Construction Of Multiple Hepatitis B Virus Core Protein Nanocage Targeted Drug Delivery System And Its Anti-tumor Application

In recent years,with the application of nanoparticle-based drug carriers,chemotherapeutic drugs have higher safety and effectiveness.Among them,virus like particles(VLPs),as a kind of protein nanocage structure with stable structure,good biocompatibility and easy functional modification,have received extensive attention in the field of biomedicine.So far,research on VLPs has focused on the field of vaccines,using their immunogenicity as an antigen presentation carrier,and a small number of people use it as a drug delivery carrier.When VLPs are used as drug carriers,their strong immunogenicity is no longer an advantage,but will hinder delivery efficiency.Based on this question,human hepatitis B virus core protein(HBc),woodchuck hepatitis B virus core protein(WHBc)and duck hepatitis B virus core protein particles(DHBc)were used in combination as a drug delivery carrier,hoping to reduce or even avoid the influence of the body’s immunity on the virus-like particle delivery carrier.he main work of this subject is as follows:First,using genetic engineering technology to functionally modify VLPs from three different sources,insert RGD peptides with tumor-targeting function on the surface of protein particles,and after later strain screening and protein purification,three target proteins are obtained:RGD-HBc,RGD-WHBc and RGD-DHBc.Through TEM and DLS analysis,the three VLPs have a complete structure,with a particle size between 24-30 nm and good particle uniformity.Secondly,this topic explored the humoral immunogenicity of the three VLPs and wild-type human-derived hepatitis B virus core protein(wtHBc)in Balb/c mice.The antibody titer level between the mouse serum and the homologous immune protein was detected by ELISA.The results showed that wtHBc can cause the strongest humoral immunity,followed by RGD-DHBc,RGD-WHBc and RGD-HBc.The antibody titer levels between mouse serum and non-homologous immune proteins were detected by ELISA,and the results showed that the three genetically modified VLPs of RGD-HBc,RGD-WHBc and RGD-DHBc were compared with non-homologous immune mice.There is no significant cross-immunity between sera.This result provides an important theoretical basis for the subject’s subsequent combined application of VLPs as carriers in anti-tumor application.Next,using the principle that VLPs can undergo depolymerization and recombination under certain conditions,this project constructed three virus-like particle nano-drug delivery systems containing the chemotherapy drug doxorubicin,DOX@RGD-HBc,DOX@RGD-WHBc and DOX@RGD-DHBc.By testing the morphology and particle size of VLPs before and after drug loading,it is proved that VLPs have the potential as drug delivery vehicles.Finally,the subject also explored the anti-tumor effects of three drug-loaded viruslike particle nano-delivery systems.Through in vitro cell experiments,it is proved that virus-like particles as a drug-carrying system can be well taken up by cells.The MTT experiment proved that the three drug-loaded VLPs delivery systems can achieve the same or even better anti-cell proliferation effect than the free DOX treatment group under the same drug concentration.In the in vivo anti-tumor experiment,the anti-tumor effects of DOX@RGD-HBc monotherapy and multiple DOX@VLPs combined treatment groups were compared by using different administration methods to mice with ectopic tumors.The tumor growth curve shows that the DOX@VLPs combined treatment group has a better anti-tumor effect than the other treatment group.The weight change of the mice during the treatment and the H&E staining results o showed that the virus-like particle nano-delivery carrier has high biological safety.