br comparison to the EGCG SLN due
comparison to the EGCG-SLN due to the presence of the targeting li-gand. These results indicate the successful and increased delivery of the cytotoxic EGCG inside the cells, thus causing a greater extent of apoptosis.
4.5. Cellular uptake studies
Uptake of nanoparticles by cancer PFK-158 was performed by using a fluorescent dye (i.e. Rhodamine-B) loaded in the lipid nanoparticles and observing fluorescence through microscopic detection as followed in previous works (Yuan et al., 2008). In this study, Rhodamine-B was chosen due to its hydrophilic nature, and therefore it can be loaded in the same phase as that of the hydrophilic drug. Uptake was studied in the MDA-MB-231 breast cancer cell line (Fig. 5). Rhodamine-B loaded SLNs (R-SLN) were prepared by the same method as EGCG-SLN, by replacing the drug with Rhodamine-B dye. R-SLN were further con-jugated with bombesin to get BBN conjugated, Rhodamine-B loaded nanoparticles (RB-SLN). Cells were observed at three-time points (0 h, 1 h and 2 h) following incubation of MDA-MB-231 cells with the dye-loaded formulations (Fig. 5). At 1 h, visibly more intense red fluores-cence was observed in cells treated with RB-SLN compared with the unconjugated nanoparticles (R-SLN). At 2 h, this diﬀerence is more pronounced with not only an increase in the number of RB-SLN treated cells showing fluorescence, but there is also visible intensification in the fluorescence of individual cells. These findings point to continual and increased uptake of nanoparticles in the cancerous cells conjugated with the targeting ligand bombesin as opposed to the unconjugated formulation.
4.6. Migration studies
Angiogenesis is the process by which a tumour develops new blood vessels from existing ones in order to maintain continual growth and nutrition (Folkman, 2002). Therefore, inhibition of angiogenesis aids in
preventing the growth and metastasis of tumours, by essentially cutting oﬀ its nutrition. EGCG has shown anti-angiogenic eﬀects in a number of cell lines (Jung and Ellis, 2002). In the present study, this eﬀect was found to be increased when it was encapsulated as EGCG-SLN and further conjugated with bombesin (Fig. 6). Wound closure is found to occur in a greater extent in control cells as compared to EGCG-treated cells. Wound closure is almost negligible in EGCG-SLN and EB-SLN. Enhanced activity in EGCG-SLN and EB-SLN could be attributed to the increased stability of the drug within the solid lipid nanoparticles and hence greater activity. Sustained release of EGCG from the nano-particles also aids in the continued anti-migratory eﬀect of the drug, leading to better wound closure in EGCG-loaded nanoparticles than pure drug (Andreani et al., 2016).
The in-vivo anti-cancer eﬃcacy of a therapeutic agent is assessed in terms of prolonging the quality of life and increasing the survival time. The anti-tumour eﬃcacy of EGCG, EGCG-SLN or EB-SLN was evaluated on tumour-bearing mice upon administration of multiple doses equivalent to 50 mg/kg EGCG. In-vivo anti-tumour eﬃcacy was eval-uated in a syngeneic C57BL/6 mouse model.
The B16F10 cancer cell line was used to develop melanoma in this study. This mouse melanoma cell line shows expression of GRPR re-ceptors and therefore was used for evaluating the eﬃcacy of the bombesin conjugated formulation (Fang et al., 2009). Mice were first injected subcutaneously with 3 × 105 B16F10 cells on the flank to grow melanomas in-situ. Forty tumour-bearing C57BL/6 mice were randomly divided into 4 groups (n = 10) and intraperitoneally administered EGCG, EGCG-SLN or EB-SLN at a dose equivalent to 50 mg/kg body weight of EGCG every third day. The control group was administered an equivalent volume of saline every third day.
The body weight, tumour volume and overall survival of the mice were recorded. The mean survival time was determined using a Kaplan-
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Fig. 5. Cellular uptake studies: Fluorescent microscopic images of MDA-MB-231 human breast cancer cells after 0 h (a), 1 h (b) and 2 h (c) of treatment with Rhodamine-loaded SLN (R-SLN) and bombesin conjugated SLN (RB-SLN).
Meier survival plot (Fig. 7). A median survival of 41, 48 and 55 days were observed for EGCG, EGCG-SLN and EB-SLN treated mice, respec-tively, compared to control mice (37 days) which were administered with normal saline. Thus, there was a significant extension in the life span of mice treated with EB-SLN.
The change in body weight of animals is an indicator of systemic toxicity caused by the formulation (Criscitiello et al., 2012). There was a rapid loss of body weight in the saline treated control group in comparison to EGCG formulation treated animals (Fig. 8). The control group receiving only saline recorded a loss of 7.01% in total body weight in 30 days whereas both EGCG-SLN groups did not show any significant change in their body weights.