Purpose: Being natural bilayer lipid particles secreted by brain endothelial cells, which are major cells composing the blood-brain barrier (BBB), exosomes present advantages of biocompatibility, stability, and BBB penetrability. These unique features contribute to a perspective nanocarrier for the brain delivery of therapeutic agents. The overall objective is to reprogram exosomes as a new therapeutic protein-enriched and productive nanoplatform. These are expected to overcome the limitations of naïve exosomes with the low amount of functional molecules and inadequate production and secretion rates. Methods: Brain-derived neurotrophic factor (BDNF) gene was transfected in mouse brain endothelial bEND.3 cells by using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas 9) activation plasmids in antibiotic-free growth media. The transfected cells were treated with exosome-depleted FBS media including different concentrations of BDNF, tumor necrosis factor α (TN-α), hydrogen peroxide, and glutathione, respectively. Exosome production media were collected 72 hours after the treatment. Cells were detached with trypsin and counted to calculate exosome secretion production per million cells. Exosomes were isolated from cell culture media using an ultracentrifuge method. Exosome particle size, morphology, surface marker, and BDNF protein were analyzed. Brain uptakes of fluorescence-labeled exosomes were assessed in living mice under an IVIS Spectrum animal imaging system. A scratching assay was used to evaluate the neuronal cell growth effect of BDNF-engineered exosomes [Exo/BDNF(+)] in a human neuroblastoma SH-SY5Y cell line. Exo/BDNF(+) protection on the integrity of the BBB transwell monolayer was tested by measuring transendothelial electrical resistance (TEER) and recovering tight junctional protein levels of Zonula occludens‑1 (ZO‑1) and Claudin-5 after 4 hours of oxygen‑glucose deprivation/ reoxygenation (OGD/R). Results: BDNF gene was successfully transfected in bEND.3 cells by using CRISPR/Cas 9 activation plasmid systems. MTT assay confirmed the formulation had no toxic effect on cell viability. BDNF expression was significantly increased in the transfected cells. The treatment of cells with growth factors robustly increased exosome production by 6.1±0.5 times. BDNF transfected cells-secreted exosomes [Exo/BDNF(+)] showed round-shaped morphologies. The particle size of exosomes was about 50 nm in diameter and there was no significant difference between naïve and programmed exosomes (p >0.05). Both exosomes expressed high levels of transmembrane protein CD63, as determined by both Western blot and ELISA. The level of BDNF in Exo/BDNF(+) was 499±50 pg/mg (quantified by the total protein in exosomes), significantly higher compared to naïve exosomes (p < 0.05). A significant increase in fluorescence intensity was observed at about 0.25 hours in the brain after IV injection in mice with an elimination half-life of 18.6±2.8 hours. The scratching assay showed that the motility invasion of SH-SY5Y neuronal cells was significantly increased following the treatments with Exo/BDNF(+) (397.9±43.7%) when compared to BDNF control (241.6±44.2%). Exo/BDNF(+) treatment (30 ng/mL) significantly recovered decreased TEER and tight junction ZO‑1 and Claudin‑5 proteins induced by OGD/R (p < 0.05). Conclusion: Our preliminary data confirmed that CRISPR/Cas 9 plasmid transfected the gene in the bEND.3 cells and then protein was highly bio-encapsulated in the transfected cell-secreted exosomes during the biogenesis. BDNF use best promotes exosome secretion compared to inflammation, oxidative stress, and antioxidants. The methodology and research discussed are directed toward addressing unsolved questions and characterizing novel exosomes that serve as a feasible nanocarrier for loading and delivering therapeutic protein in the brain.
