Development of Polymer Gel-Supported Lipid Bilayer Using Capillary-Assisted Assembly

Abstract

The modern view of the plasma membrane is that of a complex, highly dynamic, compartmentalized system that critically impacts multiple important cellular functions. Supported model membranes of well-defined compositions have emerged as attractive experimental platforms to determine the underlying molecular processes that regulate membrane-associated cellular functions using advanced biophysical detection methods with up to single molecule resolution. However, membrane properties of previously employed supported membrane systems, such as solid-supported lipid bilayer (SLB) and polymer-supported lipid bilayer with a polymer layer thickness of several nm, were found to be perturbed by the nearby solid substrate. To overcome this limitation, the present work describes for the first time the capillary-assisted formation of a lipid bilayer (CA-PGB) on the surface of a fully hydrated, several micrometers thick polyacrylamide gel. CA-PGB formation can be accomplished by physisorption or specific chemical linkages (tethering) between polymer gel and bilayer. Not dissimilar to conditions found in plasma membranes, membrane properties of CA-PGB are found to be solely influenced by the attached polymer layer. The successful formation and lipid fluidity of CA-PGB is confirmed using confocal microscopy and fluorescence correlation spectroscopy (FCS). Lipid bilayer spreading on the hydrogel surface by capillary-assisted assembly is not altered when the polymer gel stiffness or bilayer bending stiffness are varied, illustrating the robustness and versatility of the assembly process. This work also shows that, unlike other supported membrane systems, the capillary-assisted assembly approach causes the formation of a lipid reservoir at the edge of the capillary. This lipid reservoir provides a lipid supply for the CA-PGB, enabling bilayer self-healing and superior bilayer stability relative to SLB. Experimental data are presented that support an assembly process, in which bilayer spreading on the gel surface inside the water capillary between two substrates is caused by monolayer collapse of suddenly accumulated lipids at the air-water interface of the capillary during sandwiching. A key aspect of the monolayer collapse-induced bilayer spreading is its rapid kinetics, which appears to be faster than the polymer gel swelling kinetics. The importance of the fast kinetics of bilayer spreading during capillary-assisted assembly is supported by the observation that attempts to build polymer gel-supported lipid bilayer using traditional lipid assembly methods [i. e., Langmuir-Blodgett (LB)/Langmuir-Schaefer (LS), LB- vesicle fusion, and spontaneous bilayer spreading from a hydrated lipid source], characterized by slower bilayer spreading kinetics, are unable to form a homogeneous fluid lipid bilayer on the polymer gel surface. The experimental results obtained in this work strongly suggest that the CA-PGB not only represents a powerful experimental model membrane platform for the analysis of membrane-associated processes relevant in cellular membranes, but also serves as promising cell surface mimetic to probe the cellular mechanosensitivity of adherend cells.