br Migration studies br Angiogenesis is the process
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 L-NAME 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-
R. Radhakrishnan, et al. Chemistry and Physics of Lipids xxx (xxxx) xxx–xxx
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.
The change in tumour volume was also observed and results re-vealed that the tumour growth was inhibited in the mice receiving the EGCG formulations (Fig. 9). The percent increase in tumour volume in mice treated with EGCG-SLN and EB-SLN was 3.78% and 0.78%, re-spectively, as compared to mice receiving pure EGCG and the control group which were 5.45% and 7.97%, respectively.