The Construction And Evaluation Of Acid-sensitive Lipid Carriers That Modified By Polyarginine

Objective:In recent years, tumor targeting agents for cancer treatment has become a research focus, this kind of formulation can deliver drugs to the target tissue as much as possible to play a therapeutic role. Liposomes have a structure similar to biological membranes, as a non-viral vector of nano-administered agent, because of its low immunogenicity, low toxicity and targeting, etc, have been widely used as important carrier of tumor targeting agents.However, there are still problems such as reticule endothelial system clears, poor stability,low uptake efficiency, etc. Therefore, designing an improved liposomes which has been modified has significant meaning on biological macromolecules effectively delivered to the target organ.In this study, cell-penetrating peptide CPP and DSPE-PEG are connected with hydrazone, and form a functional complex pleiotropic lipid material DSPE-PEG-Hz-CPP. DSPE-PEG make liposomes have long circulation fuction and CPP can carry the drug liposomes penetrate the cell membrane into the cell, and make si RNA escape from endosomes to play interference gene in the cytoplasm.We constructed three PEG liposomes with different functional lipid material: ordinary drug-loaded liposomes, CPP modified drug-loaded liposomes and containing hydrazone CPP modified drug-loaded liposomes. The three liposomes were characterized use potential and particle size, evaluated and compared the cell uptake efficiency of three liposomes use MCF-7 and A549 cells as model cells.Methods:1 Succinimidyl- on formylbenzoate(SFB) and 3 – maleimidopropionic acid hydrazide(MPH) were dissolved. Purged with nitrogen, stirred at room temperature for 24 h, volatile organic solvent by rotary evaporation, and dried to give a yellow solid S-M.2 Homemade S-M and DSPE-PEG-NH2 were dissolved, purged with nitrogen with stirring at room temperature for 48 h, to give the intermediate DSPE-PEG-Hz.3 5 mg CPP room temperature balance for 30 min, and slowly added to DSPE-PEG-Hz under stirring, purged with nitrogen, dark reaction at room temperature for 48 h, then organic solvent evaporated to dryness by rotary evaporation to acquire yellow solid, the final product are mixture which comprising target product DSPE-PEG-Hz-CPP.4 Constructing drug-loaded liposomes(NL), CPP modified drug-loaded liposomes(CL) and containing hydrazone bond CPP modified drug-loaded liposomes(CHL), and characterization of potential and particle use Malvern particle size analyzer.Operation methods as follows:Respectively using SPC/DC-chol/Chol/DSPE-PEG-NH2,SPC/DC-chol/ Chol/DSPE-PEG-NH2/DSPE-PEG-CPP and SPC/DC-chol/Chol/DSPE-PEGHz-CPP as lipid material to form film,using 2ml DEPC(containing 3.75 nmol si RNA) hand hydration,water bath ultrasonic,then obtained translucent system NL,CL and CHL.5 Analyze MCF-7 and A549 cells which were treated by NL,CL and CHL via flow cytometry and confocal microscopy technology.In terms of flow cytometric analysis intracellular mean fluorescence intensity as index to evaluate the efficiency of cellular uptake.Confocal microscopy to visually display the location of the si RNA in the cells which were delivered into cells by three formulations to evidence flow cytometry results.6 Analyze A549 cells which were treated by CL and NL via confocal microscopy technology,and observe whether si RNA escape from endosomes to play gene silencing effect.Results:1 Synthesis S-M: to obtained yellow solid powder,the M/Z 413.6 in the spectrum is consistent with S-M theory calculated value 413.36(M + H +),proved the product structure is correct.2 Synthesis of DSPE-PEG-Hz: intermediateds average ion peak molecular is 3103.41,which is consistent with the theoretical design peptides average molecular, confirmed that the product has the target intermediate.3 Synthesis of DSPE-PEG-Hz-CPP:the product average ion peak molecular is 4611.08,which is consistent with the theoretical design peptides average molecular,proved synthesis of the target product.But there is a impurity peak average molecular is 2832.19 in the spectrum,which is the raw material of second-recation.Thus, product synthesis methods and purification steps should be improved.4 The potential and particle results of drug-loaded normal liposomes(NL),CPP modified drug-loaded liposomes(CL) and containing hydrazone bond CPP modified drug-loaded liposomes(CHL) as fllows:NL,CL,CHL respectively particle size was 124.9nm,145.6nm,156.1nm, polydispersity index average respectively was 0.302,0.246,0.284.Prepared liposomes are in line with the requirements of the particle size and particle size distribution is concentrated,the system was stabilized.5 By flow cytometry and confocal analysis the cells treated by three groups of preparations.Compared to the negative control group,the intracellular mean fluorescen-ce intensity has greatly improved by common drug-loaded formulation treated.It is show that DSPE-PEG-modified PEG liposomes improves the cellular uptake efficiency.Compared to the NL,CPP modified drug-loaded liposomes(CL) and containing hydrazone bond CPP modified drug-loaded liposomes(CHL) has greatly increased the mean fluorescence intensity.It’s indicated that PEG-modified liposome technology to further enhance the efficiency of cellular uptake.Confocal microscopy results showed that all three formulations successfully transported si RNA to the cytoplasm.CPP-modified liposomes CL and CHL transport si RNA into the cytoplasm of the number(green fluorescence) compared to ordinary liposomes NL increased significantly,and has the corresponding results with flow cytometric analysis.6 By confocal microscopy analysis the A549 cells treated by CL and CHL preparations.The two formulations were successful delivery si RNA into the cytoplasm, and the longer duration of action, the more the number of the si RNA in cytoplasm, The results showed that by CPP modified liposomes effectively promoted si RNA intracellular escape.Conclusion:The experimental results show the synthetic route of DSPE-PEG-Hz-CPP is simple, the synthesis process is not highly toxic reagents and the methods have good reproducible.Successfully prepared containing hydrazone bond CPP modified drug-loaded liposomes(CHL), and compares the efficiency of cellular uptake with the normal drug-loaded liposome(NL) and CPP modified drug-loaded liposomes(CL). All of the three formulations increase the cellular uptake efficiency, and CPP modified drug-loaded liposomes such as CL and CHL have higer cellular uptake efficiency.