Abstract
The delivery of therapeutics to the central nervous system remains a major challenge in part due to the presence of the blood–brain barrier (BBB). Recently, cell-derived vesicles, particularly exosomes, have emerged as an attractive vehicle for targeting drugs to the brain, but whether or how they cross the BBB remains unclear. Here, we investigated the interactions between exosomes and brain microvascular endothelial cells (BMECs) in vitro under conditions that mimic the healthy and inflamed BBB in vivo. Transwell assays revealed that luciferase-carrying exosomes can cross a BMEC monolayer under stroke-like, inflamed conditions (TNF-α activated) but not under normal conditions. Confocal microscopy showed that exosomes are internalized by BMECs through endocytosis, co-localize with endosomes, in effect primarily utilizing the transcellular route of crossing. Together, these results indicate that cell-derived exosomes can cross the BBB model under stroke-like conditions in vitro. This study encourages further development of engineered exosomes as drug delivery vehicles or tracking tools for treating or monitoring neurological diseases.
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References
Abbott, N. J., L. Ronnback, and E. Hansson. Astrocyte-endothelial interactions at the blood–brain barrier. Nat. Rev. Neurosci. 7:41–53, 2006.
Alvarez-Erviti, L., Y. Seow, H. Yin, C. Betts, S. Lakhal, and M. J. Wood. Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nat. Biotechnol. 29:341–345, 2011.
Andreone, B. J., B. Lacoste, and C. Gu. Neuronal and vascular interactions. Annu. Rev. Neurosci. 38:25–46, 2015.
Banks, W. A. From blood–brain barrier to blood–brain interface: new opportunities for CNS drug delivery. Nat. Rev. Drug Discov. 15:275–292, 2016.
Chen, J., Z. G. Zhang, Y. Li, L. Wang, Y. X. Xu, S. C. Gautam, M. Lu, Z. Zhu, and M. Chopp. Intravenous administration of human bone marrow stromal cells induces angiogenesis in the ischemic boundary zone after stroke in rats. Circ. Res. 92:692–699, 2003.
Colombo, M., G. Raposo, and C. Thery. Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles. Annu. Rev. Cell Dev. Biol. 30:255–289, 2014.
Deli, M. A., L. Descamps, M. P. Dehouck, R. Cecchelli, F. Joo, C. S. Abraham, and G. Torpier. Exposure of tumor necrosis factor-alpha to luminal membrane of bovine brain capillary endothelial cells cocultured with astrocytes induces a delayed increase of permeability and cytoplasmic stress fiber formation of actin. J. Neurosci. Res. 41:717–726, 1995.
Dendrou, C. A., L. Fugger, and M. A. Friese. Immunopathology of multiple sclerosis. Nat. Rev. Immunol. 15:545–558, 2015.
Descamps, L., R. Cecchelli, and G. Torpier. Effects of tumor necrosis factor on receptor-mediated endocytosis and barrier functions of bovine brain capillary endothelial cell monolayers. J. Neuroimmunol. 74:173–184, 1997.
Dobson, P. D., and D. B. Kell. Carrier-mediated cellular uptake of pharmaceutical drugs: an exception or the rule? Nat. Rev. Drug Discov. 7:205–220, 2008.
Dulamea, A. O. The potential use of mesenchymal stem cells in stroke therapy-From bench to bedside. J. Neurol. Sci. 352:1–11, 2015.
El-Andaloussi, S., I. Mager, X. O. Breakefield, and M. J. Wood. Extracellular vesicles: biology and emerging therapeutic opportunities. Nat. Rev. Drug Discov. 12:347–357, 2013.
Etame, A. B., R. J. Diaz, C. A. Smith, T. G. Mainprize, K. Hynynen, and J. T. Rutka. Focused ultrasound disruption of the blood–brain barrier: a new frontier for therapeutic delivery in molecular neurooncology. Neurosurg. Focus 32:E3, 2012.
Faille, D., F. El-Assaad, A. J. Mitchell, M. C. Alessi, G. Chimini, T. Fusai, G. E. Grau, and V. Combes. Endocytosis and intracellular processing of platelet microparticles by brain endothelial cells. J. Cell Mol. Med. 16:1731–1738, 2012.
Fischer, S., M. Wiesnet, H. H. Marti, D. Renz, and W. Schaper. Simultaneous activation of several second messengers in hypoxia-induced hyperpermeability of brain derived endothelial cells. J. Cell. Physiol. 198:359–369, 2004.
Fruhbeis, C., D. Frohlich, and E. M. Kramer-Albers. Emerging roles of exosomes in neuron-glia communication. Front Physiol. 3:119, 2012.
Gabathuler, R. Approaches to transport therapeutic drugs across the blood–brain barrier to treat brain diseases. Neurobiol. Dis. 37:48–57, 2010.
Ge, Q. Y., Y. X. Zhou, J. F. Lu, Y. F. Bai, X. Y. Xie, and Z. H. Lu. miRNA in plasma exosome is stable under different storage conditions. Molecules 19:1568–1575, 2014.
Hsu, J., J. Rappaport, and S. Muro. Specific binding, uptake, and transport of ICAM-1-targeted nanocarriers across endothelial and subendothelial cell components of the blood–brain barrier. Pharm. Res. 31:1855–1866, 2014.
Imai, T., Y. Takahashi, M. Nishikawa, K. Kato, M. Morishita, T. Yamashita, A. Matsumoto, C. Charoenviriyakul, and Y. Takakura. Macrophage-dependent clearance of systemically administered B16BL6-derived exosomes from the blood circulation in mice. J. Extracell. Vesicles 4:26238, 2015.
Kalani, A., A. Tyagi, and N. Tyagi. Exosomes: mediators of neurodegeneration, neuroprotection and therapeutics. Mol. Neurobiol. 49:590–600, 2014.
Komarova, Y., and A. B. Malik. Regulation of endothelial permeability via paracellular and transcellular transport pathways. Annu. Rev. Physiol. 72:463–493, 2010.
Kourembanas, S. Exosomes: vehicles of intercellular signaling, biomarkers, and vectors of cell therapy. Annu. Rev. Physiol. 77:13–27, 2015.
Lai, C. P., O. Mardini, M. Ericsson, S. Prabhakar, C. A. Maguire, J. W. Chen, B. A. Tannous, and X. O. Breakefield. Dynamic biodistribution of extracellular vesicles in vivo using a multimodal imaging reporter. ACS Nano 8:483–494, 2014.
Lasser, C. Exosomes in diagnostic and therapeutic applications: biomarker, vaccine and RNA interference delivery vehicle. Expert Opin. Biol. Ther. 15:103–117, 2015.
Lasser, C., S. E. O’Neil, L. Ekerljung, K. Ekstrom, M. Sjostrand, and J. Lotvall. RNA-containing exosomes in human nasal secretions. Am. J. Rhinol. Allergy 25:89–93, 2011.
Lee, C. C., J. A. MacKay, J. M. Frechet, and F. C. Szoka. Designing dendrimers for biological applications. Nat. Biotechnol. 23:1517–1526, 2005.
Lee, R. H., A. A. Pulin, M. J. Seo, D. J. Kota, J. Ylostalo, B. L. Larson, L. Semprun-Prieto, P. Delafontaine, and D. J. Prockop. Intravenous hMSCs improve myocardial infarction in mice because cells embolized in lung are activated to secrete the anti-inflammatory protein TSG-6. Cell Stem Cell 5:54–63, 2009.
Liao, W., V. Pham, L. Liu, M. Riazifar, E. J. Pone, S. X. Zhang, F. Ma, M. Lu, C. M. Walsh, and W. Zhao. Mesenchymal stem cells engineered to express selectin ligands and IL-10 exert enhanced therapeutic efficacy in murine experimental autoimmune encephalomyelitis. Biomaterials 77:87–97, 2016.
Liu, L., M. A. Eckert, H. Riazifar, D. K. Kang, D. Agalliu, and W. Zhao. From blood to the brain: can systemically transplanted mesenchymal stem cells cross the blood–brain barrier? Stem Cells Int. 2013:435093, 2013.
Liu, L., S. X. Zhang, R. Aeran, W. Liao, M. Lu, G. Polovin, E. J. Pone, and W. Zhao. Exogenous marker-engineered mesenchymal stem cells detect cancer and metastases in a simple blood assay. Stem Cell Res. Ther. 6:181, 2015.
Manders, E. M. M., F. J. Verbeek, and J. A. Aten. Measurement of colocalization of objects in dual-color confocal images. J. Microsc.-Oxford. 169:375–382, 1993.
Mathivanan, S., H. Ji, and R. J. Simpson. Exosomes: extracellular organelles important in intercellular communication. J. Proteomics 73:1907–1920, 2010.
Mulcahy, L.A., R.C. Pink, and D.R. Carter. Routes and mechanisms of extracellular vesicle uptake. J. Extracell. Vesicles 3, 2014.
Obermeier, B., R. Daneman, and R. M. Ransohoff. Development, maintenance and disruption of the blood–brain barrier. Nat. Med. 19:1584–1596, 2013.
Ohno, S., M. Takanashi, K. Sudo, S. Ueda, A. Ishikawa, N. Matsuyama, K. Fujita, T. Mizutani, T. Ohgi, T. Ochiya, N. Gotoh, and M. Kuroda. Systemically injected exosomes targeted to EGFR deliver antitumor microRNA to breast cancer cells. Mol. Ther. 21:185–191, 2013.
Ostrowski, M., N. B. Carmo, S. Krumeich, I. Fanget, G. Raposo, A. Savina, C. F. Moita, K. Schauer, A. N. Hume, R. P. Freitas, B. Goud, P. Benaroch, N. Hacohen, M. Fukuda, C. Desnos, M. C. Seabra, F. Darchen, S. Amigorena, L. F. Moita, and C. Thery. Rab27a and Rab27b control different steps of the exosome secretion pathway. Nat. Cell Biol. 12:19–30, 2010; (sup pp 1-13).
Pardridge, W. M. The blood–brain barrier: bottleneck in brain drug development. NeuroRx. 2:3–14, 2005.
Pardridge, W. M. Drug transport across the blood–brain barrier. J. Cereb. Blood Flow Metab. 32:1959–1972, 2012.
Pardridge, W. M. Targeted delivery of protein and gene medicines through the blood–brain barrier. Clin. Pharmacol. Ther. 97:347–361, 2015.
Pardridge, W. M. Blood–brain barrier endogenous transporters as therapeutic targets: a new model for small molecule CNS drug discovery. Expert Opin. Ther. Targets 19:1059–1072, 2015.
Peer, D., J. M. Karp, S. Hong, O. C. Farokhzad, R. Margalit, and R. Langer. Nanocarriers as an emerging platform for cancer therapy. Nat. Nanotechnol. 2:751–760, 2007.
Petros, R. A., and J. M. DeSimone. Strategies in the design of nanoparticles for therapeutic applications. Nat. Rev. Drug. Discov. 9:615–627, 2010.
Pols, M. S., and J. Klumperman. Trafficking and function of the tetraspanin CD63. Exp. Cell Res. 315:1584–1592, 2009.
Raposo, G., and W. Stoorvogel. Extracellular vesicles: exosomes, microvesicles, and friends. J. Cell Biol. 200:373–383, 2013.
Ridder, K., S. Keller, M. Dams, A. K. Rupp, J. Schlaudraff, D. Del Turco, J. Starmann, J. Macas, D. Karpova, K. Devraj, C. Depboylu, B. Landfried, B. Arnold, K. H. Plate, G. Hoglinger, H. Sultmann, P. Altevogt, and S. Momma. Extracellular vesicle-mediated transfer of genetic information between the hematopoietic system and the brain in response to inflammation. PLoS Biol. 12:e1001874, 2014.
Rochfort, K. D., L. E. Collins, R. P. Murphy, and P. M. Cummins. Downregulation of blood–brain barrier phenotype by proinflammatory cytokines involves NADPH oxidase-dependent ROS generation: consequences for interendothelial adherens and tight junctions. PLoS One 9:e101815, 2014.
Rubin, L. L., and J. M. Staddon. The cell biology of the blood–brain barrier. Annu. Rev. Neurosci. 22:11–28, 1999.
Sandoval, K. E., and K. A. Witt. Blood–brain barrier tight junction permeability and ischemic stroke. Neurobiol. Dis. 32:200–219, 2008.
Schiera, G., C. M. Di Liegro, and I. Di Liegro. Extracellular membrane vesicles as vehicles for brain cell-to-cell interactions in physiological as well as pathological conditions. Biomed. Res. Int. 2015:152926, 2015.
Schnitzer, J. E., P. Oh, E. Pinney, and J. Allard. Filipin-sensitive caveolae-mediated transport in endothelium: reduced transcytosis, scavenger endocytosis, and capillary permeability of select macromolecules. J. Cell Biol. 127:1217–1232, 1994.
Shlosberg, D., M. Benifla, D. Kaufer, and A. Friedman. Blood–brain barrier breakdown as a therapeutic target in traumatic brain injury. Nat. Rev. Neurol. 6:393–403, 2010.
Song, J. Ischemic apoplexy-induced sequelae treated by penetrating puncture with long needles. J. Tradit. Chin. Med. 22:200–202, 2002.
Sordi, V., M. L. Malosio, F. Marchesi, A. Mercalli, R. Melzi, T. Giordano, N. Belmonte, G. Ferrari, B. E. Leone, F. Bertuzzi, G. Zerbini, P. Allavena, E. Bonifacio, and L. Piemonti. Bone marrow mesenchymal stem cells express a restricted set of functionally active chemokine receptors capable of promoting migration to pancreatic islets. Blood 106:419–427, 2005.
Svensson, K. J., H. C. Christianson, A. Wittrup, E. Bourseau-Guilmain, E. Lindqvist, L. M. Svensson, M. Morgelin, and M. Belting. Exosome uptake depends on ERK1/2-heat shock protein 27 signaling and lipid Raft-mediated endocytosis negatively regulated by caveolin-1. J. Biol. Chem. 288:17713–17724, 2013.
Takahashi, Y., M. Nishikawa, H. Shinotsuka, Y. Matsui, S. Ohara, T. Imai, and Y. Takakura. Visualization and in vivo tracking of the exosomes of murine melanoma B16-BL6 cells in mice after intravenous injection. J. Biotechnol. 165:77–84, 2013.
Takeshita, Y., B. Obermeier, A. Cotleur, Y. Sano, T. Kanda, and R. M. Ransohoff. An in vitro blood–brain barrier model combining shear stress and endothelial cell/astrocyte co-culture. J. Neurosci. Methods 232:165–172, 2014.
Tannous, B. A., D. E. Kim, J. L. Fernandez, R. Weissleder, and X. O. Breakefield. Codon-optimized Gaussia luciferase cDNA for mammalian gene expression in culture and in vivo. Mol. Ther. 11:435–443, 2005.
Thery, C., S. Amigorena, G. Raposo, and A. Clayton. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr. Protoc. Cell Biol. Chapter 3: Unit 3 22, 2006.
Thery, C., M. Ostrowski, and E. Segura. Membrane vesicles as conveyors of immune responses. Nat. Rev. Immunol. 9:581–593, 2009.
Thery, C., L. Zitvogel, and S. Amigorena. Exosomes: composition, biogenesis and function. Nat. Rev. Immunol. 2:569–579, 2002.
Thompson, A. G., E. Gray, S. M. Heman-Ackah, I. Mager, K. Talbot, S. E. Andaloussi, M. J. Wood, and M. R. Turner. Extracellular vesicles in neurodegenerative disease—pathogenesis to biomarkers. Nat. Rev. Neurol. 12:346–357, 2016.
Tian, T., Y. L. Zhu, Y. Y. Zhou, G. F. Liang, Y. Y. Wang, F. H. Hu, and Z. D. Xiao. Exosome uptake through clathrin-mediated endocytosis and macropinocytosis and mediating miR-21 delivery. J. Biol. Chem. 289:22258–22267, 2014.
Torchilin, V. P. Recent advances with liposomes as pharmaceutical carriers. Nat. Rev. Drug Discov. 4:145–160, 2005.
Torchilin, V. P. Multifunctional, stimuli-sensitive nanoparticulate systems for drug delivery. Nat. Rev. Drug Discov. 13:813–827, 2014.
Trumpp, A., M. Essers, and A. Wilson. Awakening dormant haematopoietic stem cells. Nat. Rev. Immunol. 10:201–209, 2010.
Upadhyay, R. K. Drug delivery systems, CNS protection, and the blood brain barrier. Biomed. Res. Int. 2014:869269, 2014.
Valadi, H., K. Ekstrom, A. Bossios, M. Sjostrand, J. J. Lee, and J. O. Lotvall. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat. Cell Biol. 9:654–659, 2007.
Vercauteren, D., R. E. Vandenbroucke, A. T. Jones, J. Rejman, J. Demeester, S. C. De Smedt, N. N. Sanders, and K. Braeckmans. The use of inhibitors to study endocytic pathways of gene carriers: optimization and pitfalls. Mol. Ther. 18:561–569, 2010.
Wajant, H., K. Pfizenmaier, and P. Scheurich. Tumor necrosis factor signaling. Cell Death Differ. 10:45–65, 2003.
Whalen, G. F., Y. Shing, and J. Folkman. The fate of intravenously administered bFGF and the effect of heparin. Growth Factors 1:157–164, 1989.
Wolburg, H., and A. Lippoldt. Tight junctions of the blood–brain barrier: development, composition and regulation. Vascul. Pharmacol. 38:323–337, 2002.
Xin, H., Y. Li, and M. Chopp. Exosomes/miRNAs as mediating cell-based therapy of stroke. Front. Cell Neurosci. 8:377, 2014.
Xin, H., Y. Li, Y. Cui, J. J. Yang, Z. G. Zhang, and M. Chopp. Systemic administration of exosomes released from mesenchymal stromal cells promote functional recovery and neurovascular plasticity after stroke in rats. J. Cereb. Blood Flow Metab. 33:1711–1715, 2013.
Yang, T., P. Martin, B. Fogarty, A. Brown, K. Schurman, R. Phipps, V. P. Yin, P. Lockman, and S. Bai. Exosome delivered anticancer drugs across the blood–brain barrier for brain cancer therapy in Danio rerio. Pharm. Res. 32:2003–2014, 2015.
Zhang, Y., M. Chopp, Y. Meng, M. Katakowski, H. Xin, A. Mahmood, and Y. Xiong. Effect of exosomes derived from multipluripotent mesenchymal stromal cells on functional recovery and neurovascular plasticity in rats after traumatic brain injury. J. Neurosurg. 122:856–867, 2015.
Zhuang, X., X. Xiang, W. Grizzle, D. Sun, S. Zhang, R. C. Axtell, S. Ju, J. Mu, L. Zhang, L. Steinman, D. Miller, and H. G. Zhang. Treatment of brain inflammatory diseases by delivering exosome encapsulated anti-inflammatory drugs from the nasal region to the brain. Mol. Ther. 19:1769–1779, 2011.
Acknowledgments
We thank Dr. W. Liao and M. Lu for discussion and technique training. We thank C. Deighan from Malvern Instruments for assistance with the use of NanoSight NS300 instrument for NTA analysis. This work was supported by the National Institute of Health (1DP2CA195763-01), American Heart Association (13BGIA17140099) and UC Irvine Academic Senate Council on Research, Computing, and Libraries (CORCL) Research Grant (SIIG-2013-2014-25). CCC was supported by National Science Foundation Graduate Research Fellowship (NSF GRFP). SXZ was supported by a Cardiovascular Applied Research and Entrepreneurship fellowship (NIH/NHLBI T32).
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The authors (Claire C. Chen, Linan Liu, Fengxia Ma, Chi W. Wong, Xuning E. Guo, Jenu V. Chacko, Henry P. Farhoodi, Shirley X. Zhang, Jan Zimak, Aude Ségaliny, Milad Riazifa, Victor Pham, Michelle A. Digman, Egest J. Pone, and Weian Zhao) declare that they have no conflicts of interest.
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Claire C. Chen and Linan Liu contributed equally to this work.
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Chen, C.C., Liu, L., Ma, F. et al. Elucidation of Exosome Migration Across the Blood–Brain Barrier Model In Vitro . Cel. Mol. Bioeng. 9, 509–529 (2016). https://doi.org/10.1007/s12195-016-0458-3
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DOI: https://doi.org/10.1007/s12195-016-0458-3