| Hydroxycamptothecin(HCPT),derived from plant,is a major antitumor agent used in clinic.It has many advantages,such as high efficiency,low toxicity,no cross drug resistance and broad-spectrum anticancer activity.It had demonstrated that the complete lactone ring of HCPT was essential for its antitumor activity.However,the lactone form of HCPT is insoluble in water and unstable in physiological environment.Although the carboxylic acid form of HCPT is soluble in water,it shows less antitumor activity and several unpredictable side effects.Recent studies have indicated that HCPT induce apoptosis via inducing mitochondrial dysfunctions including loss of mitochondrial membrane potential(??m)and release of cytochrome C from mitochondria into cytosol except for interfering with DNA replication of nucleus.Recent years,drug delivery systems caused more attention because they could improve the water solubility and the therapeutic effect of chemotherapeutic drugs.Therefore,it's significant to establish a nucleus and mitochondria dual-targeted nanoparticle not only can enhance the accumulation of HCPT in the tumor tissue through enhanced permeability and retention(EPR)effect,but also increases the distribution of HCPT in mitochondrion and nucleus to increase the antitumor activity of HCPT.Objective: In this study,in order to enhance the stability of the lactone form of HCPT,increase the specific distribution of HCPT in tumor tissue,facilitate the cell uptake of HCPT,and finally enhance the antitumor activity of HCPT,p H responsive and redox responsive nanoparticles with nucleus and mitochondria dual-targeted were prepared,respectively.When nucleus and mitochondria dual-targeted nanoparticles were prepared,folate acid(FA)was used as the tumor cell targeting ligand,triphenylphosphine(TPP)was used as a mitochondria and nucleus targeting molecule,poly(lactide-co-glycolide)acid(PLGA)was used as the hydrophobic segment,and PEG was used as hydrophilic segment.Methods: 1.The structure of synthesized PLGA-hyd-PEG4k-FA,PLGA-SS-PEG4k-FA and PLGAPEG2k-TPP was confirmed by 1H NMR and IR.2.HCPT@PLGA-PEG2k-TPP/PLGA-hyd-PEG4k-FA and HCPT@PLGA-PEG2kTPP/PLGA-SS-PEG4k-FA were prepared by emulsion solvent evaporation method.The prescription of the nanoparticles was optimized by particle size,zeta potential,polydispersity index(PDI)and drug loading.3.The drug loading of the nanoparticles was determined by fluorescence spectrophotometer.Particle size,zeta potential and stability were measured by zeta potential and nano-particle size analyzer.Nanoparticles morphology was observed by transmission electron microscope(TEM).4.The drug release profile of HCPT@PLGA-PEG2k-TPP/PLGA-hyd-PEG4k-FA and HCPT@PLGA-PEG2k-TPP/PLGA-SS-PEG4k-FA was conducted in different release medium by dialysis method.The zeta potential of HCPT@PLGA-PEG2k-TPP/PLGA-hydPEG4k-FA and HCPT@PLGA-PEG2k-TPP/PLGA-SS-PEG4k-FA was measured in different p H medium and different GSH concentration medium,respectively.5.The cytotoxicity of HCPT loaded nanoparticles against Hep G2 cells and SW620 cells was detected by MTT assay.The cellular uptake and intracellular drug distribution in Hep G2 cells and SW 620 cells were observed through confocal scanning laser microscopy(CSLM).The mitochondrial membrane potential of SW620 cells was tested by JC-1 assay.The cell apoptosis induced by HCPT loaded nanoparticles on SW620 cells was assayed by caspase-3 kits.The effect of HCPT loaded nanoparticles on SW620 cell cycle was measured by flow cytometry.6.The SW620 cells in logarithmic growth phase were inoculated subcutaneously into the dorsal flank area of the nude mice.The bio-distribution of HCPT after treatment with HCPT@PLGA-PEG2k-TPP/PLGA-hyd-PEG4k-FA and HCPT@PLGA-PEG2k-TPP/PLGASS-PEG4k-FA was observed by the in vivo imaging and tissue section staining.The therapeutic effect of HCPT@PLGA-PEG2k-TPP/PLGA-hyd-PEG4k-FA and HCPT@PLGA-PEG2k-TPP/PLGA-SS-PEG4k-FA on tumor bearing nude mice was evaluated by measuring the tumor volume,body weight and tumor inhibition rate.H&E staining was used to observe the morphological changes of normal organs and tumors tissues in nude mice.Results: 1.1H NMR and IR spectrums showed the synthesized PLGA-hyd-PEG4k-FA,PLGA-SSPEG4k-FA and PLGA-PEG2k-TPP were the designed polymeric materials.2.HCPT@PLGA-PEG2k-TPP/PLGA-hyd-PEG4k-FA and HCPT@PLGA-PEG2kTPP/PLGA-SS-PEG4k-FA were prepared by the optimized materials ratio at 4:16(PLGAPEG2k-TPP:PLGA-hyd-PEG4k-FA)and 12:8(PLGA-PEG2k-TPP:PLGA-SS-PEG4k-FA),respectively.3.TEM image showed the two kinds of HCPT loaded nanoparticles were spherical with uniform size distribution.The average size of HCPT@PLGA-PEG2k-TPP/PLGA-hydPEG4k-FA was 104 nm,zeta potential was-8.1 m V,and the drug loading was 6.56%.The average size of HCPT@PLGA-PEG2k-TPP/PLGA-SS-PEG4k-FA was 98 nm,zeta potential was-18.6 m V,and the drug loading was 6.54%.In addition,the two nanoparticles had a good stability in 8 days in p H7.4 PBS.4.The in vitro drug release profile showed the accumulative HCPT release from HCPT@PLGA-PEG2k-TPP/PLGA-hyd-PEG4k-FA increased with the decrease of the medium p H value.The accumulative release of HCPT from HCPT@PLGA-PEG2k-TPP/PLGA-SS-PEG4k-FA was more in higher GSH concentration medium.Furthermore,the charge of HCPT@PLGA-PEG2k-TPP/PLGA-hyd-PEG4k-FA was reversed when the medium p H value changed from 7.4 to 5.0.The charge of HCPT@PLGA-PEG2kTPP/PLGA-SS-PEG4k-FA was also reversed when the medium GSH concentration change from 0.1?M to 10 m M.5.HCPT@PLGA-PEG2k-TPP/PLGA-hyd-PEG4k-FA showed the significantly stronger cytotoxicity than free HCPT and HCPT@PLGA-hyd-PEG4k-FA on Hep G2 cells and SW620 cells,HCPT@PLGA-PEG2k-TPP/PLGA-SS-PEG4k-FA also showed the significantly greater cytotoxicity than free HCPT and HCPT@PLGA-SS-PEG4k-FA on Hep G2 cells and SW620 cells.6.The cellular uptake of HCPT loaded nanoparticles showed a time-dependent manner.After incubation with Hep G2 cells and SW620 cells for 4 h,the cellular uptake of HCPT loaded nanoparticles was apparently increased as compared with free HCPT.Compared with HCPT@PLGA-PEG2k-TPP/PLGA-hyd-PEG4k-FA,the cellular uptake of HCPT@PLGA-PEG2k-TPP/PLGA-SS-PEG4k-FA was obviously higher.Compared with HCPT@PLGA-hyd-PEG4k-FA and HCPT@PLGA-SS-PEG4k-FA,HCPT@PLGA-PEG2kTPP/PLGA-hyd-PEG4k-FA and HCPT@PLGA-PEG2k-TPP/PLGA-SS-PEG4k-FA transported more amount of HCPT to mitochondrion and nucleus.7.HCPT loaded nanoparticles obviously reduced the mitochondrial membrane potential compared with free HCPT.Compared with HCPT@PLGA-PEG2k-TPP/PLGA-hyd-PEG4 kFA,HCPT@PLGA-PEG2k-TPP/PLGA-SS-PEG4k-FA showed greater capacity in the decrease of mitochondrial membrane potential.The HCPT loaded nanoparticles could increase the level of caspase-3 in SW620 cells than free HCPT.HCPT@PLGA-PEG2kTPP/PLGA-SS-PEG4k-FA exhibited stronger effect on caspase-3 than HCPT@PLGAPEG2k-TPP/PLGA-hyd-PEG4k-FA.In addition,HCPT loaded nanoparticles arrested SW620 cells in S phase.8.Compared with the free HCPT treated tumor-bearing mice,there was much stronger fluorescence signal in tumor tissue in which tumor-bearing mice were treated with HCPT loaded nanoparticles.HCPT loaded nanoparticles significantly inhibited the tumor growth, and almost showed no effect on the body weight of the tumor bearing nude mice.H&E staining showed that HCPT loaded nanoparticles had no dramatically toxic effects on the major normal organs,but showed great toxicity in tumor tissue.In addition,HCPT@PLGAPEG2k-TPP/PLGA-SS-PEG4k-FA exhibited higher therapeutic effect than HCPT@PLGAPEG2k-TPP/PLGA-hyd-PEG4k-FA in tumor-bearing mice.Conclusion: HCPT@PLGA-PEG2k-TPP/PLGA-hyd-PEG4k-FA and HCPT@PLGA-PEG2kTPP/PLGA-SS-PEG4k-FA showed an excellent p H-responsive and redox-responsive property,respectively.HCPT@PLGA-PEG2k-TPP/PLGA-hyd-PEG4k-FA and HCPT@PLGA-PEG2k-TPP/PLGA-SS-PEG4k-FA could efficiently enhance the cellular uptake of HCPT by tumor cells,improve the distribution of HCPT in nucleus and mitochondria,consequently increased the cytotoxicity of HCPT.HCPT@PLGA-PEG2kTPP/PLGA-hyd-PEG4k-FA and HCPT@PLGA-PEG2k-TPP/PLGA-SS-PEG4k-FA significantly increased the accumulation of HCPT in tumor tissue,consequently enhanced the in vivo antitumor activity of HCPT.In addition,compared with HCPT@PLGA-PEG2kTPP/PLGA-hyd-PEG4k-FA,HCPT@PLGA-PEG2k-TPP/PLGA-SS-PEG4k-FA exhibited higher therapeutic effect in vivo. |
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