Signals were then converted into HRP using antifluorescein selleck kinase inhibitor anti body and visualized by 3,30diaminobenzidine coloration as recommended by the manufacturer. Tissues were counter stained with methylgreen and TUNEL positive cells were counted in five randomly selected �� 200 high power fields under microscopy. The apoptosis index was calculated according to the following Inhibitors,Modulators,Libraries formula, number of apoptotic cells total number of nucleated cells �� 100%. Blood biochemical analysis At the endpoint of the above experiment, all blood sam ples were collected for toxicity testing. By retro orbital puncture, blood was collected into heparinized tubes. Within 1 h, samples were centrifuged at 5000 �� g for 10 min. The plasma was taken for biochemical parameters analysis, carried out with an automatic analyzer.

Liver enzymes, including aspartate amino transferase, and alanine aminotransferase, were measured. Urea and creatinine were also mea sured for renal function evaluation. Immunogenicitiy assay SiRNA EGFR or non specific siRNA NEG, both complexed with LPEI in 5% glucose solution, were administrated by i. p. injection to 8 week old Inhibitors,Modulators,Libraries male BALB C nude or immunocompetent Inhibitors,Modulators,Libraries mouse, and 500 ul of 5% glucose solution was Inhibitors,Modulators,Libraries given as a negative control. After 2 days, the injection was repeated at 6 h before the end of the experiment. As a positive control based on a previous study, 40 ug of lipopolysaccharide in 500 ul of phosphate buffered saline was intraperitoneally administrated to immuno competent mouse and the experiment terminated after 2 h. Mice blood was taken by retro orbital puncture and allowed to clot overnight at 4 C.

Samples were centri fuged at 5000 �� g for 10 min, and the supernatants col lected. Serum concentrations of TNF and IFN were determined by enzyme linked immunosorbent assay kits according to manufacturers Inhibitors,Modulators,Libraries instructions. The amount of cytokine was determined on 100 ul of �� 5 diluted serum, loaded in duplicate. Statistical analysis Data were presented as the means standard deviation. Statistical differences among multiple groups were calcu lated by one way analysis of variance. If a ANOVA was statistically significant, an unpaired two tailed Students t test was used for between group com parisons. P values of less than 0. 05 were considered statistically significant. Results In vitro optimizing of LPEI siRNA EGFR complexes We first performed an in vitro experiment to test whether LPEI could deliver unmodified siRNA efficiently into human lung cancer cells and exert a specific selleckchem gene silencing effect. The N P ratio, which indicates quotient of the nitrogen atoms of LPEI to siRNA phosphates in the complexes, will determine particle size and zeta potential, thus influencing the efficiency of siRNA deliv ery.