CellTiter-Glo Luminescent Cell Viability Assay kit (Gly-Phe-AFC) (Cat# AFC033) was purchased from Promega Corporation or MP Biomedicals. The appropriately protected amino acids, O-(1H-6-chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate

(HCTU) (Cat# 851012) and NovaPEG rink amide resin (Cat# 855047) were all obtained from EMD Biosciences. The preparation, purification, and characterization of the α1(IV)1263–1277 THP [(Gly-Pro-Hyp)4-Gly-Val-Lys-Gly-Asp-Lys-Gly-Asn-Pro-Gly-Trp-Pro-Gly-Ala-Pro-(Gly-Pro-Hyp)4-NH2] PA possessing a C16 tail have been described previously Inhibitors,research,lifescience,medical [48]. 2.2. Cell Culture Conditions The M14#5 and M14#11 human metastatic melanoma cell lines were generously provided by Dr. Barbara Mueller. Inhibitors,research,lifescience,medical The BJ foreskin fibroblasts from a melanoma patient were obtained from the American Type Culture Collection (ATCC) (Cat# CRL-2522). Cell media (Cat# MT10-013-CV) and trypan blue (Cat# ICN1691049) were obtained from Fisher Scientific or CellGro, and all reagents required for cell culture Inhibitors,research,lifescience,medical were purchased from Invitrogen. Cells were maintained in DMEM supplemented with 10% fetal bovine serum (Cat# 10437028), 50 units/mL penicillin, and 0.05mg/mL streptomycin

(Cat# 15140163). Cells were cultured with selleck chem Gemcitabine complete medium at 37°C in a humidified atmosphere of 5% CO2 in air. For all experiments cells were harvested Inhibitors,research,lifescience,medical from subconfluent (<80%) cultures using a trypsin-EDTA (Cat# 15400054) solution and then resuspended in fresh medium. Preparations of cells with a >90% viability, as determined by trypan blue exclusion, were used. 2.3. Preparation of DOX-Loaded Liposomes The phospholipids and cholesterol were combined

in fixed ratios Inhibitors,research,lifescience,medical (Table 1) and dissolved in an organic phase mixture of methanol, methyl tert-butyl ether, and chloroform (1:2:2.4) Cilengitide by vortexing for 0.5h at room temperature. At this stage, if PA-targeted liposomes were the desired product (Table 1), the α1(IV)1263–1277PA was added to the lipid organic phase mixture. The organic phase was then removed under reduced pressure by rotary evaporation, leaving a thin lipid film at the bottom of the flask which was dried overnight in vacuo. The phospholipid film was then rehydrated in ammonium sulfate (125mM), and the resulting dispersion was vortexed extensively. The dispersion was then stirred for 30min at 60°C. The maintenance of this temperature for a sustained time was necessary as the lipid tails were mobilized and thus allowed the aqueous medium to traverse the lipid bilayers.