The anti-cancer effect in a model of non-small cell lung cancer. A detailed analysis of the plasma and the elimination kinetics of caspase organs was carried out to study the in vivo behavior. Second Materials and methods 2.1. Materials PGA sodium salt, D penicillamine trisphosphine, a 3-ethylcarbodiimide hydrochloride, glutathione and N hydroxysuccinimide were purchased from Sigma Aldrich. Lyzer dialysis cassette Slide a, 4 maleimidophenylbutyric hydrochloride S Urehydrazid Sepharose 4B Cl, buffers and all L Solvents were purchased from Thermo Fisher Scientific. Ida hydrochloride was purchased from Pharma Synbias. 2.2. Synthesis of IDA and IDA MPBH MPBH hydrochloride were dissolved in anhydrous methanol St. A few drops of trifluoroacetic Acid was added and the reaction was for 96 h at room temperature under an argon Re stirred. The mixture was concentrated to 2 ml and centrifuged in anhydrous ethyl acetate and. The precipitate was repeated three times and the product was dried under vacuum. 1H NMR: 1.17, 1.93, 2.03, 2.32, 2.65, 3.0 3.12, 3.52, 6.96, 7.21, 7.89, 8, 29, m / e: 753.37. 2.3. Synthesis of 2 DDPC ethylamine was synthesized as previously described. PGA-sodium was dissolved in 5 ml of cold deionized water St. 1 N HCl was added dropwise and the precipitate was washed with water until the pH was 5.0. The precipitate was lyophilized on a VirTis Advantage lyophilizer. Lyophilized PGA was dissolved in anhydrous dimethylformamide St. Then, EDC, NHS and PDE were added. The mixture was 12 h at room temperature under an argon Stirred re. The PDE-PGA conjugate was dissolved in ether and then Executed careful washing with water Filled. The conjugate was dissolved in 0.5 M sodium bicarbonate St and the pH was adjusted to 7.4 with 0.1 M PBS. The degree of conjugation was thione spectrophotometrically by measuring the absorbance of the two pyridine in the reduction in the presence of 25 mM TCEP is released for 1 h. Pen D was added and the mixture was stirred for 12 h at room temperature.
The PGA-pin D-conjugate was dialyzed against 10 mM PBS for 48 h using a Slide A Lyzer dialysis cassette. The extent Pen D of conjugation was determined as described by HPLC as above. To synthesize CSD pin D-PGA conjugate was reduced in the presence of TCEP for 5 min. TCEP was removed by centrifugation with Amicon Ultra-15 centrifugal filter and reacted with Ida MPBH 12 h at room temperature. The CSD was by filtration through an S Column of Sepharose CL 4B using PBS pH 7.4 as eluent, lyophilized, and at 20 until use. 1H NMR: 1.23 1.43, 1.83 2.38, 3.31 3.49, 3.56 3.67, 7.86 8.26. The molecular weight of PGA and the viscosity t by multi-angle laser light scattering, as measured by the manufacturer, was 50 to 70 kDa. We Hedgehog Pathway determined the molecular weight before and after conjugation with dynamic light scattering using a Nano ZS Zetasizer. Toluene was used as a broadcast standard. The increment of the refractive index of 0.176 was used to separate ten standards in the range of the concentration of 5 mg / ml to 0.05 mg / ml carried out. CDD concentration within the standard curve was analyzed to obtain the molecular weight after conjugation. The average molecular weight of from DLS of CSD found 688.9 kDa was determined prior to conjugation.