ClC-3 Silencing Mediates Lysosomal Acidification Arrest And Autophagy Inhibition To Sensitize Chemo-photothermal Therapy

Objective: Protective autophagy can be activated by external stimuli such as CT and PTT etc.,leading to tumor resistance.As a key subcellular for autophagy,lysosomal dysfunction is crucial for extensive autophagy suppression.Furthermore,lysosomal drug sequestration enhances basic drug resistance such as DOX as DOX is trapped away from its target site,the nucleus.Moreover,it is often ignored that most of nanodrug delivery systems are internalized to lysosome for degradation,which further leads to basic chemotherapeutic drugs resistance.Lysosome works as an essential organelle in both drug resistance mechanisms,whose acidification arrest offers a potential strategy to inhibit autophagy and lysosomal drug sequestration simultaneously.The ClC-3 protein is known as an important Cl--H+ transporter to maintain lysosomal p H at low values of various human cells.Thus,based on the essential role of lysosomes in autophagy and drug sequestration,this thesis constructed the GT/CT/PTT three-in-one combination therapy platform BP-A-S@D.This method used HeLa cells as a model to verify the effect of ClC-3 in tumor lysosomal acidification and autophagy regulation and its sensitizing effect on CT and PTT.Methods: 1.Preparation and characterization of BP-A-S@D: A GT/CT/PTT three-in-one combination therapy platform BP-A-S@D was constructed by hierarchically self-assembling DOX,AE105 and si ClC-3 on BPNSs.Then,BP-A-S@D was characterized by high resolution transmission electron microscope(HRTEM),atomic force microscopy(AFM),dynamic light scattering(DLS),X-ray photoelectron spectroscopy(XPS)and energy dispersive X-ray spectroscopy(EDS),and the photothermal conversion efficiency was also assessed.Furthermore,CCK-8 method was used to detect the viability of HeLa cells treated with PPBP.2.The uptake,distribution and gene silencing effect of BP-A-S@D in HeLa cells were examined by flow cytometry,transmission electron microscopy(TEM),laser confocal microscopy(CLSM),quantitative real-time polymerase chain reaction(q PCR)and WB.The effect of BP-A-S mediated ClC-3 silencing on lysosomal acidification was examined by Lysosenser Green DND-189.3.WB was utilized to detect the expression level of p62 and LC3 B proteins in HeLa cells treated with GT,CT and PTT alone and in combination.MDC staining was used to assess the effect of BP-A-S on autophagy of HeLa cells.The Lyso Tracker Green DND-26 staining was utilized to observe lysosomes of HeLa cells treated with GT,CT and PTT alone and in combination.And bio-transmission electron microscopy(bio-TEM)was used to observe autophagosome and autolysosomes in HeLa cells with different treatments.4.The cytotoxicities of si ClC-3-mediated lysosomal acidification arrest and autophagy inhibition in combination with CT and PTT on Hela cells were detected by CCK-8 assay and Annexin V-FITC/PI apoptosis assay.The effect of ClC-3 silencing on migratory and invasive abilities of HeLa cells was examined by Transwell migration and invasion assay and cell scratch healing assay.Results: 1.The multifunctional nanodrug delivery system of BP-A-S@D was successfully constructed.This nanodrug delivery system showed the low contrast and good dispersion.The average size of BP-A-S@D was approximately 185 nm,fall into the range of size that favorable for targeting to tumor via EPR effect.Furthermore,PPBP at tested concentration exhibited good biocompatibility.Due to its excellent photothermal conversion efficiency and high specific surface area,BP-A-S@D can be used for PTT,CT and GT.2.The key role of ClC-3 in lysosomal acidification of HeLa cells was revealed.1)The conjugation of AE105 peptide enhanced the internalization of BP-A-S in HeLa cells through its nano-molar affinity with urokinase-type plasminogen activator receptor(u PAR).2)The m RNA and protein levels of ClC-3 were down-regulated obviously with increasing concentration of BP-A-S.3)si ClC-3 blocked the formation of lysosome by inhibiting lysosomal acidification,which not only prompted the escape of the nanodrug delivery system from lysosome,but also reduced the lysosomal drug sequestration to improve the bioavailability of DOX.3.The essential role of si ClC-3-mediated ClC-3 silencing in autophagic regulation of HeLa cells was revealed.1)BP-A-S mediated ClC-3 silencing reduced the formation of autolysosomes in the autophagic process by inhibiting lysosomal acidification,which blocked the autophagic process at the stage of autophagic cargo degradation.3)ClC-3 silencing reversed BPNSs,CT and PTT-induced autophagic activity,which was expected to further improve the anticancer efficiency of CT and PTT.4.si ClC-3-mediated lysosomal acidification arrest and autophagy inhibition combined with BPmediated PTT and DOX-mediated CT significantly enhanced anticancer efficiency.si ClC-3-mediated GT combined with CT and PTT enhanced the anti-proliferation efficiency of HeLa cells.In addition,ClC-3 silencing inhibited the migration and invasion of HeLa cells.Conclusion: In this thesis,a multifunctional nanodrug delivery system of BP-A-S@D was successfully constructed to reveal the effect of ClC-3 silencing in suppressing lysosomal acidification and autophagic cargo degradation of Hela cells.Firstly,si ClC-3 blocked the formation of lysosome by inhibiting lysosomal acidification,which not only prompted the escape of nanodrug delivery systems from lysosome,but also reduced the lysosomal drug sequestration to improve the bioavailability of DOX.Secondly,si ClC-3 can inhibit protective autophagy induced by CT and PTT to achieve effective chemosensitization and enhanced thermal ablation.Finally,ClC-3 silencing strengthened the anti-proliferative and anti-invasive effects on Hela cells at the same time.Overall,ClC-3 silencing presented a promising strategy to simultaneously inhibit autophagy and lysosomal drug sequestration to sensitize chemo-photothermal therapy,which improved antiproliferative and anti-invasive efficiency of Hela cells.