Pharmaceutical Research

, Volume 23, Issue 7, pp 1491–1501

A Novel Tool to Characterize Paracellular Transport: The APTS–Dextran Ladder

  • Winfried Neuhaus
  • Elisabeth Bogner
  • Michael Wirth
  • Joanna Trzeciak
  • Bodo Lachmann
  • Franz Gabor
  • Christian R. Noe
Research Paper

DOI: 10.1007/s11095-006-0256-z

Cite this article as:
Neuhaus, W., Bogner, E., Wirth, M. et al. Pharm Res (2006) 23: 1491. doi:10.1007/s11095-006-0256-z

Purpose

The aim of this work was to develop an easy, manageable, and precise analytic tool to describe the tightness of cell layers by a molecular weight ladder.

Methods

Dextrans were labeled by reductive amination with fluorescent 8-aminopyrene-1,3,6-trisulfonate (APTS). This mixture, including the internal standard diazepam, was used for transport studies in Transwell models using Caco-2, ECV304, and PBMEC/C1–2 cell lines. Samples were analyzed by fluorimetry, capillary electrophoresis, and reverse-phase high-performance liquid chromatography.

Results

Following this approach, a logarithm correlation of R2 = 0.8958 between transepithelial electrical resistance (TEER) and APTS–dextran permeability was shown. In addition, a TEER-dependent permeability pattern could be observed including each single fraction from free APTS, APTS–glucose up to APTS–dextran consisting of 35 glucose units. The TEER-independent permeability coefficients of diazepam and confocal laser scanning microscopy images confirmed the paracellular transport of APTS–dextran.

Conclusions

All in all, the developed APTS–dextran ladder is a useful tool to characterize cell layer tightness and especially to describe paracellular transport ways and the extent of leakiness of cell layers (for blood–brain barrier or intestinal studies) over time—applying a wide array from smaller to larger molecules at the same time to refine TEER, sucrose, or Evans blue measurements.

Key words

APTS–dextranBBBblood–brain barriercell layer tightnessparacellular transport

Abbreviations

ACM

astrocyte conditioned medium

APTS

8-aminopyrene-1,3,6-trisulfonate

BBB

blood–brain barrier

BMECs

brain microvascular endothelial cells

CLSM

confocal laser scanning microscopy

FD

FITC dextran

FITC

fluorescein isothiocyanate

LIF detector

laser-induced fluorescence detector

P-gP

P-glycoprotein

TEER

transendothelial electrical resistance for studies with cell lines ECV304 and PBMEC/C1–2, transepithelial electrical resistance in case of Caco-2

vWF

von Willebrand factor

γ-GT

γ-glutamyltransferase

Copyright information

© Springer Science + Business Media, Inc. 2006

Authors and Affiliations

  • Winfried Neuhaus
    • 1
  • Elisabeth Bogner
    • 2
  • Michael Wirth
    • 2
  • Joanna Trzeciak
    • 1
  • Bodo Lachmann
    • 1
  • Franz Gabor
    • 2
  • Christian R. Noe
    • 1
  1. 1.Department of Medicinal ChemistryUniversity of Vienna, Pharmacy CenterViennaAustria
  2. 2.Department of Pharmaceutical Technology and BiopharmaceuticsUniversity of Vienna, Pharmacy CenterViennaAustria