Afterwards, cells received the same wash ing process and fixed in 0. 5 mL of 4% paraformaldehyde. The EGFR numbers were Y-27632 129830-38-2 analyzed on a Becton Dickinson FACScan flow cytometry with excitation and emission set tings of 488 nm and 575 nm, respectively. To determine the change of EGFR numbers in SPC A1 cells, we used positive cell percentage �� mean intensity to evaluate the intensity of fluorescence. For specificity evaluation, non specific siRNA NEG was complexed by LPEI at corresponding N P ratios and thereafter transfered into SPC A1 cells. Lipofectamine 2000 complexed siRNA EGFR transfecting group was set as a positive control. The non treatment group was taken as a blank control. EGFR mRNA or protein expression levels were normalized by setting the amount of blank control at 1. 0.

Size and zeta potential measurements Dynamic light scattering was employed to deter mine hydrodynamic diameters of the complexes, and laser Doppler Inhibitors,Modulators,Libraries anemometry was utilized to meas ure their zeta potentials. LPEI complexed Inhibitors,Modulators,Libraries siRNA nano plex was prepared as above. Plasmid DNA provided by pSilencerTM 2. 1 U6 neo kit was complexed with LPEI at the same N P ratio of 5 as the manufacturers instructions. When the LPEI siRNA and LPEI DNA complexes were formed, their mean particle size and zeta potential distribution were measured using a Zeta potential particle Sizer NicompTM 380 ZLS. Position and attenuator were optimized by the de vice. Measurements were conducted in quadruplicates. Inhibitors,Modulators,Libraries LPEI siRNA complexes transfection in vivo by intraperitoneal injection SPC A1 cell suspension was prepared and inoculated subcutaneously into flanks of nude mice.

As grown up Inhibitors,Modulators,Libraries tumors were removed and divided into uniform masses of 1 mm �� 1 mm, and implanted subcutaneously into the right flank of the untreated mice. When tumors reached 6 mm �� 6 mm, the SPC A1 xenografted mice model was success Inhibitors,Modulators,Libraries fully established and i. p. treatment started. For i. p. injection, the in vivo jetPEITM transfection reagent, also from Polyplus transfection, was used, which provided as a ready to use solution at 150 mM nitrogen concen tration and less than 0. 1EU mL endotoxin. SiRNA EGFR and LPEI were dissolved in 250ul of 5% glucose solution, respectively. The latter was pipetted into the siRNA solution 10 minutes later. After vortexing and incubating at room temperature for 20 minutes, stable LPEI siRNA EGFR complexes under N P ratio of 5 were formed in 5% GS at a final vol ume of 500ul.

Afterwards, they were intraperitoneally administrated to each mouse in 1h. At the same time, a corresponding dose of LPEI complexed non specific siRNA NEG or 5% GS were given toward to the other two groups as controls. In the experiment of discontinued administration, i. p. injection was started when tumors had reached 6 mm �� 6 mm, and repeated 2 3 times per week until the 22nd day, 24 h after the last injection.