Biomimetic Nanovesicles As Drug Delivery Systems For Targeted Therapy Of Glioblastoma

Background and objective :Glioblastoma multiforme(GBM)is the most common malignant brain tumor with bleak prognosis and limited clinical treatment options.Chemotherapy is effective for tumor therapy,but for GBM,very few chemotherapeutics can breach the blood-brain barrier(BBB)to reach effective therapeutic concentrations at the tumor site,leading to limited options for chemotherapy.Nano-drug delivery systems are emerging as effective strategies for enhancing the ability of drugs to cross the BBB and improving the therapeutic effect.Exosomes are effective mediators of intercellular communication and also excellent natural carriers for drug delivery.Exosomes exhibit intrinsic tissue or cell-targeting properties owing to their particular surface structures like tetraspanins and integrins.However,natural exosomes have many disadvantages,such as difficulty in extraction and purification,high cost,low yield,high heterogeneity and low drug encapsulation efficiency.Therefore,developing biomimetic exosomes as a substitute for natural exosomes is an effective strategy to solve the above problems.To this end,we firstly extracted and purified exosomes secreted from b End.3 cells as a model,and investigated their stability when stored under different conditions.Then,we used a top-down approach to produce biomimetic nanovesicles(BNV)by extruding cells through filters of different pore sizes.BNV would have similar characterization and BBBcrossing ability and drug-loading capacity to natural exosomes,with scalable production possibility for targeted brain drug delivery for GBM therapy.To develop biomimetic exosomes with higher stability,targeting ability,drug-loading capacity and encapsulation efficiency,we used liposomes as basic nanocarriers and modified angiopep-2(Ang)on the surface of liposomes,for the similarity between the morphology and structure of liposomes and exosomes.Also,the membrane protein of U87-MG cells which expressed high level of CD63,CD9,and CD47 were extracted and chimerized on the liposome by extrusion to develop angmodified exosome-mimetics(Ang-EM).Ang-EM is supposed to have improved stability and biocompatibility.Furthermore,Ang-EM can escape macrophage phagocytosis,reduce the protein corona effect and cross the BBB,thus targeting GBM efficiently.Encapsulation of the poorly soluble chemotherapeutic drug docetaxel can effectively treat glioblastoma and reduce systemic toxicity.Methods:Firstly,the exosomes secreted by b End.3 cells were extracted by ultracentrifugation and characterized by NTA,TEM,and Western-Blot.Fresh exosomes were stored at 4°C,-20°C,and-80°C.Particle size and number were measured by NTA after storage.Furthermore,the effect of freeze-thawing circles on the quantity of Exo from-20°C,-80°C,or liquid nitrogen to 4°C was assessed and compared via NTA.Changes in total protein and RNA levels in Exo after preservation for a certain time were evaluated by the BCA method and RNA extraction kit method.Labelling Exo with PKH67 or Di R,cellular uptake of exosomes by b End.3 cells and in vivo biodistribution in nude mice after storage at different time points were investigated at different temperatures.b End.3 cells were continuously extruded through 10,5,1,0.4 μm filters and ultracentrifuged to obtain BNVs.Their similarities to Exo were characterized by NTA,TEM and Western-Blot,and the yields of the two were compared by quantitative protein amount and particle number.The uptake and lysosomal escape by U87-MG cells and b End.3 cells were investigated by labeling exosomes or BNV with PKH67,and then adding different inhibitors and quantifying the fluorescence in cells by flow cytometry to investigate the uptake mechanism.Doxorubicin(DOX)was encapsulated into exosomes and BNVs by ultrasound,then the drug loading efficiency,in vitro release behavior and cytotoxicity of Exo/DOX and BNV/DOX were compared.BBB-crossing ability and cell viability of Exo and BNV were also investigated.Next,the BBB-crossing ability and tumor-targeting ability of Exo/DOX and BNV/DOX were evaluated in zebrafish and orthotopic nude mice GBM models.Besides,the antitumor effects of Exo/DOX and BNV/DOX were evaluated in various GBM models.The tumor-killing efficiency and in vivo safety of Exo/DOX and BNV/DOX were further measured by histopathological staining,TUNEL staining,serum biochemistry,and cardiac ultrasound.Lipo,Ang-Lipo,and Ang-EM were developed by the thin layer evaporation(TLE)followed by the extrusion method.DLS,TEM,and Western-blot were applied to investigate the similarity of Ang-EM and Exo,evaluating the biocompatibility and short-term stability.By labeling Lipo,Ang-Lipo and Ang-EM with Di L,the uptake and BBB-crossing ability were investigated.Docetaxel was encapsulated in Lipo,Ang-Lipo and Ang-EM,and the in vitro release efficiency was measured.The in vitro tumor-killing ability was investigated by CCK8 assay,cell cycle,and 3D tumor spheroids experiments.Lipo,Ang-Lipo and Ang-EM were incubated with fetal bovine serum respectively and then isolated by ultracentrifugation.DLS,TEM and BCA assay were used to measure the particle size,morphology and absorbed proteins level respectively.PAGE gel staining and proteomics were performed to investigate the influence of the protein corona effect on the carriers.Cellular uptake and macrophage phagocytosis before and after protein corona formation were obtained by fluorescence microscope.The orthotopic GBM mice model was established to investigate the in vivo targeting,tumor tissue penetration and antitumor efficacy.The tumorkilling ability and in vivo safety were further verified by HE staining,TUNEL staining and serum biochemistry.Results:The Exo secreted by b End.3 cells showed an average particle size of119 nm,with vesicle morphology under TEM.Exo expressed protein markers including CD63,Tsg101 and Alix.For all storage temperatures,cellular uptake and in vivo distribution would be influenced by the increase in storage time.Also,freeze-thawing cycles decreased the number of particles significantly.Stored at 4℃,the total protein or total RNA substantially dropped.However,the effect is not significant within 14 days at-20°C or-80°C.When Exo was stored at 4℃,-20℃ and-80℃,the cellular uptake and brain distribution of Exo decreased significantly during storage.However,when stored at-80°C for no more than 7 days,the cellular uptake and distribution of Exo in the brain did not decrease significantly.The b End.3 cell biomimetic nanovesicles(BNV)had an average particle size of 141 nm.The image showed similar morphology of Exo and BNV.Also,western blot showed similar levels of protein markers tsg101,CD9 and CD63 in Exos and BNVs,but the yield of BNVs was c 500-fold compared to cell-derived Exo.Exo and BNV had similar encapsulation efficiency to DOX,and the particle size did not increase significantly after encapsulation.Moreover,the cumulative release rate of Exo/DOX and BNV/DOX was similar,72 h cumulative release rate was 51.41±3.98% and46.23±2.12%,respectively.Exo and BNV could both cross the BBB and were of good tumortargeting capability.To be more specific,Exo was mainly taken up by b End.3 through caveolin-mediated endocytosis and BNV was taken up through macropinocytosis,while Exo and BNV were taken up by U87-MG cells through clathrin and caveolin-mediated endocytosis.Despite different endocytosis mechanisms,both Exos and BNVs showed the ability for lysosomal escape in b End.3 cells and U87-MG cells for intracellular cargo delivery.Also,Exo/DOX and BNV/DOX showed superior therapeutic effects of GBM and could significantly reduce the cardiotoxicity of DOX.Lipo,Ang-Lipo and Ang-EM showed average particle sizes of 127.1nm,128.2 nm and 94.1 nm,and Zeta potentials of-37.7 m V,-31.2 m V and-28.7 m V,respectively.Exo secreted by U87-MG cells had an average particle size of 111.2 nm.Ang-EM had a similar particle size and morphology to Exo.Also,Ang-EM expressed CD63 and CD9 proteins and had good short-term storage stability.Compared with Lipo and Ang-Lipo,Ang-EM showed better tumor cell uptake and BBB-crossing ability.After loading with DTX,they showed a promising ability to kill tumor cells.After incubation with serum,Lipo and Ang-Lipo adsorbed more proteins in terms of types and quantities and the particle size increased significantly.Moreover,a large number of protein impurities around the particles were observed by TEM.However,Ang-EM adsorbed fewer proteins in terms of types and quantities,therefore the increase in particle size was slight.Also,the cellular uptake by tumor cells was less affected before and after the formation of the protein corona.With CD47 on the surface of Ang-EM,the phagocytosis by macrophages was significantly prohibited compared with Lipo and Ang-Lipo.Ang-EM showed better GBM targeting and tumor tissue penetration in orthotopic GBM mice models.Ang-EM loaded with DTX suppressed GBM progression,showing superior therapeutic effects.Conclusion:1.The stability of Exo is poor.Freshly isolated Exo is recommended to be used for experiments as soon as possible.For short-term storage,Exo can be stored at 4 °C for particle size analysis.For content analysis and biological analysis,Exo is suggested to be stored at-80°C,avoiding freezing and thawing.2.BNV produced by continuous extrusions of b End.3 cells have similar particle size,morphology and protein expression as Exo,but a nearly 500-fold increase in yield.BNV/DOX and Exo/DOX showed similar drug-loading capacity,BBB-crossing ability,GBM-targeting ability and antitumor effects in various GBM models.Also,Exos and BNVs were both delivery vehicles for effectively reducing the cardiotoxicity of DOX.b End.3 cells-derived BNV provides a promising and scalable nanocarrier for targeted brain drug delivery for GBM therapy.3.The multifunctional exosome-mimetics Ang-EM constructed by integrating cell membrane proteins on the surface of targeted liposomes had similar particle size,morphology and protein expression as the Exo secreted by U87-MG cells,with increased stability and prominent drugloading capacity of insoluble drug.Protein corona formation had a minor influence on the properties of Ang-EM when exposed to the biological environment.Ang-EM reduced phagocytosis of macrophage and had superior BBB-crossing ability and GBM-targeting capability compared to Exos.Ang-EM provides a promising delivery platform for targeted brain drug delivery and GBM therapy.There are 70 figures,7 tables and 163 references in this thesis.