The Reversible Effects And Underlying Mechanisms Of MDR1 And Autophagy Directed Dual Targeted Nanoparticles On EGFR-TKI Resistance

Objective The epidermal growth factor receptor(EGFR)is a membrane-surface protein with tyrosine kinase activity.Studies have shown that it is highly expressed in most cancer patients,and that abnormal EGFR signaling pathways play an important role in tumorigenesis,tumor progression,and metastasis.Tyrosine kinase inhibitors(TKI)that acted against the EGFR(EGFR-TKI),such as geftinib,the frst selective EGFR-TKI domain,could efectively prevent tumor growth,metastasis,and angiogenesis,and promoted tumor cell apoptosis.EGFR-TKI were typically successful in the treatment of malignancies,especially for non-small cell lung cancer.However,after a certain period of drug exposure,tumor cells gradually becomed insensitive to EGFR-TKI,ultimately surviving following exposure to chemotherapy drugs.In this way,cells developed acquired chemoresistance,thus signifcantly reducing the therapeutic efect of EGFR-TKI and limiting their clinical applications.Multiple targeted therapy has been enjoyed as a possible strategy to overcome acquired resistance to EGFR-TKI.Previous studies have demonstrated that combination of blocking cell surface MDR1 and inhibiting autophagy sensitizes tumor cells to EGFR-TKI.Based on these findings,we established a novel MDR1 antibody modified gifitinib and autophagy inhibitor chloroquine loaded chitosan nanoparticles,and examined whether it could revert EGFR-TKI resistance in vivo and in vitro.We further explored the reversible effects and underlying mechanisms of MDR1 and autophagy directed dual targeted nanoparticles on EGFR-TKI resistance.Our results will provide the theoretical and experimental basis for overcoming EGFR-TKI resistance.Methods The linearity,precision and recovery of the UV quantitative analysis method of gefitinib and CQ established by the study were in line with the measurement requirements,and could be used for the determination of the in vitro content of two drugs.Gefitinib/CQ NPs were prepared by ion-gel method using gefitinib,CQ,CS,TPP.The mAb MDR1 was modified to the surface of the nanoparticles by the interaction of positive and negative charges,and detected by fluorescence microscopy.The optimal prescription of gefitinib/CQ mAb MDR1-NPs was screened by orthogonal experiment.The parameters such as morphology,particle size,Zeta potential,encapsulation efficiency and the in vitro drug-release pattern of NPs were investigated.The cellular uptake of mAb MDR1-NPs loaded with Rhodamine B were detected by laser confocal microscopy,and assessing the targeting of antibody-modified nanoparticles.The uptake mechanism of cells was determined by endocytosis inhibition assay.MTT assay and flow cytometry for detection of gefitinib,gefitinib+mAb MDR1,gefitinib+CQ,gefitinib+mAb MDR1+CQ,gefitinib NPs,gefitinib/CQ NPs,gefitinib mAb MDR1-NPs,gefitinib/CQ mAb MDR1-NPs on proliferation and apoptosis of SMMC-7721/gefitinib cells.Western blot was used to detect the expression of apoptosis-related proteins bax,cleaved caspase 3,and cleaved PARP,and autophagy-associated protein LC3 and multidrug resistant protein MDR1.Results The linearity,precision and recovery of the UV quantitative analysis methods of gefitinib and CQ established in this study were in line with the determination requirements.So,it could be used for the determination of in vitro content of two drugs.Through orthogonal experiment,the optimal prescription of gefitinib/CQ mAb MDR1-NPs was screened,and the prepared nanoparticles exhibited a spherical morphology with a narrow size distribution.The release behavior of gefitinib/CQ NPs and gefitinib mAb MDR1-NPs were not significantly different,showing a biphasic release pattern.The uptake of NPs involved time-dependent internalization,and that more NPs were internalized and spread into the cytoplasm.mAb MDR1-NPs efectively enhanced the drug accumulation within the cell surface of MDR1-overexpressed SMMC-7721/gefitinib cells.During the endocytosis inhibition test,both caveolae and macropinocytosis mainly mediated the endocytic pathways of NPs.MTT and flow cytometry revealed that gefitinib/CQ mAb MDR1-NPs significantly induced apoptosis.Western blot analysis showed that after treatment of gefitinib/CQ mAb MDR1-NPs,the protein expression levels of cleaved caspase3,cleaved PARP and bax were significantly increased in SMMC-7721/gefitinib cells,while LC3 and MDR1 were decreased,indicated that CQ and mAb MDR1 can synergistically promote the anti-tumor effect of gefitinib.Conclusions mAb MDR1-modifed CS NPs,when combined with the co-delivery of gefitinib and CQ,showed targeting and therapeutic potential on enhancing the delivery of anticancer drugs and inducing signifcant cell apoptosis against acquired EGFR-TKI resistance through the modulation of autophagy and blocking the activity of MDR1.This fnding implied that autophagy and MDR1 may serve as dual targets to overcome acquired resistance in SMMC-7721/gefitinib cells,and the clinical application of autophagy and MDR1 inhibition might be one of the important strategies for overcoming acquired EGFR-TKI resistance in SMMC-7721/gefitinib cells.