Preparation Of Novel Self-Assembly Materials For Cytosolic Protein Delivery

In recent years,with the development of cell engineering,gene engineering and enzyme engineering,biomacromolecule drugs have become a new hotspot in drug research and development.Compared with small molecule chemical drugs,biomacromolecules have many advantages,such as precise target specificity,good biocompatibility,extremely low side effects and high biological activity.However,drug biomacromolecules are easy to degrade and inactivate,and their membrane permeability is very poor,which limits their further development and application in clinical trials.In order to solve these difficult problems,this paper designed and synthesized several new carrier materials,which can deliver biomacromolecular drugs into cells efficiently,and then achieve effective treatment of common malignant tumors at the animal level.The main contents are as follows:(1)Multiarm amphiphilic cyclodextrin(CDEH)was synthesized from?-cyclodextrin(?-CD)as the backbone and the secondary amine group modified long alkyl chain as the side chain.CDEH can self assemble into nanoparticles in aqueous solution.It only needs to be mixed with protein solution without introducing any subsequent steps that may lead to protein inactivation,so it can achieve high efficiency of protein encapsulation(encapsulation ratio>30%,w/w).More importantly,CDEH nanocarriers can be modified with various target groups by host guest package,so we designed two kinds of targeted CDEH nanocarriers for experiments.First,Saporin(SA)was used as therapeutic protein,(CDEH-AP)CDEH modified by AS1411 aptamer(AP)can not only efficiently deliver saporin into MDA-MB-231 breast cancer cells in vitro,lead to apoptosis of a large number of cancer cells.But also,in vivo experiments of MDA-MB-231 breast cancer model mice,CDEH-AP/SA can gather in tumor tissues and inhibit the growth of tumor cells efficiently.Second,Folate(FA)targeting CDEH nanocarrier(CDEH-FA)can also codeliver Cas9 nuclease and sg RNA targeting polo like kinase(Plk1)gene into cervical cancer cells.In vitro He La cell experiment,CDEH-FA/Cas9/sg RNA(CFCs)can lead to 47.1%Plk1 Gene knockout.In vivo experiment of He La xenograft nude mice model,it can cause 64.1%Plk1 protein down-regulation in tumor tissue,which greatly limits the the proliferation of cervical cancer cells.Therefore,these experimental results show that targeted CDEH nanocarriers can promote the delivery efficiency of proteins in the cytoplasm,which is very helpful to improve the therapeutic effect of protein drugs.(2)Based on the different chiral properties of L-arginine,we have constructed two small molecular compounds,L-arginine modified decan-1-ol(LC10A)and D-arginine modified decan-1-ol(DC10A).In order to play an active role in the immunotherapy of tumor,we applied these two small molecular compounds to the efficient delivery of antigen protein in dendritic cells(DCs)in vitro and in vivo.The results showed that LC10A or DC10A were assembled into two different nanovaccines(LC10A+OVA+Cp G,DC10A+OVA+Cp G)with ovalbumin(OVA)and unmethylated cytosine phosphate guanine(Cp G)in aqueous solution,respectively.Compared with DC10A+OVA+Cp G,LC10A+OVA+Cp G can promote the uptake of antigen presenting cells(APCs),the production of cytokines and the maturation of DCs.In addition,the nanovaccine LC10A+OVA+Cp G can stimulate the strong immune response in C57BL/6 mice,improve the content of various cytokines and corresponding antibodies(anti-OVA)in the blood,induce them to produce more CD8~+T cells,thus preventing the appearance of E.G7-OVA lymphoma in vivo and prolonging the survival time of mice.More importantly,LC10A+OVA+Cp G combined with Immunocheckpoint blocker(anti-PD1)has a significant effect on the established E.G7-OVA tumor model in mice.These results show that the small molecular nanocarriers designed based on arginine can promote the delivery of intracellular antigen protein,and provide a great prospect for immunotherapy of tumor.