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ZnCo2O4/ZnO nanocomposite: Facile one-step green solid-state thermal decomposition synthesis using Dactylopius Coccus as capping agent, characterization and its 4T1 cells cytotoxicity investigation and anticancer activity
⁎Corresponding authors at: Institute of Nano Science and Nano Technology, University of Kashan, Kashan, P. O. Box. 87317-51167, Iran. Tel.: +98 31 55912383; fax: +98 31 55913201. ma.amiri@kmu.ac.ir (Mahnaz Amiri), salavati@Kashanu.ac.ir (Masoud Salavati-Niasari)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Abstract
Although, using plant extracts for the green synthesis of various nanoparticles attracted the attention of researchers and scientists due to easy availability and wide distribution of plants as well as their safety to use, in the present work, ZnCo2O4/ZnO nanoparticles synthesized via the green chemistry technique using Dactylopius Coccus, the scale insect. Dactylopius Coccus plays the role of an active reducing and capping agent for synthesis of NPs with well-organized biological properties. Various formulations synthesized in order to investigate the effect of different calcination time and temperatures on the morphology of NPs. The synthesized NPs were characterized by several techniques like XRD, SEM, TEM, HRTEM, and VSM. The XRD result revealed that the particle size of the NPs prepared was in the range of 40 ± 2 nm that was confirmed through HRTEM analysis. Moreover, the cytotoxicity of the NPs was investigated to determine its anti-proliferative impact against 4T1 cancer cell lines. Effective in-vitro and in-vivo death induction in breast cancer cells in the presence of magnetic nanostructures was discussed. The results confirmed that the NPs significantly decreased the percentage of 4T1 cells survival with an IC50 of 0.3 mg/ml. Finally, the dominant mechanism of cell death was investigated that is apoptosis along with an increase in cellular oxidative stress.
Keywords
Anti-cancer activities
Cytotoxicity
Green synthesis
Magnetic ZnCo2O4/ZnO nanoparticles
Nanocomposite
Nanostructures
1 Introduction
“Nanotechnology deals with the processing of separation, consolidation, and deformation of materials by one atom or by one molecule” was well defined by Professor Norio Taniguchi, Tokyo Science University, for the term “nanotechnology.” It is well known that nanostructured materials have been taken into consideration; because, they possess special properties affected by their size, magnetic property, mechanical, and chemical properties that are greatly different from their respective bulk counterparts (Goudarzi et al., 2014; Amiri et al., 2017; Goudarzi and Salavati-Niasari, 2019). Spinel structures are amongst the NPs, which have been examined lately owing to their wide-ranging availability in chemical sensors, microwave absorbers, permanent magnets (Amiri et al., 2017), and in bio-medical applications like drug delivery (Amiri et al., 2017) and in cancer thermotherapy (Goudarzi et al., 2019; Goudarzi et al., 2019; Goudarzi et al., 2019). Binary transition metal oxides like ZnCo2O4 (Zhang and Zhu, 2019; Han et al., 2018), NiCo2O4 (Cheng et al., 2017; Tao et al., 2019), CuCo2O4 (Kamari Kaverlavani et al., 2017), and ZnFe2O4 (Li et al., 2021; Koo et al., 2017) have received much attention over the past decade, since they possess different oxidation states for various types of metallic cations with an improved electrochemical action resulting from richer redox reactions. Occasionally a conspicuous synergistic effect resulting from dissimilar metals has taken place as well. Out of the semiconductors of p-type, zinc cobaltite (ZnCo2O4) designates spinel-type structure, which has been largely applied in Li-ion batteries as electrode (Liu et al., 2012) and as supercapacitors (Zhou et al., 2014) owing to its greater and superior electrochemical and conductivities performances. Numerous approaches such as combustion (Sharma et al., 2007), thermal decomposition (Mariappan et al., 2015), co-precipitation/digestion (Vijayanand et al., 2011), W/O (water in oil) microemulsion (Niu et al., 2004), hydrothermal (Huang et al., 2015), sol–gel (Wei et al., 2007), and surfactant-mediated method (Wang et al., 2015) have been adopted to prepare ZnCo2O4. Studies showed that Zn2+ release from ZnO NPs is the reason for Schwann CD (Yin et al., 2012). Zn2+ is an imperative factor in enzymes and protein structure and displays an ionic hint among numerous intracellular organelles (Frederickson et al., 2005). To overcome the problem of toxicity, nanotechnology and green chemistry merge to fabricate nature-friendly nanoparticles via plants, microbes, etc. (Frederickson et al., 1988). Researchers have developed many synthetic routes for nanoparticle fabrication which unveiled a notable benefit to nature & environment via clean, nontoxic, and environmentally adequate “green chemistry” methods which include organisms such as bacteria, fungi, plants (Frederickson et al., 1989). The three foremost conditions for the synthesis of nanoparticles are the selection of green or environment-friendly solvent, a good reducing agent, and a harmless material for stabilization. For the synthesis of nanoparticles, extensive synthetic routes have been applied in which physical, chemical, and biosynthetic routes are very common. Generally, the chemical methods used are too expensive and incorporate the uses of hazardous and toxic chemicals answerable for various risks to the environment (Tønder et al., 1990). The biosynthetic route is a safe, biocompatible, environment-friendly green approach to synthesize nanoparticles using plants and microorganisms for biomedical applications, this synthesis can be carried out with fungi, algae, bacteria, and plants, etc. Some parts of plants such as leaves, fruits, roots, stem, seeds have been used for the synthesis of various nanoparticles due to the presence of phytochemicals in its extract which acts like stabilization and reducing agent (Premanathan et al., 2011). It is worth knowing that extra free Zn2+ ions are cytotoxic and can inspire severe neuronal wounds. Zinc oxide NPs have shown excellent toxicity for cancerous cell lines in comparison to normal cell lines (Premanathan et al., 2011). Different studies documented that ZnO NPs are toxic because of reactive oxygen species generation (ROS). The connection between toxicity and ZnO physicochemical properties have been measured as well (Sharma et al., 2012). Currently ZnO-NSts have been focused for various biological applications due to their biocompatible nature6. In the area of biological applications, there are enough quantity of research have been published towards the application of NPs and their role to control cancer cells growth but mechanism of cytotoxicity caused through ZnO-NSts has remained obscure. Accumulating evidences suggested the reasons of cytotoxicity of ZnO-NSts through reactive oxygen species (ROS) and genotoxicity in cancer cells. A recent report showed that the toxicity of cancer cells happens due to release of Zn2 + ions in zinc oxide solution. Sharma et al. (Sharma, 2009)reported that the nanoscale zinc oxide induced DNA damage through lipid peroxidation and oxidative stress in human epidermal cells. Among various types of cancers, brain, lung and human thyroid carcinomas cancer are commonly affected and considered as one of the main reason for cancer deaths. The symptoms of lung cancer are caused in the patients by primary tumor (metastasis) formation in the form of cough, chest pain, haemoptysis, dyspnea and recurrent pneumonia or bronchitis. Towards this area, it has been shown that lung cancer can be successfully reduced via the utility of nanostructured materials due to the role of nanoparticles as a drug delivery carrier which reduces the nonspecific toxicity of potent anticancer drugs. Higher tumor malignancy could be frequently deteriorated after cancer treatment procedures. To overcome these problems, several therapies, such as chemotherapy, radiotherapy, immune therapy, etc have been implemented to protect the cancer but the success rate of therapeutic outcomes is still not up to date (Wahab et al., 2013).
Therefore, in this study we presented a novel synthesis using a scale insect found on the pads of prickly pear cacti with many advantages (formed at ambient temperatures, neutral pH, low costs and environmentally friendly fashion) that mitigates the above-mentioned issues and challenges (too expensive and uses of hazardous and toxic chemicals with various risks to the environment, needs high temperature). Magnetic ZnCo2O4/ZnO NPs were synthesized by thermal decomposition method using Dactylopius Coccus; the impacts of various calcination time and temperatures, Dactylopius Coccus concentration, and salt source were investigated to reach the optimum nanocomposite structure. The anti-cancer effect of NP's against 4T1 cell lines was examined as well.
2 Experimental
2.1 Materials
All materials used in the study were analytical grade without further purification. No hazardous chemicals and solvents were used. The solvent used was deionized water. In the present paper all the metric units were used.
2.2 Preparation of Dactylopius coccus
Carmine natural dye is a derivative of the cochineal insect (Dactylopius coccus) that feeds on plant moisture and nutrients. The insects are found on the cacti pads, they are often found on the cactus during times of the year, gathered by brushing them off the plants. Carminic acid was removed from the female insects and preserved to yield carmine. The insect body contains 19–22% carminic acid (Bandyopadhyay et al., 2017). Coccus pigments were created after aeration of the female scale insect bodies at a temperature of 70 °C until a constant weight; finally, beached and kept for use.
2.3 Preparation of ZnCo2O4/ZnO nanocomposite
ZnCo2O4/ZnO nanocomposite was synthesized by using a solid-state thermal decomposition procedure. As follows: Zn (NO3)2·5H2O and Co (NO3)2·5H2O with Stoichiometric 2:1 M ratio and Dactylopius coccus were weighed. All of the materials were ball milled for 30 min. The above mixture was heated at 500 °C via furnace (10 °C/min). After 3.0 h, NPs were synthesized. Finally, the NPs were washed with deionized water and absolute ethanol to eliminate all possible contaminants if any, at a minimum of four times desiccated at room temperature and named the blank sample. Green (environmentally friendly) chemical procedures are ones that focus on developing free organic solvents and lowering energy taking methods. Due to the outstanding possessions of environmentally friendly alkalinized agents with less toxicity but more biodegradability as well as an active surface, in the present work, cochineal was applied for preparing ZnCo2O4/ZnO NPs for the first time. The cochineal played alkaline role and alkalinized the reaction media while added to the salt mixtures. The effect of various temperatures, Zn salt and Zn complex source, time and cochineal concentration was observed on morphology and size of the NPs. Fig. 1a designates the cochineal insect picture and Fig. 1b represents the schematic of solid-state thermal decomposition for nanocomposite synthesis. The variations in temperature, time, Zn Source and the amount of cochineal for different formulations are listed in Table 1.
The Cochineal insect picture (a) and the schematic of solid-state thermal decomposition for nanocomposite synthesis (b).
Sample Number
Cochineal (g)
Zn Source
Temperature (°C)
Time (h)
1
–
Zn(NO3)2
500
3
2
0.25
Zn(NO3)2
500
3
3
0.50
Zn(NO3)2
500
3
4
0.75
Zn(NO3)2
500
3
5
0.50
Zn(NO3)2
400
3
6
0.50
Zn(NO3)2
600
3
7
0.50
Zn(NO3)2
500
1
8
0.50
Zn(NO3)2
500
5
9
0.50
ZnSO4
500
3
10
0.50
Zn(OAc)2
500
3
11
0.50
Zn(OH)2
500
3
12
0.50
Zn(Ox)
500
3
13
0.50
Zn(gly)2
500
3
14
0.50
Zn(gly)2*
500
3
2.4 Characterization
The following instruments were used to characterize the synthesized NPs X-ray diffraction (XRD), Fourier transform infrared (FT-IR), transmission electron microscopy (TEM, Philips EM208 200 kV) and (FE-SEM, ZEISS, SIGMA VP-500, Germany), vibrating sample magnetometer (VSM, Meghnatis Kavir Kashan Co., Kashan, Iran).
2.5 Statistical analysis
All experiments were conducted in triplicate; thee data are presented as a mean ± SD. A P value less than 0.05 was considered significant. Statistical package for social science (SPSS) was utilized for statistical analysis (Amiri et al., 2018; LeBel et al., 1992; Gawande et al., 2015).
2.6 Cell culture and treatment of cells by magnetic ZnCo2O4/ZnO
Change in cellular characteristics was examined by AO/EB double staining and nuclear morphology was determined by DAPI staining on highly metastasis breast cancer 4T1cell line that was resulted from a naturally arising BALB/c mammary tumor (Bailey-Downs et al., 2014). The cell lines were obtained from the National Cell Bank of Iran (Pasteur Institute of Iran, Tehran, Iran). 4T1 cell line was selected because it is a proper animal model for human mammary cancer that designates transplant for BALB/c mice. Acridine orange (AO)/ethidium bromide (EB) double staining was used to characterize transformation characteristic (Alak, 1993). Finally, the image of the nuclear morphology was taken with fluorescence microscopy (Zeiss, Germany) after addition of the EB/AO solution to the cell suspensions. The nuclear morphology was determined using DAPI nuclear staining. The cells were incubated at 37 °C, 5% CO2 for 24 h. The cell lines were fixed in 4% paraformaldehyde-PBS solution for 15 min and stained with DAPI (0.3 µM) at 37 °C, 5% CO2 for 30 min at room temperature. Fluorescence microscopy was used for imaging the cells nuclear morphology and photography. To observe the presence of reduced, fragmented, and corrupted nuclei, which is the sign of apoptotic cells.
2.7 Cell viability
The percentage of the viable cells was determined by MTT assay (Mosmann, 1983). According to the method considered by Sladowski et al. (Sladowski et al., 1993). An increasing concentrations of sample 14 (0, 0.5, 1, 2, 4, 8, 16, 32 mg/ml) was used for treating cancerous cells as described in our previous work (Amiri et al., 2018; LeBel et al., 1992) . The dose that produced 50% inhibition of the viability (IC50) was calculated via variance between untreated and treated cells viability.
2.8 Intracellular ROS measurement via annexin and flow cytometric
The DCFH-DA, a non-fluorescence dye, was used to count the oxidative cell stress of the cells treated with sample 14. It helps to measures the hydroxyl, peroxyl, and other reactive oxygen species (ROS) activity inside the cell. DCFH-DA interacts with intracellular ROS to produce fluorescent 2,7- dichlorofluorescein (DCF) (LeBel et al., 1992). The quantity of fluorescent emission DCF is unswervingly related to the content of intracellular ROS. The maximum wavelength (λ max) of excitation and emission of DCF are at 495 nm and 529 nm, respectively; 5 × 105 4T1 cells was located in 6-well plates at 37 °C, 5% CO2 for 24 h. Then the cell lines were incubated for 24 h at 37 °C, 5% CO2 after treatment with 0.3 mg/ml of sample 14. The positive control was treatment with 0.1 mM PBS / hydrogen peroxide for 20 min. The cell was treated with 10 μM DCFH-DA and incubated at 37 °C, 5% CO2 for 1 h, then washed twice with PBS, and suspended in 500 μl PBS. Finally, the fluorescent intensity was determined using fluorescence spectrophotometer (a Varian Cary Eclipse fluorescence spectrophotometer).
2.9 Experimental design and in vivo anticancer study
25 mice of six to eight weeks old female BALB/c with an initial weight of about 20 ± 2.0 g were obtained from Tehran Small Animal Research and Teaching Hospital, Faculty of Veterinary Medicine, University of Tehran, Iran. The mice were kept in polycarbonate cages at 22 ± 2 °C under medical care, 85% relative humidity, and a 12-h light–dark cycle with standard water and food, wished with the pathogen-free environment; for one week before treatment for better adaptation. The mice were grouped in to five. 1 × 106 cells/100 µl 4T1 cells were injected subcutaneously in to the mammary gland to induce tumors in mice. Group 1 was used as the negative control without any 4T1 cell injection or drug treatment. Group 2 was injected with 1 × 106 cells/100 µl 4T1 cell line as the turmeric control. Groups 3 and 4 were the main test groups. Group 3 was injected with 4T1 and 30 mg/ml of sample 14 (intraperitoneal injection) simultaneously. Group 4 was treated by 4T1 cells and sample no.14 Group 5 was injected with only 30 mg/ml of sample 14. After one week, all the mice were weighed and sacrificed by the spinal process, and the solid tumor was separated aseptically and measured with caliper. All experiment were as per the guidelines of ethical standards accepted by the Animal Care and Ethics Committee of the University of Tehran.
3 Result and discussion
3.1 Characterization of ZnCo2O4/ZnO NPs
The XRD pattern for the magnetic nanocomposites is demonstrate in Figs. 2 and 3. The * and ♦ denote the related diffraction standard cards of ZnO and ZnCo2O4/ZnO, respectively. The crystal structure of ZnCo2O4 with lattice constants a ¼ b ¼ c ¼ 8.095 A, was confirmed via 220, 331, 222, 400, 422, 511, and 440 peaks (spinel cubic ZnCo2O4, ICDD No. 23–1390); the residual peaks are indexed to the hexagonal structure of ZnO which is consistent with the standard data no. 36–1451. (Gawande et al., 2015). The XRD results demonstrated that the Zn–Co precursors have converted totally into ZnO and ZnCo2O4 crystals totally after the calcination (Bandyopadhyay et al., 2017; Van Dam and May 2012). The strong and sharp peaks indicate that the crystallized samples were well ordered. Furthermore, no diffraction peaks derived from any other impurities could be detected, which indicates the high purity of the sample. As calculated by Debye- Scherrer’s formula, the average size of nanocrystals is 22 ± 1.23 nm (Goudarzi and Salavati-Niasari, 2018) as D = kλ/βcosθ; Where ‘D’ is NPs crystallite size, ‘k’ is the shape factor (0.9), ‘λ’ is the wavelength of X-ray (1.54 Å) Cu Kα radiation, ‘θ’ is the Bragg angle form 2θ value of intensity peak from XRD pattern, and ‘β’ is the full width half maximum of the diffraction from XRD pattern of NPs. The SEM image as shown in Figs. 4-8 demonstrates the morphologies of pure MNPs that were synthesized under various conditions. As shown in Fig. 4a, uniform nanospheres were well synthesized in the absence of cochineal without nano-range size. The effect of adding different amounts of cochineal was examined in Fig. 4b-4d, and the optimum amount was found to be 0.5 g, resulting in spherical and non-agglomerated particles. To produce smaller nanocomposite structures, different zinc salts like zinc acetate and zinc sulfate were used, resulted in the agglomerated particles (Fig. 5a and b). Additionally, to prepare a zinc source, Zn (OH) 2 was synthesized with a co-precipitation reaction using zinc nitrate and NaOH. The ZnO/ZnCo2O4 particles in the presence of Zn(OH)2 as a new precursor will develop rapidly and form nano-size particles with an average diameter of 30 ± 1.25 (Fig. 5c). After the formation of smaller NPs, Zn (OX) and Zn(gly) were synthesized and used as the Zn precursor for auxiliary confirmation of the morphology and structure of MNPs. Sample 14 was with the smallest and non– agglomerated particles and was selected for further biomedical applications (Fig. 6a-c). Similarly, the effect of temperature and calcination time on the synthesis of NPs was determined as shown in the SEM images from Figs. 7 and 8. The optimal calcination temperature and the finest calcination time were 500 °C 3hs; these conditions resulted in the smallest nano-size particles. TEM micrographs (Fig. 9 (a-c)) of the NPs indicated spherical shaped particles with a diameter of less than 60 nm. High-resolution TEM images of ZnO/ZnCo2O4 NPs are depicted in Fig. 9d. High-resolution TEM image with the polycrystalline structure of ZnO/ZnCo2O4 NPs are clearly observed in Fig. 10. The crystalline planes recognized by the parallel lines indicated the high degree of crystallinity of the composite. The lattice fringes are displayed by spacing fringes of 0.170 nm and 0.146 nm, and identified by the crystal planes (1 0 0) and (3 1 1) of wurtzite ZnO and spinel cubic ZnCo2O4 crystals, respectively. EDS analysis was useful for controlling the stoichiometry and purity portion of nanocomposites (sample 2). Fig. 10a demonstrates the EDS pattern that indicates the existence of Zn, Co and O elements; likewise, no impurity peaks are observed. Nanocrystal magnetic stuff was investigated by a vibrating sample magnetometer at 300 K as shown in Fig. 10b. The saturation magnetization (Ms) is 3.49 emu g−1. Generally, ZnCo2O4, at room temperature, proves the paramagnetic nature as a result of its normal spinel structure. There is always an opportunity of partial inversion amongst ZnCo2O4 NPs leading to a coupling between the cations of both tetrahedral and octahedral sites, thus increasing the occurrence of superparamagnetic coupling (Wang et al., 2005). These coupling effects are well known to be size-dependent (Liu et al., 2003).
XRD patterns of ZnCo2O4/ZnO nanoparticles: (a) sample no. 1, (b) sample no. 2, (c) sample no. 3, (d) sample no. 4.
XRD patterns of ZnCo2O4/ZnO nanoparticles: (a) sample no. 6, (b) sample no. 5, (c) sample no. 11, (d) sample no. 9, (e) sample no. 12, (f) sample no. 13, (g) sample no. 14.
SEM images of ZnCo2O4/ZnO nanoparticles: in the absence of Cochineal (a), in the presence of different amount of Cochineal based on Table 1. Sample no. 2 (b) sample no. 3 (c) and sample no. 4 (d).
SEM images of ZnCo2O4/ZnO nanoparticles synthesized in the presence of different zinc source; sample no. 9 (a), sample no. 10 (b) and sample no. 11 (c).
SEM images of ZnCo2O4/ZnO nanoparticles synthesized in the presence of different zinc source; sample no. 12 (a), sample no. 13 (b) and sample no. 14 (c).
SEM images of ZnCo2O4/ZnO nanoparticles synthesized at different temperatures sample no. 5 (a) and sample no. 6 (b).
SEM images of ZnCo2O4/ZnO nanoparticles synthesized at different time of reaction; sample no. 7 (a) and sample no. 8 (b).
(a-c) TEM images of optimum formulation of ZnCo2O4/ZnO nanoparticles (sample no.14) with different magnification, and d) High resolution TEM images of optimum formulation of ZnCo2O4/ZnO nanoparticles (sample no.14).
EDS pattern (a) and VSM curve (b) of ZnCo2O4/ZnO nanoparticles.
3.2 Anticancer evaluation of the sample
The anti-proliferative effect of ZnCo2O4/ZnO NPs (sample no.14) against 4T1 cell lines was examined by MTT assay. Fig. 11a displayed that ZnCo2O4/ZnO nanocrystals meaningfully inhibited the cancerous cell lines 4T1 cell proliferation. The cytotoxicity of the samples indicated a dose-dependent pattern with IC50 of 0.3 mg/ml. Zn based NPs consistently have been reported to selectively (or preferentially) induce apoptosis in cancer cells rather than normal cells (Hanley et al., 2008) (see Fig. 12).
Percent of the 4T1 cell viability (a) and ROS generation (b) of ZnCo2O4/ZnO nanoparticles (sample no.14) on 4T1 cell line.
Images of fluorescent microscopy of 4T1 cells stained with AO/EB, Un-treated cells (a) and treated cell (b) by and Images of 4T1 cells with DAPI staining Un-treated cells (c) and treated cell (d) by nanoparticles.
3.3 Effect of ZnCo2O4/ZnO on cellular ROS
To examine the AP probable triggering parameter, the ROS was determined. H2DCF-DA assay was used for measuring the level of intracellular ROS in cell lines after the addition of ZnCo2O4/ZnO NPs (sample no.14). The association of intracellular ROS increment with the cytotoxicity of nano-sized materials was confirmed by literature (Nel et al., 2006). ROS have multifunction application in both normal and stress states. In the normal condition, ROS is an activator of cell growth and development; and in the stress situation, it might lead to not only pathological possessions but also to CD (cell death). The advanced levels of intracellular ROS lead to the apoptotic pathway (Hu et al., 2009).
Furthermore, it was observed that the AP (apoptosis) mechanism in cells treated with heavy-metal NPs might associate with augmented ROS levels. It was recognized that sample 14 managed the increment of intracellular ROS level. As illustrated in Fig. 11b, the ROS level produced by the reaction of ZnCo2O4/ZnO (sample no.14) with treated cells is meaningfully higher than that of normal cell lines.
3.4 Mechanism of anticancer effect
The CD mechanism in 4T1 cell lines treated by ZnCo2O4/ZnO NPs was observed by investigating 1) cellular characteristics, 2) nuclear morphology, 3) annexin V/7-AAD staining checked by flow cytometry, and 4) cell cycle arrest. For studying the cellular characteristics, 4T1 cell lines treated by IC50 values of ZnCo2O4/ZnO NPs and untreated 4T1 cells were used as negative control groups. Then stained with AO/EB and detected by a fluorescence microscope (Sharma et al., 2012). Green and red fluorescence are related to living and apoptotic cells, respectively. As depicted in Fig. 13a and b, the 4T1 cells treated by ZnCo2O4/ZnO (sample no.14) showed extended AP in comparison to the untreated 4T1 cells. For studying the morphological variations in nucleus alterations in the treated 4T1 cells by ZnCo2O4/ZnO, treated and untreated cells were stained by DAPI. As shown in Fig. 13c and d, the untreated cell lines designated homogeneous nuclei, however sample 14 treated cells display fragmentized nuclei. Quantification of AP was determined using flow cytometry. Fig. 13a and b display that 0.3 mg/ml of ZnCo2O4/ZnO NPs induced 15% early AP, 40% late AP, and 10% necrosis in 4T1 cells after 24 hs treatment. Due to the fact that the higher portion of mortality is apoptotic (55%) and necrotic death constituted a small part (10%), ZnCo2O4/ZnO (sample no.14) NPs can be taken as an anticancer for invasive breast cancer cells. Fig. 13a and b undoubtedly illustrated that the main CD mechanism induced by ZnCo2O4/ZnO NPs is AP.
Two-dimensional contour density plots. Un-treated cells (a) and treated cell (b) by nanoparticles. Flow cytometry based assay of cell cycle of control. Treated cell (c) and Un-treated cells (d) by nanoparticles.
3.5 Effect of ZnCo2O4/ZnO NPs on cell cycle
The cell cycle arrest effect of ZnCo2O4/ZnO (sample no.14) NPs on 4T1 cells was also investigated by PI staining. Flow cytometry data (13c and d) suggested that 0.3 mg/ml ZnCo2O4/ZnO NPs treatment after 24 h induced variations in 4T1 cell cycle distributions (38% in G1 phase, 38% in S phase, and 23% in G2 phase) in comparison to controlled untreated cell lines (58% in G1 phase, 32% in S phase, and 10% in G2 phase). A drastic decrease in G1 phase cell abundance and a drastic increment in G2 phase cell abundance were observed in treated cells. Moreover, 7.91% of cells were in sub-G1 that is correlated to the apoptotic population. Accordingly, analysis of cell cycle data confirmed that CD mechanism could be AP and G2 phase cell arrest; it could be due to capturing of the cells at cell division stage owing to both DNA cleavage and cell division machinery damage.
3.6 Effect of ZnCo2O4/ZnO NPs on mice body weight and tumor size
The anti-cancer effect of ZnCo2O4/ZnO (sample no.14) NPs was studied in vivo against BALB/c mice. The effect on mice weight and tumor size was determined and summarized in Fig. 14 a and b. To do this, we grouped the mice in to 5 groups; group 1: control group (neither injected with 4T1 cell nor treated with NPs), group 2: Mice injected with 4T1 cells, group 3: Mice after 7 days of injection with 4T1 cells (this group, when primary tumors reached a mean diameter of 3–4 mm, they were treated by NPs), group 4: Mice injected with 4T1 cells and treated with NPs, group 5: Mice treated with NPs without 4T1 cell injection. Among the groups, a significant reduction of weight was observed in group 2 due to 4T1 cells (Fig. 14a group 2), and the size of tumor was the biggest in this group (Fig. 14 b group 2).
Effect of nanoparticles on the mice’s weight (a) and tumor size (b). Group 1 untreated mice as control, Group 2 treated only by 4T1 cells, Group 3 treated by 4T1 cells and after 7 days, Group 4 treated by 4T1 cells and nanoparticles simultaneously, Group 5 treated only by nanoparticles.
Group 3 showed a little loss of weight in comparison with the control (Fig. 14a group3), and the size of tumor is the smallest of all due to ROS effect of ZnCo2O4/ZnO NPs (Fig. 14b group3). This indicates that, the prepared NPs have a great effect against growing tumor cells. The loss of weight by group 4 was more than group 3 because of prolonged contact time of NPs with mice cells, (Fig. 14a group4). The tumor size in this group was larger than group 3 (Fig. 14b group 4). Therefore, treatment of cells with NPs after 4T1 cells injection have not enough tumorigenesis prevention effect. As shown in Fig. 14a group 5 showed loss of weight, but lower than weight loss by group 2 (mice injected with 4T1 cells without any NPs treatment). There were no tumor sign detected on mice in groups 1 and 5. Tumor-bearing mice without any ZnCo2O4/ZnO NPs treatment showed the maximum tumor size (Fig. 14b, Group 2). By comparing the tumor size between groups 3 and 4, we concluded that, synthesized nanomaterials could protect tumorigenesis. Group 3 revealed the most significant decrease in tumor size associated with ZnCo2O4/ZnO NPs (sample no.14) treatment of tumor-bearing mice. Hence, the synthesized NPs have anti-tumor effect.
4 Conclusion
In this work, a novel original biosynthetic method for the synthesis of Sub 40 nm uniform magnetic ZnCo2O4/ZnO NPs in the presence of cochineal dye, a green and ecological friendly precursor, as a green capping and as a reducing agent via a solid-state thermal decomposition process is introduced for the first time. It is a solitary step for the NPs synthesis that appears to be proper for bulk scale production as it is an effortless, cost-effective, non-toxic, and eco-friendly manner, exhibiting several properties. The magnetic NPs designated significant cytotoxic and dose-dependent CD effect against 4T1 cancer cell lines. The in vitro and in vivo result reviled, that ZnCo2O4/ZnO NPs are promising chemotherapeutic agents against invasive breast cancer cells, and had great potential for anticancer activities; though, more pharmacological investigations are certainly needed.
Acknowledgments
Authors are grateful to the council of Iran National Science Foundation (97017837) and the University of Kashan for supporting this work with grant No (159271/MG6).
References
- Synthesis, characterization and degradation of organic dye over Co3O4 nanoparticles prepared from new binuclear complex precursors. RSC Adv.. 2014;4(87):46517-46520.
- [Google Scholar]
- Sol–gel auto-combustion synthesize and characterization of a novel anticorrosive cobalt ferrite nanoparticles dispersed in silica matrix. J. Mater. Sci.: Mater. Electron.. 2017;28(14):10495-10508.
- [Google Scholar]
- Synthesis, characterization and evaluation of Co3O4 nanoparticles toxicological effect; synthesized by cochineal dye via environment friendly approach. J. Alloy. Compd.. 2019;784:676-685.
- [Google Scholar]
- A magnetic CoFe2O4/SiO2 nanocomposite fabricated by the sol-gel method for electrocatalytic oxidation and determination of L-cysteine. Microchim. Acta. 2017;184:825-833.
- [Google Scholar]
- Caffeine: A novel green precursor for synthesis of magnetic CoFe2O4 nanoparticles and pH-sensitive magnetic alginate beads for drug delivery. Mater. Sci. Eng., C. 2017;76:1085-1093.
- [Google Scholar]
- Effective induction of death in breast cancer cells with magnetite NiCo2O4/NiO nanocomposite. Compos. B Eng.. 2019;166:457-463.
- [Google Scholar]
- Green synthesis and characterization of MnCo2O4/Co2Mn3O8 ceramic nanocomposites and investigation of their cytotoxicity on the 4T1 cells. Compos. B Eng.. 2019;163:424-430.
- [Google Scholar]
- Sonochemical assisted thermal decomposition method for green synthesis of CuCo2O4/CuO ceramic nanocomposite using Dactylopius Coccus for anti-tumor investigations. J. Alloy. Compd.. 2019;788:944-953.
- [Google Scholar]
- Hollow polyhedral ZnCo2O4 anode materials for lithium-ion batteries. Mater. Lett.. 2019;236:337-341.
- [Google Scholar]
- Metal-Organic Framework Templated 3D Hierarchical ZnCo2O4@ Ni(OH)2 Core-Shell Nanosheet Arrays for High-Performance Supercapacitors, Chemistry–A. European Journal. 2018;24:18106-18114.
- [Google Scholar]
- Interconnected hierarchical NiCo2O4 microspheres as high-performance electrode materials for supercapacitors. Dalton Trans.. 2017;46(28):9201-9209.
- [Google Scholar]
- Construction of NiCo2O4 nanosheet-decorated leaf-like Co3O4 nanoarrays from metal–organic framework for high-performance hybrid supercapacitors. Dalton Trans.. 2019;48(37):14156-14163.
- [Google Scholar]
- Self-templated synthesis of uniform nanoporous CuCo2O4 double-shelled hollow microspheres for high-performance asymmetric supercapacitors. Chem. Commun.. 2017;53(6):1052-1055.
- [Google Scholar]
- MOF-derived porous hierarchical ZnCo2O4 microflowers for enhanced performance gas sensor. Ceram. Int.. 2021;47(7):9214-9224.
- [Google Scholar]
- MOF derived ZnCo2O4 porous hollow spheres functionalized with Ag nanoparticles for a long-cycle and high-capacity lithium ion battery anode. J. Mater. Chem. A. 2017;5(43):22717-22725.
- [Google Scholar]
- Hierarchical three-dimensional ZnCo2O4 nanowire arrays/carbon cloth anodes for a novel class of high-performance flexible lithium-ion batteries. Nano Lett.. 2012;12(6):3005-3011.
- [Google Scholar]
- Simple method for the preparation of highly porous ZnCo2O4 nanotubes with enhanced electrochemical property for supercapacitor. Electrochim. Acta. 2014;123:450-455.
- [Google Scholar]
- Nanophase ZnCo2O4 as a High Performance Anode Material for Li-Ion Batteries. Adv. Funct. Mater.. 2007;17(15):2855-2861.
- [Google Scholar]
- Functional properties of ZnCo2O4 nano-particles obtained by thermal decomposition of a solution of binary metal nitrates. RSC Adv.. 2015;5(34):26843-26849.
- [Google Scholar]
- Nanostructured spinel ZnCo2O4 for the detection of LPG. Sens. Actuators, B. 2011;152(1):121-129.
- [Google Scholar]
- Preparation and gas sensing properties of ZnM2O4 (M = Fe Co, Cr) Sens. Actuators, B. 2004;99:405-409.
- [Google Scholar]
- Controllable interior structure of ZnCo2O4 microspheres for high-performance lithium-ion batteries. Nano Energy. 2015;11:64-70.
- [Google Scholar]
- Preparation and characterization of the spinel oxide ZnCo2O4 obtained by sol–gel method. Mater. Chem. Phys.. 2007;103:54-58.
- [Google Scholar]
- Surfactant-mediated synthesis of ZnCo2O4 powders as a high-performance anode material for Li-ion batteries. Ionics. 2015;21:623-628.
- [Google Scholar]
- Cytotoxic effects of ZnO hierarchical architectures on RSC96 Schwann cells. Nanoscale Res. Lett.. 2012;7:439.
- [Google Scholar]
- The neurobiology of zinc in health and disease. Nat. Rev. Neurosci.. 2005;6(6):449-462.
- [Google Scholar]
- Loss of zinc staining from hippocampal mossy fibers during kainic acid induced seizures: a histofluorescence study. Brain Res.. 1988;446(2):383-386.
- [Google Scholar]
- Translocation of zinc may contribute to seizure-induced death of neurons. Brain Res.. 1989;480(1-2):317-321.
- [Google Scholar]
- Possible role of zinc in the selective degeneration of dentate hilar neurons after cerebral ischemia in the adult rat. Neurosci. Lett.. 1990;109(3):247-252.
- [Google Scholar]
- Selective toxicity of ZnO nanoparticles toward Gram-positive bacteria and cancer cells by apoptosis through lipid peroxidation, Nanomedicine: Nanotechnology. Biol. Med.. 2011;7(2):184-192.
- [Google Scholar]
- Zinc oxide nanoparticles induce oxidative DNA damage and ROS-triggered mitochondria mediated apoptosis in human liver cells (HepG2) Apoptosis. 2012;17:852-870.
- [Google Scholar]
- DNA damaging potential of zinc oxide nanoparticles in human epidermal cells. Toxicol. Lett.. 2009;185:211-218.
- [Google Scholar]
- Wahab, R., Khan, F., Musarrat, J. & Al-Khedhairy, A. A. Role of Smart Nanostructured Materials in Cancers in Responsive Materials and Methods: State-of-the-Art Stimuli Responsive Materials and Their Applications (eds Tiwari, A. & Kobayashi, H.) Ch. 9, 237–272(John Wiley & Sons, 2013).
- Impacts of SW monsoon on phytoplankton community structure along the western coastal BOB: an HPLC approach. Estuaries Coasts. 2017;40:1066-1081.
- [Google Scholar]
- Magnetic nickel ferrite nanoparticles: Green synthesis by Urtica and therapeutic effect of frequency magnetic field on creating cytotoxic response in neural cell lines. Colloids Surf., B. 2018;172:244-253.
- [Google Scholar]
- Evaluation of the probe 2', 7'-dichlorofluorescin as an indicator of reactive oxygen species formation and oxidative stress. Chem. Res. Toxicol.. 1992;5(2):227-231.
- [Google Scholar]
- Effect of zinc: Cobalt composition in ZnCo2O4 spinels for highly selective liquefied petroleum gas sensing at low and high temperatures. RSC Adv.. 2015;5:40429-40436.
- [Google Scholar]
- Development and characterization of a preclinical model of breast cancer lung micrometastatic to macrometastatic progression. PLoS ONE. 2014;9(5):e98624.
- [CrossRef] [Google Scholar]
- Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods. 1983;65(1-2):55-63.
- [Google Scholar]
- A.R. Van Dam, B. May, A new species of Dactylopius Costa (Dactylopius gracilipilus sp. nov.)(Hemiptera: Coccoidea: Dactylopiidae) from the Chihuahuan Desert, Texas, USA, Zootaxa, 3573 (2012) 33–39.
- Using pomegranate peel powders as a new capping agent for synthesis of CuO/ZnO/Al2O3 nanostructures; enhancement of visible light photocatalytic activity. Int. J. Hydrogen Energy. 2018;43(31):14406-14416.
- [Google Scholar]
- Controllable synthesis of ZnO nanocrystals via a surfactant-assisted alcohol thermal process at a low temperature. Mater. Lett.. 2005;59(23):2867-2871.
- [Google Scholar]
- Preparation and characterization of superparamagnetic functional polymeric microparticles. China Particuol.. 2003;1(2):76-79.
- [Google Scholar]
- Preferential killing of cancer cells and activated human Tcells using ZnO nanoparticles. Nanotechnology. 2008;19(29):295103.
- [CrossRef] [Google Scholar]
- Metallic nanostructures as localized plasmon resonance enhanced scattering probes for multiplex dark-field targeted imaging of cancer cells. J. Phys. Chem. C. 2009;113:2676-2684.
- [Google Scholar]
