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In vivo Evaluation of Nanostructured Lipid Carrier System in Rats Bearing Breast Tumor

The aim of this work is to study the anti-proliferative potential of two anticancer drugs loaded in nanostructured lipid carriers (NLCs).The maximal inhibition of cell growth by Raloxifene (RLX) & Curcumin (CUM) nanostructured lipid carriers (RLX-CUM-NLCs) was determined by assessing the viability of MDA-MB 231 cells. As far as we know, this is the first research to look at the effects of RLX-CUM-NLCs on DMBA-induced breast carcinogenesis in a rat model. RLX-CUM-NLCs reduced the number of tumors in an in-vivo investigation. After 14 weeks of induction, we discovered a tumor with a 100% incidence rate. The incidence of experimental breast cancer was decreased to 83.33% in the RLX-treated group. In contrast, RLX-CUM-NLCs demonstrated a significant anticancer effect with a 50% incidence in the RLX-CUM-NLCs group. Compared to controls, the RLX-CUM-NLCs therapy did not cause any toxicity in the animals in terms of food intake, body weight, or activity levels until 300 mg/kg BW. The current research shows that the RLX-CUM-NLCs has a chemopreventive impact on DMBA-induced breast cancer in rats by decreasing tumor burden and restoring marker enzymes activity.


INTRODUCTION
Breast cancer is the most often diagnosed cancer in women and the leading cause of death. Metastasis and tumor recurrence are posing new problems in the management of breast cancer [1]. Nowadays, breast cancer treatments comprise surgery, chemotherapy, radiation, hormone and immunotherapy; however, these therapies have side effects. Moreover, the 5-year disease-free survival rate of stage II breast cancer is 75-90%, ~ 30% for stage III patients and 0-10% for stage IV breast cancer [2], highlighting the need for new treatment and prevention strategies. Natural products are being used and investigated for the discovery and development of new therapeutic antineoplastic compounds [3]. Previously, several studies were done on CUM and RLX for its antitumorigenic action in a breast cancer promoting a significant reduction in tumor cells. In the study, development of nanostructured lipid carriers (NLC) as carriers for two antitumor compounds that possesses a remarkable antineoplastic activity [4]. Recently, NLC has been described as a promising drug delivery system for enhanced cancer therapy [5]. Among the advantages of the NLC is the retention of the drug in the lipid core to promote the control of release. NLCs are second-generation Solid lipid nanopaticles (SLNs) which are most effective drug delivery system for anticancer drug targeting [5]. Pure solid lipids are crystalline, and they prefer to stay in the -polymorphic state, which is low in energy and well organized [5]. The lipid re-arranges into less stable forms when it is molten and cool during the synthesis of SLNs: polymorphic shape and form. These lipids are amorphous, which allows medicines to be retained inside the lipid matrix [6]. By replacing liquid lipid for a component of pure solid lipid, strategies to keep defects in the lipid, even after lengthy storage, alleviate the issues associated with standard SLNs [7]. When a little amount of liquid lipid/oil was added to a solid lipid matrix, the crystal lattice structures were less ordered [8]. Liquid lipids contributed in widening the loading capabilities of lipid nanocarriers by increasing the number of defects wherein amorphous drug clusters might fit [9]. As a consequence, this dual lipid framework may not only be able to accommodate higher drug load, but it may also be able to reduce drugs expulsion from lipid during storage [10].
Mingzhen Lin et al. [11] formulated a folic acidloaded curcumin nanostructured lipid carrier using a solvent diffusion approach. The results revealed that FA-CUM-NLCs were efficient in selective delivery to cancer cells over expressing FA receptors (FRs). CUM is also delivered to Breast cancer cells via FA-CUM-NLCs, boosting anti-tumor action. As a consequence, FA-CUM-NLCs might be a more effective nanomedicine for tumor therapy. The dual drug delivery offers several advantages such as dose reduction, overcoming of multidrug resistance and can result in synergistic effects [12].In this paper we have discussed the in vitro cytotoxicity assay, in vivo study, acute toxicity study, Haematological and biochemical analyses and histopathological studies which shows that RLX-CUM-NLCs has a chemopreventive impact on DMBA-induced breast cancer in rats by decreasing tumor burden and restoring marker enzyme activity.

Chemicals and reagents
7, 12-Dimethylbenz(a)anthracene (DMBA) Cas No. 57-97-6 and other fine chemicals were purchased from Sigma Chemical Co. Raloxifene (RLX) was procured from Cadila Pharmaceuticals Limited, Ahmedabad, India. Curcumin was purchased from Amrita Herbal Sanwer road Indore MP. All other chemicals and solvents used were of analytical grade and highest purity.

Animals
Female rats of weighing 190±20 g was selected and procured from Animal house of PBRI. The animals were maintained under standard conditions of humidity, temperature (25±2 °C) and light (12 h light/dark). Rats were acclimatized for 7 days before the start of the experimental work. They were fed with standard rat pellet diet and water ad libitum.

In vitro cytotoxicity assay
200μl cell suspension was seeded in a 96-well plate at required cell density (20,000 cells per well), without the test agent. After 24 hrs, cell was treated with various concentrations 6.25µg/ml-100µg/ml of the RLX, CUM and RLX-CUM-NLC. Then, plates were incubated for 24 hrs at 37°C in a 5% CO 2 atmosphere [13]. After 24 h incubation, the cells were treated with 20 μl of MTT solution (0.5 mg ml−1 in PBS) before being incubated for 3 h at 37 •C. Then, 100 μl of dimethyl sufoxide was added to completely dissolve the formazan crystals, absorbance was recorded at 570 nm and % cell viability was measured.

Acute toxicity study
The acute toxicity study was conducted in accordance with Organization for Economic Cooperation and Development (OECD) 423 Guideline for Testing of Chemicals.
Twelve animals (female rats) were randomly assigned into four groups (n=3 animals each), which were at four fixed doses (5, 50, 300 and 2000mg/kg of body weight) of CUM-RLX-NLC. One normal control with three animals was also taken in study. All treatments were given orally on the first day only. Rats were monitored for 14 days for any changes in general physical conditions such as appearance, body weight changes, behaviour and mortality. The body weight was measured thrice 0, 7 and 14 day using a table top electronic balance.

Haematological and biochemical analyses
Haematological parameters were analysed using an automated haematologyanalyser(Procan-P6800). The parameters measured were red blood cells, haemoglobin concentration, lymphocytes, white blood cells, and platelet count. Biochemical analysis was performed using a chemical analyser (Star 21 E114947). For hepatic function, the levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP), total bilirubin were evaluated. For renal function, the levels of blood urea nitrogen and serum creatinine were determined.

Histopathological study
The organs, namely the liver, heart, spleen, lung, and kidney, were carefully excised and weighed. These organs were preserved in a fixation medium of 10% buffered formalin for histopathological study. The relative organ weight of each animal was calculated as follows: Relative organ weight = (organ weight (g)/body weight of the animal on sacrifice day (g)) × 100. Tissue sections were cut and fixed in 10% formalin for at least 48 hrs. The fixed samples were placed in plastic cassettes and dehydrated using automated tissue processor. The processed tissues were embedded in paraffin and the blocks trimmed and sectioned were cut using a microtome (Spencer, US Pat 2-232-003).
The tissue sections were mounted on glass slides using a hot plate (Remi) and subsequently treated in order with 100, 90, and 70% ethanol for two minutes each. Finally, the sections were rinsed with tap water and stained with haematoxylin and eosin for light microscopy.

Tumor induction
These experimental rats were housed in standard polypropylene cages having 6 animals in each cage and they were randomly distributed into control and treated groups. A single dose of 75 mg/kg DMBA in 1 ml sesame oil was given to all animals p.o. mammary carcinoma was confirmed by palpation. Rats were palpated weekly starting from 4th week after DMBA administration, to check for the tumor appearance. The first tumor appeared in the 4 months, after administration of DMBA while by 4.5 months tumor appeared in all the rats. The body weight of each animal in each group was also recorded weekly. Tumor size was measured by a verniercalliper (Aerospace) [14-17].
Group II-DMBA induced rats only with single dose of 75 mg/kg DMBA in 1 ml sesame oil.
Group III-DMBA induced rats treated with Curcumin at the dose of 40 mg/kg by oral gavage daily after 14 weeks of tumor palpation until completing 20 weeks.
Group IV: DMBA induced rats treated with RLX at a dose of 15 mg/kg by oral gavage daily after 14 wk of tumor palpation until completing 20 weeks.
Group V: DMBA induced rats treated with RLX-CUM-NLC by oral gavages daily after 14 wk of tumor palpation until completing 20 weeks.
After the completion of dosing, female rats were anaesthetized by diethyl ether and sacrificed. Blood samples were collected through the orbital puncture of the experimental rats. Serum was separated for biochemical tests. The isolated tumor was subjected to endogenous antioxidant estimation. Tissues of breast were fixed in 10% formalin for the histopathological studies.

Preliminary in vitro Cytotoxicity Screening in Human Breast Cancer Cell Lines
In

In vivo Toxicity Studies
During the 14-day observation period, no significant weight loss was noted in all treatment groups till 300 mg/kg bw. Food and water consumption was found normal till 14 days as shown in table 1. There was no sign of behavioral abnormality in the surviving female rats. So, depending on the current observed data of acute toxicity study, the final selected dose for further study were carried out using 1/20 th of 300 mg/kg bw which was 15 mg/kg bw.
A single administration of RLX-CUM-NLCs did not cause any significant changes in the organ weight and body weight as shown in Table 2. Simultaneously the biochemical parameters were studied and summarized in Table 3.

Liver
Control rats show normal architecture of the liver with hepatocytes radiating from the central vein, sinusoidal space; RLX-CUM-NLCs at 5,50,300 mg/kg bw treated rats showed normal architecture but 2000mg/kg bw treated rats show moderate parenchymal cells, dilatation of sinusoidal space with severe inflammatory cell infiltration; H&E (10X) shown in Fig. 4. Comparative IC50 values vs MDA MB 231

Kidney
H&E-stained kidney sections of normal control group looked normal with clearly visible glomeruli, basement membrane of the capsular wall. The interstitial appeared normal and with no infiltrations. In 5, 50 and 300 mg/kg bw RLX-CUM-NLC treated rats, some of the proximal and distal convoluted tubules showed hypertrophic cells, glomeruli with Bowman's space and capillary tufts, which appeared normal and vacuolization and in 2000 mg/kg bw treated rats showed a severe distortion of the glomerular architecture, with atrophic and necrotic changes, pyknotic nuclei as well as infiltrations of mesangial spaces and interstitial shown in Fig. 5.

Heart
Histological analysis of rat heart with H and E staining Normal control; most cells are elongated and rod shaped with striated muscles. The 300 mg/kg bw treated rats showed rod shaped cells which are intact and except few cells all the cells are normal but in 2000 mg/kg bw treated rats cell swelling, striated muscles and blood vessels seen ( (Fig. 6). 30 min ischemia followed by 45 min reperfusion; cell swelling and increased congested blood vessels were observed.

Lungs
Photomicrograph showed the normal histological structure of the epithelial cells in normal control rats (Fig. 7). The 5, 50, 300 mg/kgbw treated bw group showed bronchiole, alveolar sca, pulmonay vessels, septum and 2000 mg/kgbw rats showed intensive contraction of alveoli, diffuse accumulation of inflammatory cells in the pulmonary parenchyma compressed filled with necrotic debris.

Body weight changes
The body weight changes of animals in DMBA induced cancers and treated with RLX, CUM and RLX-CUM-NLC was presented in graph.
Tumor volume is a potential biomarker for breast cancer development. All the treatments group showed significant (p<0.05) reduction in tumor volume as compared to Tumor control. The RLX-CUM-NLC-treated group showed significant (p<0.05) inhibition in tumor volume as compared to other treatment groups ( Fig. 9 & 10).
Tumor volume was 82.06±7.311 mm 3 for DMBA induced group, 31.72±4.748 mm 3 for CUM, 20.22 ± 10.583 mm3 for RLX and 6.748± 1.567 for RLX-CUM-NLC-treated which was significantly (p<0.05) low as compared to tumor control on 20 th week. As expected, animals treated with RLX-CUM-NLC-treated showed a significant (p<0.05) reduction in tumor weight of (0.12±0.10 g) as compared to DMBA control animals (1.81±0.31). On the basis of assessment parameters and the results obtained from the tumors excised from all the treated rats, it was found that the tumor volume, tumor weight, tumor burden, tumor yield and tumor incidence was found to be increased in the DMBA-treated groups (Tumor control) when compared with the treatment groups. There was a significant decrease in the below mentioned tumor parameters in animals administered with RLX, CUM and RLX-CUM-NLCs. RLX-CUM-NLCs demonstrated a rapid and significant decrease in tumor volume, tumor weight, and tumor burden, and tumor incidence when compared with other treatment groups. In the tumor control group CUM treated group, the tumor incidence rate was 100%; however, in the group treated with RLX, the tumor incidence rate was markedly reduced to 83.33% and in RLX-CUM-NLCs treated group its was reduced to 50%. These data suggest that RLX-CUM-NLCs treated could prevent the chemically carcinogenesis induced by DMBA-treatment (Table: 4).Oxidative stress also plays an important role in progression of cancer breast. This study was conducted to compare the levels of superoxide dismutase (SOD), GSH and malondialdehyde (MDA) in breast cancer rats (Table: 5).In contrast, haematological study was done to study the complete blood profile in rats ( Table 6).

CONCLUSION
In the present study, no toxicity was observed in histopathology of lung, heart, spleen liver and kidney in the groups that received RLX-CUM-NLCs treatment till 300 mg/kg bw. Slides of 2000 mg/kg treated rats were also presented showing toxicity in various organs. Oxidative stress is a key mechanism inducing cytotoxicity through enhancing the production of MDA accompanied by depletion of GSH in tissues; these are considered the most important indexes of antioxidant activities. Significant reduction of oxidative stress and haematological parameters were observed in treatment groups. The levels of oxidative stress markers such as level of LPO were substantially increased by DMBA treatment in control group. However, in treatment group RLX-CUM-NLCs significantly restored the required LPO level. Oxidative stress as indicated by decrease in (GSH) and (SOD) activity compared with the tumor control group values. The present study reveals that the RLX-CUM-NLCs exert chemo preventive effect by suppressing the tumor burden and restoring the activities of marker enzymes on DMBA induced breast cancer in rats.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.