br The aim of this study was the construction of
The aim of this study was the construction of a breast-cancer tar-geted, biocompatible nanoparticle vehicle with minimum toxicity to non-specific tissues. Herein, bombesin was conjugated to the EGCG-encapsulated SLN system and bombesin-conjugated solid lipid nano-particles were then explored for their anti-cancer eﬃcacy by in-vitro cytotoxicity and uptake studies. In-vivo studies in C57BL/6 mice were performed to evaluate changes in tumour volumes and survivability.
Glycerol mono-stearate (GMS) was purchased from Alfa Aesar (Johnson Matthey Chemicals, Hyderabad, India). EGCG, bombesin acetate salt hydrate (Glp-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2), stearic acid, Pluronic F68 (P-F68), 1-ethyl-3-(3-di-methylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), 2-(n-morpho-lino)ethanesulfonic Seladelpar (MES), Roswell Park Memorial Institute (RPMI)-1640 medium, fetal bovine serum (FBS), trypsin–EDTA, phosphate buﬀered saline (Ca2+ and Mg2+ free), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and di-methyl sulfoxide (DMSO) were purchased from Sigma Aldrich (St. Louis, MO, USA). Lecithin soy was purchased from HiMedia (Mumbai, × 100
3.3. Bioconjugation of bombesin to nanoparticles
For the synthesis of targeted nanoparticles, drug-loaded nano-particles (EGCG-SLN) were dispersed in MES buﬀer (pH 6.2) and in-cubated with NHS and EDC as reported earlier ((Bartczak and Kanaras, 2011). Bombesin was added to this suspension and allowed to incubate. The nanoparticle dispersion was collected, centrifuged and washed with distilled water. The supernatant was used to estimate the con-jugation eﬃciency using a standard Bradford protein assay. The su-pernatant was diluted appropriately, and the absorbance was measured at 595 nm to determine the concentration of free or unconjugated bombesin using a calibration curve of bovine serum albumin.
The percent bombesin conjugation eﬃciency (% CE) was de-termined as follows:
BA: Amount of bombesin added, BS: Amount of bombesin present in supernatant
3.4. Physico-chemical characterization of nanoparticles
Particle dimensions and surface charge of nanoparticles were de-termined by photon correlation spectroscopy. Briefly, mean particle size, polydispersity index and surface potential of EGCG-loaded SLNs (EGCG-SLN) and bombesin-conjugated SLNs (EB-SLN) were measured using a Malvern Zetasizer Nano ZS (Malvern Instrument Ltd., Malvern, UK). Bioconjugation of bombesin on the surface of the nanoparticles was analysed by Fourier transform infrared analysis by scanning EGCG-
R. Radhakrishnan, et al.
3.5. In-vitro cytotoxicity studies
MDA-MB-231 and B16F10 cells were grown in DMEM and RPMI medium, respectively and supplemented with 10% fetal bovine serum, 100 g/mL penicillin, 200 g/mL streptomycin and 2 mM L-glutamine. The cultures were maintained in a humidified atmosphere with 5% CO2 at 37 °C.
The cytotoxicity of formulations was determined by MTT assay against MDA-MB-231 human breast cancer cells based on mitochon-drial reduction of yellow MTT tetrazolium dye to a highly coloured blue formazan product which shows absorption at 570 nm (Edmondson et al., 1988). About 1 × 104 cells were seeded (counted by the trypan blue exclusion dye method) in 96-well plates in 100 μL of medium. Cells Chemistry and Physics of Lipids xxx (xxxx) xxx–xxx
subcutaneously into the right flank via a 30 G needle. After induction of tumour, mice were randomly divided into four groups: control group, EGCG group, EGCG-SLN group and EB-SLN. (The control group was administered sterile PBS, EGCG group received pure drug at a dose of 50 mg/Kg body weight while EGCG-SLN or EB-SLN group received nanoparticle formulation containing EGCG equivalent to 50 mg/Kg body weight. PBS, EGCG, EGCG-SLN and EB-SLN were administrated independently via intra-peritoneal injection every third day from day 14 after tumour implantation.
3.8.2. Measurement of body weights and tumour volumes
The tumour size and body mass were measured regularly following the inoculation of tumour cells. Callipers were used to assess tumour growth by measuring two bisecting diameters in each tumour. The following equation was used to calculate the tumour volume:
were allowed to adhere overnight and then incubated with EGCG, EGCG-SLN or EB-SLN with a series of concentrations for 48 h at 37 °C. The above media was then replaced with fresh serum free media and 10 μL of MTT reagent (5 mg/mL), then plates were incubated at 37 °C
for 4 h. After removing the media, DMSO was added to dissolve for-mazan crystals. The absorbance was measured at 570 nm using a spectrophotometer (Spectramax Plus, Molecular Devices, USA). The half maximal inhibitory concentration (IC50) was calculated by using Probit software.