GLUT-1 Mediated,Redox-Responsive Paclitaxel Nanomicelles For Overcoming Chemoresistance In Lung Cancer

Background:With the worsening of the global ecological environment,the acceleration of people’s pace of life and the increasing pressure of work,the morbidity and mortality of malignant tumors have been increasing year by year,becoming the leading killer of human health.Among all cancers,lung cancer is one of the leading causes of death and is now the first most common type of cancer.About 1.7 million people die of lung cancer each year,and the overall five-year survival rate is only 15 percent.Because lung cancer is difficult to diagnose,the initial stage of the tumor is asymptomatic,when the patient is diagnosed with lung cancer has been advanced,surgery has no help.In addition,lung cancer treatment often encounters problems of metastasis and drug resistance.The nano-drug delivery system contains anti-tumor drugs,which can increase the solubility of drugs,change the distribution of drugs in vivo,and improve the drug targeting,thus improving the therapeutic effect and reducing the incidence of adverse reactions.At present,more and more forms such as microspheres and nanoparticles are made by grafting ligands onto drugs or carriers and then wrapping them around chemotherapeutic drugs.However,the grafting of proteins and antibodies onto polymers is complicated and easy to degrade,which is limited in practical production.Therefore,it is of great significance to search for new target heads and targets to effectively deliver drugs into cells and reverse tumor resistance in the process of drug treatment of multidrug resistance of tumors.Most malignant tumor cells have the characteristics of high metabolism and both aerobic and anaerobic metabolism coexist,so the tumor cells overexpress glucose transporter protein（GLUT-1）to absorb enough glucose.Therefore,Aminoglucose（AG）is used to specifically identify the target molecule GLUT-1 on the surface of tumor cells to achieve tumor targeting.At the same time,the concentration of glutathione（GSH）in tumor cells is much higher than that in normal cells,and the carrier containing disulfide bond is easy to break quickly in this environment,and the drug package can be quickly released,so as to achieve a better goal of cancer treatment.Based on the above,we will construct tumor targeted redox-responsive drug-carrying nano-micelles,which will enhance the endocytosis of tumor cells through GLUT-1 mediation.Drug release through redox reaction leads to disulfide bond breaking,increasing the accumulation of drugs in cells per unit of time,and ultimately better treatment of tumor drug resistance.Objective:1.A linear copolymer（AG-PEG-SS-PLA,abbreviated as AG-P-SS-P/PTX）was prepared to reverse the oxidation and reduction of lung cancer drug resistance by GLUT-1 mediated oxidation（GLUT-1）as a carrier,which was loaded with the anti-lung cancer chemotherapy drug paclitaxel（PTX）and self-assembled into nano-micelles（AG-P-SS-P/PTX）,and its physical and chemical properties were determined.2.In vitro experiments were conducted to investigate the mechanism of oxidative reduced paclitaxel nanoparticles mediated by GLUT-1 to inhibit the proliferation of lung cancer cells and reverse the multidrug resistance of A549/ADR lung cancer cells.3.In vivo experiments were conducted to investigate the anti-lung cancer resistance effect of AG-P-SS-P/PTX nano-micelle in tumor-bearing mice and its toxicity on normal tissues.Methods:1.Synthesis of AG-P-SS-P copolymer:PEG-SS-NH2 and PLA-OH were synthesized by mercaptane-ene reaction to produce PEG-SS-PLA（P-SS-P）,and AG and P-SS-P were synthesized by amide reaction to produce AG-P-SS-P.Preparation of AG-P-SS-P/PTX nanomicelles:ptx-containing nanomicelles were prepared by dialysis method,PTX was dissolved in anaqueous DMSO（10mL）,mixed overnight with P-SS-P/AG-P-SS-P,and then transferred to a dialysis bag（MWCO,1kDa）for dialysis at room temperature for 24 hours.The drug-carrying nano-micelles P-SS-P/PTX or AG-P-SS-P/PTX were obtained.The structure and molecular weight of the synthesized products were determined by¹H-NMR and GPC.The cracking of disulfide bond was detected by AG-P-SS-P with fluorescence spectrophotometer.Dynamic light scattering（DLS）was used to detect the particle size and Zeta potential of the drug-loaded micelles.The 6 morphology and appearance of the micelles were observed by transmission electron microscopy（TEM）.The encapsulation rate,drug loading and drug release of P-SS-P/PTX and AG-P-SS-P/PTX nano-micelles were detected by HPLC.2.In vitro experiments were conducted to explore the mechanism of inhibiting ADR in lung cancer cells:A549 lung cancer cells（A549）and drug-resistant A549 lung cancer cells（A549/ADR）were selected,and AG,Colchicine,PhAsO and Filinpin were added to co-incubate the cells with nano-micelles,respectively,to explore the mechanism of cell uptake.AG was added or not added to the two types of cells to verify whether the nanomicelles have in vitro cell targeting.Inhibition of proliferation of lung cancer cells by nano-micelles was detected with CCK8 kit.Apoptosis was detected using Annexin V-fitc/PI staining kit and flow cytometry.Protein levels in human lung cancer cells were detected using Westernblot.3.In vivo experiments were conducted to explore its anti-tumor effect:subcutaneous injection of A549/ADR cells into nude mice was used to construct A subcutaneous xenograft tumor model,and AG-P-SS-P/PTX nano-micellar was used to treat xenograft tumor mice,and body weight was recorded to evaluate whether the therapy had toxic effects.Finally,the in vivo biosafety of AG-P-SS-P/PTX nanomicelles was evaluated by H&E staining.Results:1.In this experiment,AG-P-SS-P was synthesized successfully,and a series of AG characteristic peaks were observed from¹H-NMR:2.01（d,1H,H11）,5.12（s,1H,H12）,2.51（m,2H,H13）,3.33（m,1H,H14）,3.49（m,1H,H15）,3.81（m,1H,H16）.The self-assembled nano-micelles（AG-P-SS-P/PTX）containing paclitaxel showed excellent physical properties.The particle diameter was 75 nm,the charge was-0.25 mV,the encapsulation rate was 84.21%,the drug loading was 8.2%,and the PDI was 0.245.Cumulative release of 70%of PTX was achieved in 10 m M GSH solution for about 20 minutes.The cumulative amount in A549/ADR cells was71.9%,much higher than that in other groups.2.Tumor targeting experiments showed that:compared with P-SS-P/Rh123 and AG-P-SS-P/Rh123+AGgroup,AG-P-SS-P/Rh123 nano micelles groupA549 green fluorescence was significantly enhanced.The reverse drug resistance experiment showed that:compared with Taxol,P-P/Rh123 and P-SS-P/Rh123 group,the green fluorescence intensity of AG-P-SS-P/Rh123 in A549/ADR cells was significantly increased,and the fluorescence intensity was similar to that of AG-P-SS-P/Rh123 in A549 cells.The tumor cell endocytosis experiment showed that the green fluorescence intensity of tumor cells added with philipine was significantly reduced compared with other groups,suggesting that the uptake of AG-P-SS-P/Rh123nanometer micelles by cancer cells was mainly dependent on the endocytosis mediated by fossa protein.Flow cytometry data showed that the green fluorescence intensity of A549/ADR cells in the control group（only AG-P-SS-P/Rh123）was higher than that of AG-P-SS-P/Rh123 with GSH-OEt,but lower than that of AG-P-SS-P/Rh123 A549/ADR cells with BSO,suggesting that AG-P-SS-P/PTX could occur in the cells.Cytotoxicity tests showed that AG-P-SS-P/PTX significantly inhibited A549/ADR cells compared with the P-P/PTX and P-SS-P/PTX groups,with IC₅₀of 3.51 uM.Apoptosis experiments showed that compared with the P-P/PTX and P-SS-P/PTX groups,AG-P-SS-P/PTX significantly induced apoptosis of A549/ADR cells.It was found that the micelles activated Caspase-9 and Caspase-3,and promoted the apoptosis of cancer cells by upregulation of the pro-apoptotic protein Bax and inhibition of the anti-apoptotic protein Bcl-2.3.In vivo anti-tumor experiments in mice showed that compared with other groups,AG-P-SS-P/PTX nano-micelles could significantly inhibit tumor growth in nude mice with xenograft tumor of A549/ADR cells（drug-resistant human non-small cell lung cancer cells）.The weight of each group did not change significantly.H&E staining showed that AG-P-SS-P/PTX micelles had no significant toxic effect on major organs such as heart,liver,spleen,lung and kidney.Conclusion:In this study,we successfully synthesized and prepared the antioxidant reduced paclitaxel nanoparticles mediated by GLUT-1,which reverse the drug resistance of lung cancer.Both in vivo and in vitro experiments have shown that AG-P-SS-P/PTX nano-micelles can enhance tumor targeting and drug release and accumulation in MDR lung cancer cells,reduce the drug resistance of lung cancer cells,inhibit tumor growth,and reduce the toxic and side effects of PTX.These results suggest that AG-P-SS-P/PTX nanomicelles have the potential to reverse multidrug resistance in tumors.