Lipid Membranes DOI: 10.1002/anie.201308990 Monitoring and Quantifying the Passive Transport of Molecules Through Patch–Clamp Suspended Real and Model Cell Membranes** Pierluca Messina, FrØdØric Lemaître, FranÅois Huet, KieuAn Ngo, Vincent Vivier, Eric LabbØ, Olivier Buriez,* and Christian Amatore* Abstract: Transport of active molecules across biological membranes is a central issue for the success of many pharmaceutical strategies. Herein, we combine the patch– clamp principle with amperometric detection for monitoring fluxes of redox-tagged molecular species across a suspended membrane patched from a macrophage. Solvent- and protein- free lipid bilayers (DPhPC, DOPC, DOPG) patched from single-wall GUV have been thoroughly investigated and the corresponding fluxes measurements quantified. The quality of the patches and their proper sealing were successfully charac- terized by electrochemical impedance spectroscopy. This procedure appears versatile and perfectly adequate to allow the investigation of transport and quantification of the trans- port properties through direct measurement of the coefficients of partition and diffusion of the compound in the membrane, thus offering insight on such important biological and pharmacological issues. T ransport of active molecules across biological membranes is a central issue for the success of many pharmaceutical strategies. However, despite this crucial importance, most approaches rely on the measurement of partition coefficients between water and hydrophobic solvent models. Further- more, this thermodynamic method is by definition blind to any kinetics. Conversely, transport through artificial mem- branes is a mature field, but most of the methods involved cannot be transposed to real cellular membranes. It is the scope of this work to establish and test the concept of a new method for investigating passive transport of molecules through real biological membranes of any drug-targeted cells. To date, the most popular approach to form suspended planar lipid bilayers remains the “painting” technique. [1] Under these conditions, the resulting artificial membrane (typically from 50 to 500 mm in diameter) may, however, incorporate solvent molecules which may alter some of the membrane’s properties. Solvent-free suspended lipid bilayers can be obtained using the Langmuir–Blodgett technique, [2] but the absence of a solvent–lipid annulus at the aperture edge destabilizes the artificial membrane. To overcome this drawback, nanometer-sized apertures have been used to paint stable suspended membranes over anodized porous alu- mina, [3] porous polycarbonate membranes, [4] and glass nano- pores. [5] These configurations are notably useful for single ion- channel recordings after ion-channel proteins are inserted across the artificial membrane. Indeed, small apertures decrease capacitance changes and allow precise measure- ments of resistive variations when ions or molecules pass across the protein channels. [5] Nevertheless, they are not well adapted to electrochemical detection of molecular species crossing the membrane considering the drastic changes incurred by the nanovolume liquid entrapped between the lipid bilayer and an electrode due to the necessity of a Faradaic process. [6] On the other hand, the painting method cannot be adapted to investigations of real cell membranes. In terms of membrane stability and electrochemical detection, the formation of lipid bilayers, suspended at glass pipettes with diameters in the range of 0.5 to 5 mm, by the tip– dip method [7] is a good compromise. However, bilayers obtained under such conditions are not always solvent-free and the access to the pipette tip is not easy, it being located under a lipid monolayer. Again, this method is not adapted for the suspension of real cell membranes. Alternatively, the patch–clamp technique (that enables the study of single or multiple ion channels in cells) [8] allows sealing a membrane detached from real cells at the tip of a glass micropipette. This technique is commonly used for electrophysiological investigations in biology, particularly for deciphering vesicular exocytotic mechanisms or character- ization of ionic channels. [8] However, to our knowledge, this approach has not been popular for investigating artificial membranes. One such investigation was reported in 1982 by Tank et al. who showed that an isolated patch of artificial membrane could be sealed onto a glass micropipette rim by ripping off a solvent-free proteoliposome in solution. [9] Furthermore, the inside-out patch mode was scarcely attempted with protein-free liposomes. Considering that proteins and cholesterol significantly increase the rigidity of cell membranes, [10] patching pure lipid liposomes remains [*] Dr. P. Messina, Dr. F. Lemaître, Prof. E. LabbØ, Dr. O. Buriez, Prof. C. Amatore Ecole Normale SupØrieure, DØpartement de Chimie UMR CNRS-ENS-UPMC 8640 “PASTEUR” 24 rue Lhomond, 75231 Paris cedex 05 (France) E-mail: olivier.buriez@ens.fr christian.amatore@ens.fr Prof. F. Huet, Dr. K.A. Ngo, Dr. V. Vivier LISE, CNRS UPR 15, UniversitØ Pierre et Marie Curie CP 133, 4 Place Jussieu, 75252 Paris Cedex 05 (France) [**] This work was supported by the CNRS (UMR 8640 and UPR 15), the French Ministry of Research, the Ecole Normale SupØrieure, UniversitØ P. et M. Curie, and the Agence Nationale de la Recherche (grant number: ANR-12-BS08-0002-01 “ELIPTIC”). We also thank Prof. D. Baigl for useful discussions about the electroformation of vesicles. Supporting information for this article (experimental section) is available on the WWW under http://dx.doi.org/10.1002/anie. 201308990. . Angewandte Communications 3192 # 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2014, 53, 3192 –3196