Development And Application Of Nano-drug Delivery Platform To Inhibit Lysosome Function

Cancer is one of the major diseases threatening human health and causing death,and the rapid division,invasion,and stress to cope with various treatments of cancer cells depend on the function of lysosomes.Lysosomals are the main breakdown centers of endocytosis of goods in cells and intracellular macromolecules,and the normal physiological processes of cells depend on the function of lysosomes.Therefore,in tumor therapy,lysosomal can be used as an ideal target.The catabolic function of lysosomals is achieved through acidases in the lysosome,and the V-ATPase is important for maintaining the acidic environment of lysosomals.In the current study,tumor treatment by inhibiting the acidic environment of lysosomals or directly inhibiting VATPase to disrupt the normal function of lysosomals has been studied.However,during treatment,the cells trigger the lysosomal stress process: the lysosomal-associated transcription factor TFEB translocation to the nucleus increases the expression of lysosomal-related genes,resulting in lysosomal biogenesis to alleviate the adverse effects of lysosomal function destruction,making the treatment effect worse.Lysosomal stress is related to the m TORC1-TFEB pathway,when the acidic environment of the lysosomal is destroyed,the decomposition of intracellular macromolecules is inhibited,resulting in a lack of nutrients such as amino acids in the cell.Inactivation of m TORC1 in response to intracellular nutrient signals,dephosphorylation of TFEB,and eventually translocation of TFEB into the nucleus,resulting in increased expression of lysosomal-associated genes to relieve the pressure of cell therapy.Therefore,for tumor treatment,how to inhibit lysosomal stress is very important to enhance the therapeutic effect.At present,there are a variety of drugs such as hydroxychloroquine,diphyllin by inhibiting the acidic environment of lysosomals for treatment,but drug toxicity and low delivery efficiency are problem that cannot be ignored.In view of the important functions of lysosomes and the special acidic environment,an environmentally responsive nano drug loading platform with acid specific drug release ability and low toxicity has been proposed.On one hand,nano drug loading platform can be designed to deliver drugs such as diphyllin,meantime phosphorylation reagent coulud be used to prevent nuclear translocation of TFEB and improve the therapeutic effect;on the other hand,a nano drug loading platform responsive to environmental pH can be designed to enhance the therapeutic effect by specifically releasing drugs in acidic environment.Based on this,this thesis mainly studies the following two aspects:1.In the second chapter,a PEG-PLGA nanoparticle carrier system was constructed to deliver the V-ATPase inhibitor diphyllin(DP),together with phosphorylation reagent 3-aminopropyl phosphate(3-APPA),to explore the synergistic therapeutic effect of destroying lysosomal function and inhibiting lysosomal stress.Firstly,a nano-drug carrier platform PEG-PLGA/DP was synthesized.Cell experiments have proved that PEG-PLGA/DP had obvious toxicity to Hep G2 cells,and cytotoxicity was positively correlated with the concentration of the drug-loading platform;the acidic environment of the lysosome was destroyed,and the intracellular autophagy body accumulated;and TFEB is more located in the nucleus instead of in the cytoplasm.Compared with the Hep G2 cells only treated with PEG-PLGA/DP,the Hep G2 cells treated with PEGPLGA/DP and 3-APPA were susceptible to apoptosis,and the destruction of lysosomal acidic environment and the accumulation of autophagosomes were significantly enhanced,and TFEB were more accumulated in the cytoplasm.PEG-PLGA/DP destroyed the acidic environment of lysosomal by inhibiting V-ATPase,leading tumor cell apoptosis and meanwhile triggering the process of lysosomal stress,then translocation of TFEB to the nucleus occurred and made lysosomal-related gene expression,which alleviated the therapeutic effect;after the lysosomal stress was triggered,the lysosomal stress process was inhibited due to the maintenance of the TFEB phosphorylation with the aid of 3-APPA,and the therapeutic effect was significantly improved.2.In the third chapter,a pH-responsive lignin nanoparticle carrier system L-DPA/HCQ was constructed to inhibit the acidic environment of lysosomal acid by specifically releasing the autophagy inhibitor hydroxychloroquine(HCQ)in an acidic environment,exploring the therapeutic effect of the nanodrug carrier platform L-DPA/HCQ.Firstly,the N,Ndiisopropylethylenediamine(DPA)was grafted on lignin by Mannich reaction to obtain the carrier L-DPA,and the nano-drug carrier platform L-DPA/HCQ was obtained by self-assembling HCQ with L-DPA.Experimental results showed that L-DPA/HCQ had good pH responsiveness and could decompose under acidic conditions,so that HCQ was specifically released.At the same time,L-DPA had good pH buffer capacity due to its protonation,and the pH of the solution decreased slowly with the increase of acidity.Cell experiments have proved that L-DPA/HCQ had obvious toxicity to Hep G2 cells,the acidic environment of the lysosome was destroyed,the number of acidic endosomes in cells was significantly reduced,and more autophagosomes accumulated in the cells,resulting in cell apoptosis.The nano-drug carrier platform achieved pH response effect by protonation in an acidic environment,so that HCQ was specifically released to play a role in achieving therapeutic purposes.