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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

Screening functionalised polymersomes on the in vitro BBB models.(a) Schematic representation of the transwell system, consisting of a transwell insert with a base made of microporous membrane separating the well into upper and lower compartments. (b) Schematic representation of cells grown on both sides of transwell insert membrane coated with rat-tail collagen. (c) 3D Z-stack confocal micrograph of transwell insert membrane plus cells treated with polymersomes. (d) Micrograph sections of top/middle/bottom view of insert membrane and cells treated with A-EP polymersomes and (arrow) 3D volume viewer. (e) Fluorescence analysis of transwell insert microporous membrane and cells after different polymersome treatments. (f) Transwell permeability assay, timecourse of fluorescence from A-EP polymersomes in cell media of upper and lower compartments. Scale bars 20 μm. One-way ANOVA was used for statistical analysis for n = 3 independent experiments, p < 0.005. Error bars: SEM.
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f1: Screening functionalised polymersomes on the in vitro BBB models.(a) Schematic representation of the transwell system, consisting of a transwell insert with a base made of microporous membrane separating the well into upper and lower compartments. (b) Schematic representation of cells grown on both sides of transwell insert membrane coated with rat-tail collagen. (c) 3D Z-stack confocal micrograph of transwell insert membrane plus cells treated with polymersomes. (d) Micrograph sections of top/middle/bottom view of insert membrane and cells treated with A-EP polymersomes and (arrow) 3D volume viewer. (e) Fluorescence analysis of transwell insert microporous membrane and cells after different polymersome treatments. (f) Transwell permeability assay, timecourse of fluorescence from A-EP polymersomes in cell media of upper and lower compartments. Scale bars 20 μm. One-way ANOVA was used for statistical analysis for n = 3 independent experiments, p < 0.005. Error bars: SEM.

Mentions: Polymersomes including POEGMA-PDPA (EP), PMPC-PDPA (Supplementary Fig. 1a and 1b) and peptide-functionalised EP were prepared via a pH-switch method; this is a ‘bottom-up’ self-assembly process that can be precisely manipulated, as reported elsewhere3536. The resulting polymersomes had a mean diameter of 100 nm (Supplementary Fig. 1c) and transmission electron microscopy (TEM) studies confirmed their vesicular morphology (Supplementary Fig. 1d). Further physicochemical characteristics, and their uptake by the mouse brain endothelial cell line bEnd.3, can be found in Supplementary Fig. 1 and Supplementary Fig. 2. The most effective formulations for cellular uptake were further tested for transcytosis efficiency. To do so, we employed a 3D in vitro BBB model where brain endothelial cells were cultured on collagen-coated trans-well microporous filter inserts in which the upper compartment is connected to the lower (basolateral) compartment via 0.4 μm pores through the filter (Fig. 1a). The underside of the filter facing the lower compartment was used to culture astrocytes (mouse astrocyte cell line) and/or ‘pericytes’ (mouse mesenchymal stem cell line with pericyte-like properties) to induce a more effective BBB37. Using this 3D model, we were able to distinguish formulations that can enter brain endothelial cells via endocytosis from those that target receptors associated with transcytosis, since the latter process involves active transport across the cell layer and consequent accumulation in the microporous membrane and/or basolateral compartment (Fig. 1b).


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)

Screening functionalised polymersomes on the in vitro BBB models.(a) Schematic representation of the transwell system, consisting of a transwell insert with a base made of microporous membrane separating the well into upper and lower compartments. (b) Schematic representation of cells grown on both sides of transwell insert membrane coated with rat-tail collagen. (c) 3D Z-stack confocal micrograph of transwell insert membrane plus cells treated with polymersomes. (d) Micrograph sections of top/middle/bottom view of insert membrane and cells treated with A-EP polymersomes and (arrow) 3D volume viewer. (e) Fluorescence analysis of transwell insert microporous membrane and cells after different polymersome treatments. (f) Transwell permeability assay, timecourse of fluorescence from A-EP polymersomes in cell media of upper and lower compartments. Scale bars 20 μm. One-way ANOVA was used for statistical analysis for n = 3 independent experiments, p < 0.005. Error bars: SEM.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Screening functionalised polymersomes on the in vitro BBB models.(a) Schematic representation of the transwell system, consisting of a transwell insert with a base made of microporous membrane separating the well into upper and lower compartments. (b) Schematic representation of cells grown on both sides of transwell insert membrane coated with rat-tail collagen. (c) 3D Z-stack confocal micrograph of transwell insert membrane plus cells treated with polymersomes. (d) Micrograph sections of top/middle/bottom view of insert membrane and cells treated with A-EP polymersomes and (arrow) 3D volume viewer. (e) Fluorescence analysis of transwell insert microporous membrane and cells after different polymersome treatments. (f) Transwell permeability assay, timecourse of fluorescence from A-EP polymersomes in cell media of upper and lower compartments. Scale bars 20 μm. One-way ANOVA was used for statistical analysis for n = 3 independent experiments, p < 0.005. Error bars: SEM.
Mentions: Polymersomes including POEGMA-PDPA (EP), PMPC-PDPA (Supplementary Fig. 1a and 1b) and peptide-functionalised EP were prepared via a pH-switch method; this is a ‘bottom-up’ self-assembly process that can be precisely manipulated, as reported elsewhere3536. The resulting polymersomes had a mean diameter of 100 nm (Supplementary Fig. 1c) and transmission electron microscopy (TEM) studies confirmed their vesicular morphology (Supplementary Fig. 1d). Further physicochemical characteristics, and their uptake by the mouse brain endothelial cell line bEnd.3, can be found in Supplementary Fig. 1 and Supplementary Fig. 2. The most effective formulations for cellular uptake were further tested for transcytosis efficiency. To do so, we employed a 3D in vitro BBB model where brain endothelial cells were cultured on collagen-coated trans-well microporous filter inserts in which the upper compartment is connected to the lower (basolateral) compartment via 0.4 μm pores through the filter (Fig. 1a). The underside of the filter facing the lower compartment was used to culture astrocytes (mouse astrocyte cell line) and/or ‘pericytes’ (mouse mesenchymal stem cell line with pericyte-like properties) to induce a more effective BBB37. Using this 3D model, we were able to distinguish formulations that can enter brain endothelial cells via endocytosis from those that target receptors associated with transcytosis, since the latter process involves active transport across the cell layer and consequent accumulation in the microporous membrane and/or basolateral compartment (Fig. 1b).

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