Storage stability All lyophilised empty and tyrphostin AG 1478 loaded NLC were stored at 0 C for 3 months in the dark. After this time, samples were dispersed in bidistilled water and characterized in terms of mean size, PDI and zeta potential. Moreover, chemical stability of tyrphostin www.selleckchem.com/products/mek162.html AG 1478 loaded into the NLC was evaluated by HPLC ana lysis, as reported above. Drug release in PBS at pH 7. 4ethanol Tyrphostin release was assayed on NLC samples at pre fixed time intervals. For this purpose, dispersions of each batch containing 5 mg of each freeze dried sample in a mixture of 9. 6 ml of PBS 0. 01 M at pH 7. 4 and 2. Inhibitors,Modulators,Libraries 4 ml of ethanol, were prepared and kept at 370. 1 C under mechanical stirring in a Benchtop 80 C incubator Orbital Shaker model 420.

At scheduled time inter vals, solution aliquots were taken out from the outside of the dialysis membrane and replaced with fresh PBS aqueous ethanolic solution. Release profile was determined by comparing the amount of released tyr phostin AG 1478 as a function of incubation time with the total amount of drug loaded into NLC. Data Inhibitors,Modulators,Libraries were corrected taking in account the dilution procedure. A control experiment to determine the release behavior of the free tyrphostin AG 1478 was also performed. A sus pension of free tyrphostin AG 1478 in PBS aqueous ethanolic solution at pH 7. 4 was prepared at the same concentration of drug entrapped in the NLC, put into a dialysis tube and immersed into the proper medium. The amount of tyrphostin AG 1478 was detected as reported above. Drug release in human plasma Tyrphostin release was assayed on NLC samples at pre fixed time intervals.

For this purpose, dispersions of each batch containing 2. 5 mg of each freeze dried sample in 2 ml of Inhibitors,Modulators,Libraries human plasma were prepared and kept at 370. 1 C under mechanical stirring in a Benchtop 80 C incubator Orbital Shaker model 420. At suitable time intervals, samples were filtered through 0. 45 um nylon filters. then acetonitrile was added and the ob tained blend was centrifuged at 4 C and 12,000 rpm for 15 min. Then the supernatant was filtered by 0. 45 um PTFE filters and analyzed by HPLC. Western blot analysis For Western blot analysis whole cellular lysates were ob tained using RIPA buffer. Protein concentrations of super natants were determined with the Bio Rad protein assay kit, and Western blotting were performed as previously described, with primary antibodies raised against B actin and EGFR.

Cell culture and clonogenic assay The human hepatocellular carcinoma HA22TVGH cell line, a poorly Inhibitors,Modulators,Libraries differentiated human hepatocellular carcin oma cell line established from a surgical specimen of hepatocellular carcinoma Inhibitors,Modulators,Libraries obtained from a 56 year old Chinese male was kindly provided by Professor Massimo Levrero. Cells were cultured in Roswell Park Memorial Institute medium supplemented namely with 10% heat inactivated fetal calf serum.