Nanoscopic lipid domains determined by microscopy and neutron scattering.

Biological membranes are highly complex supramolecular assemblies, which play central roles in biology. However, their complexity makes them challenging to study their nanoscale structures. To overcome this challenge, model membranes assembled using reduced sets of membrane-associated biomolecules have been found to be both excellent and tractable proxies for biological membranes. Due to their relative simplicity, they have been studied using a range of biophysical characterization techniques. In this review article, we will briefly detail the use of fluorescence and electron microscopies, and X-ray and neutron scattering techniques used over the past few decades to study the nanostructure of biological membranes.

Cations Control Lipid Bilayer Memcapacitance Associated with Long-Term Potentiation.

Phospholipid bilayers can be described as capacitors whose capacitance per unit area (specific capacitance, Cm) is determined by their thickness and dielectric constant─independent of applied voltage. It is also widely assumed that the Cm of membranes can be treated as a "biological constant". Recently, using droplet interface bilayers (DIBs), it was shown that zwitterionic phosphatidylcholine (PC) lipid bilayers can act as voltage-dependent, nonlinear memory capacitors, or memcapacitors. When exposed to an electrical "training" stimulation protocol, capacitive energy storage in lipid membranes was enhanced in the form of long-term potentiation (LTP), which enables biological learning and long-term memory. LTP was the result of membrane restructuring and the progressive asymmetric distribution of ions across the lipid bilayer during training, which is analogous, for example, to exponential capacitive energy harvesting from self-powered nanogenerators. Here, we describe how LTP could be produced from a membrane that is continuously pumped into a nonequilibrium steady state, altering its dielectric properties. During this time, the membrane undergoes static and dynamic changes that are fed back to the system's potential energy, ultimately resulting in a membrane whose modified molecular structure supports long-term memory storage and LTP. We also show that LTP is very sensitive to different salts (KCl, NaCl, LiCl, and TmCl3), with LiCl and TmCl3 having the most profound effect in depressing LTP, relative to KCl. This effect is related to how the different cations interact with the bilayer zwitterionic PC lipid headgroups primarily through electric-field-induced changes to the statistically averaged orientations of water dipoles at the bilayer headgroup interface.

Integrated laser ablation-dropletProbe-mass spectrometry for absolute drug quantitation, metabolite detection, and distribution in tissue.

RATIONALE: Spatially resolved and accurate quantitation of drug-related compounds in tissue is a much-needed capability in drug discovery research. Here, application of an integrated laser ablation-dropletProbe-mass spectrometry surface sampling system (LADP-MS) is reported, which achieved absolute quantitation of propranolol measured from <500 × 500 µm thin tissue samples. METHODS: Mouse liver and kidney thin tissue sections were coated with parylene C and analyzed for propranolol by a laser ablation/liquid extraction workflow. Non-coated adjacent sections were microdissected for validation and processed using standard bulk tissue extraction protocols. High-performance liquid chromatography with positive ion mode electrospray ionization tandem mass spectrometry was applied to detect the drug and its metabolites. RESULTS: Absolute propranolol concentration in ~500 × 500 µm tissue regions measured by the two methods agreed within ±8% and had a relative standard deviation within ±17%. Quantitation down to ~400 × 400 µm tissue regions was shown, and this resolution was also used for automated mapping of propranolol and phase II hydroxypropranolol glucuronide metabolites in kidney tissue. CONCLUSIONS: This study exemplifies the capabilities of integrated laser ablation-dropletProbe-mass spectrometry (LADP-MS) for high resolution absolute drug quantitation analysis of thin tissue sections. This capability will be valuable for applications needing to quantitatively understand the spatial distribution of small molecules in tissue.

Absolute quantitation of propranolol from 200-µm regions of mouse brain and liver thin tissues using laser ablation-dropletProbe-mass spectrometry.

RATIONALE: The ability to quantify drugs and metabolites in tissue with sub-mm resolution is a challenging but much needed capability in pharmaceutical research. To fill this void, a novel surface sampling approach combining laser ablation with the commercial dropletProbe automated liquid surface sampling system (LA-dropletProbe) was developed and is presented here. METHODS: Parylene C-coated 200 × 200 µm tissue regions of mouse brain and kidney thin tissue sections were analyzed for propranolol by laser ablation of tissue directly into a preformed liquid junction. Propranolol was detected by high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) in positive electrospray ionization mode. Quantitation was achieved via application of a stable-isotope-labeled internal standard and an external calibration curve. RESULTS: The absolute concentrations of propranolol determined from 200 × 200 µm tissue regions were compared with the propranolol concentrations obtained from 2.3-mm-diameter tissue punches of adjacent, non-coated sections using standard bulk tissue extraction protocols followed by regular HPLC/MS/MS analysis. The average concentration of propranolol in both organs determined by the two employed methods agreed to within ±12%. Furthermore, the relative abundances of phase II hydroxypropranolol glucuronide metabolites were recorded and found to be consistent with previous results. CONCLUSIONS: This work illustrates that depositing a thin layer of parylene C onto thin tissue prior to analysis, which seals the surface and prevents direct liquid extraction of the drug from the tissue, coupled to the novel LA-dropletProbe surface sampling system is a viable approach for sub-mm resolution quantitative drug distribution analysis.

Peptide-Induced Lipid Flip-Flop in Asymmetric Liposomes Measured by Small Angle Neutron Scattering.

Despite the prevalence of lipid transbilayer asymmetry in natural plasma membranes, most biomimetic model membranes studied are symmetric. Recent advances have helped to overcome the difficulties in preparing asymmetric liposomes in vitro, allowing for the examination of a larger set of relevant biophysical questions. Here, we investigate the stability of asymmetric bilayers by measuring lipid flip-flop with time-resolved small-angle neutron scattering (SANS). Asymmetric large unilamellar vesicles with inner bilayer leaflets containing predominantly 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and outer leaflets composed mainly of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) displayed slow spontaneous flip-flop at 37 ◦C (half-time, t1/2 = 140 h). However, inclusion of peptides, namely, gramicidin, alamethicin, melittin, or pHLIP (i.e., pH-low insertion peptide), accelerated lipid flip-flop. For three of these peptides (i.e., pHLIP, alamethicin, and melittin), each of which was added externally to preformed asymmetric vesicles, we observed a completely scrambled bilayer in less than 2 h. Gramicidin, on the other hand, was preincorporated during the formation of the asymmetric liposomes and showed a time resolvable 8-fold increase in the rate of lipid asymmetry loss. These results point to a membrane surface-related (e.g., adsorption/insertion) event as the primary driver of lipid scrambling in the asymmetric model membranes of this study. We discuss the implications of membrane peptide binding, conformation, and insertion on lipid asymmetry.