Complete release of DOX from the vesicles at each time point yields 100% dequenching and was obtained from control ethanol-treated liposome samples. The percentage release of DOX from the vesicles was determined from the fluorescence intensity of each sample relative to 100% dequenching, which can then be expressed in terms of percentage of DOX release. 2.5. Cytotoxicity Assay The cytotoxicity of all liposomal systems used in this study, as well as free DOX, on the cells was determined using the CellTiter-Glo Luminescent Cell Viability Assay. Inhibitors,research,lifescience,medical The M14#5,
M14#11, and BJ cells were plated on 96-well tissue cultured treated plates corning at a density of 5 × 103 cells per well and incubated for 24h at 37°C and 5% CO2. The culture medium was then replaced with 100μL of medium containing various concentrations of each liposomal system or free DOX. The cells were then exposed to Inhibitors,research,lifescience,medical the drug for 3h; the cells were washed twice with sterile PBS following drug exposure. Fresh culture medium was then added, and the incubation was continued for 24h. After the incubation period, 100μL CellTiter Glo reagent was added to each well. The cells were allowed to incubate for an additional 3h at 37°C and 5% CO2. The cytotoxicity assays were done in triplicate and were repeated at least twice in separate selleck chem experiments. 2.6. Tumor Growth In Vivo B16F10 murine Inhibitors,research,lifescience,medical melanoma cells
were prepared at the Washington University . C57BL/6 mice were obtained from the Harlan selleck catalog Laboratories (Indianapolis, IN). Mice were housed under pathogen-free conditions according to the guidelines of the Division of Comparative Medicine, Washington University School of Medicine. The Washington University Animal Studies Committee Inhibitors,research,lifescience,medical approved all experiments. Tumor cells (105 cells/100μL in PBS) were injected subcutaneously in the neck of C57BL/6 anesthetized mice and allowed to grow 7–14d until tumors were ~5 × 5mm. Eight mice per treatment group were inoculated with 105 tumor cells. The number of animals Inhibitors,research,lifescience,medical tested (n) was calculated by power analysis (probability
of type I error α = 0.05; probability of type II error β = 0.20) based on previous data. This was Batimastat the minimum number of animals required to achieve statistical significance. Mice inoculated with tumor cells were divided into a control (saline treated) as well as groups treated with the various DOX-loaded liposomes at doses (5mg/kg with an average mouse weighing ~20g) corresponding to those used previously for DOX-loaded liposomes in melanoma mouse models . Liposomes or saline was injected on days 0, 3, 5, 6, and 8, with day 0 being the first day of the regimen and all animals dosed on the same days. The experiment was terminated at 11d after initiation of treatment regimen. Mice were anesthetized by isoflurane (2% vaporized in O2).