was performed following the method [20].Briefly, the cells were cultured in 96-well plate and grown at 37 °C in a CO 2 incubator.After overnight, MCF-7 cells were treated with 1, 2, 5, 10, 25, 50, and 100 µg/ml of NDs-ZnONSs for 24 h.Then, cells were incubated in NR medium (50 µg/ml) for 3 h.The solution was then removed and the dye was extracted in 50% ethanol and 1% acetic acid solution.The plate was measured at 550 nm.

ROS generation
MCF-7 cells were plated in a 24-well culture plate and exposed to 25, 50, and 100 µg/ml of NDs-ZnONSs for 24 h.The ROS generation in MCF-7 cells was performed by using DCF-DA (2'-7'-Dichlorodihydrofluorescein diacetate) dye and assay was done with following method [20].After the exposure, cells were incubated in DCF-DA dye for 30 min at 37 °C.The intracellular ROS was measured using the spectroflurometer and cellular images were grabbed under a fluorescence microscope.

Gene expression study analyzed via qPCR analysis
The RNA was extracted from RNeasy mini Kit (Qiagen) as according to company protocol.The MCF-7 cells were cultured in a 6-well plates and exposed to NDs-ZnONSs at concentration of 50 µg/ml for 24 h.Initially, the cDNA was formed from treated and untreated cells and taking 1 µg of RNA by Reverse Transcriptase kit using MLV reverse transcriptase (GE Health Care, UK) as described the manufactures' protocol.The qRT-PCR was performed on Roche® Light Cycler ® 480 (96-well block) (USA) following the cycling program as recommended.2 μL (40 ng) of cDNA template included the 20 µL volume to this reaction mixture [21].

X-ray diffraction analysis
The processed white product was observed via XRD to know the crystallite size, phases of the crystals, and crystalline character of the material.The received data in the form of XRD (

Assessment of structural morphology via FESEM
The structural morphology of the prepared product was examined via FESEM and obtained images are displayed as Figure 2(a-b).The image was captured (Figure 2a) at low magnification scale, illustrate that a number of spherical and trigonal shaped disk like structures are seen in the whole surface, which are either in grouped on the surface or seems to be separated as individual sheet.For more specified detail, the images were observed at high magnified scale and illustrates that the grown structures are seems to be flower shaped structures with number of sheets arranged as petals (Figure 2b).The individual sheet width size is about 20-25 nm whereas the whole sheet diameter ranges between ~2-4 µm in size, interlinked with other sheets [9] (Figure 2c and d).The surfaces are very clean, smooth and aligned (Figure 2d).The sheets are gathered together and form a flower or bunched of nanostructures.

Chemical functional analysis via FTIR spectroscopy
The chemical functional groups in NDs-ZnONSs material were examined via FTIR spectroscopy and the recovered spectra is illustrated as Figure 3.The wide and shallow peak obtained in the FTIR spectrum between 3200-3600 cm −1 related to the H 2 O whereas the asymmetric stretching mode hydroxyl group (O-H) are centered at 1576 cm −1 (Figure 3) respectively.A small and centered peak at 2926 cm −1 is related to the -CH group in the spectrum.The pointed and long peak centered at 1598 cm −1 is associated to the carboxyl molecule (-C = C-), at 1410 cm −1 methyl group (-CH 3 ) and at 1025 cm −1 for sulfate group (-O = S-) from SDS respectively [9,22], whereas at 444 cm −1 represent the formation of ZnO in FTIR spectrum.The FTIR authenticates the formation of NDs-ZnONSs and it's analogous with available XRD pattern and FESEM data's.

Cells morphology analyzed via microscopy
The cultured cancer cells were grown in an incubator and their growth was accessed via inverted microscopy.The control cells are those which have no exposure with NDs-ZnONSs.After their optimal confluence (~90%), cells were treated with varying  concentrations of NDs-ZnONSs (1, 2, 5, 10, 25, 50, and 100 µg/ml).The results showed that at initial concentrations of the material (1, 2, 5 µg/ml) doesn't shows any change in the morphology of MCF-7 cells, whereas at higher concentrations (10, 25, 50, and 100 µg/ ml) of NDs-ZnONSs, it seems that the cells become rounded, internal organelles were damaged, and ultimately leads to cell death (Figure 4).

Cytotoxicity assessment by neutral red uptake (NRU) assay
The cytotoxic potential of NDs-ZnONSs was also assessed by NRU assay.The MCF-7 cells treated with NDs-ZnONSs at 1, 2, 5, 10, 25, 50 and 100 µg/ml for 24 h significantly reduced the viability of MCF-7 cells.The results of NRU assay have been provided in Figure 6.The results clearly showed that the viability of MCF-7 cells was decreased by 102%, 101%, 98%, 89%, 69%, 50%, and 35% at 1, 2, 5, 10, 25, 50 and 100 µg/ml concentrations.However, NDs-ZnONSs at 5 µg/ml and below concentrations did not produced any cytotoxicity to MCF-7 cells.These cytotoxicity results are also correlating with the results of cellular cytological changes in the MCF-7 cells after the contact of NDs-ZnONSs for 24 h (Figure 6).

Reactive oxygen species (ROS) generation
ROS generation in MCF-7 cells induced by NDs-ZnONSs was measured by DCF-DA dye.The results of ROS generation have been provided in Figure 7.The cells were treated with 25, 50, and 100 µg/mL concentrations of NDs-ZnONSs.The results showed that fluorescence intensity of ROS was increased in MCF-7 cells exposed to NDs-ZnONSs by 24%, 61%, and 97% at 25, 50, and 100 µg/ml of NDs-ZnONSs .The fluorescence images also showed that the exposure of NDs-ZnONSs increased the DCF fluorescence intensity in treated MCF-7 cells as compared to control (Figure 7).

Gene expression
The mRNA levels for the apoptotic genes were also studied with qPCR.To examine the mRNA expression level of genes (p53, bax and casp3), the breast cancer cells (MCF-7) were treated with NDs-ZnONSs (Figure 8) for 24 h at a concentration of 50 μg/ml.Significant changes were observed with the NDs-ZnONSs for the apoptotic genes in MCF-7 cells (p < 0.05 for each gene).The mRNA levels of tumor suppressor gene p53 (1.8), bax (2.1), and casp-3 (2.8) were upregulated with the treatment of NDs-ZnONSs with control (Figure 8).

Discussion
Among various types of nanostructures, ZnO is widely being utilized as a consumer product such as sun-screen, cosmetics, textiles, food products, and medicines [21].Their large surface area, high reactivity, small in size makes it a valuable NMs and much effective against various biological objects [23].The current work illustrates that at initial, the sheets like nanostructures were prepared with the use of Zn(CH 3 COO) 2 .2H 2 O, sodium hydroxide (NaOH) and surfactant sodium dodecyl sulfate (SDS, CH 3 (CH 2 ) 11 OSO 3 Na) through hydrothermal chemical process.The mixture was heated at ~100 °C for 8 h in a steel autoclave jacket.The product was well characterized with XRD, which displays that the nanostructures were highly pure and crystalline in nature.The morphology of the prepared structures were examined with FESEM and it discloses that the obtained images shows the sheets like structure with width size ranges from 20-25 nm and length reaches 2-3 μm.Including the basis characterization of the prepared nanostructures (NDs-ZnONSs), the biological efficacy was checked against breast cancer cells (MCF-7) accordingly at dose dependent manner.To evaluate the utility of NDs-ZnONSs, in this experiment, initially the performed MTT and NRU assays for the assessment of cytotoxic response after the acquaintance of 24 h.From the MTT and NRU experiments, it revealed that NDs-ZnONSs induces the cytotoxicity in MCF-7 cells at 1-100 μg/ml.The obtained results are fully corroborated with the previously published report related to the cytotoxic response of nanostructures in this range [9].The MTT and NRU assays displayed the NDs-ZnONSs in MCF-7 cells react as dose reliant and at initial concentrations (1 µg/ml, 2 µg/ml, 5 µg/ml) of NDs-ZnONSs no significant cytotoxicity was observed, it may be due to the size of an individual sheet like structures, concentration of the utilized NMs, pH etc [21].It postulates that, these impacts created in the cells solution and directly effect on MCF-7 cells, which is may be due to their unique structural organization of sheets and their functions in solutions [9,21].Once the concentrations (10 µg/ml, 25 µg/ml, 50 µg/ml, 100 µg/ml) of NDs-ZnONSs increases, the cytotoxicity is much influenced.The calculation of viable and non-viable cancer cells were measured and received data reveals that initially the sheets like structures not affect to the cancer cells surfaces but once their concentration increases, these sheets like structures exhibit capability to destroy the cells surfaces and can easily enter to the cells organelles and possible to damage it, due to less amount of the sheets structures doesn't affect to the cells, whereas once the amount/ concentration of the sheets like structures were increased, it directly affect to the cells.As it's known that the free radical (FRs) in the cells generated through reactive oxygen species (ROS) produced in the cells.Therefore, to explore the mechanism of cytotoxicity generated with the interaction of NDs-ZnONSs, the ROS was studied in MCF-7 cells before and after 24 h exposure with NDs-ZnONSs.The ROS is produced in cells due to the interaction of foreign particles/NDs-ZnONSs and it's a key role in the cytotoxicity and apoptosis.It's also known that the ROS is capable to disturb the function of cells organelles such as DNA, protein, lipids destroying their structure and ultimately causes, cell death [24].In this experiment, the received ROS results are fully dose dependent, which express that ROS is increase with the increase of the concentration of NDs-ZnONSs in MCF-7 cells.The results are fully substantiated as previously published literature [9,21].
From these observations, it deduced that the cell death is caused due to the production of ROS in cells with NDs-ZnONSs in MCF-7 cells.Also, the gene expression study is in accordance with the previously published literature [21], and discloses that NDs-ZnONSs are responsible in upregulation in gene expression and apoptosis in cells.In the area of cancer studies, till to date numerous treatment methods were developed and applied for the complete eradication of cancer cells for instance chemotherapy, radiotherapy etc.In these applied techniques, the cells were removed through surgery but if any cells remain it again grow and form more and more cells.Also the cost of surgery is very high and much complicated for the deprived families, therefore a detailed work is needs to establish for a successful therapy, which can cure well, efficiently and very low price.The nanotechnology provides a great contribution in cancer studies also fulfil the cost-effective ways to control and cure the cancers.It's due to the biocompatible nature of the prepared material and easy to handle, facilitates better and significant improvements as compared to the available technologies and have no any adverse effect, if used in a permissible limit.These nanostructures will reduce the cost of the drugs, will work as a nano drugs, also to minimize the fear against surgery for deprived patents [9].

Conclusion
In conclusion, the zinc oxide nano sheets shaped structures were produced via hydrothermal process.The produced structures were characterized in terms of their crystallite size, morphological structure, and chemical properties.These data show that structures are crystalline in nature with sheet shaped morphology.The NDs-ZnONSs demonstrated that synthesized structures induce the cytotoxicity in MCF-7 cells.The cells structures with and without NDs-ZnONSs were examined via microscopy and it reveals that at initially (1 µg/ml, 2 µg/ml, 5 µg/ml), no change was observed in the cells structures whereas once the concentration of NDs-ZnONSs increase (10 µg/ml, 25 µg/ml, 50 µg/ml and 100 µg/ml), morphological changes were observed in cancer cells growth.This change was also displayed in the MTT and NRU assays and it presented a concentration dependent decrease in cell viability of MCF-7 cells.The obtained results also displayed that these NDs-ZnONSs are capable to induced ROS generation in MCF-7 cells which are responsible for the cell death.The alterations in the expression of apoptotic genes of MCF-7 cells induced by NDs-ZnONSs clearly exhibited that NDs-ZnONSs are capable to induce apoptosis in MCF-7 cells.Therefore, NDs-ZnONSs can be utilize against human breast cancer cells.

Figure 1 .
Figure 1.xrD pattern shows the formation of nDs-Znonss.*show the unidentified peak.

Figure 4 .
Figure 4. cellular morphological changes in mcf-7 cells exposed to nDs-Znonss for 24 h.images were taken using phase contrast inverted microscope at 20 x magnifications.

Figure 8 .
Figure 8. mrna quantification levels, fold change of apoptotic genes (p53, bax, and casp3) were analyzed.cells treated 50 μg/ml of nDs-Znonss for 24 h.for the normalization, an internal control of GapDH used.the values are mean ± sE of three independent experiments with statistical standard error (p < 0.05 for each).