Construction And Study Of The Cellular Effects Of Cascade Nanoreactors And Its Cytological Effect

In the past several decades,the treatment based on enzyme catalysis has attracted more and more attention in biomedicine because of its high specificity,good biological tolerance and low immunotoxicity.Among them,some of the protease molecules also showed excellent anti-cancer properties,has been tried to be used in cancer treatment.Although this protease-based approach to cancer therapy is attractive,it is limited by the enzyme's inherent instability,environmental susceptibility to degradation and denaturation,and low cell penetration,the effective accumulation and function of therapeutic protease molecules in target cells are severely restricted.Therefore,it is very important to develop efficient delivery system of protein-like enzyme for promoting the research and application of enzyme-catalyzed therapy.In recent years,nanoreactors have been tried to be used in the effective delivery and activity regulation of proteases,aiming at the problem of poor accumulation of proteases in tumor cells.Although nanoreactors can reduce the hydrolysis and inactivation of protease molecules in the bloodstream and increase drug accumulation in tumor sites,however,its complex preparation process and low loading rate limit its further application.It is urgent to construct a high-loading and high-activity nanoreactor based on protein-like enzyme molecules,and to realize its effective delivery to tumor cells,and to clarify its cytological fate and activity regulation mechanism.In this paper,Glucose oxidase(GOx),Hemoglobin(Hb)and Ribonuclease A(RNase A)were selected as therapeutic proteins Zeolites Pyrimidine Framework(ZPF)was selected as the carrier of protease to construct the nano-reactor,which effectively solved the problems of complex material synthesis and low loading efficiency of protease.In addition,the uptake,transport and intracellular distribution of the nanoreactors at the subcellular level were systematically studied by real-time imaging of living cells,the process of enzyme catalysis in the nano-reactor after entering the cell and transporting to lysosome by microtubules was revealed.Through the evaluation of its killing effect on tumor cells,it is proved that this kind of nano-reactor can inhibit the growth of cancer cells.Therefore,this work builds a more potential platform for protease delivery and provides a new idea for enzyme-catalyzed therapy.This thesis mainly includes the following three aspects:1.Construction and application of GOx-based nanoreactorGlucose oxidase(GOx),a potential cancer drug,uses Glucose and oxygen to produce D-gluconic acid and hydrogen peroxide(H₂O₂);GOx not only blocks the cell's energy supply,and the H₂O₂ produced in high concentrations can promote cancer cell death.In this work,a glucose oxidase-based nanoreactor(GOx@ZPF)was prepared.The results of electron microscopy,particle size analysis and protein content determination showed that GOx@ZPF was successfully prepared,the results showed that the enzyme-catalyzed reaction efficiency of GOx@ZPF could be improved and the yield of H₂O₂ could be increased in the presence of micro-acid.The results of cell experiments showed that GOx@ZPF could be effectively ingested by cells,and the nano-reactor eventually distributed in lysosomes and played a catalytic role,producing a large amount of H₂O₂,leading to cell death.2.Application and research of separated nanoreactor on subcellular levelIn the complex organism,many kinds of enzyme cascade reactions are needed to realize many functions.Enzyme cascade reaction can improve the overall reaction efficiency and become an important means of regulating cell function.Hemoglobin(Hb)is an iron-rich protein that reacts with H₂O₂ in Fenton reagent(Fenton reaction)to produce the more toxic reactive oxygen species(·OH).Therefore,the combination of GOx and Hb can produce enzyme cascade reaction to enhance the killing effect of cancer cells.The results of TEM,FESEM and DLS show that the size of hydrated particle in the reactor is less than 250 nm,and the dispersion and stability of the reactor are good.A real-time three-color imaging system of living cells was constructed to visualize the transport,distribution and as FESEM of the separated nanoreactors in cells.The results showed that GOx@ZPF and Hb@ZPF were transported along microtubules,and then as FESEM in the process of transportation,in order to enhance the killing effect of cancer cells.This unique separate design can greatly reduce the damage to normal tissue during transport,and provide a safer method and regulatory means for enzyme-catalyzed therapy.3.Construction and application of responsive double-enzyme nanoreactorIn this part of the work,Ribonuclease A was chosen as a therapeutic protein that degrades RNA and induces apoptosis.Because RNase A has strong non-specific toxicity to normal tissues and cells,it is necessary to reversibly inhibit its activity before administration.In this part of work,4-nitrobenzene 4(4,4,5,5-tetramethyl-1,3,2-diboronic acid-2-yl)benzyl carbonate(NBC)was chosen to react with RNase A to block the protein activity Its product RNase A-NBC(RNBC)can respond to the stimulation of Reactive Oxygen Species(ROS),and in the presence of H₂O₂,RNase A can be lysed,and RNase A can be rereleased and reactivated.In order to enhance the recovery efficiency of RNase A activity,RNBC and GOx were co-loaded into ZPF to form a responsive bienzyme nanoreactor.GOx can continuously produce H₂O₂,activate RNase A,and induce cell apoptosis in glucose-dependent cancer cells.In this work,NBC was firstly synthesized and RNase A was modified to form RNBC,which blocked the activity of RNase molecule.Then,RNBC and GOx were loaded into ZPF to obtain a double enzyme nano-reactor with a diameter of about 245 nm and good dispersion and stability.Cytological tests showed that the nanoreactor could be effectively ingested by tumor cells,and the presence of GOx greatly enhanced the killing effect of cancer cells.