Baicalein (BL), a potential malignancy chemopreventative flavone, has been reported to

Baicalein (BL), a potential malignancy chemopreventative flavone, has been reported to inhibit malignancy cell growth by inducing apoptosis and causing cell cycle arrest in various human malignancy cell models. drug loading rates, as well as targeting and slow releasing actions, enhanced oral bioavailability and long-circulating properties (18C25). Despite these advantages, you will find no studies in the literature describing the use of liposomes to deliver BL to K562 cells or INCB8761 inhibitor to investigate the antitumor activities of free BL and liposomal BL on these cells. Previous investigations have shown that BL has multiple biological activities, including anti-inflammatory (26) anti-microbial (27) and antioxidant (28) properties. BL exerts an antitumor effect by promoting the apoptosis or inhibiting the proliferation of INCB8761 inhibitor malignancy cells (29C32) through multiple signalling pathways including the cell proliferation pathway, the cell apoptosis and caspase activation pathway, the tumor suppressor pathway and the protein kinase pathway (33,34). However, the exact mechanism of apoptosis and its related pathways induced by BL is not yet fully comprehended. In the present study, we evaluated different sizes of liposome formulations for the delivery of BL. We further investigated the cytotoxicity and pro-apoptotic effects of BL and liposomal BL on CML K562 cells. The mechanism involved in this process was also explored. Materials and methods Materials Soy phosphatidylcholine (PC) was purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). Meth oxypolyethyleneglycol-di-stearoyl-phosphatidylethanolamine (DSPE-PEG2000, with mPEG MW2000 Da) was obtained from Genzyme (Oxford, UK). Cholesterol (Chol), PBS, dialysis tubing, propidium iodide (PI), RNase and BL were all purchased from Sigma-Aldrich (UK). Methanol, dichloromethane, CyQUANT? Cell Proliferation Assay kit and Annexin V-FITC/PI Apoptosis Detection kit were both from Thermo Fisher Scientific (Loughborough, UK). RPMI-1640, INCB8761 inhibitor L-glutamine, penicillin-streptomycin and fetal bovine serum (FBS) were all from Invitrogen Life Technologies (UK). The CellTiter 96? AQueous One Answer Cell Proliferation Assay (MTS) kit was purchased from Promega (Southampton, UK). Liposome preparation and characterization Three types of liposomes with different diameters were prepared. Liposomes were composed of soy PC, cholesterol, and methoxypolyethyleneglycol-di-stearoyl-phosphatidylethanolamine (DSPE-PEG2000; Genzyme). Liposomes were prepared as explained elsewhere (35). Briefly, the lipids were dissolved in methanol:dichloromethane 1:2 (v/v) at INCB8761 inhibitor a PC:Cholesterol:DSPE-PEG2000 molar ratio of 78.9:19.7:1.4 at room heat. BL was dissolved in the solvent with lipid combination when formulating the liposomes. Different lipid/BL mass ratios were tested before settling on a fixed ratio of 10:1. The lipid mixtures were deposited on the side wall of the rotary glass vial by removing the solvent with nitrogen. The dried lipid films were hydrated in 10 mM sodium phosphate buffer pH 7.4. This process led to the spontaneous formation of pegylated liposomes. The liposomes were then down-sized by passing through 0.1, 0.2 or 0.4 m polycarbonate membrane syringe filters (Whatman?; Whatman, Inc., Clifton, NJ, USA) to produce lipo1, 2 and 3 suspensions, respectively. Free BL was removed by dialysis (14,000 Da cutoff membrane) against 10 mM sodium phosphate buffer pH 7.4 overnight. The size and -potential of liposomes were measured by dynamic light scattering on a Zetasizer-Nano ZS (Malvern Devices Ltd., Malvern, UK). Cell culture Human leukemia K562 cells were purchased from ATCC (UK). Cells were cultured in RPMI-1640 media made up of 10% fetal calf serum, 100 U/ml of penicillin, 100 mg/ml streptomycin in 75 cm2 flasks. The cells were grown in a humidified incubator made up of 5% CO2 and 95% air flow at 37C. Cells growing in the Defb1 log phase and free from mycoplasma was used in this study. Cytotoxicity assay K562 cells were cultured at a density of 6104 cells/well in 96-well plates overnight.

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