Inhibition Of SARS-CoV-2 Infection Through Extracellular Vesicles And The Effect Of Curcumin On Extracellular Vesicles

Virus-virus receptor interaction is the crucial step for the body to be infected.According to this strategy,the design and preparation of inhibitors have been verified in the interventional treatment of various diseases.Angiotensin converting enzyme(ACE2),as the receptor of SARS-CoV-2,its interaction with the virus is an essential step for the occurrence and development of the Corona Virus Disease 2019(COVID-19).Inhibit the combination of SARS-CoV-2 and ACE2,which can reduce the ability of the virus to infect.ACE2 is abnormally expressed in many tumor cells,and the immortalization of tumor cells makes it a suitable cell line to study the inhibition of SARS-CoV-2 infection.Based on the way SARSCoV-2 binds to ACE2,there are two strategies to design ACE2 inhibitors,including extracting cell membranes or extracellular vesicles and utilizing ACE2 on them to neutralize the virus in advance.Compared with the cell membrane,ACE2 on extracellular vesicles has a higher affinity for SARS-CoV-2.However,the current challenges include 1)Information in the form of proteins,lipids,DNA molecules,miRNAs,mRNAs,non-coding RNAs,and other forms of tumor cell-related vesicle carriers of tumor cells overexpressing ACE2 has a potential carcinogenic risk;2)SARS-CoV-2 is extremely contagious,and it is difficult to directly conduct related suppression experiments against the virus;3)It is difficult for extracellular vesicles to stay at the site of administration for a long time;4)The extraction process of extracellular vesicles is complicated and the output is low.Using this as an entry point,we constructed the genetically engineered cell overexpressing ACE2,HEK293TACE2 as the parent cell for extracting extracellular vesicles.At the same time,a pseudovirus that can bind to ACE2 was constructed as a viral template through gene transfection technology.The temperature-sensitive hydrogel is used as a carrier to load extracellular vesicles to achieve the purpose of staying at the site of administration for a long time.Besides,using curcumin to stimulate parental cells,extracellular vesicles can carry more ACE2,which can inhibit the ability of virus infection.Object:In this project,extracellular vesicles overexpressing ACE2 were extracted and loaded on a thermosensitive hydrogel as an inhibitor of SARS-CoV-2 virus infection,and curcumin stimulated parental cells to increase the ACE2 expression level of the extracellular vesicle.Method:In this project,the human embryonic kidney cell overexpressing ACE2,HEK293TACE2 has constructed as the parent cell of extracellular vesicles,and the extracellular vesicles(EVs-ACE2)has extracted by differential ultracentrifugation.After being applied to the administration site,a thin layer of gel containing EVs-ACE2 has formed,which neutralizes SARS-CoV-2 in advance to achieve the effect of inhibiting virus infection.To be able to bind to ACE2 as a simulation object,prepare a pseudovirus as a template for SARSCoV-2.Through the combination of key sites,it is hoped that an inhibitor that can effectively inhibit SARS-CoV-2 infection has been obtained.Subsequently,HEK293T-ACE2 is stimulated by curcumin,a traditional Chinese medicine component,so that the secreted extracellular vesicles carry more ACE2 and enhance the effect of extracellular vesicles.Result:1.Constructed human embryonic kidney cells overexpressing ACE2,HEK293T-ACE2,and investigated the particle size and ACE2 protein expression of its extracellular vesicles.The experimental results show that the extracellular vesicle secreted by HEK293T-ACE2 cells have a uniform particle size in the range of 40-100nm,which belongs to the category of exosomes,and EVs-ACE2 stably carries ACE2.2.A pseudo virus carrying Luciferase and expressing pTagRFP protein and Spike protein had constructed.Experimental results show that the pseudovirus can bind to the ACE2 receptor on extracellular vesicles and can use as a viral template.3.By pre-treating the pseudovirus with EVs-ACE2,the infection of the virus to the cell is reduced,then build a hydrogel drug-carrying system loaded with EVs-ACE2.when the virus invades,it can first come into contact with the hydrogel drug-carrying system,and after the neutralization of EVs-ACE2,the pseudo virus infection was blocked.4.The effect of curcumin on the physical and chemical properties of extracellular vesicles had studied.Experimental results show that the stimulation of curcumin on parental cells will enable secreted extracellular vesicles to express more ACE2.Conclusion:Extracellular vesicles carrying ACE2 can bind to the Spike protein on the pseudovirus,so that the virus loses the binding site and inhibits the virus’s ability to infect.The temperature-sensitive hydrogel carrying the extracellular vesicles can maintain a solid-state under body temperature and extend the time that EVs-ACE2 stays at the administration site.At the same time,the stimulation of curcumin,a traditional Chinese medicine component,on parent cells can make extracellular vesicles carry more ACE2.