| Small interfering RNA (shRNA) has promisingapproach as a therapeutic agent for Castration-Resistant prostate cancer (PCa). As a safety and efficacy of new therapeutic delivery vehicles, nanotechnology provide prospect for the clinical applications of shRNA. In this study, we describe a novelnanoparticles (mPEG-PEI) as an efficient non-viral carrier and found these copolymers displayed enhanced efficiency in shRNA-mediated knockdown of target genes.Previous studies has indicated that enhancer of zeste homolog 2 (EZH2) is often elevated in Castration-Resistant PCa and has been implicated in human PCa progression. Targeting EZH2 may have therapeutic efficacy for treating metastatic, hormone-refractory prostate cancer. mPEG-PEI binds plasmid DNA yielding nanoparticles and this complexes exhibits low cytotoxicity and high gene transfection efficiency. Taken together, mPEG-PEI would be a promising nonviral gene carrier for delivering EZH2 shRNA to PC3 cells for advanced prostate cancer therapy.Methods:1. Synthesised mPEG-PEI copolymer and exosyndromed it.2. Measurement of particle size and zeta potential of mPEG-PEI/pDNA complexes,Attachment of mPEG-PEI/pDNA complexes was assessed using Gel retardation assay, MTT assay and Hemagglutination assay for the The cytotoxicity and the efficiency of transfection,studied the N/P of the best efficiency and the character of mPEG-PEI.3. In vitro transfection experiments. Real-Time qPCR and Western Blot Analysis were tested to determine the expression of EZH2 mRNA.Tested the gene silencing effect of EZH2-shRNA.Results:1. We successfully synthesised and exosyndromed mPEG-PEI copolymer.2. mPEG-PEI could completely retard the migration of DNA when the N/P ratio was 10,and the ability of mPEG-PEI to bind DNA inhanced with increasing ratio of N/P.3. Cytotoxicity was different at different concentrations of the mPEG-PEI nanoparticles and different N/P ratios. PC3 cell viability displayed a decreasing trend with increasing mPEG-PEI polymer concentration, when mPEG-PEI nanoparticles concentrations was upper 50 μg/ml it showed a significant increase in toxicity,with the cells death rate of upper 50%. The cytotoxicity of the mPEG-PEI/pDNA complexes increased as the N/P ratio increased. mPEG-PEI/pDNA complexes showed very small or negligible cytotoxicity when N/P ratio were below 50. Cell viabilities were 94.13±3.42% and 78.6±3.94% at N/P ratio were 3 and 20, respectively. However, the cytotoxicity of mPEG-PEI/pDNA increased drastically at N/P ratio over 50. The agglutinating activity was different of at different N/P ratios.4. The gene transfection efficiency correspondingly increased with increased N/P ratio from 3 to 20, and the transfection efficiencies increased from 12.10±1.61% to 64.67±3.92%. The highest transfection activity of mPEG-PEI/pDNA was obtained at the N/P ratio of 20 in PC3 cells.5. mPEG-PEI/pDNA at N/P ratio 20 was selected for transfection of PC3 cells. EZH2 gene expression was measured by real-time RCR as well as western blot analysis. The result demonstrates that EZH2 mRNA expression decreased after transfection of mPEG-PEI/pDNA particles to PC3 cells.and EZH2 shRNA has gene silencing effect.Conclusions:1. As a non-viral carrier, mPEG-PEI showed high efficient of transfection and low cytotoxicity. The transfection efficiency of mPEG-PEI/pDNA related with different concentrations and N/P ratio of mPEG-PEI/pDNA, and cytotoxicity was different with different N/P ratio.2. EZH2 mRNA expression decreased after transfection of EZH2 shRNA with mPEG-PEI particles to PC3 cells,and which could be applied to treatment of PCa... |
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