Study On Cholesterol Exhaustion In Cell Membrane For Overcoming "Lipid Rafts"-related Multidrug Resistance

In order to explore the relationship between the formation of lipid rafts and multidrug resistance,and to solve the problems of damaged membrane endocytosis caused by high-density lipid accumulation in drug-resistant cells,in this study,a double-loaded drug delivery system was constructed,and then its response characteristics,the relationship between lipid rafts and drug resistance and anti-tumor activity were studied systematically.The research contents were as follows:(1)Construction and characterization of nanoparticles Firstly,TPGS2k-NH2 was synthesized by amide reaction of α-VES and PEG2k-diamine,and then TPGS2k was successfully synthesized by FT-IR and 1H NMR.After dehydrochloricating DOX·HCl to DOX,SV solution and DOX solution were mixed and then mixed with PLGA solution,and slowly dropped the mixed solution into the TPGS2k aqueous solution to synthesize the nanoparticles double-loaded DOX and SV(SV/DOX@TPGS2k-PLGA NPs).UV-Vis spectroscopy and HPLC were used to establish in vitro methods for drugs analysis.The particle size potential and morphological characteristics of these nanoparticles were investigated by using dynamic light scattering(DLS)and transmission electron microscope(TEM).The DLS results showed that the particle sizes of SV/DOX@TPGS2k-PLGA NPs were about 150 nm and the potentials were about-23.1 mV,which were uniform and stable.TEM results showed that the nanoparticles were spherical and no obvious agglomeration,indicating that SV/DOX@TPGS2k-PLGA NPs were suitable as a nano delivery system.(2)The study of the characteristics of drug released in vitro The acid-responsive release characteristics of DOX and SV were investigated by UV-Vis spectroscopy and HPLC at different pH in vitro.The results showed that the cumulative release rate of DOX and SV under simulated acidic environments in tumors was 80%and 85%,respectively,which were significantly higher than normal physiological conditions.At the same time,the cumulative release rates of DOX and SV were still increasing after 7 days,indicating that these drugs might still be released continuously,which might be due to the slow-release characteristics of PLGA and TPGS2k improving the stability of the carriers.(3)Antitumor activity,cellular uptake and correlation between lipid rafts and drug resistance in vitro The biological safety of the blank vector and the inhibitory effect of this nanosystem on SW620/AD300 cells were investigated by MTT.Fluorescence microscope and flow cytometry were used to investigate the cell uptake and the drug resistance of SW620/AD300 cells.In addition,cholesterol kit and immunofluorescence were used to verify whether S V/DOX@TPGS2k-PLGANPs could block the formation of lipid rafts by reducing the cholesterol content in lipid rafts.The JC-1 mitochondrial kit and western-blot were used to investigate the effect of the nano-controlled release system on the expression of MDR-related proteins and the function of mitochondrial membrane potential.In vitro cell experiments showed that the blank vector had no obvious toxic,and SV/DOX@TPGS2k-PLGA NPs could significantly inhibit the proliferation of cancer cells.Compared with free DOX,the uptake rate of SV/DOX@TPGS2k-PLGA NPs was significantly increased,which was beneficial to reverse the drug resistance.Meanwhile,we demonstrated that the presence of lipid rafts in SW620/AD300 cells and lower cholesterol content in lipid rafts blocked the synthesis of lipid rafts,and further studied the mechanisms of reversing MDR after lipid rafts were destroyed.(4)The studies of targeting and anti-tumor activity in vivo BALB/c nude mice bearing SW620/AD300 cells were used as animal models.The nude mice were labeled with free IR783 and IR783@TPGS2k-PLGA NPs,the targeting ability and tissue distribution of them were investigated by in vivo fluorescence imaging system.Besides,the anti-tumor effect and side effect of the delivery system were analyzed by body weight,relative tumor volume,H&E staining and Tunel staining of the nude mice.In vivo activity imaging results showed that the delivery system could be efficiently delivered to tumor sites due to the EPR effect,and had no significant toxic effects on other major organs.In vivo pharmacodynamic results showed that SV/DOX@TPGS2k-PLGANPs reversed the tumor drug resistance and significantly inhibited tumor growth.In short,SV/DOX@TPGS2k-PLGANPs reduced the cholesterol content in lipid rafts to block the formation of lipid rafts,resulting in enhanced lipid membrane fluidity and restored membrane endocytosis.Moreover,the drug resistance of SW620/AD300 cells was successfully reversed by decreased activity of P-gp and changed the mitochondrial membrane potential,which led to reduced drug efflux and enhanced apoptosis.