Study on the Improvement of Transfection Efficiency of Antisense Oligonucleotides and Its in Vivo Anti-tumor Effect by Reduction Responsive Micelle Based on Polyethyleneimine
OBJECTIVE To synthesize a polymer PEI-ss-PEG2000-DSPE containing disulfide bond and to prepare as cationic micelle(P-ss-PD)based on branched polyethyleneimine(PEI).To investigate the ability of P-ss-PD micelle to reduce cytotoxicity and improve the transfection efficiency of antisense oligonucleotide(ASO)in human breast cancer cell lines,and to study the anti-tumor effect of P-ss-PD micelle in nude mice.METHODS PEI-ss-PEG2000-DSPE was synthesized by grafting PEG2000-DSPE onto branched PEI with disulfide bond as a connecting arm.P-ss-PD micelle was prepared by ethanol injection method and P-ss-PD/ASO nanocomplex was obtained by combining P-ss-PD micelle with ASO.The particle size and zeta potential of P-ss-PD/ASO nanocomplex at various mass ratios were determined by laser particle size analyzer.Agarose gel retardation assay was used to investigate the binding degree of P-ss-PD/ASO nanocomplex and determine the optimal mass ratio.At the same time,the reduction responsive ability of P-ss-PD micelle was investigated.The cytotoxicity of P-ss-PD micelle was detected by CCK8 kit.The transfection efficiency of P-ss-PD micelle was investigated by flow cytometry and high content cell imaging analysis system in MDA-MB-231 cells.The anti-tumor effect of P-ss-PD micelle was investigated by tumor-bearing nude mice models.RESULTS When the mass ratio was 300∶1,the particle size of P-ss-PD/ASO nanocomplex was the smallest and had a good stability.The average particle size was(58.90±4.08)nm,the average zeta potential was(16.80±1.23)mV,and the morphology was uniform spherical.P-ss-PD/ASO nanocomplex had the reduction responsive ability and could release ASO under highly reductive conditions.In vitro,compared with unmodified branched PEI,the cytotoxicity of P-ss-PD micelle was significantly reduced and the transfection efficiency was significantly increased.In vivo,the tumor growth inhibition rate of P-ss-PD/ASO nanocomplex in tumor-bearing nude mice was more than 50％.CONCLUSION The P-ss-PD micelle prepared in this study is a kind of low toxicity and high transfection efficiency non-viral vector,which has the characteristics of reduction responsive releasing,and shows a promising application in ASO drug delivery.
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