Co-Delivery Of Doxorubicin And Temozolomide Using HBc Virus-like Particles For Synergistic Glioma Therapy

Glioblastoma is a malignant tumor of the central nervous system.Its incidence in the normal population is 0.002%.The survival rate of patients is very low.The 10 year survival rate after diagnosis is only 2.8%.Current treatment methods mainly include surgery,chemotherapy,and radiotherapy.Due to the existence of the blood-brain barrier and the blood-brain tumor barrier,most of the chemotherapeutic drugs are restricted from reaching the tumor site,so it is difficult for the drug treatment to achieve the desired therapeutic effect,and the toxic and side effects and systemic distribution of most drugs further limit The clinical efficacy of chemotherapy drugs.In recent years,the application of drug carriers such as nanometers has provided prospects for the clinical application of chemotherapy drugs.Among them,virus-like particles(VLPs)have received extensive attention in the field of biomedicine due to their advantages such as good structural stability,high absorption efficiency,and good biocompatibility.So far,most studies have applied it to the field of vaccines,and only a few people have tried to apply it to cancer chemotherapy.VLPs are nanostructures,which have the ability to encapsulate or attach proteins,nucleic acids or other small molecules.At the same time,they can be modified and attached to various targeting carriers so that they can be specifically targeted to cells,tissues or organs,thereby increasing The specificity of the drug and reduce systemic side effects.This project uses genetic engineering methods to modify the target group of hepatitis B virus core protein virus-like particles to obtain tumor-targeting RGD-HBc VLPs and brain-targeting TGN-HBc VLPs.The hybrid recombination allows the nanocarriers to have dual targeting and better reach the tumor site.This article mainly does the following parts:First,the relevant plasmids of virus-like particles to be used are introduced into the E.coli prokaryotic system for expression and purification.After expanded culture and induced expression of E.coli,1.5L bacterial solution can obtain virus-like particles with a content of up to 20-30 mg,and after preliminary purification and later purification,the purity of the protein is higher.Through the TEM and DLS particle size characterization of virus-like particles,it can be seen that the VLPs are complete in shape and uniform in size.The measured particle size of TGN VLPs is 28.61 nm,and the particle size of RGD VLPs is 26.69 nm.Secondly,a nano drug system containing two chemotherapeutic drugs(adriamycin and temozolomide)was constructed,and the morphological changes and particle size of VLPs before and after drug loading were characterized.DOX@TGN/RGD VLPs particle size It is 29.33 nm;TMZ@TGN/RGD VLPs particle size is 27.37 nm.The structure of VLPs before and after drug loading is relatively stable,with almost no change in morphology and particle size.DOX@TGN/RGD VLPs and TMZ@TGN/RGD VLPs contained DOX and TMZ with drug loadings of 28.87%and 1.72%,respectively.Thirdly,in vitro anti-tumor research was conducted on the successfully constructed nano-pharmaceutical system.In the concentration range of blank VLPs of 50-500 mg/mL,the cell viability is above 85%,which can prove that the VLPs as a drug carrier have good biocompatibility.The MTT results of drug-loaded VLPs showed that the DOX@TGN/RGD VLPs+TMZ@TGN/RGD VLPs group in the co-administered group and the DOX@TGN/RGD VLPs group in the single-administered group were better than the free DOX.Group,and when the concentration of DOX is in the range of 0.001-0.1 μg/mL,the synergistic administration group is more effective in inhibiting tumor viability than the single administration group,proving that the nano-drug system has more anti-tumor cell viability than free drugs.Better and combined drugs have better ability to inhibit the survival viability of tumor cells.The results of subsequent laser confocal experiments show that the nanocarriers can be taken up by the cells at 1 h,and most of the carriers have entered the cytoplasm at 6 h.Finally,in vivo anti-tumor properties of nano-pharmaceutical systems were studied.After successfully constructing an in situ glioma model in nude mice,the targeting of blank TGN/RGD VLPs labeled with Cy5.5 was studied to verify that the dual targeting vector has a strong targeting ability for brain tumors;anti-tumor The experiment showed that after 10 days of administration,the tumor growth inhibition rate of the DOX@TGN/RGD VLPs+TMZ@TGN/RGD VLPs group in the targeted administration group could reach 91.65%,and the therapeutic effect was significantly better than other groups.