Polymeric Nanoassemblies Based On Autophagy Modulation For Tumor Multidrug-resistance

Multidrug resistance(MDR)is well recognized as one of the major obstacles that deteriorate the clinic effect of chemotherapy for malignant tumor.The development of MDR is complex,which can be generally divided into two major types,“pump”resistance and“non-pump”resistance.For“pump”resistance,it is raised by the overexpression of drug-efflux pumps to reduce intercellular drug concentration.P-glycoprotein(P-gp),coded by the mdr1 gene,has been reported that its overexpression would induce MDR in many cancer cells.RNA interference(RNAi)technology is frequently combined with chemotherapy to suppress the expression of P-gp.It takes advantage of small interfering RNA(siRNA)molecules to silence specific gene,which shows high specificity and excellent treatment effect in tumor therapy.There are many other mechanisms,expect drug-efflux pump,that are closely associated with MDR,for instance,MDR-related genes participating in repairing of DNA,alterations of the drug targeting,and reductions of drug uptake through solute carriers.Recent evidences have shown that autophagy plays a significant role in the development of MDR.Autophagy is activated in limited growth conditions such as hypoxia,nutrient starvation,and chemo-radiotherapy.Autophagy degrades cytoplasmatic materials(such as damaged organelles,obsolete proteins,and incading pathogens)and recycles energy to maintain homeostasis in cells.Inhibiting presurvival autophagy could resensitize chemotherapeutic drugs in MDR cell.Herein,in order to reverse overall MDR,a novel hyaluronic acid(HA)-coated siRNA/Paclitaxel(PTX)co-delivery nanoassemblies were designed to suppress P-gp level,block autophagic flux,and efficiently deliver siRNA and paclitaxel.We synthesized the polymer-drug conjugates of low molecular weight polyethyleneimine(1.8k PEI)and PTX,named PEI-PTX(PP),which has high drug loading content(～25.2%)and can encapsulate siRNA for gene therapy,of which structure was confirmed by FTIR,~1H NMR,and GPC.Subsequently,the optimum weight of PP/siRNA/HA was 3:1:6 after formulation optimization.PP/siRNA/HA nanoassemblies were core-shell nanoparticles with 144.4±1.0nm and-9.03±0.33 m V,of which drug loading content was 6.99±0.15%.PP/siRNA/HA nanoassembles have excellent stability and biocompatibility,which could protect siRNA from anionic environment in physiological fluids and enzymatic challenge in FBS.PP/siRNA/HA also exhibited excellent cellular internalization and endo/lysosomal escape ability,meanwhile the internalization level was related with CD44 expression on the membrane of cells.In the study of reversing MDR in vitro,PP/siRNA/HA could efficiently reduce the expression of P-gp and mdr-1 gene of A549/T cells by WB and RT-PCR analysis.The cell viability/cytotoxic potential of individual formulation were performed by MTT assay.PP/siRNA/HA unfolded excellent anticancer effects on A549 cells and A549/T cell.The cell apoptosis study and cell cycles distribution were measured by flow cytometry,and the results showed that PP/siRNA/HA exhibited better cytotoxicity and reversal of drug resistance on A549/T cells.Further researches showed that high dose of PP could block autophagic flux via alkalizing lysosomes.Bio-TEM image further confirmed the effects of autophagy inhibition of PP and PP/siRNA/HA.The bio-distribution and antitumor effect in vivo were further evaluated in A549/T tumor-bearing nude mice.The results of bio-distribution showed that PP/siRNA/HA nanoassemblies could enrich in tumor tissue.PP/siRNA/HA exhibited a superior antitumor efficiency with the tumor growth inhibition rates of 63.57%.There was no significant physiological morphology abnormality in main organs,confirming the safety of PP/siRNA/HA.The tumor tissues treated with PP/siRNA/HA exhibited a more obviously decline in P-gp protein expression and mdr1 gene expression.The PP/siRNA/HA effectively induced more apoptosis and inhibited the cellular proliferation in tumor tissues by TUNEL and Ki67 assay.For further confirming the autophagy blockade effect of nanocomplexes,the expression of autophagy-associated proteins(LC3 and p62)in tumor was monitored at the end of the treatments.The LC3B-I/II and p62 protein expressions in tumor tissues were evaluated by western blot and immunofluorescence staining,which confirmed the efficiency of blocking autophagic flux.In summary,the polymeric nanoassemblies provided a potential strategy for combating both pump and nonpump resistance via the synergism of RNAi and autophagy modulation.Our work developed a novel strategy for MDR that could block autophagic flux,and achieved overcoming pump and nonpump resistance when combined with RNAi,autophagy inhibition and chemotherapy.