Metal-Organic Framework-Based Confined Cascade Catalytic Nanoreactor For Alcoholism Therapy

Alcoholism and the resulting liver damage have become a non-negligible threat to human health,resulting in 6%of all deaths worldwide,and have been recognized as a global health priority by the World Health Organization(WHO).Current clinical treatments for alcoholism mainly play a supportive role;the degradation and metabolism of alcohol still rely on the body’s own ethanol dehydrogenase(ADH)and acetaldehyde dehydrogenase(ALDH).However,the low expression of ADH and ALDH in hepatocytes cannot rapidly eliminate the alcohol that invades the body;moreover,the diffusion of toxic intermediates(acetaldehyde,reactive oxygen species,etc.)during alcohol metabolism and the large consumption of intracellular energy metabolism cofactors(NAD+)can cause structural and functional damage to the liver.Therefore,it is urgent to develop a method to rapidly degrade alcohol and mitigate the continued deterioration of alcoholic liver injury.Inspired by the ethanol oxidation system(which consumes oxygen to degrade ethanol),ethanol oxidase(AOx)-based multi-enzyme confined cascade catalytic system can effectively reduce the consumption of NAD+,significantly improve the catalytic efficiency of the cascade reaction and avoid the diffusion of toxic intermediates,which is an effective strategy for the treatment of alcoholism.In addition,with the continuous development of nanotechnology,metal-organic frameworks(MOFs)have been widely used as enzyme immobilization carriers for the construction of domain-limited cascade systems by virtue of their porous structure and enzyme-like activity.Based on the above considerations,in this thesis,a domain-limited cascade nanoreactor(named AA@mMOF)was synthesized by assembling AOx and ALDH in the pore channels of mesoporous metal-organic frameworks(mMOF)with peroxidase-like(CAT)activity and used for the treatment of acute alcoholism and alleviating the deterioration of chronic alcoholic liver injury.The main research contents and findings are as follows:1.Metal-organic framework NH2-MIL-101(Fe)was synthesized by hydrothermal method and its pore size was broadened by hydrochloric acid etching.Subsequently,AA@mMOF nanoreactors were successfully synthesized by coupling AOx,ALDH and PEG molecules to mMOF using glutaraldehyde as a coupling agent and the covalent reaction of aldehyde and amino groups.The material characterization results demonstrated the successful synthesis of AA@mMOF nanoreactors with good water solubility and stability.Fluorescence experiments demonstrated that AOx and ALDH were successfully coupled in mMOF in close spatial proximity.In addition,mMOF can effectively protect AOx and ALDH from environmental inactivation.2.Based on the successful synthesis of AA@mMOF,its in vitro alcohol degradation ability,biocompatibility and ability to protect cells from alcohol were evaluated.Firstly,it was verified that AA@mMOF can rapidly degrade alcohol while avoiding the massive accumulation of acetaldehyde and hydrogen peroxide through in vitro catalytic assays.The results of cytotoxicity assays demonstrated that AA@mMOF has good biocompatibility.In vitro cellular uptake and detoxification experiments demonstrated that hepatocytes could uptake AA@mMOF into cells through various uptake pathways and avoid lysosomal degradation,which effectively alleviated the oxidative stress of cells in the alcohol environment and significantly improved the cell survival rate.3.Based on AA@mMOF’s good in vitro alcohol degradation ability,excellent biocompatibility and ability to protect cells from alcohol,we constructed a mouse model of acute alcoholism and a model of chronic alcoholic liver injury to evaluate the ability of AA@mMOF nanoreactors to treat alcoholism in vivo,and also investigated its vivo biocompatibility and metabolic pathways.The experimental results showed that AA@mMOF could rapidly reduce the concentration of ethanol and acetaldehyde in their blood with its efficient alcohol degradation ability and liver targeting,which effectively reduced the intoxication time of mice.The reduction of alcohol concentration effectively relieved the metabolic stress of the liver,reduced the oxidative stress state of the liver,inhibited the accumulation of lipids in the liver sites,and finally alleviated the further deterioration of alcoholic liver injury.In addition,the potential in vivo degradation ability of AA@mMOF makes it not stay in the body for a long time,and it can be metabolized out of the body gradually without causing serious damage to organs,which has good in vivo biocompatibility.In summary,inspired by the ethanol oxidation system and the domain-limited cascade catalytic system,we successfully prepared nanoreactors that can rapidly remove alcohol and its metabolites,and achieved excellent therapeutic effects for alcoholism in vivo.The results of this thesis provide new ideas and effective references for the design and development of multienzyme catalytic systems that can be used for the treatment of alcoholism and alcoholic liver injury diseases.