Construction And Evaluation Of Environmental Sensitive Nano-lipid Carriers

In recent years,with the rapid development of nanotechnology,environmental sensitive nanocarriers have attracted attention.In biological systems,physiological and biochemical differences between pathological tissues and normal tissues are triggers for drug release.On the other hand,external stimuli,such as light,magnetic field and ultrasound can induce the release of drug in a specific location.Compared with traditional nanocarriers,environmental sensitive nanocarrers have the characteristics of precise release and well controllability.Warburg effect is that tumor cells proliferate rapidly and local hypoxia occurs,tumor tissues produce acidic metabolites,the tumor microenvironment is weakly acidic.Using this characteristic,a pH sensitive liposome was designed to release drugs in tumor tissues to achieve the active target.In this study,siRNA and DTX were used as antitumor agents,and the liposome modified by pH sensitive peptide was used as drug carriers to realize the effective uptake of siRNA and DTX.In the end,a new type of acid sensitive functional peptide was synthesized,which was used for the construction of subsequent targeted vectors.Part one Preparation and characterization of environmental sensitive liposomesObjective:The liposomes were modified with low pH insertion peptides（pHLIPs）to prepare p H-Lip/DiD/si and DTX-PL.Methods:Preparation and characterization of pH-Lip/DiD/si:Liposome was prepared using film dispersion method.The pH low insertion peptides（pHLIPs）was modified by the post insertion method on the liposome surface.The particle size and PDI were measured.Preparation and characterization of DTX-PL:Liposome was prepared using film dispersion method.pHLIPs was modified by the post insertion method on the liposome surface.The particle size and PDI were measuredResults:The particle size of pH-Lip/Di D/si were around 160 nm and PDI≤0.3.The particle size of DTX-PL were within 200 nm and PDI≤0.4.Conclusion:pH-Lip/DiD/si and DTX-PL were in line with the requirements of the particle and PDI,and particle size distribution were concentrated.Part two In vitro cellular uptake studyObjective:MCF-7 cells and Hela cells were selected as models to investigate the uptake and distribution of p H-Lip/DiD/si in cells.Methods:pH-Lip/DiD/si was incubated with MCF-7 and Hela cells,where siRNA has green fluorescence,DiD has red fluorescence,Hoechst33258 was stained with the nucleus.The distribution of pH-Lip/Di D/si in cells was imaged using laser scanning confocal microscope.Results:The experiment of cell uptake and distribution showed that pH-Lip/DiD/si was able to penetrate into the cell,and siRNA,DiD were located in the cytoplasm.Conclusion:p H-Lip/DiD/si were able to allow siRNA and Di D cytoplasmic release.Part three In vitro tumor spheroids antitumor efficacy studyObjective:MCF-7 tumor spheroids and Hela tumor spheroids were selected as the model to investigate the antitumor efficacy of docetaxel loaded liposomes on tumor spheroids.Methods:Tumor spheroids were constructed by hanging-drop method,docetaxel loaded liposomes were incubated with tumor spheroids for 0⁵ days,and the size of tumor spheroids was measured every day,and the tumor spheroids volume was calculated.The inhibition effect of different liposomes on tumor spheroids was investigated.Results:Toxicity test of tumor spheroids showed that docetaxel loaded liposomes can effectively inhibit the growth of tumor spheroids.Docetaxel loaded liposomes with pHLIPs presents the strongest antitumor efficacy.Conclusion:The nano-lipid carrier with p HLIPs had significant tumor microacid environment sensitivity,which can realize the effective transfection of docetaxel and inhibit tumor growth.Part four Study on acid sensitive functional peptide.Objective:By using the reaction of Schiff’s base,the NP peptide was coupled with the PP peptide,synthesizing the acid sensitive peptide NP-PP.Methods:On the basis of literatures and several preliminary experiments,several synthesis methods of NP-PP were determined.Under the condition of weak acid and 30oC water bath,the reaction system was stirred for 32 h with N₂,The synthetic products were characterized by MS.Results:In view of MS analysis,the practical molecular weight of NP-PP was consistent with the theoretical data.Acid sensitive peptide was successfully synthesized.Conclusion:Acid sensitive peptide was successfully synthesized.The method was simple and reproducible.