Doxorubicin Nanomedicine With Lysosomal PH-triggered Drug Release And Lysosome Escape Potential

In recent years,nanomedicines play more and more important role in the biopharmaceutical fields.Compared with the traditional chemotherapeutic drugs,intelligent nanomedicine could passive target to tumor tissue and reduce side effects through high permeability and retention effect?EPR effect?.Researches show that nanomedicines can enter the tumor cells lysosomes through endocytosis,then escape from lysosomes into nucleus to enhance drug efficiency.Therefore,it's very important for nanomedicines to escape from lysosomes into nucleus which could improve drug efficacy and achieve cellular targeting.At present,the nanomedicines with proton sponge effect may realize the lysosomal escape function by increasing lysosomal osmotic pressure and finally leading to lysosome rupture.For example,nanomedicines with dendritic structure can absorb plenty of hydrogen ions at acidic pH in the lysosomal.Then lysosomal proton pump remains open,and chlorine ion flowed into lysosomes causing lysosomal osmotic pressure increasing rapidly.Finally,the lysosomes broke down owing to influx of water and nanomedicines escape into the cytoplasm.In addition,the acidic pH of tumor cell lysosomes is quite different from that of normal tissues.Therefore,acid sensitive nanomedicines based on tumor microenvironment have attracted wide attention from researchers.Acid sensitivity nanomedicines are stable in normal tissues.But in tumor site,it will rapidly degrade in the acidic pH and release drugs.The nanomedicines with acid pH-triggered drug release and lysosome escape function have enhanced curative effect and reduced side effects on normal tissues.Therefore,they have broad application prospects.In the first chapter,we introduced the research progress of nanomedicine,and discussed the acid sensitivity and proton sponge effect of nanomedicines in detail.In the second chapter,the synthesis methods and structure characterizations of two block polymer PEG-block-PGAG2-Hpa containing?-carboxylic amide units and dendritic structures are introduced.The degradations of the polymers in different pH buffer solutions were monitored by NMR spectrometer.The results showed that the polymer had obvious acidic-degradation ability.According to the kinetic equations of degradations,the degradation half-life(t_1/2)of polymers were obtained to be 105.30 h?pH=7.4?,35.26 h?pH=6.5?,10.07 h?pH=5.0?.The copolymer PEG-block-PGAG2 and G2 containing dendritic structures were synthesized,and the proton absorptive capacity of the polymers were investigated though non-aqueous titrations.The results showed that the two block polymer PEG-block-PGAG2 had significant proton sponge effect.Inthethirdchapter,PEG-block-PGAG2-Hpamicellesand PEG-block-PGAG2-Hpa·DOX micelles were prepared.Then the physicochemical properties of the micelles were characterized.For example,particle size,zeta potential and morphology of the micelles were investigated by dynamic light scattering?DLS?and atomic force microscopy?AFM?.The critical concentration of aggregation?CAC?was measured by dynamic light scattering and CAC was 0.0696 mg/mL.In vitro drug release studies of PEG-block-PGAG2-Hpa·DOX micelles were investigated in pH 7.4,pH 6.5 and pH 5.The experimental results showed that,the drug release of PEG-block-PGAG2-Hpa·DOX micelles increased with the decrease of pH value,especially in lysosomal pH value?pH=5?.Within 48 h,the total drug release of drug loaded micelles were 78%in pH 5.0.In the fourth chapter,the cytotoxicity of PEG-block-PGAG2-Hpa·DOX micelles and DOX·HCl on A549 cells were studied.The phagocytosis of cells were qualitatively characterized by inverted fluorescence microscope,and the phagocytosis of cells were quantitatively characterized by flow cytometry.According to the literatures,NH₄Cl could increase the pH value of lysosome.So the A549 cells were cultured in the medium containing 40 mM NH₄Cl as a negative control.In cytotoxicity experiments,survival rate of cells cultured with 40 mM NH₄Cl medium increased significantly,showing that drug release decreased with lysosome pH value increasing.Theresultsfurtherindicatingthatdrugreleaseof PEG-block-PGAG2-Hpa·DOX micelles depended on lysosome acidic environment.The phagocytosis of cells were observed by inverted fluorescence microscope.The fluorescence photos showed that PEG-block-PGAG2-Hpa·DOX could help DOX escape from lysosome into nucleus to kill cancer cells.The above results showed that PEG-block-PGAG2-Hpa·DOX micelles were stable in neutral blood circulation.But lysosome acidic pH could trigger drug release of PEG-block-PGAG2-Hpa·DOX micelles.In addition,the micelles could help DOX escape from lysosomes and reduce toxicity of normal tissue.