Confocal imaging to quantify passive transport across biomimetic lipid membranes.
The ability of a molecule to pass through the plasma membrane without the aid of any active cellular mechanisms is central to that molecule's pharmaceutical characteristics. Passive transport has been understood in the context of Overton's rule, which states that more lipophilic molecules cross membrane lipid bilayers more readily. Existing techniques for measuring passive transport lack reproducibility and are hampered by the presence of an unstirred layer (USL) that dominates transport across the bilayer. This report describes assays based on spinning-disk confocal microscopy (SDCM) of giant unilamellar vesicles (GUVs) that allow for the detailed investigation of passive transport processes and mechanisms. This approach allows the concentration field to be directly observed, allowing membrane permeability to be determined easily from the transient concentration profile data. A series of molecules of increasing hydrophilicity was constructed, and the transport of these molecules into GUVs was observed. The observed permeability trend is consistent with Overton's rule. However, the values measured depart from the simple partition-diffusion proportionality model of passive transport. This technique is easy to implement and has great promise as an approach to measure membrane transport. It is optimally suited to precise quantitative measurements of the dependence of passive transport on membrane properties.
Li
Su
S
Mork Family Department of Chemical Engineering and Materials Science, University of Southern California, California 90089, USA.
Hu
Peichi
P
Malmstadt
Noah
N
D009327:4-Chloro-7-nitrobenzofurazan / Q000737:chemistry; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism*; D005027:Ethylene Oxide / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D057927:Hydrophobic and Hydrophilic Interactions; D018613:Microscopy, Confocal / Q000379:methods*; D010539:Permeability; D011092:Polyethylene Glycols / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D053835:Unilamellar Liposomes / Q000378:metabolism*
D005456:Fluorescent Dyes; D053835:Unilamellar Liposomes; D011092:Polyethylene Glycols; D009327:4-Chloro-7-nitrobenzofurazan; D005027:Ethylene Oxide