A passive diffusion model of fluorescein derivatives in an in vitro human brain microvascular endothelial cell (HBMEC) monolayer

Jamelle M Simmons,Luke Achenie,Yong Woo Lee

doi:10.21595/jme.2018.20186

Abstract

Eukaryotic cells have a protective plasma membrane, which restricts the free movement of molecules from the external environment to the internal environment. This study aims to computationally model the transport of fluorescein derivatives across the monolayer of human brain microvascular endothelial cells (HBMEC). The determination of plausible effective diffusion constants (Deff) will allow models to be built that could be useful beyond in vitro experimentation. Fluorescein-5-isothiocyanate (FITC) modeling produced a Deff range of 1E-20 to 5E-20 cm2/s at a 1 µm cell monolayer thickness and a Deff constant near 5E-29 cm2/s at a 5 µm cell monolayer thickness. Both fluorescein and sodium fluorescein (NaFl) modeling at the 1 and 5 µm thicknesses did not produce simulations that closely resembled the HBMEC in vitro model. Overall, it is possible that the fluorescent intensity noted with fluorescein and NaFl may be better explained by a mechanism other than passive diffusion. Simulations of FITC diffusion produced a narrow range of Deff constants that closely matched the in vitro HBMEC fluorescent intensity.

Highlights

Eukaryotic cells are enclosed in a plasma membrane which separates the external and internal environments that serves as protection for the cells
The aim of the present study was to create a simple approach that relies on fluorescence to determine an effective diffusion constant (D ) of FITC, fluorescein, and NaFl transport across human brain microvascular endothelial cells (HBMEC) by passive diffusion
With a 5 μm monolayer thickness there is a decrease in all time points when the effective diffusion constant (D ) is increased from 5E-19 to 5E-18 cm2/s. For both fluorescein and NaFl tracers the in vitro data showed high fluorescence compared to FITC time points with few statistically significant increases in tracer uptake over the 24-hour period

Summary

Introduction

Eukaryotic cells are enclosed in a plasma membrane which separates the external and internal environments that serves as protection for the cells. The lipid bilayer of the plasma membrane is composed of hydrophilic head groups which are directed towards the aqueous exterior and interior portions of cells and hydrophobic tails separating the hydrophilic groups. The plasma membrane is mostly composed of lipids and proteins that help it form a barrier around the cell’s internal environment [1,2,3]. Isothiocyanates (ITCs) are small molecules found in vegetables that are capable of binding to amino groups and proteins and passively diffusion through gastrointestinal epithelium and capillary endothelium [4, 5]. Phenethyl isothiocyanate (PEITC) has been shown to be absorbed into human non-small lung carcinoma (A549) cells and ITCs bind to thiol groups at a faster rate compared to amino groups under basic conditions [6,7,8,9]. Research on the cellular accumulation of ITCs have found that the uptake was dependent on intracellular levels of glutathione (GSH) and not dependent on the lipophilicity [10,11,12]

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