Construction And Evaluation Of Anti-tumor Drug Nano Delivery System Based On Autophagy Inhibition

Autophagy is a unique lysosome-dependent metabolic pathway in eukaryotic cells.It is a dynamic process in which cells could degrade damaged organelles and macromolecules through membrane structure under energy stress such as starvation and hypoxia.Emerging studies have revealed that autophagy usually promotes the development and metastasis of advanced cancer.In addition,cytoprotective autophagy is frequently induced in cancer cells under treatment stress,weakening the efficacy of multiple therapeutic options.Therefore,inhibiting autophagy of tumor cells provides a potential way to develop new therapeutic strategies for refractory and drug-resistant cancer.Lysosomal alkalization could weaken the activity of proteolytic enzymes and inhibit the degradation of autophagosomes,thus cutting off the way of autophagy providing nutrients for tumor cells.Currently,lysosomal alkalizers,such as chloroquine and matrine,can effectively improve the therapeutic efficacy of chemotherapy agents through autophagy inhibition,but with some side effects caused by their pharmacological activities.In this manuscript,the p H-sensitive polycationic polymer of methoxy polyethylene glycol-b-poly(diisopropylethylmethacrylate-b-dimethylaminoethylmethacrylate)(m PEG-b-P(DPA-b-DMAEMA))was synthesized by Reversible Addition-Fragmentation Chain Transfer Polymerization reaction(RAFT),and the chemotherapeutic agent epirubicin(EPI)was loaded to form micelles(Ms)by self-assembly.The self-assembled nanomicelles with polyethylene glycol as hydrophilic shell could selectively accumulate in tumors through the enhanced permeability and retention(EPR)effect,and finally enter into lysosomes of tumor cells.In lysosomal acidic environment(p H 4.5～5.5),the rapid protonation of tertiary amines of P(DPA-b-DMAEMA)triggers the irreversible alkalization of lysosomes and eventually results in causing impairment of lysosomal capacity to degrade autophagosomes,thus inhibiting autophagic flux.Meanwhile,the nanomicelles could effectively release EPI after dissociation,which could promote cell death more effectively while inhibiting autophagy of tumor cells.The successful synthesis of polymer m PEG-b-P(DPA-b-DMAEMA)was confirmed by Gel Permeation Chromatography(GPC)and ~1H-NMR.Blank nanomicelles(PEDD-Ms)and EPI-loaded nanomicelles(PEDD/EPI-Ms)were prepared by dialysis method,and their particle size,zeta potential and morphological feature were characterized via Malvern Zetasize Nano ZS90 and transmission electron microscope(TEM).The results showed that both PEDD-Ms and PEDD/EPI-Ms clearly appeared uniform spherical morphology with particle sizes of 165.3±1.8 nm and 185.8±0.3 nm,and Zeta potentials of 34.1±0.1 m V and 32.4±0.6 m V,respectively.TEM and Malvern Zetasize Nano ZS90 were performed to detect the p H-triggered size change of PEDD-Ms in different buffer solutions with p H 7.4 and 5.5,which indicated that it had obvious p H sensitivity.Meanwhile,the EPI-loading efficiency and entrapment efficiency of PEDD/EPI-Ms were determined using a fluorescence spectrophotometer,and the entrapment efficiency was 50.730±0.012%with the drug loading efficiency of4.830±0.001%.Cellular uptake and intracellular distribution studies showed that PEDD/EPI-Ms could be internalized into cells via endocytic pathway after treating Hep G2 cells for 1～4 h,and effectively release the loaded EPI through the endo/lysosomal drug release procedure.Western blotting,acridine orange(AO)staining and GFP-LC3dot formation were performed to identify autophagy behaviors mediated by PEDD-Ms in Hep G2 cells.The results showed that Hep G2 cells treated with PEDD-Ms showed large-scale accumulation of autophagosomes and LC3II,and the degradation of autophagy substrate p62 was blocked,which indicated that PEDD-Ms could effectively inhibit autophagy.Moreover,Lyso Tracker Red(LTR)staining,Lyso Sensor Green DND-189 staining and acid phosphatase activity assay were used to analyze the effects of PEDD-Ms on lysosomal stability,which indicated the degradation ability of lysosomes was impaired by lysosomal alkalization in a dose-dependent manner.In addition,the effect of PEDD/EPI-Ms on the viability of Hep G2 cells was studied by MTT assay,and compared with free EPI to evaluate the sensitizing effect of PEDD-MS on EPI.The results showed that compared with the cells treated with free EPI,the survival rate of cells treated with PEDD/EPI-Ms was significantly decreased,and the combination index of free EPI and PEDD/EPI-Ms calculated by Chou-Talalay method was 0.83±0.12,showing obvious synergistic effect.In vivo imaging showed that the loaded nanomicelles have specific tumor-targeting capacity through EPR effect based on the nanoscale size.The volume of tumors,body weight curves and immunohistochemical staining showed that compared with the saline group,the individual EPI-treated group exhibited a slight inhibition in tumor growth,and PEDD-Ms also exhibited certain anti-tumor efficacy due to autophagy inhibition.Furthermor,the PEDD/EPI-Ms group showed the best therapeutic efficacy with good biological safety during the whole treatment.In addition,immunofluorescence staining results showed that the levels of autophagy proteins LC3and p62 in tumor tissues treated with PEDD-Ms and PEDD/EPI-Ms were significantly increased,indicating that the nanomicelles could effectively inhibit autophagy in tumor sites.The above results confirmed that PEDD/EPI-Ms could inhibit autophagy by alkalizing lysosomes and impairing the degradation ability of hydrolases,thus enhancing the anti-tumor efficacy of EPI,which have great potential in improving the efficacy of traditional chemotherapy,and may provide a new idea for the application of autophagy mediated by polycationic nanomaterials in cancer treatment.