Design And Evaluation Of Lipoplexes Modified By D-α-Tocopheryl Poly (2-Ethyl-2-Oxazoline) Succinate For SiRNA Delivery

In this paper,to obtain p H-sensitive small interfering RNA(siRNA)nano preparation,D-α-tocopheryl poly(2-ethyl-2-oxazoline)succinate(TPOS)was modified on the surface of siRNA lipoplexes which was constructed by 3β-[N-(N’,N’-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride(DC-Chol).The fluorescence spectrophotometry for the determination of FAM-siRNA was established.Blank cationic liposomes(CLs)was prepared by the thin film dispersion method,CLs/siRNA was prepared by mixing CLs and siRNA directly.Polyethylene glycol-distearoyl phosphatidyl ethanolamine(PEG-DSPE)or TPOS was modified on the surface of CLs/siRNA by the post-insertion method to obtain PEGylated CLs/siRNA(PEG-L/siRNA)and TPOSylated CLs/siRNA(TPOS-L/siRNA).The encapsulation efficiency of CLs/siRNA was(86.68±1.41)%,the modification of PEG and TPOS had no significant effect on the encapsulation efficiency of CLs/siRNA.The lipid bilayer structure of the lipoplexes was observed by transmission electron microscopy,and the particle size of the lipoplexes was less than 200 nm.The transmittance of PEG-L/siRNA and TPOS-L/siRNA were remained above 80%compared with blank CLs and CLs/siRNA when the preparations were incubated with 10%(v/v)fetal bovine serum(FBS)for 24 h,which illustrated that PEG-DSPE or TPOS endowed lipoplexes with good stability.CLs/siRNA,PEG-L/siRNA and TPOS-L/siRNA were diluted in different times in PBS still had good stability.The results of agarose gel electrophoresis test showed that the combination of DC-Chol and siRNA had strong stability when the weight ratio of DC-Chol and siRNA was not less than 5:1.And the binding stability of DC-Chol and siRNA in TPOS-L/siRNA was still better than that of PEG-L/siRNA when the weight ratio of DC-Chol to siRNA was 5:1.The results of acid-base test showed that TPOS had a good buffer capacity of H~+in the simulation of tumor microenvironment.The results of in vitro release test showed that the accumulative drug release rates of TPOS-L/siRNA was significantly increased at 24 h compared with CLs/siRNA when the p H was decreased from 7.4 to 6.4,which indicated that TPOS could endow good p H-sensitive properties with lipoplexes.The observation of inverted fluorescence microscope showed that TPOS-L/siRNA could significantly enhance the green fluorescence intensity compared with CLs/siRNA and PEG-L/siRNA when the p H was decreased from 7.4 to 6.4.The results of cell inhibition test showed that blank TPOSylated CLs(TPOS-L)had the highest survival rate compared with blank CLs and PEGylated CLs(PEG-L),indicating that TPOS could be constructed for subsequent drug delivery systems to provide safety because of its good biocompatibility.