The Preparation And In Vitro Study Of PH-sensitive Liposome

Objective:Since the tumor extracellular environment are slightly acidic, a nano device was developed which is p H-sensitive to selectively deliver the nucleic acid si RNA into tumor tissue,cell or even organelles. In order to target the acidic environment of the tumor cell membrane and help endosomal escape DSPE-PEG-p HLIP was synthesized and arginine octamer positively charged can largely improve si RNA entrapment. In this study, a cationic polymer SA-R8 was synthesized by using the arginine octamer and stearic acid, electrostatic interaction between multiple sites of arginine octamer and si RNA helps to form SA-R8/si RNA complexes. Liposomes were prepared by film dispersion method and modified by adding the condensed material and DSPE-PEG-p HLIP followed by incubation finally obtained the p H-sensitive liposome formulations. The loading efficiency of cationic SA-R8/si RNA complexes was evaluated by agarose gel electrop Horesis and its various nano characterizations were showed by Malvern particle size analyzer; The human breast cancer cells(MCF-7) were used as the cell model and quantified the uptake rate at p H6.5 and p H7.4 environment respectively by flow cytometry and confocal laser scanning microscope; At the same time, in order to monitor the distribution and location of the multifunctional liposome, confocal laser scanning microscope was used. Thereby a more profound study and discussion of the vitro activity were showed.Methods:According to the literature and preliminary experiment, a method to synthesize cationic si RNA condenser SA-R8 was constructed. At room temperature, after stirring 24 h in the dark with N2, the target product was identified by MS.The reaction of DSPE-PEG-p HLIP was completed by stirring 48 h at room temperature in the dark with N2 and the target product was verified by MS. By the way the residual sulfhydryl content of DMF,DMSO,HBS three different solvents was measured.Through referring to the relevant literature, the liposome with film dispersion method was prepared, using si RNA as model drug, the average particle sizes and the result of agarose gel electrophoresis test were conducted as reference index to obtain optimum formulation. What’s more, the appearance and particle size can be considered as the index of the stability of liposome.The human breast cancer cells(MCF-7) were picked as the cell model. In order to quantitatively evaluate the cellular uptake rate, the percentage of positive cells and the mean fluorescence intensity in cells by flow cytometry were detected. To monitor the distribution and location of the p H-sensitive liposome, confocal laser scanning microscope was used.Results:By MS analysis, the product of SA-R8 and DSPE-PEG-p HLIP were obtained. After freeze drying they are all white flocculent solid; The residual sulfhydryl content of the reaction after 24 h in three different solvent such as DMF, DMSO, HBS were 8.42%, 2.81%, 5.61% respectively, to react continually the residual sulfhydryl content is substantially unchanged; The particle size of the p H-sensitive liposome preparation is uniform and it can exist for a certain time stably.The compressed material SA-R8 contributes to the behavior of si RNA entrapment in the Agarose gel electrophoresis experiments.Compared to the ordinary liposome, the cellular uptake of the modified condensed liposomes were significantly increased and under p H6.5 circumstance the cellular uptake of condensed SA-R8 liposomes was significantly higher than at p H7.4, in other words, it has a special p H responsive. Confocal experiments showed:si RNA with green fluorescence is in cell and can be well located in the cytoplasm.Conclusions:It is confirmed that, when si RNA as model drug, cationic polymer SA-R8 as si RNA carrier, DSPE-PEG-p HLIP modified the liposome surface, with film dispersion method, we prepared liposomes which can exist for a certain time stably.Breast cancer MCF-7 cells were as model cells, p HLIP modified the surface as a leading transmembrane material, at p H6.5, the uptake rate of MCF-7 cells was significantly higher than that unmodified liposomes at p H7.4, The compressed material SA-R8 can improve si RNA entrapment which significantly improves the release of si RNA.