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LRP-1-mediated intracellular antibody delivery to the Central Nervous System.

Tian X, Nyberg S, S Sharp P, Madsen J, Daneshpour N, Armes SP, Berwick J, Azzouz M, Shaw P, Abbott NJ, Battaglia G - Sci Rep (2015)

Bottom Line: We show that LRP-1 is associated with endothelial transcytosis that does not involve acidification of cargo in membrane-trafficking organelles.By contrast, this receptor is also associated with traditional endocytosis in CNS cells, thus aiding the delivery of relevant cargo within their cytosol.We prove this using IgG as a model cargo, thus demonstrating that the combination of appropriate targeting combined with pH-sensitive polymersomes enables the efficient delivery of macromolecules into CNS cells.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Chemistry, University College London, London, UK [2] The MRC/UCL Centre for Medical Molecular Virology, University College London, London, UK.

ABSTRACT
The blood-brain barrier (BBB) is by far the most important target in developing new approaches to improve delivery of drugs and diagnostic tools into the Central Nervous System (CNS). Here we report the engineering of pH- sensitive polymersomes (synthetic vesicles formed by amphiphilic copolymers) that exploit endogenous transport mechanisms to traverse the BBB, enabling delivery of large macromolecules into both the CNS parenchyma and CNS cells. We achieve this by targeting the Low Density Lipoprotein Receptor-Related Protein 1 (LRP-1) receptor. We show that LRP-1 is associated with endothelial transcytosis that does not involve acidification of cargo in membrane-trafficking organelles. By contrast, this receptor is also associated with traditional endocytosis in CNS cells, thus aiding the delivery of relevant cargo within their cytosol. We prove this using IgG as a model cargo, thus demonstrating that the combination of appropriate targeting combined with pH-sensitive polymersomes enables the efficient delivery of macromolecules into CNS cells.

No MeSH data available.


Related in: MedlinePlus

In vitro BBB model assessment with A-EP polymersomes.(a) bEnd.3 monolayer on transwell insert treated with A-EP polymersomes (3D z-stack and top/middle/bottom section micrographs). (b) bEnd.3 co-cultured with pericytes on transwell insert treated with A-EP polymersomes (3D z-stack and top/middle/bottom section micrographs). Scale bars 20 μm.
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f2: In vitro BBB model assessment with A-EP polymersomes.(a) bEnd.3 monolayer on transwell insert treated with A-EP polymersomes (3D z-stack and top/middle/bottom section micrographs). (b) bEnd.3 co-cultured with pericytes on transwell insert treated with A-EP polymersomes (3D z-stack and top/middle/bottom section micrographs). Scale bars 20 μm.

Mentions: Quantification of A-EP fluorescence in the culture media of both compartments revealed rapid apical-to-basolateral compartment transport, which was markedly faster in the presence of cells relative to the cell-free control (Fig. 1f). Most of the polymersome signal in the upper compartment had disappeared by 60 minutes, suggesting that polymersome transport occurs via an active process. The signal from the lower compartment slowly reached a plateau in the astrocyte co-culture; this may indicate intracellular uptake by the astrocytes, rather than the polymersomes moving freely into the lower compartment medium as observed the endothelial cell monoculture. With counter-staining of the 3D model for endothelial tight junction protein Zonula Occludens 1 (ZO-1) as an indicator of barrier formation and integrity, A-EP was found to penetrate the endothelial monolayer across its whole surface (Fig. 2a). In fact, transcytosis was still observed after enhancing the tightness of our 3D model by the introduction of pericytes38, as confirmed by the increased TEER and positive expression of PDGFR-β/CD140 marker (Fig. 2b and Supplementary Fig. 6) hence unlikely to be via paracellular transport through the tight junctions. Finally, a ‘reverse’ model with endothelial cells on the underside of the transwell filter (pericytes on top, α-SMA, smooth muscle actin) also showed effective transcytosis (Supplementary Fig. 7). Taken together, these data indicate the transcellular movement of polymersomes across a tight endothelial layer via an active transport mechanism, rather than passive diffusion.


LRP-1-mediated intracellular antibody delivery to the Central Nervous System.

Tian X, Nyberg S, S Sharp P, Madsen J, Daneshpour N, Armes SP, Berwick J, Azzouz M, Shaw P, Abbott NJ, Battaglia G - Sci Rep (2015)

In vitro BBB model assessment with A-EP polymersomes.(a) bEnd.3 monolayer on transwell insert treated with A-EP polymersomes (3D z-stack and top/middle/bottom section micrographs). (b) bEnd.3 co-cultured with pericytes on transwell insert treated with A-EP polymersomes (3D z-stack and top/middle/bottom section micrographs). Scale bars 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4507173&req=5

f2: In vitro BBB model assessment with A-EP polymersomes.(a) bEnd.3 monolayer on transwell insert treated with A-EP polymersomes (3D z-stack and top/middle/bottom section micrographs). (b) bEnd.3 co-cultured with pericytes on transwell insert treated with A-EP polymersomes (3D z-stack and top/middle/bottom section micrographs). Scale bars 20 μm.
Mentions: Quantification of A-EP fluorescence in the culture media of both compartments revealed rapid apical-to-basolateral compartment transport, which was markedly faster in the presence of cells relative to the cell-free control (Fig. 1f). Most of the polymersome signal in the upper compartment had disappeared by 60 minutes, suggesting that polymersome transport occurs via an active process. The signal from the lower compartment slowly reached a plateau in the astrocyte co-culture; this may indicate intracellular uptake by the astrocytes, rather than the polymersomes moving freely into the lower compartment medium as observed the endothelial cell monoculture. With counter-staining of the 3D model for endothelial tight junction protein Zonula Occludens 1 (ZO-1) as an indicator of barrier formation and integrity, A-EP was found to penetrate the endothelial monolayer across its whole surface (Fig. 2a). In fact, transcytosis was still observed after enhancing the tightness of our 3D model by the introduction of pericytes38, as confirmed by the increased TEER and positive expression of PDGFR-β/CD140 marker (Fig. 2b and Supplementary Fig. 6) hence unlikely to be via paracellular transport through the tight junctions. Finally, a ‘reverse’ model with endothelial cells on the underside of the transwell filter (pericytes on top, α-SMA, smooth muscle actin) also showed effective transcytosis (Supplementary Fig. 7). Taken together, these data indicate the transcellular movement of polymersomes across a tight endothelial layer via an active transport mechanism, rather than passive diffusion.

Bottom Line: We show that LRP-1 is associated with endothelial transcytosis that does not involve acidification of cargo in membrane-trafficking organelles.By contrast, this receptor is also associated with traditional endocytosis in CNS cells, thus aiding the delivery of relevant cargo within their cytosol.We prove this using IgG as a model cargo, thus demonstrating that the combination of appropriate targeting combined with pH-sensitive polymersomes enables the efficient delivery of macromolecules into CNS cells.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Chemistry, University College London, London, UK [2] The MRC/UCL Centre for Medical Molecular Virology, University College London, London, UK.

ABSTRACT
The blood-brain barrier (BBB) is by far the most important target in developing new approaches to improve delivery of drugs and diagnostic tools into the Central Nervous System (CNS). Here we report the engineering of pH- sensitive polymersomes (synthetic vesicles formed by amphiphilic copolymers) that exploit endogenous transport mechanisms to traverse the BBB, enabling delivery of large macromolecules into both the CNS parenchyma and CNS cells. We achieve this by targeting the Low Density Lipoprotein Receptor-Related Protein 1 (LRP-1) receptor. We show that LRP-1 is associated with endothelial transcytosis that does not involve acidification of cargo in membrane-trafficking organelles. By contrast, this receptor is also associated with traditional endocytosis in CNS cells, thus aiding the delivery of relevant cargo within their cytosol. We prove this using IgG as a model cargo, thus demonstrating that the combination of appropriate targeting combined with pH-sensitive polymersomes enables the efficient delivery of macromolecules into CNS cells.

No MeSH data available.


Related in: MedlinePlus