Angiopep-2 And TAT Peptides Dual-functionalized Exosomes For Efficient And Targeted Glioma Therapy

Glioma is one of the most common types of primary intracranial brain tumor in the central nervous system,characterized by rapid and invasive growth and highly angiogenic and poor prognosis.Chemotherapy is still the most direct and effective means in glioma therapy over a long duration of time.However,due to the low blood-brain barrier(BBB)permeability,traditional chemotherapeutic drugs are cannot effectively enter the brainand kill glioma cells.Exosomes,a type of extracellular vesicle with diameters of 30～150 nm,are derived from all types of cells that carry protein,lipids,RNA and DNA.Taken the advantages including high biocompatibility,low immunogeniccity and accessible modification of membrane surface,exosomes have been developed as excellent drug carriers.The angiopep-2 is a targeted peptide that specifically binds to low-density lipoprotein receptor-related protein-1(LRP-1),which is overexpressed in both brain capillary endothelial cells and glioma cells.Trans-activator of transcription activator(TAT)peptide is an effective cell-penetrating peptide,which can highly permeate the BBB and penetrate dense tumor tissues.Doxorubicin(Dox),a highly effective anticancer drug with broad spectrum activity,has a certain effect on glioma.In the present study,Angiopep-2 and TAT peptides were modified to the membrane surface of exosomes to obtain engineered exosomes with double targeting functions,the Dox loaded engineered exosomes were then constructed for the application in targeted treatment of glioma.Firstly,we designed Ang-lamp2b-HA fusion protein and TAT-lamp2b-EGFP(EGFP,Enhanced Green Fluorescent Protein)fusion protein,Ang peptide can be specific to target LRP-1,TAT peptide can play an efficient penetrate-membrane.Then,stable Ang-lamp2b-HA and TAT-lamp2b-EGFP overexpression cell lines were obtained by infecting 293T cells with purified lentivirus particles.Exosomes with LRP-1 targeting and cell penetration were obtained by gradient centrifugation,ultrafast separation and purification after expanding culture of stable cell lines and collecting the cell culture supernatant.Through molecular biology and cell biology,it has been proved that gene engineering of cells can realize exosome modification targeting peptide and transmembrane peptide,which can effectively improve the targeting and penetration of exosome as the carrier,and also provide more possibilities and novel perspectives for exosome as the platform of drug carrier.Secondly,co-culture of engineered exosomes with U87MG glioma cells and bend.3 brain microvascular endothelial cells confirmed the efficient targeting of double-targeted exosomes to glioma and BBB,and the efficient permeability of double-targeted exosomes to BBB was validated by constructing BBB model in vitro.In addition,electroporation technology can achieve the loading of therapeutic drug Dox into exosomes,and the encapsulation rate can reach upto 20%.Drug loaded engineered exosomes can effectively deliver drugs to LRP-1 overexpressed cells and inhibit the growth of tumor cells.Thirdly,we demonstrated the efficient targeting of engineered exosomes with dual targeting functions to endothelial cells of BBB and glioma,high permeability to BBB and high penetration to glioma tissues by constructing the subcutaneous tumor and brain orthotopic tumor model in vivo.These results indicate that Ang peptide modified exosomes can play a multistage targeting function,and TAT peptide not only plays a membrane-penetrating function but also overcomes the saturation of Ang receptor to a certain extent,which further improves the targeting efficiency of engineered exosomes.Finally,electroporation technique was utilized to load Dox into the engineered exosomes with dual targeting functions.The specificity of the dual targeted drug delivery system can penetrate blood-brain barrier,target glioma and penetrate into the tumor,achieving the effective drug delivery,through the tail vein medication in situ glioma model in mice.Compared with the control group,the anti-tumor effect was increased by about 5.8 times.The drug delivery was highly efficient,thus significantly improving the survival of the glioma model mice with minimal side effects.In conclusion,the dissertation demonstrates a dual modification of exosome with targeting peptide and transmembrane peptide through engineering transformation,endowing exosomes with multistage targeting function.In addition,Dox mediated by engineered exosomes can achieve effective inhibition of glioma,revealing that engineered exosomes with dual targeting function can be used as an excellent carrier for the treatment of glioma diseases and highly targeted drug delivery,providing a unique/novel and efficient drug delivery strategy for the treatment of brain glioma diseases.