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APTS-labeled dextran ladder: a novel tool to characterize cell layer tightness
Winfried Neuhaus1, Joanna Trzeciak, Regina Lauer
1Department of Medicinal/Pharmaceutical Chemistry, University of Vienna, Pharmacy Center, Althanstrasse 14, A-1090 Vienna, Austria.
Journal of Pharmaceutical and Biomedical Analysis
|October 26, 2005
Summary
A new APTS-dextran ladder system precisely measures cell layer tightness and blood-brain barrier permeability for various molecular sizes. This advanced method offers a more accurate assessment of paracellular transport and leakiness than existing techniques.
Area of Science:
- Biomedical Engineering
- Pharmacology
- Cell Biology
Background:
- Accurate characterization of cell layer tightness is crucial for understanding barrier functions, particularly the blood-brain barrier (BBB).
- Existing methods for assessing barrier permeability may lack precision or the ability to analyze a wide range of molecular sizes simultaneously.
Purpose of the Study:
- To develop a precise and manageable analytical system for evaluating cell layer tightness in a molecular size-dependent manner.
- To create a novel APTS-dextran ladder for characterizing paracellular transport and BBB leakiness.
Main Methods:
- Labeling dextrans with fluorescent 1-aminopyrene-3,6,8-trisulfonate (APTS) to create a molecular ladder.
- Utilizing a transwell BBB model with porcine brain microvascular endothelial cells (PBMEC/C1-2).
- Analyzing sample permeability using fluorescence measurements, capillary electrophoresis (CE), and reversed-phase liquid chromatography (RP-LC).
Main Results:
- Successfully generated an APTS-dextran ladder encompassing sizes from APTS to dextran with 31 glucose units.
- Calculated permeability coefficients, demonstrating a decrease in permeability with increasing dextran size (e.g., APTS: 16.38±3.79 µm/min, APTS-dextran (31 units): 6.07±1.23 µm/min).
- Internal standard diazepam showed higher permeability (67.97±7.32 µm/min).
Conclusions:
- The developed APTS-dextran ladder is a valuable tool for assessing cell layer tightness and BBB leakiness.
- This system enables simultaneous evaluation of permeability across a broad spectrum of molecular sizes.
- It offers a more refined approach compared to traditional methods like TEER, sucrose, or Evans blue assays for characterizing paracellular pathways.