Preparation Of DOX Brain-targeted Micelles Based On Rabies Virus-derived Peptide And Its Anti-glioma Activity

In recent years,malignant brain tumors?such as glioma?,are at a high incidence,and the incidence is increasing year by year.Drugs for the treatment of brain diseases are generally facing an unsatisfactory situation because of the low efficiency of penetrating the blood-brain barrier,and are associated with systemic toxicity and side effects.We connected RVG-15?Rabies Virus Glycoprotein-derived Peptide?to PEG-DSPE?Polyethylene glycol-Distearoyl phosphoethanolamine?and synthesized RVG-15-PEG-DSPE.We hypothesize that using RVG-15 as a carrier may solve the bottleneck problem of small molecule chemical drugs entering the brain and reduce their systemic toxicity and side effects.Firstly,doxorubicin particle micells were prepared by one-step self-assembly using brain targeting vector DSPE-PEG-RVG-15 to encapsulate doxorubicin.The size was 14.19±0.21nm with a entrapment efficiency of 94.76%,and Drug loading was14.43%.In vitro release studies suggest that the polymer is more efficient in microenvironments with lower pH of tumors than other normal environments in releasing drugs.The uptake efficiency of doxorubicin?DOX?RVG-15-PMs in glioma C6 cells was significantly higher than that in DOX solution.The uptake of DOX RVG-15-PMs by C6 cells was time-dependent.The cytotoxicity of DSPE-PEG₂₀₀₀-RVG-15 on C6 cells was inspected by CCK-8 assay and cell viabilities of all the groups were above 80%The when concentration of the sample ranged from 0.01 to 1000ng/ml in 24 hours or 48 hours after culture.Scratch test found that the order of scratch width was DOX RVG-15-PMs>DOX Solution>blank RVG-15-PMS,which approximately equals to control group.In vivo brain targeting function showed that the fluorescence intensity of mouse brain increased significantly with time after 0,4 and 24 hours of injection of DSPE-PEG-RVG-15.Twenty-five mice with glioma were randomly divided into saline group,blank RVG-15-PMs group,DOX solution group,DOX RVG-15-PMs group and control group.Except the control mice without tumors,the weight loss of DOX RVG-15-PMs group was significantly lower than that of the other three groups.On the 14th day after administration,except for the control group,brain edema in DOX-RVG-15-PMs group was significantly lower than that in the other three groups.HE staining showed that there were some vascular infiltration and metastasis in lung and liver tissues of saline group,blank RVG-15-PMs group and DOX solution group,but there was no abnormality in DOX-RVG-15-PMs group.TUNEL assay showed that most of the tumor cells in DOX RVG-15-PMs group had apoptosis,while a few apoptotic cells in DOX group,while few apoptotic cells in blank group and RVG-15-PMs group.Taken together,we found that the nanomicelles prepared by encapsulating doxorubicin with brain targeting vector DSPE-PEG₂₀₀₀-RVG-15 had no significant effect on organisms,and could effectively pass through BBB.The amount of drug released could effectively kill tumor cells and penetrate into tumor cells.