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Targeting choroid plexus epithelia and ventricular ependyma for drug delivery to the central nervous system.

Gonzalez AM, Leadbeater WE, Burg M, Sims K, Terasaki T, Johanson CE, Stopa EG, Eliceiri BP, Baird A - BMC Neurosci (2011)

Bottom Line: Because the choroid plexus (CP) is uniquely suited to control the composition of cerebrospinal fluid (CSF), there may be therapeutic benefits to increasing the levels of biologically active proteins in CSF to modulate central nervous system (CNS) functions.Furthermore, after i.c.v. injection into rat brains, each peptide was found to target phage to epithelial cells in CP and to ependyma lining the ventricles.These data demonstrate that ligand-mediated targeting can be used as a strategy for drug delivery to the central nervous system and opens the possibility of using the choroid plexus as a portal of entry into the brain.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Experimental Medicine and Dentistry, University of Birmingham, Edgbaston, UK.

ABSTRACT

Background: Because the choroid plexus (CP) is uniquely suited to control the composition of cerebrospinal fluid (CSF), there may be therapeutic benefits to increasing the levels of biologically active proteins in CSF to modulate central nervous system (CNS) functions. To this end, we sought to identify peptides capable of ligand-mediated targeting to CP epithelial cells reasoning that they could be exploited to deliver drugs, biotherapeutics and genes to the CNS.

Methods: A peptide library displayed on M13 bacteriophage was screened for ligands capable of internalizing into CP epithelial cells by incubating phage with CP explants for 2 hours at 37C and recovering particles with targeting capacity.

Results: Three peptides, identified after four rounds of screening, were analyzed for specific and dose dependent binding and internalization. Binding was deemed specific because internalization was prevented by co-incubation with cognate synthetic peptides. Furthermore, after i.c.v. injection into rat brains, each peptide was found to target phage to epithelial cells in CP and to ependyma lining the ventricles.

Conclusion: These data demonstrate that ligand-mediated targeting can be used as a strategy for drug delivery to the central nervous system and opens the possibility of using the choroid plexus as a portal of entry into the brain.

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Related in: MedlinePlus

Internalization and specificity of PhD-targeted peptide phage in CP cells in culture. TR-CSFB cells were incubated with untargeted or targeted phage (5 × 1010pfu/ml) for 2 h at 37C, acid washed to remove any particles on the cell surface, fixed and then immunostained with antibodies against M13 phage and subsequently Alexa594-labeled goat anti rabbit antibodies. Little internalization is observed when the cells were treated with control phage (Panel A) and while cells tended to clump together upon staining, significant intracellular immunoreactivity was nevertheless observed in TR-CSFB cells after incubation with Peptide B (Panel B), Peptide C (Panel C) or Peptide D (Panel D) targeting of phage. M13 phage: red; Cell nuclei (DAPI): white. To demonstrate that internalization was ligand-mediated, TR-CSFB cells were incubated with targeted phage (1 × 1010pfu/ml) for 2 h at 37C in the absence (Panel A) or presence of an excess (200 ng) of Peptide C (Panel E) or Peptide D (Panel F). M13 phage: red; Cell nuclei (DAPI): white.
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Figure 2: Internalization and specificity of PhD-targeted peptide phage in CP cells in culture. TR-CSFB cells were incubated with untargeted or targeted phage (5 × 1010pfu/ml) for 2 h at 37C, acid washed to remove any particles on the cell surface, fixed and then immunostained with antibodies against M13 phage and subsequently Alexa594-labeled goat anti rabbit antibodies. Little internalization is observed when the cells were treated with control phage (Panel A) and while cells tended to clump together upon staining, significant intracellular immunoreactivity was nevertheless observed in TR-CSFB cells after incubation with Peptide B (Panel B), Peptide C (Panel C) or Peptide D (Panel D) targeting of phage. M13 phage: red; Cell nuclei (DAPI): white. To demonstrate that internalization was ligand-mediated, TR-CSFB cells were incubated with targeted phage (1 × 1010pfu/ml) for 2 h at 37C in the absence (Panel A) or presence of an excess (200 ng) of Peptide C (Panel E) or Peptide D (Panel F). M13 phage: red; Cell nuclei (DAPI): white.

Mentions: To confirm epithelial cell targeting and to determine whether there existed specificity for epithelial cell subpopulations, rat choroid plexus epithelial cells from the TR-CSFB cell line were incubated with Peptide-A (control), Peptide-B, Peptide-C or Peptide-D targeted phage (5 × 1010pfu/ml) for 2 h at 37C, then acid washed to remove any particles on the cell surface, fixed and immunostained with antibodies against M13 phage (Figure 2). Binding of primary antibodies was detected using Alexa594-goat anti rabbit antibodies. No internalization is observed when the cells are treated with control PhD A phage (Panel A). Significant intracellular immunoreactivity is observed in TR-CSFB cells after incubation with PhD B (Panel B), PhD C (Panel C) or PhD D (Panel D). M13 phage, red; Cell nuclei (DAPI), white. The TR-CSFB cells were also co-incubated with excess of free Peptide-C (Panel E) or Peptide-D (Panel F) and cells were washed with 50 mM Glycine, pH 2.8, fixed and then immunostained with antibodies against M13 phage. In both instances, internalization is almost completely blocked when the targeted particles are competing for binding to cell surface receptors with an excess of ligand.


Targeting choroid plexus epithelia and ventricular ependyma for drug delivery to the central nervous system.

Gonzalez AM, Leadbeater WE, Burg M, Sims K, Terasaki T, Johanson CE, Stopa EG, Eliceiri BP, Baird A - BMC Neurosci (2011)

Internalization and specificity of PhD-targeted peptide phage in CP cells in culture. TR-CSFB cells were incubated with untargeted or targeted phage (5 × 1010pfu/ml) for 2 h at 37C, acid washed to remove any particles on the cell surface, fixed and then immunostained with antibodies against M13 phage and subsequently Alexa594-labeled goat anti rabbit antibodies. Little internalization is observed when the cells were treated with control phage (Panel A) and while cells tended to clump together upon staining, significant intracellular immunoreactivity was nevertheless observed in TR-CSFB cells after incubation with Peptide B (Panel B), Peptide C (Panel C) or Peptide D (Panel D) targeting of phage. M13 phage: red; Cell nuclei (DAPI): white. To demonstrate that internalization was ligand-mediated, TR-CSFB cells were incubated with targeted phage (1 × 1010pfu/ml) for 2 h at 37C in the absence (Panel A) or presence of an excess (200 ng) of Peptide C (Panel E) or Peptide D (Panel F). M13 phage: red; Cell nuclei (DAPI): white.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Internalization and specificity of PhD-targeted peptide phage in CP cells in culture. TR-CSFB cells were incubated with untargeted or targeted phage (5 × 1010pfu/ml) for 2 h at 37C, acid washed to remove any particles on the cell surface, fixed and then immunostained with antibodies against M13 phage and subsequently Alexa594-labeled goat anti rabbit antibodies. Little internalization is observed when the cells were treated with control phage (Panel A) and while cells tended to clump together upon staining, significant intracellular immunoreactivity was nevertheless observed in TR-CSFB cells after incubation with Peptide B (Panel B), Peptide C (Panel C) or Peptide D (Panel D) targeting of phage. M13 phage: red; Cell nuclei (DAPI): white. To demonstrate that internalization was ligand-mediated, TR-CSFB cells were incubated with targeted phage (1 × 1010pfu/ml) for 2 h at 37C in the absence (Panel A) or presence of an excess (200 ng) of Peptide C (Panel E) or Peptide D (Panel F). M13 phage: red; Cell nuclei (DAPI): white.
Mentions: To confirm epithelial cell targeting and to determine whether there existed specificity for epithelial cell subpopulations, rat choroid plexus epithelial cells from the TR-CSFB cell line were incubated with Peptide-A (control), Peptide-B, Peptide-C or Peptide-D targeted phage (5 × 1010pfu/ml) for 2 h at 37C, then acid washed to remove any particles on the cell surface, fixed and immunostained with antibodies against M13 phage (Figure 2). Binding of primary antibodies was detected using Alexa594-goat anti rabbit antibodies. No internalization is observed when the cells are treated with control PhD A phage (Panel A). Significant intracellular immunoreactivity is observed in TR-CSFB cells after incubation with PhD B (Panel B), PhD C (Panel C) or PhD D (Panel D). M13 phage, red; Cell nuclei (DAPI), white. The TR-CSFB cells were also co-incubated with excess of free Peptide-C (Panel E) or Peptide-D (Panel F) and cells were washed with 50 mM Glycine, pH 2.8, fixed and then immunostained with antibodies against M13 phage. In both instances, internalization is almost completely blocked when the targeted particles are competing for binding to cell surface receptors with an excess of ligand.

Bottom Line: Because the choroid plexus (CP) is uniquely suited to control the composition of cerebrospinal fluid (CSF), there may be therapeutic benefits to increasing the levels of biologically active proteins in CSF to modulate central nervous system (CNS) functions.Furthermore, after i.c.v. injection into rat brains, each peptide was found to target phage to epithelial cells in CP and to ependyma lining the ventricles.These data demonstrate that ligand-mediated targeting can be used as a strategy for drug delivery to the central nervous system and opens the possibility of using the choroid plexus as a portal of entry into the brain.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Experimental Medicine and Dentistry, University of Birmingham, Edgbaston, UK.

ABSTRACT

Background: Because the choroid plexus (CP) is uniquely suited to control the composition of cerebrospinal fluid (CSF), there may be therapeutic benefits to increasing the levels of biologically active proteins in CSF to modulate central nervous system (CNS) functions. To this end, we sought to identify peptides capable of ligand-mediated targeting to CP epithelial cells reasoning that they could be exploited to deliver drugs, biotherapeutics and genes to the CNS.

Methods: A peptide library displayed on M13 bacteriophage was screened for ligands capable of internalizing into CP epithelial cells by incubating phage with CP explants for 2 hours at 37C and recovering particles with targeting capacity.

Results: Three peptides, identified after four rounds of screening, were analyzed for specific and dose dependent binding and internalization. Binding was deemed specific because internalization was prevented by co-incubation with cognate synthetic peptides. Furthermore, after i.c.v. injection into rat brains, each peptide was found to target phage to epithelial cells in CP and to ependyma lining the ventricles.

Conclusion: These data demonstrate that ligand-mediated targeting can be used as a strategy for drug delivery to the central nervous system and opens the possibility of using the choroid plexus as a portal of entry into the brain.

Show MeSH
Related in: MedlinePlus