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      Elucidation of Exosome Migration across the Blood-Brain Barrier Model In Vitro

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          Abstract

          The delivery of therapeutics to the central nervous system (CNS) 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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          Most cited references36

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          Dynamic biodistribution of extracellular vesicles in vivo using a multimodal imaging reporter.

          Extracellular vesicles (EVs) are nanosized vesicles released by normal and diseased cells as a novel form of intercellular communication and can serve as an effective therapeutic vehicle for genes and drugs. Yet, much remains unknown about the in vivo properties of EVs such as tissue distribution, blood levels, and urine clearance, important parameters that will define their therapeutic effectiveness and potential toxicity. Here we combined Gaussia luciferase and metabolic biotinylation to create a sensitive EV reporter (EV-GlucB) for multimodal imaging in vivo, as well as monitoring of EV levels in the organs and biofluids ex vivo after administration of EVs. Bioluminescence and fluorescence-mediated tomography imaging on mice displayed a predominant localization of intravenously administered EVs in the spleen followed by the liver. Monitoring EV signal in the organs, blood, and urine further revealed that the EVs first undergo a rapid distribution phase followed by a longer elimination phase via hepatic and renal routes within six hours, which are both faster than previously reported using dye-labeled EVs. Moreover, we demonstrate systemically injected EVs can be delivered to tumor sites within an hour following injection. Altogether, we show the EVs are dynamically processed in vivo with accurate spatiotemporal resolution and target a number of normal organs as well as tumors with implications for disease pathology and therapeutic design.
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            Macrophage-dependent clearance of systemically administered B16BL6-derived exosomes from the blood circulation in mice

            Previous studies using B16BL6-derived exosomes labelled with gLuc–lactadherin (gLuc-LA), a fusion protein of Gaussia luciferase (a reporter protein) and lactadherin (an exosome-tropic protein), showed that the exosomes quickly disappeared from the systemic circulation after intravenous injection in mice. In the present study, the mechanism of rapid clearance of intravenously injected B16BL6 exosomes was investigated. gLuc-LA-labelled exosomes were obtained from supernatant of B16BL6 cells after transfection with a plasmid DNA encoding gLuc-LA. Labelling was stable when the exosomes were incubated in serum. By using B16BL6 exosomes labelled with PKH26, a lipophilic fluorescent dye, it was demonstrated that PKH26-labelled B16BL6 exosomes were taken up by macrophages in the liver and spleen but not in the lung, while PKH26-labelled exosomes were taken up by the endothelial cells in the lung. Subsequently, gLuc-LA-labelled B16BL6 exosomes were injected into macrophage-depleted mice prepared by injection with clodronate-containing liposomes. The clearance of the intravenously injected B16BL6 exosomes from the blood circulation was much slower in macrophage-depleted mice than that in untreated mice. These results indicate that macrophages play important roles in the clearance of intravenously injected B16BL6 exosomes from the systemic circulation.
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              Approaches to transport therapeutic drugs across the blood-brain barrier to treat brain diseases.

              The central nervous system is protected by barriers which control the entry of compounds into the brain, thereby regulating brain homeostasis. The blood-brain barrier, formed by the endothelial cells of the brain capillaries, restricts access to brain cells of blood-borne compounds and facilitates nutrients essential for normal metabolism to reach brain cells. This very tight regulation of the brain homeostasis results in the inability of some small and large therapeutic compounds to cross the blood-brain barrier (BBB). Therefore, various strategies are being developed to enhance the amount and concentration of therapeutic compounds in the brain. In this review, we will address the different approaches used to increase the transport of therapeutics from blood into the brain parenchyma. We will mainly concentrate on the physiologic approach which takes advantage of specific receptors already expressed on the capillary endothelial cells forming the BBB and necessary for the survival of brain cells. Among all the approaches used for increasing brain delivery of therapeutics, the most accepted method is the use of the physiological approach which takes advantage of the transcytosis capacity of specific receptors expressed at the BBB. The low density lipoprotein receptor related protein (LRP) is the most adapted for such use with the engineered peptide compound (EPiC) platform incorporating the Angiopep peptide in new therapeutics the most advanced with promising data in the clinic.
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                Author and article information

                Journal
                101468590
                36341
                Cell Mol Bioeng
                Cell Mol Bioeng
                Cellular and molecular bioengineering
                1865-5025
                1865-5033
                13 July 2016
                7 July 2016
                December 2016
                01 December 2017
                : 9
                : 4
                : 509-529
                Affiliations
                [1 ]Department of Pharmaceutical Sciences, Sue and Bill Gross Stem Cell Research Center, Chao Family Comprehensive Cancer Center and Edwards Life sciences Center for Advanced Cardiovascular Technology, 845 Health Sciences Road, University of California-Irvine, Irvine, California, 92697, USA
                [2 ]Department of Biomedical Engineering, University of California-Irvine, Irvine, California, 92697, USA
                [3 ]State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin 300020, China
                [4 ]Department of Molecular Biology & Biochemistry, University of California-Irvine, Irvine, California, 92697, USA
                [5 ]Laboratory for Fluorescence Dynamics, University of California-Irvine, California 92697, USA
                [6 ]Centre for Bioactive Discovery in Health and Ageing, School of Science and Technology, University of New England, Armidale, New South Wales 2351, Australia
                Author notes
                Address correspondence to Weian Zhao, Department of Pharmaceutical Sciences, 845 Health Sciences Road, University of California-Irvine, Irvine, California, 92697, USA. weianz@ 123456uci.edu . Contact telephone number: +01 (949) 824-9744
                [7]

                These authors contributed equally to this work.

                Article
                NIHMS801559
                10.1007/s12195-016-0458-3
                5382965
                28392840
                c04eb06b-3071-4934-bbd9-e05c35e23f3a

                OPEN ACESS. This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.

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                Categories
                Article

                Biomedical engineering
                drug delivery,blood-brain barrier (bbb),exosome,humanized gaussia luciferase (hgluc),stroke,inflammation,endocytosis,exocytosis,transcytosis

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