Preparation Of PEG-CS/DNA-NC By Optimizing Volume Ratio And Transfection To Rat Aortic Smooth Muscle Cells

Objective:①To prepare PEGylated Chitosan Nanoparticles(PEG-CS/NPs) by modifing chitosan with PEG as gene vector and loading it with pACE-shRNA, synthesize PEGylated Chitosan/DNA Nanoparticle Complex (PEG-CS/DNA-NC) and investigate its biological characteristics.②By optimizing transfection condition to observe the transfection efficiency of the PEG-CS/DNA -NC in Rat Aortic Smooth Muscle Cells (RASMC) in Vitro and evaluate the expression level of ACE mRNA before and after transfection.Methods:1. Preparation of PEG-CS/NPs: The chitosan nanoparticles were prepared with ion exchange method and modified by PEG, then its diameter, shape and distribution were measured by scanning electron microscope.2. Construction of eukaryotic expression vector for shRNA targeting ACE gene: Firstly, RNAi site targeting rat ACE gene in GenBank was selected according to the guidelines for the selection of highly effective siRNA sequence and then BLAST test was performed. Secondly, the oligonuleotide fragment was synthesized and annealed to double strands. Thirdly, the double strands were cloned into plasmid vector pGenesil-1. Finally, the recombinant plasmids were identified by restriction enzyme digestion and assayed sequence of them. The recombinant plasmid was named pACE-shRNA.3. Preparation and Characterization of PEG-CS/DNA-NC:①The combinations of CS/NPs with pACE-shRNA and PEG-CS/NPs with pACE-shRNA ( concentration: 0.5ug/ul ) were at different volume ratios (10:10,10:20,10:30,10:40,10:50; unit: ul ). The binding ability was evaluated by agarose gel electrophoresis analysis, the capsulating rate was determined by fluorospectrophotometr and the optimal ratio of pACE-shRNA to PEG-CS/NPs was selected.②Diameter, shape and distribution of the CS/DNA-NC and PEG-CS/DNA-NC were measured by scanning electron microscope.③The complex in the optimal ratio of pACE-shRNA to PEG-CS/NPs and pDNA were digested by DnaseⅠ. The effect of PEG-CS/NPs protecting pDNA was evaluated by agarose gel electrophoresis analysis.④The releasing of DNA from the complex was observed in phosphate buffered solution at four different pH and the releasing rate were determined by fluorospectrophotometr.4. Optimization of the transfection condition of tranfecting PEG-CS/DNA-NC to RASMC:①The rat RASMC were obtained from thoracic aortas and cultured by tissue explant method. The morphology of RASMC was studied by phase contramicroscope. Its molecular markers were observed by alpha-actin factor immunocytochemistry . Passage 3～4 cells were used in experiment.②RASMC were respectively transfected with the optimal ratio CS/DNA-NC and PEG-CS/DNA-NC of different amount (60ul,80ul,100ul,120ul ). Transfection efficiency was determined by using fluorescence microscope and cell viability was calculated by using MTT assay after 24h,48h,72h of transfection.5. Study on the inhibition of ACE mRNA expression in RASMC: RASMC were transfected with PEG-CS/DNA-NC of the optimal ratio and optimal amount. Total RNA were extracted from RASMC before and after 24h,48h,72h of transfection. The expression level of ACE mRNA was evaluated by RT-PCR. RASMC were divided into three group:①blank control group,②plasmid control group,③PEG-CS/DNA-NC group.Results:1. CS-NPs were found to be evenly distributed as spherical particles by scanning electron microscope with the diamete mean of about 5 nm. Its diameter,shape and distribution were not found apparent change after binding with PEG.2. Restriction enzyme digestion analysis showed that eukaryotic expression vector of shRNA targeting ACE gene had been constructed successfully. Sequencing results revealed that the sequence of the vector was right.3.①Agarose gel electrophoresis showed that CS/NPs and PEG-CS/NPs could combine with pDNA effectively. The optimal encapsulation efficiency of CS/NPs with pACE-shRNA combinded at the ratio of 10:10 was 96.8%, and the PEG-CS/NPs with pACE-shRNA combinded at the same ratio was 98.7% by fluorospectrophotometr.②CS/DNA-NC and PEG-CS/DNA-NC were found to be evenly distributed as spherical particles by scanning electron microscope with the diamete mean of about 80nm and 100 nm respectively.③Agarose gel electrophoresis showed that PEG-CS/NPs could protect pDNA from degradating by 0.1u DnaseⅠeffectively, while pDNA were digested completely.④The PEG-CS/DNA-NC being stable in vitro and the gradual release 100h of DNA was confirmed in PBS with pH<7.5 by fluorospectrophotometr. The releasing curve rised linearly within 50h and then gradually stable.4.①The passaged RASMC developed typically"peak and valley"growth pattern. Most cells displayed green fluorescence under fluorescent microscopy, which accords with the characteristics of RASMC.②According to transfection efficiency and cell viability, the optimal ratio of pACE-shRNA to PEG-CS/NPs was 10:10 and the optimal complex amount was 100ul. At this transfection condition, transfection efficiency was 55.2% and cell viability was 90.3% after 72h of transfection. 5. Compared with control group, the expression level of ACE mRNA of PEG-CS/DNA-NC 100ul was significantly suppressed after 48h of transfection (p<0.05) and was lower 72h later (p<0.05) . There was no significant difference about expression level of ACE mRNA in blank control group and plasmid control group before and after transfection.Conclusion:1. Construct successfully PEG-CS/NPs evenly distributed as spherical particles.2. Construct successfully eukaryotic expression vector pACE-shRNA.3. PEG-CS/NPs could combine with pDNA effectively. The diamete mean of CS/DNA-NC and PEG-CS/DNA-NC were about 80nm and 100 nm respectively and meet the request of transfection. The complex can protect itself from degradating by nuclease. The gradual release 100h of DNA from the complex was confirmed in PBS with pH<7.5.4. By explant technique, we successfully accomplished the isolation,primary culture and passaged culture of RASMC. The cells purity was above 90%. The optimal transfection efficiency could be obtained by optimizing the parameters: the ratio of pACE-shRNA to PEG-CS/NPs was 10:10 and the complex amount was 100ul, at the same time, with high cells viability.5. Transfection of pACE-shRNA to rat aortic smooth muscle cells coule initiate sequence-specific post-transcriptional gene silencing and inhibit the expression of ACE mRNA.