| 1. Background and objective:An novel PEG-detachable PEI-based polymer, mPEG-SeSe-PEI, has been developed. By taking advantage of higher intracellular glutathione (GSH) levels, the diselenide bond between PEG and PEI can be easily cleaved in the cytoplasm to facilitate the release of DNA. The novel redox-responsive polycation with minimized cytotoxicity allows easier endosomal escape and excellent transfection efiBcacy for delivering pDNA in HepG2 cells. We intended to evaluate the properties and efficiency of mPEG-SeSe-PEI to deliver siRNA and apply it for our subsequent research.2. Experimental methods:2.1 The mPEG-SeSe-PEI and PEG11.8-PEI copolymers were synthesized, and different siGADPH complexes were prepared according to protocol.2.2 The agarose gel retardation assay was conducted to determine the ability of mPEG-SeSe-PEI, PEG11.8 s-PEI and PEI to condense siGADPH molecules.2.3 The transfection efficiency of different vectors were evaluated using FCM.2.4 The particle sizes and Zeta-potentials of complexes dissolved in ddH2O at different N/P ratios were determined by DLS analysis with a Zetasizer nanoparticle analyzer.2.5 The cytotoxicity of PEG11.6-SeSe-PEI, PEG11.8-PEI, PEI and LiPo2000 in PANC-1 cells was assessed in the MTT assay.2.6 The internalization efficiency of different polyplexes in PANC-1 cells was confirmed with CLSM.2.7 The RT-PCR assay was conducted to investigate the effects of different siGADPH complexes on GADPH mRNA levels in PANC-1 cells.3. Results:3.1 The mPEG-SeSe-PEI and PEG11.8-PEI copolymers were formulated, The different siGADPH complexes at various N/P ratios were then synthesized.3.2 The siGADPH could be completely condensed by PEI at the N/P ratio of 5, while PEG11.8-PEI, PEG5-SeSe-PEI, PEG6.9-SeSe-PEI and PEG11.6-SeSe-PEI condensed all the siGADPH at the N/P ratio of 10,8,8 and 10, respectively.3.3The maximum transfection efficiencies of PEI/siRNA(N/P=20), PEG5-SeSe-PEI/siRNA(N/P=5), PEG69-SeSe-PEI/siRNA (N/P=15) PEG11.6-SeSe-PEI/siRNA (N/P=10) and PEG11.8-PEI (N/P=10) were 54.1%,43.6%, 59.8%,73.8% and 75.7%, respectively.3.4 The sizes and Zeta-potentials of siGADPH complexes were successfully detected.3.5 Cell viability decreased dramatically when the N/P ratio increased and the toxicity of the complexes increased in an N/P ratio-dependent manner. The survival rate of cells treated with Lipo2000/siRNA was lower then that of cells exposed to PEGn 6-SeSe-PEI/siRNA (N/P=10).3.6 SiRNA accumulation within the cytoplasm of cells incubated with Lipo2000/siRNA, PEG11.6 6-SeSe-PEI/siRNA and PEG11.8-PEI/siRNA complexes was markedly greater than that in cells transfected with PEI/siRNA.3.7 The degree of inhibition induced by PEG11.6-SeSe-PEI/siGADPH was higher than that induced by other complexes.4. Conclusions:4.1 PEG11.6-SeSe-PEI can completely encapsulate the siRNA at N/P ratios greater than 10 and condense siRNA into small spherical nanoparticles with good distribution.4.2 PEG11.6-SeSe-PEI showed reduced cytotoxicity, excellent cell internalization efficiency and ability to mediate significant gene suppression in PANC-1 cells even in the presence of serum protein.4.3 Therefore, PEG11.6-SeSe-PEI is a superior siRNA carrier compared with PEI, PEG11.8-PEI or Lipo2000 and holds tremendous potential for clinical therapy applications. |
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