br Cellular uptake studies br Rhodamine B loaded nanoparticl
3.6. Cellular uptake studies
Rhodamine-B loaded nanoparticles (R-SLN) were prepared by re-placing EGCG with Rhodamine B and conjugated with BBN (RB-SLN) for comparative cellular uptake studies (Xu et al., 2009). For quanti-tative studies, 1 × 105 cells per well were seeded in 12-well plates and allowed to adhere for 24 h. Cells were incubated with R-SLN or RB-SLN formulations for time intervals of 12 and 24 h. After removal of the culture media, the cells were washed twice with cold PBS and observed under a fluorescence microscope.
3.7. Migration assay
The eﬀect of EGCG formulations on migration of the cells was de-termined by wound healing assay. MDA-MB-231 (5 × 105 cells/well) were seeded in petri dishes and allowed to grow to 80% confluent monolayer. Wounds were created through sterile 250 μL pipette tip by scratching the monolayer. Cells debris were removed by washing the cells twice with PBS. Cells were incubated with EGCG, EGCG-SLN or EB-SLN, equivalent to 100 μg/mL EGCG. The zone of wound healing was observed at 0 and 24 h using a bright field microscope. The per-centage of wound closure was determined by measuring the wound area using Image J analysis software
The animal experimental protocol for this study was approved by the Institutional Animal Ethics Committee of the CSIR-Indian Institute of Chemical Technology, Hyderabad (approval no. IICT/PHARM/SRK/ FEB/2013/8). All the animal studies were performed in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). Forty female C57/ BL6 mice (6–8 weeks), weighing between 20–25 g, were used for the survival studies. Animals were kept in polypropylene cages under standard laboratory conditions (12:12 h light/dark cycles) at 24 °C. The animals were housed five per cage and had free access to food and water.
The administration of formulations to the groups of mice was con-tinued until the animals died.
The body weight, tumour volume and overall survival of the mice were recorded, and the mean survival time was determined using a Kaplan-Meier survival plot.
4. Results and discussion
4.1. Particle size and surface charge
EGCG was encapsulated in the lipid matrix of GMS and stearic SCR-7 by a double emulsification method. Bombesin was conjugated on the surface of EGCG-SLN by EDC/NHS reaction. The bombesin-conjugated nanoparticles (EB-SLN) had a hydrodynamic diameter of 163.4 ± 3.2 nm, PD index of 0.341 ± 0.15 and a negative surface charge of -25.2 ± 2.8 mV (Table 1). There was a decrease in negative zeta potential in comparison to EGCG-SLN (−37.2 mV, owing to the free carboxylic group in the nanoparticle matrix), indicating the con-jugation of bombesin on the surface of nanoparticles. The EE of EGCG within SLNs was approximately 67.2 ± 3.5%, which shows an eﬃcient loading of EGCG within the lipid core of nanoparticles.
BBN peptide conjugation eﬃciency calculated by the standard Bradford assay showed a conjugation eﬃciency of about 92.4 ± 2.4%. The conjugation of bombesin was further confirmed by FTIR studies. EGCG-SLN showed peaks at 3358 cm−1 for OeH stretching in GMS, peaks at 2917 cm−1 for the OH stretch and 1731 cm−1 for the C–O stretch in stearic acid and at 1468 cm−1 for the C–H bend in alkanes. EB-SLN did not show the peaks at 1730 cm−1 for carboxylic acid groups, suggesting the conjugation of the carboxyl groups due to the formation of an amide bond with the amine group of bombesin. Furthermore, a prominent peak at 1640 cm−1, characteristic of C–N stretching of an amide bond, was observed in the spectrum for EB-SLN. These changes point to the formation of amide bonds between the constituent lipids of the nanoparticle matrix and the peptide ligand.
Physicochemical parameters of bombesin conjugated, EGCG-loaded solid lipid nanoparticles (EB-SLN).
R. Radhakrishnan, et al.
Fig. 1. Percent cell viability of MDA-MB-231 human breast cancer cells after 48 h of treatment with pure EGCG, EGCG-loaded SLN (EGCG-SLN) and bom-besin-conjugated EGCG-SLN (EB-SLN).
4.2. In-vitro cytotoxicity
The in-vitro cytotoxicity of EGCG, EGCG-SLN or EB-SLN against MDA-MB-231 human breast cancer cells and B16F10 mouse melanoma cells was evaluated by MTT assay (Figs. 1 and 2). The cell viability of EGCG, EGCG-SLN and EB-SLN-treated cells after 48 h of treatment at a concentration range of 5–100 μg/mL was determined. The observed IC50 values for EGCG, EGCG-SLN and EB-SLN in MDA-MB-231 were 65.4 ± 4.9 μg/mL, 6.9 ± 1.1 μg/mL and 3.2 ± 1.7 μg/mL, respec-tively (Table 2). The cell viability of MDA-MB-231 following treatment with EGCG was 93.9% at the lowest tested concentration of 5 μg/mL. At the same concentration, EGCG-SLN and EB-SLN showed approximately 62% and 54.5% viability, respectively, demonstrating the comparative eﬀectiveness of the nanoformulations. Similarly, these formulations were also more eﬀective against B16F10 cells with IC50 values for EGCG, EGCG-SLN and EB-SLN found to be 59.3 ± 6.4 μg/mL, 28.2 ± 1.9 μg/mL and 15.6 ± 1.3 μg/mL, respectively. The results indicate that the activity of EGCG increased more than 9 times after encapsulation in SLNs and more than 20 times post conjugation with bombesin against MDA-MB-231. Similarly, against B16F10, the cyto-toxicity increased more than 2 times for EGCG-SLN and almost 4 times for EB-SLN. We hypothesize that the increased cytotoxicity of EGCG in the nanoparticle formulations to the decreased degradation, and hence prolonged eﬃciency of EGCG because of the protection provided by the lipid core. Comparing targeted and non-targeted formulations, EB-SLN showed more cytotoxicity may be due to the interaction between BBN and GRPR receptors present in these cells, which enhance the inter-nalization through receptor-mediated endocytosis.