Diffusion of small molecules between intra- and extra-cellular spaces across lipid layers is instrumental to the understanding of fundamental biological transport processes and the improvement in drug delivery schemes. In natural systems, organisms develop intricate ion channels and stimuli-responsive gating machineries for signal transduction, cell identification, and toxin removal, while still allowing the selective and proficient uptake of vital nutrients that are critical to survival, growth, and reproduction. Understanding membrane function is a colossal step in developing cutting-edge drug therapies in a rational way. Lipinski’s rule of five describes the molecular properties essential for the bioavailability and pharmacokinetics of candidate drugs inside human bodies and thus serves as a first-order consideration for drug design.Altering the length of an alkyl tail is thus a viable method to tune the lipophilicity of a compound, thus modulating the transport properties of the drug.

In this presentation, we report on our efforts to interrogate the “flip-flop” diffusion mechanism of alkyl proton carriers traversing the lipid layer of a hybrid bilayer membrane (HBM) using electrochemical techniques including but not limited to electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), and pulse techniques. The 4-nm thick HBM electrochemical platform consists of a lipid monolayer appended on top of a self-assembled monolayer (SAM) containing a dinuclear Cu-triazole complex for O₂ reduction reaction (ORR) on a Au electrode prepared using electron-beam evaporation (Scheme 1a). The “flip-flop” diffusion rates of the proton carriers dictate the O₂ reduction turnover frequency by the Cu-based electrocatalyst. By varying both the tail lengths of the proton carriers and the lipids, we find that a difference between the lipid tail lengths and the proton carrier tail lengths of approximately 6-10 Å results in a maximum “flip-flop” diffusion rate.

We evaluate these empirical findings using a biophysical model that contains parameters considering changes in Coulombic interactions, stress buildup, surface tension, and dielectric constants across the lipid-water interface. The model correctly mimics the relative alterations in the energy barriers associated with the rate-determining step (RDS) for the “flip-flop” diffusion process (Scheme 1b). Studies with a rigid proton carrier further substantiate findings that the RDS involves the bending of the alkyl tail of the proton carrier as it moves across the hydrophobic interior of the lipid layer. We envision that the methodologies developed here will ultimately lead to improved understanding of the mechanismof “flip-flop” diffusion in lipid bilayers and aid in the development of next-generation targeted drug delivery schemes.

Acknowledgements. E.C.M.T. acknowledges a Croucher Foundation Scholarship. We thank the US Department of Energy (DE-FG02-95ER46260) for support of this research.

Scheme 1 is presented here.