Abstract
We investigated the permeation of molecules across lipid membranes on an open microfluidic platform. An array of droplet pairs was created by spotting aqueous droplets, dispersed in a lipid oil solution, onto a plate with cavities surrounded by a hydrophobic substrate. Droplets in two adjacent cavities come in contact and form an artificial lipid bilayer, called a droplet interface bilayer (DIB). The method allows for monitoring permeation of fluorescently tagged compounds from a donor droplet to an acceptor droplet. A mathematical model was applied to describe the kinetics and determine the permeation coefficient. We also demonstrate that permeation kinetics can be followed over a series of droplets, all connected via DIBs. Moreover, by changing the lipid oil composition after spotting donor droplets, we were able to create asymmetric membranes that we used to mimic the asymmetry of the cellular plasma membrane. Finally, we developed a protocol to separate and extract the droplets for label-free analysis of permeating compounds by liquid chromatography–mass spectrometry. Our versatile platform has the potential to become a new tool for the screening of drug membrane permeability in the future.
Highlights
Most pharmaceutical small-molecule drugs are primarily administered orally and mainly absorbed in the small intestine.[1]As the transcellular route is the most relevant for the absorption of these drugs, assays for predicting membrane permeability play an important role during the drug discovery and development process.[2−5] A commonly used cell-free in vitro permeability testing technique is the parallel artificial membrane permeability assay (PAMPA).[3,4,6,7] In this method, two-layered multiwell plates are used to measure permeation through barriers formed between the top and bottom wells
PAMPA is suitable for predicting purely lipoidal permeation; this is not possible in cellbased assays, in which carrier-mediated and lipoidal permeation coexist.[2−4,6,7] In PAMPA, the barriers formed between the donor and acceptor wells consist of porous filters with a typical thickness of ∼10 to 100 μm soaked in a mixture of lipid and hydrocarbon oil or pure hydrocarbon oil.[3,4,7]
The barriers in PAMPA are much thicker than a lipid bilayer (∼5 nm), and their structure is dissimilar to actual biological membranes
Summary
Most pharmaceutical small-molecule drugs are primarily administered orally and mainly absorbed in the small intestine.[1]. PEG4-NBD and fluorescein permeated across the DIB (Figures S2 and S3), while no permeation was observed for calcein (Figure S4) This observation is in good agreement with the very low permeability coefficients (10−10 to 10−11 cm/s) reported for calcein permeation across liposome membranes.[37] For isolated droplets that contain one of these fluorophores but are not connected to an acceptor droplet, the fluorescence intensity is constant, and we assume to have no leakage of permeants into the oil phase. The results confirm that our method facilitates the determination of the permeation coefficient by either fluorescent microscopy or mass spectrometry
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