Safety and Efficacy of Resveratrol-ZnO nanoconjugate in DMBA-induced ovarian cancer in murine model

Safety and Efficacy of Resveratrol-ZnO nanoconjugate in DMBA-induced ovarian cancer in murine model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Safety and Efficacy of Resveratrol-ZnO nanoconjugate in DMBA-induced ovarian cancer in murine model Mahamuda khatun, Santasree Mazumder, Dr Soumendra Darbar This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6187379/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Resveratrol (3,5,4-trihydroxy-transstilbene), a natural phytoalexin is well known for its anticancer and chemo-preventive effect. To overcome stability and solubility issue of Resveratrol (RSV), a conjugation of RSV with ZnO nanoparticles was prepared to improve the efficacy and reduce the toxicity. Thus objective of the present study was to evaluate the potential effect of RSV-ZnO nano-conjugate in DMBA-induced ovarian cancer in a defined rat model. The acute and sub-acute toxicity was carried as per OECD guidelines 423 and 407, respectively. In the acute toxicity study, nano-conjugate drug was administered via single Intraperitoneally (IP) injection at 200 mg/kg and 400 mg/kg body weight doses in female albino Wistar rats while in sub-acute toxicity study, female Wistar rats was administered daily IP injection of 50 mg/kg, 100 mg/kg and 200 mg/kg body weight for 28 days. At the end of the study, the animals were sacrificed and evaluate the effect of RSV-ZnO nano-drug on body and organ weight and hematological biochemical parameters and histopathological findings. DMBA was used as inducing agent to develop ovarian cancer. Rats were treated by DMBA (50 mg/kg body weight) via IP route once a week up to 3 weeks and kept animals for 3 months for development and progression of carcinogenesis in ovary. After 3 months, we treated the induced animals by RSV-ZnO nano-conjugate in three different doses (50 mg/kg, 100 mg/kg and 150 mg/kg body weight by IP) weekly for one month. After that, we sacrificed the animals and taken out the ovaries for histopathology for tumor subtyping and immunohistochemistry to evaluate the markers such as CA125, CK7, Vimentin. RSV-ZnO nano-conjugate showed significant reduction of expression of cancer bio-markers and size of tumor. Besides, in acute and sub-acute toxicity study, it exhibited no untoward effects in animals. The findings demonstrated that RSV ZnO nano-conjugate drug is safe and may be used for the treatment of ovarian cancer with more efficacies and less toxicities. Toxicology Cancer Biology Safety study of the nano-drug Ovarian cancer induction by DMBA Histopathological analysis Immunohistochemistry CA125 CK7 Vimentin cancer biomarker Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Introduction Ovarian cancer is one of the leading causes of cancer-related deaths among women [1]. Effective detection and treatment of ovarian cancer remains a significant clinical challenge. Approximately 90% of ovarian tumors derive from an epithelial origin, most often considered to be the ovarian surface epithelium; 80% of these originated in the ovarian surface epithelium [2–4]. Incessant ovulation, postmenopausal increase of gonadotropin hormone levels, chronic inflammation and environmental carcinogens are assumed to play a key role in ovarian carcinogenesis [5–9]. When identified as stage I disease, 5 years disease free survival approaches 95%, although 20% of these patients will eventually relapse to a treatment failure overall. In early stage ovarian tumors lack specific clinical symptoms, which hampering early detection and delaying diagnosis in some cases. Recent use of lysophosphatidic acid and the a-folate receptor as biomarkers, in combination with CA125, have improved the specificity for detection of early stage malignant gynecologic tumors [10] and will probably be further advanced by the addition of proteomic screening of low molecular weight serum proteins using Surface-Enhanced Laser Desorption and Ionization Time-off Light (SELDI-TOF) mass spectroscopy [11]. Incidence of ovarian adenocarcinoma has varied between 10% and 45%, perhaps, owing to the strain of rat employed or the chemical form of the DMBA utilized and the fact that different studies have observed a wide variance in the incidence of tumors of stromal origin [12,13]. To prevent or reverse the progression of initiated cells to malignant disease chemoprevention is an alternative approach. Clinical trials of preventive agents have demonstrated an efficacy in patients at risk for both breast and colon cancer [14]. The first prevention trials for women shows high risk for the development of ovarian cancer and hence prophylactic oophorectomy have now been initiated to evaluate biomarker modulation e.g. ovarian surface metaplasia, cellular atypia and cortical inclusion cysts following short term treatment with fenretinide, oral contraceptive therapy or placebo [15]. Spontaneous rodent ovarian tumors of epithelial origin have been reported but these do not appear to occur in experimentally significant numbers [16]. Ovarian cancers are divided into type I or type II by histologically and genetically. Type I cancers are of low histological grade such as endometrioid, mucinous, and clear-cell carcinomas. Type II cancers are of higher histological grade and include serous carcinoma and carcinosarcoma [17]. Treatment of ovarian cancer are still not clear, there is a continuing need for a validated animal model possessing histological characteristics appropriate to the presentation of human ovarian tumors to evaluate the efficacy of newly developed chemo preventive compounds. 7,12-Dimethylbanz(a)anthracene (DMBA) is a well-known polycyclic aromatic carcinogen that is capable of inducing the initiation, promotion and progression of tumor importantly the histological type of experimental ovarian tumors may vary based on the animal strain used, use of carcinogen and age of animals. Use of carcinogen means amount and route of administration and method of induction. So, treatment and effective detection of ovarian cancer still a significant clinical challenge. The direct implantation of chemical carcinogen DMBA in the rat ovary [18] leads to the induction of ovarian tumors at an incidence of 37% these include adenocarcinomas, as well as stroma and mesothelialtumors [19–21]. There is however, lack of information regarding the sequence and nature of events elicited by DMBA and leading to ovarian cancer development. To improve its usage and physiologic relevance to the human disease, the DMBA model of ovarian cancer was modified by significantly decreasing the DMBA dose, thereby preserving maximally the integrity of the organ and by incorporating multiple gonadotropin hormone treatments, thus introducing an additional risk factor associated with human ovarian cancer, known also to induce hyper ovulation and enhanced mitogenesis of the ovarian surface epithelium [22]. Nanomaterial toxicity is a matter of concern and many in vivo studies have suggested that the surface chemistry, release of metal ion, Reactive Oxygen Species (ROS) production, cell apoptosis and break down of nano-materials are the possible causes of cellular toxicity [23–25]. Current problem for nano-medicine involve understanding the issue related to toxicity, in chemotherapy, toxicity to healthy cell is associated with anticancer drugs. Nanoparticles are used as a drug carrier for the specific delivery of drugs to cancer cells because of the size of nanoparticles which is similar to that of most biological molecules and structures. Consequently nanoparticles can be useful for both in vivo and in vitro biomedical application and research. The anticancer drug is delivered specifically to the cancer cells in targeted drug delivery. This reduces the toxicity to normal cells. The physicochemical characteristics of nano carriers, such as composition, particle size, surface charge and the presence of ligands on their surface, will dictate their bio-distribution and pharmacokinetics [26,27]. Nanoparticle to be used as a carrier in drug delivery system should be nontoxic in vivo . Surface modification and nano-conjugation of nanoparticle with drugs is an aspect of drug delivery system design for biomedical applications. Modulation of nanoparticle by conjugation with drugs can influence particle uptake, biological responses, bio-distribution and its efficacy also. Surface functionalization can be utilized to increase the drug circulation time in blood, reduce non-specific distribution or specific targeting of tissue or cells by using a targeting ligands [28]. In vivo , nanoparticles will be entrapped in and translocated to other tissues or organs along the blood circulation. The small size and large surface area equip them with an active group or intrinsic toxicity. Even at a very small cellular level, they may constitute potential long-term health hazards [29,30]. Toxicity indicates the disadvantageous effects due to the interaction between nano-materials and cells. This interaction may differ depending on the chemical properties of the toxicants and the cell membrane, as it may take place on the cell surface or within the cell body. The evaluation of toxic properties of nano-materials is pivotal when considering them for use in biomedical science. In practice, the evaluation typically includes acute, subchronic and chronic studies [31]. The current status of toxicology due to nanoparticles has been reported by Becker [32]. Toxicity of NPs has been evaluated in different biological systems, such as bacteria, mammalian cells and in vivo model [33–35]. The safeties of RSV are well known, but the combined effects of this nano-conjugate are unclear. Thus, it becomes essentials to evaluate the safety and toxicity of the conjugate, before their use. Therefore the objective of the present study, to assess the safety of nano-conjugate by acute and sub-acute toxicity of nano-conjugate in Wistar rats, the study was done on the report of the Organization for Economic Co-operation and Development (OECD) guidelines 423 and 407 respectively. After completing the safety study we evaluate characterization of ovarian tissue by two different study of this modified animal model. One is induction of ovarian cancer by IP dosage of DMBA and another is treatment of ovarian carcinoma by RSV-ZnO nano-conjugate drug (100 mg/wk for 3 weeks) with the immunohistochemistry study of some cancer biomarker such as CA125, Vimentin and CK7. Cancer induction method by using DMBA is well established method but in this study we used IP injecton of DMBA with particular dose as an induction method which is very easy and time consuming compare to the other published methods. So this is one of new findings of our research. Confirm the induction of ovarian cancer by measuring ovary size and histological study of ovarian tissue which shows different type of carcinoma arise in tissue such as serous, clear cell, sarcoma, epithelial carcinoma. Materials and Methods In our study there is two methods are used one is study safety of the drug. Then study the efficacy of the drug on ovarian cancer induced rat. So firstly we explain the methods for safety study of drug and the methods are described in below. Animals Healthy female swiss albino mice and Wistar rats (8–10 weeks old)(approximately 30–35 g body weight mice, 150–155 g body weight rats, Calcutta university, India) were housed in polypropylene cages with a maximum of three animals of the same sex per cage and placed in a ventilated, temperature-controlled room. The standard conditions were maintained at 20°C ± 2°C, 60% ± 10% relative humidity and a 12 hr light/dark cycle. The cages composed autoclaved paddy husk as bedding that was replaced twice in a week. The commercial pellet diet and distilled water for mice were available at lab of Calcutta University. The experimental protocol involving animals was carefully reviewed and approved Animal House of Dey’s medical. Approval No. IAEC/s/PHARM/16/2016 by the Institutional Animal Ethics Committee, India, where the studies were carried out in compliance with the guidelines of the Committee for the Purpose of Control and Supervision of Experimental Animals (CPCSEA), India. Animals were acclimated to their environment for 7 days prior to treatment. Synthesis and characterization of RSV-ZnO nano-hybrid ZnO NPs were prepared by the co-precipitation technique using highly pure zinc acetate dehydrate (Zn [CH 3 COO] 2 H 2 O, 99% pure) as a precursor with sodium hydroxide solution following the procedures reported earlier [36]. Required amount of RSV was added into 20 ml of ZnO NPs solution and kept it in a shaker for overnight to formation of nano-conjugate. Characterization of nano-hybrid confirmed by TEM, FTIR study, Picosecond resolved spectroscopy study and also by Raman scattering experiment [37–39]. The HRTEM image clearly reflects crystalline nature of ZnO Np [40]. The drug used in this study RSV, contain three hydroxyl groups, which possesses binds ability to the surface of ZnO [41]. All data are published in our previous paper [42]. Drug administration We choose the administration of drug (Res-ZnO nano-conjugate) by intraperitoneal injection as it is predominantly used for it’s easy compared to other parental methods during animal testing for the administration of systemic drugs and fluids. Additionally, one can administer a large volume of nano-conjugate suspension (400 mg/kg) to the mice and rats if one choose Intraperitoneal (IP) route compared to the intravenous method. This much drug cannot inject via the intravenous route. Wistar rats (nine females) were assigned to the following test groups: Group I (200 mg/kg body weight of Res-ZnO nano-conjugate, IP); and group II (400 mg/kg body weight) and control. The respective doses were suspended in DMSO and were administered Intraperitoneally (IP). After that, the animals were observed for 14 days for clinical signs of toxicity or mortality. On completion of the treatment, the animals were sacrificed by cervical dislocation and necropsies to facilitate gross pathological examination of organs. Acute toxicity To obtain the LD 50 of nano-conjugate, the experiments and its interval were designed in accordance with the karber’s method. After 7 days acclimation, the animals (Wistar rats, 8–10 weeks) were randomly divided into 4 treatment groups and were gavages with the respective doses (control, 50 mg/kg body weight, 100 mg/kg body weight and 200 mg/kg body weight) of nano-conjugate once daily, for a period of 28 days. The central nervous system, motor nervous system, vegetative nervous system, respiratory system, digestive system, urinary system, changes of the eyes, skim, clothing hair and death situation were continuously observed and recorded during the 14 day treatment. Special attention was paid on the clinical signs of toxicity including tremors, convulsion, salivation, nausea, diarrhea, lethargy and coma. Body weights were recorded on days 0, 7, 14, 21, 27 and 28. Feed consumption was deliberated per cage over successive periods of 3 days by weighing the feeders. The animals were examined for any clinical signs of morbidity, mortality, changes in body weight and changes in food consumption throughout the dosing. At the end of the treatment, blood was collected from the animals from the ocular vein for clinical pathology assessment, which included analysis of various hematological parameters and biochemical parameters. Consequently, the animals were sacrificed by cervical dislocation and histopathological assay was done of the various organs. The heart, liver, kidney, lung, spleen, stomach were stripped out and weighed accurately. The histopathological study also included careful and consistent dissection of various target organs like liver and kidneys, Heart, lungs, spleen, stomach. The tissue of organ samples were embedded in paraffin blocks then sliced and placed onto glass slides. After H and E staining, the pathological changes were observed on microscope. Blood biomarker assay All animals were sacrificed at the same time and blood samples were collected via the ocular vein. Of this sample, 1 ml was collected in 10% EDTA for hematological assay and the remaining of blood serum was collected for biochemical assay. Blood samples were collected in tubes containing Ethylene Diamine Tetra Acetic (EDTA) acid as anticoagulant. Hemoglobin, platelets, mean corpuscular hemoglobin concentration, packed cell volume, WBC were measured using a hematology auto analyzer. Biochemical parameter analysis The serum was obtained by centrifugation of the whole blood at 3000 rpm for 15 min. Liver function was evaluated based on the serum levels of Alkaline Phosphatase (ALP), Alanine Aminotransferase (ALT) and Aspartate Aminotransferase (AST), GGT, Tbil, Dbil as well as on blood glucose and cholesterol levels. Nephrotoxicity was determined by uric acid, urea, serum creatinine, total protein, albumin and globulin. Organ weight and histopathological examination After sacrifice, organs like heart, lungs, spleen, stomach, liver and kidney were removed and adherent tissues, if any were carefully cropped. The wet weight of organs was registered right away. All histopathological tests were performed using standard laboratory procedures for pathological studies. The dissected organs were fixed in 10% neutral buffer formalin and processed adequately. The tissues were embedded in paraffin blocks, then sliced to 5 µm thickness and placed onto glass slides. After hematoxylin–eosin staining, the slides were examined and photos were taken using an optical microscope. The results were analyzed by a veterinary pathologist. Statistical analysis For statistical analysis, each of the experimental values was differentiated with its corresponding control. The results were conveying as mean ± standard deviation. Multi-group comparisons of the means were accomplished by one-way analysis of variance test. Statistical consequence for all tests was set at P-value is 0.05. Induction of ovarian cancer and efficacy of the Drug: In our study the second methods we used are how to induced ovarian cancer in rat model and also elaborate the methods about the study of efficacy of the drug on ovarian cancer induced rat. Chemicals DMBA (Sigma Chemical Co.) DMBA dissolved in 10 ul corn oil (as a vehicle). Rats were treated for 3 days during a 3 weeks period by IP injections of DMBA in corn oil at doses of 50 micrograms/kg day (150 micrograms/kg total dose). Animal selection : Wistar rats, six female, eight weeks old, 150 mg-200 mg. Rats per group(n = 6) were housed in the conventional facility in clear plastic cages with plastic lids containing filter space. Temperature, humidity and photoperiod were constant (22°C, 50–70%, and 12:12 hrs light: Dark cycle). Rats acclimated to the facility for 7 day before the experiment began. Study design For our study we take 54 rats, for three group (n = 6) with 3 times repetitions. Animals were maintained in lab conditions for a week’s time and fed Amrut Lab Rodent Chow (4.55% Fat, 20% Crude protein, 60% Carbohydrate and 5.08% Dietary fiber) during the entire course of the study. Rats were treated for 2 days (Day1, Day 14) during a 2 week period by Intraperitoneal injection or IP injections of DMBA in corn oil at doses of 50 micrograms/kg day (100 micrograms/kg total dose). Rats were palpated weekly throughout the observation period, which terminated 4 month after IP injection of DMBA. After 3 month we observe by sacrificing rats that cancer is induced by histological study of ovary tissue. Rats with palpable tumors were terminated before any morbidity and the tumors were sampled to provide both frozen and formalin-fixed tissue. We evaluate the different weight of ovaries compare to control. Ovaries of tumor bearing rats were fixed to provide histologic controls. All tissue for histologic evaluation was fixed overnight in10% neutral buffered formalin, sectioned at 5 um and stained with Hemotoxylin and eosin and processed for immunohistochemistry to confirm the induction of ovarian tumor. After 4 month of observation and conformation of half of animals with tumor rest animals were treated with our nano-conjugated drug. Animals were treated with RSV-ZnO nano-conjugate drug with dose of 100 mg/kg body weight for 3 days during a 3 weeks period by IP injection of RSV-ZnO nano-conjugate. Rats with treatment of drugs were terminated before any morbidity and abnormalities. Then we evaluate the weight and size of the ovaries compare to untreated ovaries. Treated ovaries were processed for immunohistochemistry of CA125, CK7, Vimentin to confirm the efficacy of the drug. Immunohistochemistry : Paraffin-embedded section for immunohistochemistry were dewaxed, rehydrated and processed for antigen unmasking by heating to near boiling in citrate buffer (0.01 M) in a microwave oven, then allowed to cool for 20 min. Following this and all subsequent incubations except for the blocking serum, slides were washed in PBS. Endogenous peroxidase activity was blocked by hydrogen peroxide incubation (0.3% methanol) for 20 mins. Frozen section were mounted onto slides fixed in cold acetone (-20°C) for 10 min and air dried before peroxide block incubation. After 30 min incubation in blocking serum (diluted 1:50 in PBS) appropriate to each secondary antibody, slides were incubated with primary antibody at 40°C overnight (Cytokeratin 7, CA125, Vimentin) 1 ug/ml, Santa Cruz Biotechnology Inc. All reagents except for the chromagen substrates were diluted with PBS containing 2% Bovine serum albumin. Detection of primary antibody proceeded with the binding of biotinylated secondary antibody (diluted 1:200 for 30 min) followed by avidin-biotin peroxidase complex and DAB chromogen substrate [43]. Results Safety of the drug: In the part of safety of the drug we explain the findings about is there any side effect of the drug and also find out which dose is safe for the study. Mortality and clinical observation No mortality was found in the animals in control group and any other test groups. The animals did not show any abnormal behavior throughout the 14 days of acute study and there were no treatment related mortality and clinical signs of toxicity in 28 days repeated dose sub chronic study. Clinical sign of nano-conjugate over treated and control groups were similar ( Table 1 and Fig. 1 ). The nanoparticle based drug delivery was found to be safe for a single limit dose equivalent to 100 and 200 mg/kg of nano-conjugate. Table 1 Effect of RSV-ZnO nano-conjugate on Clinical Signs of Toxicity. Clinical signs Control 50 mg/kg 100 mg/kg 200 mg/kg 400 mg/kg Hyperactivity Absent Absent Absent Absent Absent Piloerection Absent Absent Absent Absent Absent Convulsion Absent Absent Absent Absent Absent Jumping Absent Absent Absent Absent Absent Irritability Absent Absent Absent Absent Absent Reddening Absent Absent Absent Absent Absent Abnormal Absent Absent Absent Absent Absent Secretion Absent Absent Absent Absent Absent Acute toxicity analysis of nano-conjugate No mortality was observed in the animals in any test group or in the control group. The animals did not show any abnormal behaviour throughout the 14 days. Pathological examination of liver, kidney and other organs did not show any evidence of toxicity. The nanoparticle-based targeted drug delivery system was found to be safe for a single limit dose equivalent to 100–200 mg/kg of Res-ZnO and the 95% confidence limits for the LD50 are 100–200 mg/kg body weight. All clinical symptoms were evaluated by comparing treated animals with controls (n = 6). The toxicity of the nanoparticle formulation due to a single dose for less than 14 days was determined by acute toxicity analysis. The present study showed that intraperitoneal treatment of animals with Res-ZnO did not induce mortality or significant clinical symptoms of toxicity. Body weight and organ index No animal mortality was observed in any of the test groups for the entire period of subchronic study for nanoparticles. The subchronic toxicity result shows that the nanoparticle formulations did not reveal any conflicting effect with long-term administration on the general health of the animals. Body weight was weighed before the administration and on day 7 and 14 after administration, respectively. Body weight of animals from group 2, group 3, group 4 and group 5 showed a decreasing trend. These results are given in Fig. 2 . There was no change in the general systemic health of the animals ( Fig. 3 ) presents the mean organ weight and % organ weight to bodyweight for rats. Both control and treated rats did not show any significant difference in mean organ weights of kidney, liver, spleen, heart, stomach, lungs. Hematology and clinical biochemistry The normal values of hematological parameters in the control group and quantitative changes of hematological parameters in treated rats with nano-conjugate are shown in Fig. 4 . There was no significant change between the three subject groups in any of the hematological parameters studied. The Hemoglobin (Hb) levels, WBC, platelets level of treated rats were marginally lower than the control rats. In Fig. 5 A and 5 B we evaluate effect of different doses of drug on Liver enzymes. There was slight elevation in AST, ALP, ALT, and GGT which was not significant There were no significant changes in kidney parameter and lipid, blood glucose level ( Figs. 6 and 7 ) . Protein level parameter also shows normal result compare to control ( Fig. 8 ) . Histopathology : Fig. 9 represent the histopathological findings from the control and the treated rats. No marked morphological and histological changes were observed in any organ of lower (100 mg/kg) medium (200 mg/kg) higher (400 mg/kg) doses of treated rats. Sections of liver exhibited normal central/portal veins, kidney showed normal nephrons, spleen showed normal splenic follicle, heart featured normal myocardium, stomach, lungs did not show any abnormal pathology changes. Hence no toxicological differences on histopathological analysis were observed. Efficacy of the drug During the experimental period, all of the animals were observed and handled based on the same criteria. The amount of food intake did not differ between the experimental groups. No differences in the final body weights or body weight gains were observed during the experimental period. The absolute and relative weights and sizes of the left ovaries were significantly higher in the induced animals. The incidence of DMBA, IP-induced ovarian tumors (the percentage of developed tumors) was higher in animals. After sequential tumor development, the abdominal-pelvic cavity was opened to evaluate ovarian tumors. Although the ovaries with fallopian tube of the rats in the DMBA(IP) treated groups were increased in size ( Table 2 and Fig. 10 ) after 120 days post DMBA Induction.The majority of ovarian tumors were adenocarcinomas. After 120 days of ovarian tumor induction, the experimental groups displayed variations in the histotypes and grades of the tumors ( Fig. 11 ) . Serous carcinomas characterized by pailae formation with cellular budding, and clear cell carcinomas composed of numerous vacuolated cells were more prevalent in DMBA treated group of animals. Epithelial carcinomas, epithelium is composed of stratified cells with loss of nuclear polarity and nuclear pleomorphism with chromatin clumping, Sarcoma composed of non-epithelial malignant cells were also prevalent in DMBA treated ovaries tissue. To analyze the efficacy of the drug we used some cancer biomarker CA125, CK7 and Vimentin to study their expression level with immuno histopathology study ( Figs. 12 – 14 ) . Also evaluate the data by densitometry analysis which is also known as dot blot quantification analysis. Densitometry analysis is quantified by two methods one is by measure percent area value and another method is integrated density. In our study we measure densitometric analysis by compare percent area value of Ca125, CK7 and vimentin treated and induced biomarker ( Fig. 15 ) . After treatment with our drug RSV-ZnO nano-conjugate (dosage of 100 mg/kg body weight per week during 3 weeks study) we have found very positive result with lower expression level of cancer marker (CA125, CK7, Vimentin). Table 2 Ovary weight changes after induction and treatment with nano-conjugate. Sample name Ovary weight after induction Ovary weight after treatment Control (n = 6) 18 mg (normal, no induction) No treatment RSV-ZnO nano-Conjugate (Group II) n = 6 50 mg (Right ovary) 59 mg (left ovary) 20 mg (right ovary) 25 mg (left ovary) RSV (Group III) n = 6 49 mg (Right Ovary) 40 mg (left ovary) 20 mg (Right ovary) 19 mg (left ovary) Discussion Physiochemical properties, such as size surface areas, shapes, solvent and media could affect the toxicity of nanoparticle. Colvin reported that the toxic effects of nanoparticles showed variation depending upon the medium composition in which the nanoparticles are suspended. Hou, et al. reported that the same nanoparticles exhibited different toxic manifestation when dissolved indifferent mediums. Thus suspending agent need to be considering when an in vivo toxicity of nanoparticles was carried on. In our study we conjugate nanoparticle with well-known drug Resveratrol. After conjugation the risk of toxicity of free nanoparticle cannot affect and we found this IP administration of nano-conjugate is very safe. However result of other research show that the target organs for ZnO nanoparticle were the liver, spleen, heart, kidney and lung. In the present study, histopathological damages of the liver kidney, spleen, heart, lungs were observed only in high dose (400 mg/kg). The histopathological analysis of RSV-ZnO-treated (100 mg/kg and 200 mg/kg) animals is depicted in Fig. 9 . In the control, 100 and 200 mg/kg body weight nano-conjugate groups, there was no change in hepatocytes, portal area, or integrated hepatic side effects. Similar to the observation in liver, heart, lungs, kidney, stomach, spleen did not show any abnormal pathology changes at 100 and 200 mg/kg. There were no lesions in tissue of the stomachs, livers, spleens, and lungs, heart. But at higher dose (400 mg/kg body weight) the colour of heart and spleen was slightly changed and also found enlarged spleen and swollen stomach. But with the dose of 50 mg/kg, 100 mg/kg, 200 mg/kg body weight is safe. There were no such lesions found in these organs. In previous research they published that oral administration of ZnO nanoparticle has effect on this organ but in our work result shows the IP doses (50 mg/kg, 100 mg/kg, 200 mg/kg) of our drug is absolutely safe [44]. Hematology and clinical biochemistry Changes in hematological parameters are used to detect the physiological and pathological changes in animals and humans. Figure 4 shows the hematological parameters. There were changes in MCH, Hemoglobin and packed cells volume. There was a significant decrease (P-value 0.05) in the lymphocyte count in the animals. Lymphocytes circulate in the blood and migrate to injured tissues [45]. A dose-dependent decrease in the WBC and platelet may be due to daily dose intake and may be attributed to local reactions at the injection site [46]. There was no significant change in the hematological parameters of the treated group when compared with the control group in animals. This indicates that the nanoparticle formulations are safe at 50 mg/kg, 100 mg/kg body weight and 200 mg/kg body weight. The biochemical parameters for animals are given in Fig. 5 . There were no significant changes in blood glucose and cholesterol in female animals. There was a slight change in AST values and ALP values in females (P-value 0.05) at a higher dose (200 mg/kg body weight). But the result that all the animals survived throughout the investigation period indicated that the liver was not seriously damaged and neither formulation was lethal. The ALP levels were male different only at a higher dose, indicating that bone metabolism was not disturbed with the usage of ZnO in the targeted drug delivery system at a low dose. In our findings, the elevated AST and ALT levels indicated that it is not fatal, and all the animals survived through the whole experiment. The conjugation reduces the toxicity of the nanoparticle. The ALP levels are changed only at a higher dose. This shows that the metabolism was not disturbed with the usage of ZnO in a targeted drug delivery system at a low dose. There were no changes in the serum creatinine values Fig. 6 . The lipid profile study shows in Fig. 6 indicates there was no significant changes in animals compare to control. The globulin levels and albumin-to-globulin ratio are given in Fig. 8 for animals. Liver abolished the side effects induced by nanoparticles; some proportion of these particles should be excreted by the kidneys. The major route of excretion of nanoparticle is liver and kidney but at high concentration it is difficult to clear and this would result in high AST, ALP. When the liver cells get damaged, liver enzyme present in liver cell get spilled into blood which causes an increase in the enzyme level of albumin, the primary component of total protein, is made by liver. The change in albumin level was significant (P-value 0.05) at higher dosage in female animals Fig. 8 . There was no change in albumin level neither in animals nor at low doses in the female animals when compared to untreated rats. It confirms that there was no liver damage. Total protein and blood clotting factors are also measure the liver function. In our findings there was no change of total protein plasma proteins (albumin and globulins) between the control and nano-conjugate-treated animals, indicating no abnormalities in liver even at the highest dose of nano-conjugate treatment. Similarly, there was no change in blood sugar levels, which shows there were no dietary changes [47]. The toxicity associated with ZnO as reported previously was not found in our study, which may be due to the conjugation formation [48]. Histopathological analysis Diminution of internal organ weight in general is very sensible measure of toxicity after exposes of toxic substance [49]. In this study organ weights were present as a potential indicator of the toxic effect of Resveratrol-ZnO nano-conjugate on rats. There were no significant changes in the organ weights between treated and control animals. When diet contain toxic agent, the adverse effect always results in organ damage leading to histopathological changes. In our results the inability to observe any lesion, change of color, texture in the organs in histopathological study, so the study could probably attributed to the nontoxic nature of the nano-conjugate drug (100 mg/kg body weight, 200 mg/kg body weight). Even though slight changes in histopathological study of some organs was noted in only higher dose of the drug (400 mg/kg body weight) treated group. It was not that significant to be presented in histology. Overall, the histopathological result in Fig. 9 , of the nano-conjugate treated rats showed no such adverse changes in the vital organs which include heart, liver, kidney, spleen and corroborates to the biochemical analysis. As shown in current study, a primary cancer of the ovary developed in approximately half of the rats that received DMBA, IP treatment. This incidence was similar to previous study [50, 51]. There is still controversy regarding the origin of DMBA-induced cancer. Tunca, et al. observed a high incidence of ovarian epithelial-lined cysts after DMBA treatment these findings recommend that the surface epithelium of rat ovary contains the progenitor of cancer [18]. Sekiya, et al. believed that the induced tumor arise from epithelial proliferation [20]. The majority of tumors were adenocarcinomas; in our study we represent serous carcinoma, clear cell carcinoma, epithelial carcinoma and sarcoma Fig. 3 . In summary, DMBA consumption by IP enhanced the ovarian carcinomas in animals. Furthermore, the association of DMBA caused an increase in ovarian tumor mass with a high prevalence of malignant histologic subtypes Fig. 10 .These data represent an important benchmark for understanding ovarian cancer development in a model of IP injected DMBA rats. To confirm histologic evaluations, we employed a number of immune histochemical studies for epithelial or mesenchymal cell type Fig. 11 . For this study antibodies to intermediate filament protein are often useful. It’s differentiating cytokeratin positive cells of epithelial origin from Vimentin positive cells of mesenchymal origin. The rat ovarian mesothelium is reported to cytokeratin expression while only Vimentinis weakly expressed in granulose cells and strongly expressed in stromata cell. Antibodies with specificity for high molecular weight cytokeratin are not reactive in granulose cell tumors [52]. This cell of origin expression pattern is maintained not only in most primary tumors but in most solid metastatic tumors as well [53]. In our study, rat ovarian mesothelium is reported as high expression of CK7, CA125 ( Fig. 12 , 13 ) and Vimentin, where the treated ovarian mesothelium treated with our drug shows weakly expression of this markers, which represent positive efficacy of the drug against tumor ( Fig. 14 ). In DMBA induced rat ovarian adenocarcinomas, neoplastic epithelial cells reveal a positive staining for cytokeratin distributed diffusely throughout the cytoplasm and also appeared as localized to luminal boundary ( Fig. 12 ) . Staining for Vimentin was positive in cells and distribution pattern was either strongly perinuclear, polar and weakly diffuse ( Fig. 14 ) . After conformation of induction next evaluation of our study is efficacy of the drug. To obtain the results we evaluated immune-histochemical study by tumor biomarker (CA125, CK7, Vimentin) with reported less expression. In all treated ovaries cases markers were identified by a weak expression compare to induced ovaries and we confirm the result by Dot blot quantification or Densitometric analysis by measuring area value in Image J ( Fig. 15 ). The results of this study suggest that IP of DMBA induced epithelial carcinoma, where in maximum previous research published suture based DMBA induction. However our methods are much easier and it is established proper epithelial carcinoma which found in our results. In Fig. 15 study we have evaluated the efficacy of our drug (RSV-ZnO nano-conjugate) by densitometric study, it shows a remarkable changes in percent area value which denotes dot blot quantification of biomarker by using Image J and plot an excel file to compare the percent area value of biomarker[54]. These data represent an important benchmark for understanding ovarian cancer induction only by IP dosage of DMBA and also suggest that RSV-ZnO nano-conjugate our interested drug may be potentially used as adjuvant therapy. Conclusion In this present study, the toxicological aspects of nanoparticles were analyzed. From the toxicity studies it can be understood that Resveratrol ZnO nano-conjugate is neither toxic nor does it cause adverse effect. The attachment of RSV to ZnO nanoparticles will improve the pharmacological effects of the free Resveratrol. In our study we find a different method to induce epithelial tumor by Intraperitonial (IP) injection of DMBA. Although a majority of these induced tumor were reported to show the histology of epithelial tumor. In our study after induction by IP of DMBA we collect the ovary and study the histopathology of ovary tissue for tumor subtyping and immuno-histochemical study of different marker such as CA125, CK7, Vimentin, which confirm the induction of tumor. We examined antitumor efficacy of RSV-ZnO nano-conjugate by using 3 doses in a month with compared control. After treatment with our drug RSV-ZnO nano-conjugate (dosage of 100 mg/kg body weight per week during 3 weeks study) we have found very positive result with lower expression level of cancer marker (CA125, CK7, Vimentin). In much previous research we have found that free Resveratrol at dose of 150 mg/kg body weight shows adverse effect on ovarian cancer. Thus the results suggest the potential utility of these conjugate for medical applications. Declarations Conflict of Interest The authors state that there are no conflicts of interest. Acknowledgement The author Mahamuda Khatun (MK) is thankful to the UGC-MANF for fellowship grant during the period of study. The author would also like to thank Dey’s Medical Stores, Quality Control Department for Research Instrumentation Facility, Dr. Soumendra Darbar, Sr. Executive Q.C of Dey’s Medical stores, Mr. A Garai, Assistant Manager Q.C of Dey’s Medical stores. Also like to thanks Partho Pratim Roy for Histopathological analysis and Department of Biochemistry, Calcutta University for laboratory support during work. Ethical approval and consent to participate: Experiment was carried out under registered animal house of Dey’s Medical Stores, Kolkata. The project approval No-IAEC/s/PHARM/DEY’S/16/2016 by the Institutional Animal Ethics Committee (IAEC), India, according to CPCSEA’s guidelines. The studies were carried out in compliance with the all statutory guidelines of the Committee for the Purpose of Control and Supervision of Experimental Animals (CPCSEA). Competing Interest The authors state that they have no competing interest. Authors Contributions MK (Dr. Mahamuda Khatun) carried out biochemical, chemical, statistical analysis and histological experiments. She wrote the main manuscript and designed the experiments, coordination between the authors; she is 1st author and Corresponding Author. RM (Ritwik Maity) prepared Figure 11-14 and helps in animal handling and considers him as 2nd Author. SM (Prof. Santasree Mazumder helped in experiment with chemical supply and departmental instrument, consider her as 3rd author. SD (Dr. Soumendra Darbar) conceived and guide the experiment, conducted and supervised the animal experiment, review and the approved the final manuscript. Consider as 4 th Author. Funding UGC-MANF 2013 (Doctoral Fellowship). 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Data are expressed as mean ± standard deviation (n=6).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6187379/v1/2d9482eab62b217e6d364386.png"},{"id":78250733,"identity":"f874ea3e-e32b-48a9-a3a1-2f3db78b5d3b","added_by":"auto","created_at":"2025-03-11 09:54:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":42659,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA:\u003c/strong\u003eEffect of Zinc Oxide Nanoparticles (ZnO NPs) on Liver Enzymes \u0026nbsp;AST, ALT \u0026amp; ALP in different groups. Data are expressed as mean ± standard deviation (n=6).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB:\u003c/strong\u003e Effect of Zinc Oxide Nanoparticles (ZnO NPs) on Liver Parameters Total bilirubin, Direct Bilirubin and GGT Enzyme in different groups. Data are expressed as mean ± standard deviation (n=6)\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6187379/v1/fd632c39e01da01e46a067c8.png"},{"id":78250730,"identity":"13a613a8-aace-44da-bbf5-ef1c4f7ee20a","added_by":"auto","created_at":"2025-03-11 09:54:07","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":39772,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of Zinc Oxide Nanoparticles (ZnO NPs) on kidney parameters in different groups. Data are expressed as mean ± standard deviation (n=6).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6187379/v1/32333986c690632e7844fa4e.png"},{"id":78251604,"identity":"bee8a6c3-c38d-4972-a284-0b7fad9a2658","added_by":"auto","created_at":"2025-03-11 10:02:07","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":42122,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of nanoparticles on lipid profile and blood glucose level in different groups Data are expressed as mean ± standard deviation (n=6).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6187379/v1/93f20f1cc0024d439075787c.png"},{"id":78251612,"identity":"6c93775b-74e9-44fc-b5a9-875881e0e3ee","added_by":"auto","created_at":"2025-03-11 10:02:08","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":27583,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of nanoparticles on protein in different groups. Data are expressed as mean ± standard deviation (n=6).\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6187379/v1/563844eff225cce675aaccff.png"},{"id":78250766,"identity":"a07b68df-248b-45ba-aa7f-42eed5b86f79","added_by":"auto","created_at":"2025-03-11 09:54:09","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":394754,"visible":true,"origin":"","legend":"\u003cp\u003eHistopathological analysis of A) Heart B) kidney and (\u003cstrong\u003eC\u003c/strong\u003e) Liver \u003cstrong\u003eD\u003c/strong\u003e) Lungs (\u003cstrong\u003eE\u003c/strong\u003e) Stomach (\u003cstrong\u003eF\u003c/strong\u003e) Spleen of Res-ZnO nano-hybrid treated animals. Results show no changes in treated tissue samples compare to control.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-6187379/v1/d062d63ad11fa0270220b942.png"},{"id":78250742,"identity":"929afbe8-4f83-47e4-ba7f-a406458be4c6","added_by":"auto","created_at":"2025-03-11 09:54:08","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":202006,"visible":true,"origin":"","legend":"\u003cp\u003eIncreased Size of Fallopian tube and Ovary after 120 days post DMBA Induction.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-6187379/v1/713eb225ada58ed7fe6c3998.png"},{"id":78252914,"identity":"cdcc280f-fa8c-4a47-85f2-b80a7f42e5fc","added_by":"auto","created_at":"2025-03-11 10:10:08","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":502580,"visible":true,"origin":"","legend":"\u003cp\u003eDifferent type of carcinoma, Serous carcinoma characterized by cellular papillae formation with cellular budding (arrows), Clear cell carcinoma: Composed of numerous vaculated cells (arrows) ovarian carcinoma, the epithelium is composed of stratified cells with loss of nuclear polarity, sarcoma, composed of non-epithelial malignant cells.\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-6187379/v1/23676801b436318c18482f65.png"},{"id":78250747,"identity":"96d39542-7767-4538-b2c0-465211838bac","added_by":"auto","created_at":"2025-03-11 09:54:08","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":544955,"visible":true,"origin":"","legend":"\u003cp\u003eEpithelial cell staining of CA125 (Cancer Antigen 125) appeared as lower level in case of treated sample compare to induce.\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-6187379/v1/098c5e64e38a7f62d62aa859.png"},{"id":78250782,"identity":"e144ce8f-73e3-4968-b0f7-936d27bafcee","added_by":"auto","created_at":"2025-03-11 09:54:10","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":663481,"visible":true,"origin":"","legend":"\u003cp\u003eCytokeratin staining of epithelial cells express in lower level in case of treated sample compare to induced sample.\u003c/p\u003e","description":"","filename":"13.png","url":"https://assets-eu.researchsquare.com/files/rs-6187379/v1/432db2f4ae91c50567cf5295.png"},{"id":78250752,"identity":"ffa7c9ff-6adf-47be-9beb-25a916dbbf01","added_by":"auto","created_at":"2025-03-11 09:54:08","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":540985,"visible":true,"origin":"","legend":"\u003cp\u003eEpithelial cell staining with Vimentin appeared as perinuclear polar, diffuse in undifferentiated areas and lower level in case of treated sample compare to induced one.\u003c/p\u003e","description":"","filename":"14.png","url":"https://assets-eu.researchsquare.com/files/rs-6187379/v1/c9f2f441672777b594d983f0.png"},{"id":78251615,"identity":"59100de9-d271-48fc-b801-01864b43222a","added_by":"auto","created_at":"2025-03-11 10:02:08","extension":"png","order_by":15,"title":"Figure 15","display":"","copyAsset":false,"role":"figure","size":16475,"visible":true,"origin":"","legend":"\u003cp\u003eDensitometry analysis or Dot blot quantification by measure percent area value of cancer biomarkers by using Image J. (CK7, Vimentin, CA125).\u003c/p\u003e","description":"","filename":"15.png","url":"https://assets-eu.researchsquare.com/files/rs-6187379/v1/f16ff47cc519d4d767b17ecc.png"},{"id":78253404,"identity":"daa58a79-35c0-4a08-8b56-355ae0c6ac5c","added_by":"auto","created_at":"2025-03-11 10:18:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4546350,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6187379/v1/6dce536f-ede5-4188-9310-e3948d53cfc0.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eSafety and Efficacy of Resveratrol-ZnO nanoconjugate in DMBA-induced ovarian cancer in murine model\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOvarian cancer is one of the leading causes of cancer-related deaths among women [1]. Effective detection and treatment of ovarian cancer remains a significant clinical challenge. Approximately 90% of ovarian tumors derive from an epithelial origin, most often considered to be the ovarian surface epithelium; 80% of these originated in the ovarian surface epithelium [2\u0026ndash;4]. Incessant ovulation, postmenopausal increase of gonadotropin hormone levels, chronic inflammation and environmental carcinogens are assumed to play a key role in ovarian carcinogenesis [5\u0026ndash;9]. When identified as stage I disease, 5 years disease free survival approaches 95%, although 20% of these patients will eventually relapse to a treatment failure overall. In early stage ovarian tumors lack specific clinical symptoms, which hampering early detection and delaying diagnosis in some cases. Recent use of lysophosphatidic acid and the a-folate receptor as biomarkers, in combination with CA125, have improved the specificity for detection of early stage malignant gynecologic tumors [10] and will probably be further advanced by the addition of proteomic screening of low molecular weight serum proteins using Surface-Enhanced Laser Desorption and Ionization Time-off Light (SELDI-TOF) mass spectroscopy [11]. Incidence of ovarian adenocarcinoma has varied between 10% and 45%, perhaps, owing to the strain of rat employed or the chemical form of the DMBA utilized and the fact that different studies have observed a wide variance in the incidence of tumors of stromal origin [12,13]. To prevent or reverse the progression of initiated cells to malignant disease chemoprevention is an alternative approach. Clinical trials of preventive agents have demonstrated an efficacy in patients at risk for both breast and colon cancer [14]. The first prevention trials for women shows high risk for the development of ovarian cancer and hence prophylactic oophorectomy have now been initiated to evaluate biomarker modulation \u003cem\u003ee.g.\u003c/em\u003e ovarian surface metaplasia, cellular atypia and cortical inclusion cysts following short term treatment with fenretinide, oral contraceptive therapy or placebo [15]. Spontaneous rodent ovarian tumors of epithelial origin have been reported but these do not appear to occur in experimentally significant numbers [16]. Ovarian cancers are divided into type I or type II by histologically and genetically. Type I cancers are of low histological grade such as endometrioid, mucinous, and clear-cell carcinomas. Type II cancers are of higher histological grade and include serous carcinoma and carcinosarcoma [17]. Treatment of ovarian cancer are still not clear, there is a continuing need for a validated animal model possessing histological characteristics appropriate to the presentation of human ovarian tumors to evaluate the efficacy of newly developed chemo preventive compounds. 7,12-Dimethylbanz(a)anthracene (DMBA) is a well-known polycyclic aromatic carcinogen that is capable of inducing the initiation, promotion and progression of tumor importantly the histological type of experimental ovarian tumors may vary based on the animal strain used, use of carcinogen and age of animals. Use of carcinogen means amount and route of administration and method of induction. So, treatment and effective detection of ovarian cancer still a significant clinical challenge. The direct implantation of chemical carcinogen DMBA in the rat ovary [18] leads to the induction of ovarian tumors at an incidence of 37% these include adenocarcinomas, as well as stroma and mesothelialtumors [19\u0026ndash;21]. There is however, lack of information regarding the sequence and nature of events elicited by DMBA and leading to ovarian cancer development. To improve its usage and physiologic relevance to the human disease, the DMBA model of ovarian cancer was modified by significantly decreasing the DMBA dose, thereby preserving maximally the integrity of the organ and by incorporating multiple gonadotropin hormone treatments, thus introducing an additional risk factor associated with human ovarian cancer, known also to induce hyper ovulation and enhanced mitogenesis of the ovarian surface epithelium [22].\u003c/p\u003e \u003cp\u003eNanomaterial toxicity is a matter of concern and many \u003cem\u003ein vivo\u003c/em\u003e studies have suggested that the surface chemistry, release of metal ion, Reactive Oxygen Species (ROS) production, cell apoptosis and break down of nano-materials are the possible causes of cellular toxicity [23\u0026ndash;25]. Current problem for nano-medicine involve understanding the issue related to toxicity, in chemotherapy, toxicity to healthy cell is associated with anticancer drugs. Nanoparticles are used as a drug carrier for the specific delivery of drugs to cancer cells because of the size of nanoparticles which is similar to that of most biological molecules and structures. Consequently nanoparticles can be useful for both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e biomedical application and research. The anticancer drug is delivered specifically to the cancer cells in targeted drug delivery. This reduces the toxicity to normal cells. The physicochemical characteristics of nano carriers, such as composition, particle size, surface charge and the presence of ligands on their surface, will dictate their bio-distribution and pharmacokinetics [26,27]. Nanoparticle to be used as a carrier in drug delivery system should be nontoxic \u003cem\u003ein vivo\u003c/em\u003e. Surface modification and nano-conjugation of nanoparticle with drugs is an aspect of drug delivery system design for biomedical applications. Modulation of nanoparticle by conjugation with drugs can influence particle uptake, biological responses, bio-distribution and its efficacy also. Surface functionalization can be utilized to increase the drug circulation time in blood, reduce non-specific distribution or specific targeting of tissue or cells by using a targeting ligands [28].\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn vivo\u003c/em\u003e, nanoparticles will be entrapped in and translocated to other tissues or organs along the blood circulation. The small size and large surface area equip them with an active group or intrinsic toxicity. Even at a very small cellular level, they may constitute potential long-term health hazards [29,30]. Toxicity indicates the disadvantageous effects due to the interaction between nano-materials and cells. This interaction may differ depending on the chemical properties of the toxicants and the cell membrane, as it may take place on the cell surface or within the cell body. The evaluation of toxic properties of nano-materials is pivotal when considering them for use in biomedical science. In practice, the evaluation typically includes acute, subchronic and chronic studies [31]. The current status of toxicology due to nanoparticles has been reported by Becker [32]. Toxicity of NPs has been evaluated in different biological systems, such as bacteria, mammalian cells and \u003cem\u003ein vivo\u003c/em\u003e model [33\u0026ndash;35]. The safeties of RSV are well known, but the combined effects of this nano-conjugate are unclear. Thus, it becomes essentials to evaluate the safety and toxicity of the conjugate, before their use. Therefore the objective of the present study, to assess the safety of nano-conjugate by acute and sub-acute toxicity of nano-conjugate in Wistar rats, the study was done on the report of the Organization for Economic Co-operation and Development (OECD) guidelines 423 and 407 respectively.\u003c/p\u003e \u003cp\u003eAfter completing the safety study we evaluate characterization of ovarian tissue by two different study of this modified animal model. One is induction of ovarian cancer by IP dosage of DMBA and another is treatment of ovarian carcinoma by RSV-ZnO nano-conjugate drug (100 mg/wk for 3 weeks) with the immunohistochemistry study of some cancer biomarker such as CA125, Vimentin and CK7. Cancer induction method by using DMBA is well established method but in this study we used IP injecton of DMBA with particular dose as an induction method which is very easy and time consuming compare to the other published methods. So this is one of new findings of our research. Confirm the induction of ovarian cancer by measuring ovary size and histological study of ovarian tissue which shows different type of carcinoma arise in tissue such as serous, clear cell, sarcoma, epithelial carcinoma.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eIn our study there is two methods are used one is study safety of the drug. Then study the efficacy of the drug on ovarian cancer induced rat. So firstly we explain the methods for safety study of drug and the methods are described in below.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eHealthy female swiss albino mice and Wistar rats (8\u0026ndash;10 weeks old)(approximately 30\u0026ndash;35 g body weight mice, 150\u0026ndash;155 g body weight rats, Calcutta university, India) were housed in polypropylene cages with a maximum of three animals of the same sex per cage and placed in a ventilated, temperature-controlled room. The standard conditions were maintained at 20\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, 60% \u0026plusmn; 10% relative humidity and a 12 hr light/dark cycle. The cages composed autoclaved paddy husk as bedding that was replaced twice in a week. The commercial pellet diet and distilled water for mice were available at lab of Calcutta University. The experimental protocol involving animals was carefully reviewed and approved Animal House of Dey\u0026rsquo;s medical. Approval No. IAEC/s/PHARM/16/2016 by the Institutional Animal Ethics Committee, India, where the studies were carried out in compliance with the guidelines of the Committee for the Purpose of Control and Supervision of Experimental Animals (CPCSEA), India. Animals were acclimated to their environment for 7 days prior to treatment.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSynthesis and characterization of RSV-ZnO nano-hybrid\u003c/h3\u003e\n\u003cp\u003eZnO NPs were prepared by the co-precipitation technique using highly pure zinc acetate dehydrate (Zn [CH\u003csub\u003e3\u003c/sub\u003eCOO] \u003csub\u003e2\u003c/sub\u003e H\u003csub\u003e2\u003c/sub\u003eO, 99% pure) as a precursor with sodium hydroxide solution following the procedures reported earlier [36]. Required amount of RSV was added into 20 ml of ZnO NPs solution and kept it in a shaker for overnight to formation of nano-conjugate. Characterization of nano-hybrid confirmed by TEM, FTIR study, Picosecond resolved spectroscopy study and also by Raman scattering experiment [37\u0026ndash;39]. The HRTEM image clearly reflects crystalline nature of ZnO Np [40]. The drug used in this study RSV, contain three hydroxyl groups, which possesses binds ability to the surface of ZnO [41]. All data are published in our previous paper [42].\u003c/p\u003e\n\u003ch3\u003eDrug administration\u003c/h3\u003e\n\u003cp\u003eWe choose the administration of drug (Res-ZnO nano-conjugate) by intraperitoneal injection as it is predominantly used for it\u0026rsquo;s easy compared to other parental methods during animal testing for the administration of systemic drugs and fluids. Additionally, one can administer a large volume of nano-conjugate suspension (400 mg/kg) to the mice and rats if one choose Intraperitoneal (IP) route compared to the intravenous method. This much drug cannot inject \u003cem\u003evia\u003c/em\u003e the intravenous route. Wistar rats (nine females) were assigned to the following test groups: Group I (200 mg/kg body weight of Res-ZnO nano-conjugate, IP); and group II (400 mg/kg body weight) and control. The respective doses were suspended in DMSO and were administered Intraperitoneally (IP). After that, the animals were observed for 14 days for clinical signs of toxicity or mortality. On completion of the treatment, the animals were sacrificed by cervical dislocation and necropsies to facilitate gross pathological examination of organs.\u003c/p\u003e\n\u003ch3\u003eAcute toxicity\u003c/h3\u003e\n\u003cp\u003eTo obtain the LD 50 of nano-conjugate, the experiments and its interval were designed in accordance with the karber\u0026rsquo;s method. After 7 days acclimation, the animals (Wistar rats, 8\u0026ndash;10 weeks) were randomly divided into 4 treatment groups and were gavages with the respective doses (control, 50 mg/kg body weight, 100 mg/kg body weight and 200 mg/kg body weight) of nano-conjugate once daily, for a period of 28 days. The central nervous system, motor nervous system, vegetative nervous system, respiratory system, digestive system, urinary system, changes of the eyes, skim, clothing hair and death situation were continuously observed and recorded during the 14 day treatment. Special attention was paid on the clinical signs of toxicity including tremors, convulsion, salivation, nausea, diarrhea, lethargy and coma. Body weights were recorded on days 0, 7, 14, 21, 27 and 28. Feed consumption was deliberated per cage over successive periods of 3 days by weighing the feeders. The animals were examined for any clinical signs of morbidity, mortality, changes in body weight and changes in food consumption throughout the dosing. At the end of the treatment, blood was collected from the animals from the ocular vein for clinical pathology assessment, which included analysis of various hematological parameters and biochemical parameters. Consequently, the animals were sacrificed by cervical dislocation and histopathological assay was done of the various organs. The heart, liver, kidney, lung, spleen, stomach were stripped out and weighed accurately. The histopathological study also included careful and consistent dissection of various target organs like liver and kidneys, Heart, lungs, spleen, stomach. The tissue of organ samples were embedded in paraffin blocks then sliced and placed onto glass slides. After H and E staining, the pathological changes were observed on microscope.\u003c/p\u003e\n\u003ch3\u003eBlood biomarker assay\u003c/h3\u003e\n\u003cp\u003eAll animals were sacrificed at the same time and blood samples were collected \u003cem\u003evia\u003c/em\u003e the ocular vein. Of this sample, 1 ml was collected in 10% EDTA for hematological assay and the remaining of blood serum was collected for biochemical assay. Blood samples were collected in tubes containing Ethylene Diamine Tetra Acetic (EDTA) acid as anticoagulant. Hemoglobin, platelets, mean corpuscular hemoglobin concentration, packed cell volume, WBC were measured using a hematology auto analyzer.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eBiochemical parameter analysis\u003c/h2\u003e \u003cp\u003eThe serum was obtained by centrifugation of the whole blood at 3000 rpm for 15 min. Liver function was evaluated based on the serum levels of Alkaline Phosphatase (ALP), Alanine Aminotransferase (ALT) and Aspartate Aminotransferase (AST), GGT, Tbil, Dbil as well as on blood glucose and cholesterol levels. Nephrotoxicity was determined by uric acid, urea, serum creatinine, total protein, albumin and globulin.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eOrgan weight and histopathological examination\u003c/h3\u003e\n\u003cp\u003eAfter sacrifice, organs like heart, lungs, spleen, stomach, liver and kidney were removed and adherent tissues, if any were carefully cropped. The wet weight of organs was registered right away. All histopathological tests were performed using standard laboratory procedures for pathological studies. The dissected organs were fixed in 10% neutral buffer formalin and processed adequately. The tissues were embedded in paraffin blocks, then sliced to 5 \u0026micro;m thickness and placed onto glass slides. After hematoxylin\u0026ndash;eosin staining, the slides were examined and photos were taken using an optical microscope. The results were analyzed by a veterinary pathologist.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eFor statistical analysis, each of the experimental values was differentiated with its corresponding control. The results were conveying as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Multi-group comparisons of the means were accomplished by one-way analysis of variance test. Statistical consequence for all tests was set at P-value is 0.05.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eInduction of ovarian cancer and efficacy of the Drug:\u003c/h2\u003e \u003cp\u003eIn our study the second methods we used are how to induced ovarian cancer in rat model and also elaborate the methods about the study of efficacy of the drug on ovarian cancer induced rat.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eChemicals\u003c/strong\u003e \u003cp\u003eDMBA (Sigma Chemical Co.) DMBA dissolved in 10 ul corn oil (as a vehicle). Rats were treated for 3 days during a 3 weeks period by IP injections of DMBA in corn oil at doses of 50 micrograms/kg day (150 micrograms/kg total dose).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAnimal selection\u003c/b\u003e: Wistar rats, six female, eight weeks old, 150 mg-200 mg. Rats per group(n\u0026thinsp;=\u0026thinsp;6) were housed in the conventional facility in clear plastic cages with plastic lids containing filter space. Temperature, humidity and photoperiod were constant (22\u0026deg;C, 50\u0026ndash;70%, and 12:12 hrs light: Dark cycle). Rats acclimated to the facility for 7 day before the experiment began.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStudy design\u003c/h2\u003e \u003cp\u003eFor our study we take 54 rats, for three group (n\u0026thinsp;=\u0026thinsp;6) with 3 times repetitions. Animals were maintained in lab conditions for a week\u0026rsquo;s time and fed Amrut Lab Rodent Chow (4.55% Fat, 20% Crude protein, 60% Carbohydrate and 5.08% Dietary fiber) during the entire course of the study. Rats were treated for 2 days (Day1, Day 14) during a 2 week period by Intraperitoneal injection or IP injections of DMBA in corn oil at doses of 50 micrograms/kg day (100 micrograms/kg total dose). Rats were palpated weekly throughout the observation period, which terminated 4 month after IP injection of DMBA. After 3 month we observe by sacrificing rats that cancer is induced by histological study of ovary tissue. Rats with palpable tumors were terminated before any morbidity and the tumors were sampled to provide both frozen and formalin-fixed tissue. We evaluate the different weight of ovaries compare to control. Ovaries of tumor bearing rats were fixed to provide histologic controls. All tissue for histologic evaluation was fixed overnight in10% neutral buffered formalin, sectioned at 5 um and stained with Hemotoxylin and eosin and processed for immunohistochemistry to confirm the induction of ovarian tumor.\u003c/p\u003e \u003cp\u003eAfter 4 month of observation and conformation of half of animals with tumor rest animals were treated with our nano-conjugated drug. Animals were treated with RSV-ZnO nano-conjugate drug with dose of 100 mg/kg body weight for 3 days during a 3 weeks period by IP injection of RSV-ZnO nano-conjugate. Rats with treatment of drugs were terminated before any morbidity and abnormalities. Then we evaluate the weight and size of the ovaries compare to untreated ovaries. Treated ovaries were processed for immunohistochemistry of CA125, CK7, Vimentin to confirm the efficacy of the drug.\u003c/p\u003e \u003cp\u003e \u003cb\u003eImmunohistochemistry\u003c/b\u003e: Paraffin-embedded section for immunohistochemistry were dewaxed, rehydrated and processed for antigen unmasking by heating to near boiling in citrate buffer (0.01 M) in a microwave oven, then allowed to cool for 20 min. Following this and all subsequent incubations except for the blocking serum, slides were washed in PBS. Endogenous peroxidase activity was blocked by hydrogen peroxide incubation (0.3% methanol) for 20 mins. Frozen section were mounted onto slides fixed in cold acetone (-20\u0026deg;C) for 10 min and air dried before peroxide block incubation. After 30 min incubation in blocking serum (diluted 1:50 in PBS) appropriate to each secondary antibody, slides were incubated with primary antibody at 40\u0026deg;C overnight (Cytokeratin 7, CA125, Vimentin) 1 ug/ml, Santa Cruz Biotechnology Inc. All reagents except for the chromagen substrates were diluted with PBS containing 2% Bovine serum albumin. Detection of primary antibody proceeded with the binding of biotinylated secondary antibody (diluted 1:200 for 30 min) followed by avidin-biotin peroxidase complex and DAB chromogen substrate [43].\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSafety of the drug:\u003c/h2\u003e \u003cp\u003eIn the part of safety of the drug we explain the findings about is there any side effect of the drug and also find out which dose is safe for the study.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eMortality and clinical observation\u003c/strong\u003e \u003cp\u003eNo mortality was found in the animals in control group and any other test groups. The animals did not show any abnormal behavior throughout the 14 days of acute study and there were no treatment related mortality and clinical signs of toxicity in 28 days repeated dose sub chronic study. Clinical sign of nano-conjugate over treated and control groups were similar \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e \u003cb\u003eand\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e The nanoparticle based drug delivery was found to be safe for a single limit dose equivalent to 100 and 200 mg/kg of nano-conjugate.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of RSV-ZnO nano-conjugate on Clinical Signs of Toxicity.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClinical signs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50 mg/kg\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100 mg/kg\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e200 mg/kg\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e400 mg/kg\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHyperactivity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePiloerection\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eConvulsion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eJumping\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIrritability\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eReddening\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAbnormal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSecretion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eAcute toxicity analysis of nano-conjugate\u003c/strong\u003e \u003cp\u003eNo mortality was observed in the animals in any test group or in the control group. The animals did not show any abnormal behaviour throughout the 14 days. Pathological examination of liver, kidney and other organs did not show any evidence of toxicity. The nanoparticle-based targeted drug delivery system was found to be safe for a single limit dose equivalent to 100\u0026ndash;200 mg/kg of Res-ZnO and the 95% confidence limits for the LD50 are 100\u0026ndash;200 mg/kg body weight. All clinical symptoms were evaluated by comparing treated animals with controls (n\u0026thinsp;=\u0026thinsp;6). The toxicity of the nanoparticle formulation due to a single dose for less than 14 days was determined by acute toxicity analysis. The present study showed that intraperitoneal treatment of animals with Res-ZnO did not induce mortality or significant clinical symptoms of toxicity.\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eBody weight and organ index\u003c/h2\u003e \u003cp\u003eNo animal mortality was observed in any of the test groups for the entire period of subchronic study for nanoparticles. The subchronic toxicity result shows that the nanoparticle formulations did not reveal any conflicting effect with long-term administration on the general health of the animals. Body weight was weighed before the administration and on day 7 and 14 after administration, respectively. Body weight of animals from group 2, group 3, group 4 and group 5 showed a decreasing trend. These results are given in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. There was no change in the general systemic health of the animals \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e presents the mean organ weight and % organ weight to bodyweight for rats. Both control and treated rats did not show any significant difference in mean organ weights of kidney, liver, spleen, heart, stomach, lungs.\u003c/p\u003e\u003cp\u003e \u003cstrong\u003eHematology and clinical biochemistry\u003c/strong\u003e \u003cp\u003eThe normal values of hematological parameters in the control group and quantitative changes of hematological parameters in treated rats with nano-conjugate are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. There was no significant change between the three subject groups in any of the hematological parameters studied. The Hemoglobin (Hb) levels, WBC, platelets level of treated rats were marginally lower than the control rats. In Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eB we evaluate effect of different doses of drug on Liver enzymes. There was slight elevation in AST, ALP, ALT, and GGT which was not significant There were no significant changes in kidney parameter and lipid, blood glucose level \u003cb\u003e(\u003c/b\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Protein level parameter also shows normal result compare to control \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eHistopathology\u003c/b\u003e: Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e9\u003c/span\u003e represent the histopathological findings from the control and the treated rats. No marked morphological and histological changes were observed in any organ of lower (100 mg/kg) medium (200 mg/kg) higher (400 mg/kg) doses of treated rats. Sections of liver exhibited normal central/portal veins, kidney showed normal nephrons, spleen showed normal splenic follicle, heart featured normal myocardium, stomach, lungs did not show any abnormal pathology changes. Hence no toxicological differences on histopathological analysis were observed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eEfficacy of the drug\u003c/h2\u003e \u003cp\u003eDuring the experimental period, all of the animals were observed and handled based on the same criteria. The amount of food intake did not differ between the experimental groups. No differences in the final body weights or body weight gains were observed during the experimental period. The absolute and relative weights and sizes of the left ovaries were significantly higher in the induced animals. The incidence of DMBA, IP-induced ovarian tumors (the percentage of developed tumors) was higher in animals. After sequential tumor development, the abdominal-pelvic cavity was opened to evaluate ovarian tumors. Although the ovaries with fallopian tube of the rats in the DMBA(IP) treated groups were increased in size \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e \u003cb\u003eand\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e10\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e after 120 days post DMBA Induction.The majority of ovarian tumors were adenocarcinomas. After 120 days of ovarian tumor induction, the experimental groups displayed variations in the histotypes and grades of the tumors \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e11\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Serous carcinomas characterized by pailae formation with cellular budding, and clear cell carcinomas composed of numerous vacuolated cells were more prevalent in DMBA treated group of animals. Epithelial carcinomas, epithelium is composed of stratified cells with loss of nuclear polarity and nuclear pleomorphism with chromatin clumping, Sarcoma composed of non-epithelial malignant cells were also prevalent in DMBA treated ovaries tissue. To analyze the efficacy of the drug we used some cancer biomarker CA125, CK7 and Vimentin to study their expression level with immuno histopathology study \u003cb\u003e(\u003c/b\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e14\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Also evaluate the data by densitometry analysis which is also known as dot blot quantification analysis. Densitometry analysis is quantified by two methods one is by measure percent area value and another method is integrated density. In our study we measure densitometric analysis by compare percent area value of Ca125, CK7 and vimentin treated and induced biomarker \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e15\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. After treatment with our drug RSV-ZnO nano-conjugate (dosage of 100 mg/kg body weight per week during 3 weeks study) we have found very positive result with lower expression level of cancer marker (CA125, CK7, Vimentin).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eOvary weight changes after induction and treatment with nano-conjugate.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOvary weight after induction\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOvary weight after treatment\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl (n\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18 mg (normal, no induction)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo treatment\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRSV-ZnO nano-Conjugate (Group II) n\u0026thinsp;=\u0026thinsp;6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50 mg (Right ovary)\u003c/p\u003e \u003cp\u003e59 mg (left ovary)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20 mg (right ovary)\u003c/p\u003e \u003cp\u003e25 mg (left ovary)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRSV (Group III) n\u0026thinsp;=\u0026thinsp;6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e49 mg (Right Ovary)\u003c/p\u003e \u003cp\u003e40 mg (left ovary)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20 mg (Right ovary)\u003c/p\u003e \u003cp\u003e19 mg (left ovary)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePhysiochemical properties, such as size surface areas, shapes, solvent and media could affect the toxicity of nanoparticle. Colvin reported that the toxic effects of nanoparticles showed variation depending upon the medium composition in which the nanoparticles are suspended. Hou, et al. reported that the same nanoparticles exhibited different toxic manifestation when dissolved indifferent mediums. Thus suspending agent need to be considering when an \u003cem\u003ein vivo\u003c/em\u003e toxicity of nanoparticles was carried on. In our study we conjugate nanoparticle with well-known drug Resveratrol. After conjugation the risk of toxicity of free nanoparticle cannot affect and we found this IP administration of nano-conjugate is very safe. However result of other research show that the target organs for ZnO nanoparticle were the liver, spleen, heart, kidney and lung. In the present study, histopathological damages of the liver kidney, spleen, heart, lungs were observed only in high dose (400 mg/kg). The histopathological analysis of RSV-ZnO-treated (100 mg/kg and 200 mg/kg) animals is depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e9\u003c/span\u003e. In the control, 100 and 200 mg/kg body weight nano-conjugate groups, there was no change in hepatocytes, portal area, or integrated hepatic side effects. Similar to the observation in liver, heart, lungs, kidney, stomach, spleen did not show any abnormal pathology changes at 100 and 200 mg/kg. There were no lesions in tissue of the stomachs, livers, spleens, and lungs, heart. But at higher dose (400 mg/kg body weight) the colour of heart and spleen was slightly changed and also found enlarged spleen and swollen stomach. But with the dose of 50 mg/kg, 100 mg/kg, 200 mg/kg body weight is safe. There were no such lesions found in these organs. In previous research they published that oral administration of ZnO nanoparticle has effect on this organ but in our work result shows the IP doses (50 mg/kg, 100 mg/kg, 200 mg/kg) of our drug is absolutely safe [44].\u003c/p\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eHematology and clinical biochemistry\u003c/h2\u003e \u003cp\u003eChanges in hematological parameters are used to detect the physiological and pathological changes in animals and humans. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e shows the hematological parameters. There were changes in MCH, Hemoglobin and packed cells volume. There was a significant decrease (P-value 0.05) in the lymphocyte count in the animals. Lymphocytes circulate in the blood and migrate to injured tissues [45]. A dose-dependent decrease in the WBC and platelet may be due to daily dose intake and may be attributed to local reactions at the injection site [46]. There was no significant change in the hematological parameters of the treated group when compared with the control group in animals. This indicates that the nanoparticle formulations are safe at 50 mg/kg, 100 mg/kg body weight and 200 mg/kg body weight.\u003c/p\u003e \u003cp\u003eThe biochemical parameters for animals are given in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e. There were no significant changes in blood glucose and cholesterol in female animals. There was a slight change in AST values and ALP values in females (P-value 0.05) at a higher dose (200 mg/kg body weight). But the result that all the animals survived throughout the investigation period indicated that the liver was not seriously damaged and neither formulation was lethal. The ALP levels were male different only at a higher dose, indicating that bone metabolism was not disturbed with the usage of ZnO in the targeted drug delivery system at a low dose. In our findings, the elevated AST and ALT levels indicated that it is not fatal, and all the animals survived through the whole experiment. The conjugation reduces the toxicity of the nanoparticle. The ALP levels are changed only at a higher dose. This shows that the metabolism was not disturbed with the usage of ZnO in a targeted drug delivery system at a low dose. There were no changes in the serum creatinine values Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e. The lipid profile study shows in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e indicates there was no significant changes in animals compare to control. The globulin levels and albumin-to-globulin ratio are given in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003e for animals. Liver abolished the side effects induced by nanoparticles; some proportion of these particles should be excreted by the kidneys. The major route of excretion of nanoparticle is liver and kidney but at high concentration it is difficult to clear and this would result in high AST, ALP. When the liver cells get damaged, liver enzyme present in liver cell get spilled into blood which causes an increase in the enzyme level of albumin, the primary component of total protein, is made by liver. The change in albumin level was significant (P-value 0.05) at higher dosage in female animals Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003e. There was no change in albumin level neither in animals nor at low doses in the female animals when compared to untreated rats. It confirms that there was no liver damage. Total protein and blood clotting factors are also measure the liver function. In our findings there was no change of total protein plasma proteins (albumin and globulins) between the control and nano-conjugate-treated animals, indicating no abnormalities in liver even at the highest dose of nano-conjugate treatment. Similarly, there was no change in blood sugar levels, which shows there were no dietary changes [47]. The toxicity associated with ZnO as reported previously was not found in our study, which may be due to the conjugation formation [48].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eHistopathological analysis\u003c/h2\u003e \u003cp\u003eDiminution of internal organ weight in general is very sensible measure of toxicity after exposes of toxic substance [49]. In this study organ weights were present as a potential indicator of the toxic effect of Resveratrol-ZnO nano-conjugate on rats. There were no significant changes in the organ weights between treated and control animals. When diet contain toxic agent, the adverse effect always results in organ damage leading to histopathological changes. In our results the inability to observe any lesion, change of color, texture in the organs in histopathological study, so the study could probably attributed to the nontoxic nature of the nano-conjugate drug (100 mg/kg body weight, 200 mg/kg body weight). Even though slight changes in histopathological study of some organs was noted in only higher dose of the drug (400 mg/kg body weight) treated group. It was not that significant to be presented in histology. Overall, the histopathological result in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e9\u003c/span\u003e, of the nano-conjugate treated rats showed no such adverse changes in the vital organs which include heart, liver, kidney, spleen and corroborates to the biochemical analysis.\u003c/p\u003e \u003cp\u003eAs shown in current study, a primary cancer of the ovary developed in approximately half of the rats that received DMBA, IP treatment. This incidence was similar to previous study [50, 51]. There is still controversy regarding the origin of DMBA-induced cancer. Tunca, et al. observed a high incidence of ovarian epithelial-lined cysts after DMBA treatment these findings recommend that the surface epithelium of rat ovary contains the progenitor of cancer [18]. Sekiya, et al. believed that the induced tumor arise from epithelial proliferation [20]. The majority of tumors were adenocarcinomas; in our study we represent serous carcinoma, clear cell carcinoma, epithelial carcinoma and sarcoma Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. In summary, DMBA consumption by IP enhanced the ovarian carcinomas in animals. Furthermore, the association of DMBA caused an increase in ovarian tumor mass with a high prevalence of malignant histologic subtypes Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e10\u003c/span\u003e.These data represent an important benchmark for understanding ovarian cancer development in a model of IP injected DMBA rats. To confirm histologic evaluations, we employed a number of immune histochemical studies for epithelial or mesenchymal cell type Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e11\u003c/span\u003e. For this study antibodies to intermediate filament protein are often useful. It\u0026rsquo;s differentiating cytokeratin positive cells of epithelial origin from Vimentin positive cells of mesenchymal origin. The rat ovarian mesothelium is reported to cytokeratin expression while only Vimentinis weakly expressed in granulose cells and strongly expressed in stromata cell. Antibodies with specificity for high molecular weight cytokeratin are not reactive in granulose cell tumors [52]. This cell of origin expression pattern is maintained not only in most primary tumors but in most solid metastatic tumors as well [53]. In our study, rat ovarian mesothelium is reported as high expression of CK7, CA125 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e12\u003c/span\u003e, \u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e13\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e and Vimentin, where the treated ovarian mesothelium treated with our drug shows weakly expression of this markers, which represent positive efficacy of the drug against tumor \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e14\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn DMBA induced rat ovarian adenocarcinomas, neoplastic epithelial cells reveal a positive staining for cytokeratin distributed diffusely throughout the cytoplasm and also appeared as localized to luminal boundary \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e12\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Staining for Vimentin was positive in cells and distribution pattern was either strongly perinuclear, polar and weakly diffuse \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e14\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. After conformation of induction next evaluation of our study is efficacy of the drug. To obtain the results we evaluated immune-histochemical study by tumor biomarker (CA125, CK7, Vimentin) with reported less expression. In all treated ovaries cases markers were identified by a weak expression compare to induced ovaries and we confirm the result by Dot blot quantification or Densitometric analysis by measuring area value in Image J \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e15\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e The results of this study suggest that IP of DMBA induced epithelial carcinoma, where in maximum previous research published suture based DMBA induction. However our methods are much easier and it is established proper epithelial carcinoma which found in our results. In Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e15\u003c/span\u003e study we have evaluated the efficacy of our drug (RSV-ZnO nano-conjugate) by densitometric study, it shows a remarkable changes in percent area value which denotes dot blot quantification of biomarker by using Image J and plot an excel file to compare the percent area value of biomarker[54]. These data represent an important benchmark for understanding ovarian cancer induction only by IP dosage of DMBA and also suggest that RSV-ZnO nano-conjugate our interested drug may be potentially used as adjuvant therapy.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this present study, the toxicological aspects of nanoparticles were analyzed. From the toxicity studies it can be understood that Resveratrol ZnO nano-conjugate is neither toxic nor does it cause adverse effect. The attachment of RSV to ZnO nanoparticles will improve the pharmacological effects of the free Resveratrol.\u003c/p\u003e \u003cp\u003eIn our study we find a different method to induce epithelial tumor by Intraperitonial (IP) injection of DMBA. Although a majority of these induced tumor were reported to show the histology of epithelial tumor. In our study after induction by IP of DMBA we collect the ovary and study the histopathology of ovary tissue for tumor subtyping and immuno-histochemical study of different marker such as CA125, CK7, Vimentin, which confirm the induction of tumor. We examined antitumor efficacy of RSV-ZnO nano-conjugate by using 3 doses in a month with compared control. After treatment with our drug RSV-ZnO nano-conjugate (dosage of 100 mg/kg body weight per week during 3 weeks study) we have found very positive result with lower expression level of cancer marker (CA125, CK7, Vimentin). In much previous research we have found that free Resveratrol at dose of 150 mg/kg body weight shows adverse effect on ovarian cancer. Thus the results suggest the potential utility of these conjugate for medical applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors state that there are no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author Mahamuda Khatun (MK) is thankful to the UGC-MANF for fellowship grant during the period of study. The author would also like to thank Dey\u0026rsquo;s Medical Stores, Quality Control Department for Research Instrumentation Facility, Dr. Soumendra Darbar, Sr. Executive Q.C of Dey\u0026rsquo;s Medical stores, Mr. A Garai, Assistant Manager Q.C of Dey\u0026rsquo;s Medical stores. Also like to thanks Partho Pratim Roy for Histopathological analysis and Department of Biochemistry, Calcutta University for laboratory support during work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval and consent to participate:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eExperiment was carried out under registered animal house of Dey\u0026rsquo;s Medical Stores, Kolkata. The project approval No-IAEC/s/PHARM/DEY\u0026rsquo;S/16/2016 by the Institutional Animal Ethics Committee (IAEC), India, according to CPCSEA\u0026rsquo;s guidelines. The studies were carried out in compliance with the all statutory guidelines of the Committee for the Purpose of Control and Supervision of Experimental Animals (CPCSEA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors state that they have no competing interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMK (Dr. Mahamuda Khatun) carried out biochemical, chemical, statistical analysis and histological experiments. She wrote the main manuscript and designed the experiments, coordination between the authors; she is 1st author and Corresponding Author. RM (Ritwik Maity) prepared \u003cstrong\u003eFigure 11-14\u003c/strong\u003e and helps in animal handling and considers him as 2nd Author. SM (Prof. Santasree Mazumder helped in experiment with chemical supply and departmental instrument, consider her as 3rd author. SD (Dr. Soumendra Darbar) conceived and guide the experiment, conducted and supervised the animal experiment, review and the approved the final manuscript. Consider as 4\u003csup\u003eth\u003c/sup\u003e Author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUGC-MANF 2013 (Doctoral Fellowship).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFellowship ID\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eF1-17.1/12-13 MANF-2012-13-MUS-WES-17574.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of Data and Material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data were generated during the experiment. The nano-conjugate drug (RSV-ZnO) is synthesized in our laboratory. Cancer induction process designed in our lab.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eGabra H, Smyth J (1997) Biology of female cancers. 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Int J Biol Sci. 2021; 17(2): 574\u0026ndash;588.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"University of Calcutta","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Safety study of the nano-drug, Ovarian cancer induction by DMBA, Histopathological analysis, Immunohistochemistry, CA125, CK7, Vimentin cancer biomarker","lastPublishedDoi":"10.21203/rs.3.rs-6187379/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6187379/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eResveratrol (3,5,4-trihydroxy-transstilbene), a natural phytoalexin is well known for its anticancer and chemo-preventive effect. To overcome stability and solubility issue of Resveratrol (RSV), a conjugation of RSV with ZnO nanoparticles was prepared to improve the efficacy and reduce the toxicity. Thus objective of the present study was to evaluate the potential effect of RSV-ZnO nano-conjugate in DMBA-induced ovarian cancer in a defined rat model. The acute and sub-acute toxicity was carried as per OECD guidelines 423 and 407, respectively. In the acute toxicity study, nano-conjugate drug was administered \u003cem\u003evia\u003c/em\u003e single Intraperitoneally (IP) injection at 200 mg/kg and 400 mg/kg body weight doses in female albino Wistar rats while in sub-acute toxicity study, female Wistar rats was administered daily IP injection of 50 mg/kg, 100 mg/kg and 200 mg/kg body weight for 28 days. At the end of the study, the animals were sacrificed and evaluate the effect of RSV-ZnO nano-drug on body and organ weight and hematological biochemical parameters and histopathological findings. DMBA was used as inducing agent to develop ovarian cancer. Rats were treated by DMBA (50 mg/kg body weight) \u003cem\u003evia\u003c/em\u003e IP route once a week up to 3 weeks and kept animals for 3 months for development and progression of carcinogenesis in ovary. After 3 months, we treated the induced animals by RSV-ZnO nano-conjugate in three different doses (50 mg/kg, 100 mg/kg and 150 mg/kg body weight by IP) weekly for one month. After that, we sacrificed the animals and taken out the ovaries for histopathology for tumor subtyping and immunohistochemistry to evaluate the markers such as CA125, CK7, Vimentin. RSV-ZnO nano-conjugate showed significant reduction of expression of cancer bio-markers and size of tumor. Besides, in acute and sub-acute toxicity study, it exhibited no untoward effects in animals. The findings demonstrated that RSV ZnO nano-conjugate drug is safe and may be used for the treatment of ovarian cancer with more efficacies and less toxicities.\u003c/p\u003e","manuscriptTitle":"Safety and Efficacy of Resveratrol-ZnO nanoconjugate in DMBA-induced ovarian cancer in murine model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-11 09:53:52","doi":"10.21203/rs.3.rs-6187379/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ff6dfb4b-9424-49c3-925c-36ce61c4aa53","owner":[],"postedDate":"March 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":45411518,"name":"Toxicology"},{"id":45411519,"name":"Cancer Biology"}],"tags":[],"updatedAt":"2025-03-11T09:53:52+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-11 09:53:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6187379","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6187379","identity":"rs-6187379","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}