Effects of Ion Concentration and Headgroup Chemistry on Thin Lipid Film Drainage.

While the use of lipid nanoparticles in drug delivery applications has grown over the past few decades, much work remains to be done toward the characterization and rational design of the drug carriers. A key feature of delivery is the interaction of the exterior leaflet of the LNP with the outer leaflet of the cell membrane, which relies in part on the fusogenicity of the lipids and the ionic environment. In this paper, we study the interactions between two lipid monolayers using a thin film balance to create lipid thin films and interferometry to measure film evolution. We probe the role of lipid headgroup chemistry and charge, along with ionic solution conditions, in either promoting or hindering film drainage and stability. Specific headgroups phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and phosphatidylserine (PS) are chosen to represent a combination of charge and fusogenicity. We quantify each film's drainage characteristics over a range of capillary numbers. Qualitatively, we find that films transition from drainage via a large dimple to drainage via channels and vortices as the capillary number increases. Additionally, we observe a transition from electrostatically dominated film drainage at low CaCl2 concentrations to fusogenic-dominated film drainage at higher CaCl2 concentrations for anionic fusogenic (PS) films. Understanding the role of headgroup composition, ionic composition, and ionic concentration will pave the way for the design of tunable vesicle and buffer systems that behave desirably across a range of ex vivo and in vivo environments.

Determining the Bending Rigidity of Free-Standing Planar Phospholipid Bilayers.

We describe a method to determine membrane bending rigidity from capacitance measurements on large area, free-standing, planar, biomembranes. The bending rigidity of lipid membranes is an important biological mechanical property that is commonly optically measured in vesicles, but difficult to quantify in a planar, unsupported system. To accomplish this, we simultaneously image and apply an electric potential to free-standing, millimeter area, planar lipid bilayers composed of DOPC and DOPG phospholipids to measure the membrane Young's (elasticity) modulus. The bilayer is then modeled as two adjacent thin elastic films to calculate bending rigidity from the electromechanical response of the membrane to the applied field. Using DOPC, we show that bending rigidities determined by this approach are in good agreement with the existing work using neutron spin echo on vesicles, atomic force spectroscopy on supported lipid bilayers, and micropipette aspiration of giant unilamellar vesicles. We study the effect of asymmetric calcium concentration on symmetric DOPC and DOPG membranes and quantify the resulting changes in bending rigidity. This platform offers the ability to create planar bilayers of controlled lipid composition and aqueous ionic environment, with the ability to asymmetrically alter both. We aim to leverage this high degree of compositional and environmental control, along with the capacity to measure physical properties, in the study of various biological processes in the future.

Fabrication and electromechanical characterization of freestanding asymmetric membranes.

All biological cell membranes maintain an electric transmembrane potential of around 100 mV, due in part to an asymmetric distribution of charged phospholipids across the membrane. This asymmetry is crucial to cell health and physiological processes such as intracell signaling, receptor-mediated endocytosis, and membrane protein function. Experimental artificial membrane systems incorporate essential cell membrane structures, such as the phospholipid bilayer, in a controllable manner in which specific properties and processes can be isolated and examined. Here, we describe an approach to fabricate and characterize planar, freestanding, asymmetric membranes and use it to examine the effect of headgroup charge on membrane stiffness. The approach relies on a thin film balance used to form a freestanding membrane by adsorbing aqueous phase lipid vesicles to an oil-water interface and subsequently thinning the oil to form a bilayer. We validate this lipid-in-aqueous approach by analyzing the thickness and compressibility of symmetric membranes with varying zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and anionic 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) sodium salt (DOPG) content as compared with previous lipid-in-oil methods. We find that as the concentration of DOPG increases, membranes become thicker and stiffer. Asymmetric membranes are fabricated by controlling the lipid vesicle composition in the aqueous reservoirs on either side of the oil. Membrane compositional asymmetry is qualitatively demonstrated using a fluorescence quenching assay and quantitatively characterized through voltage-dependent capacitance measurements. Stable asymmetric membranes with DOPC on one side and DOPC-DOPG mixtures on the other were created with transmembrane potentials ranging from 15 to 80 mV. Introducing membrane charge asymmetry decreases both the thickness and stiffness in comparison with symmetric membranes with the same overall phospholipid composition. These initial successes demonstrate a viable pathway to quantitatively characterize asymmetric bilayers that can be extended to accommodate more complex membranes and membrane processes in the future.