The current study reports on the layer-by-layer assembly of a polymer-tethered lipid multi-bilayer stack using the iterative addition and roll out of giant unilamellar vesicles (GUVs) containing constituents with thiol and maleimide functional groups, respectively. Confocal microscopy and photobleaching experiments confirm stack integrity and stability over time, as well as the lateral fluidity of individual bilayers within the stacks. Complementary wide-field single molecule fluorescence microscopy and atomic force microscopy experiments show that increasing bilayer-substrate distances are associated with changes in lipid lateral mobility and bilayer morphology. Importantly, the described iterative approach can be employed to assemble multi-bilayer stacks with more than two bilayers, thus further reducing the influence of the underlying solid substrate on membrane behavior. Furthermore, the presence of lipopolymers within the multi-bilayer stacks results in fascinating membrane dynamics and organization properties, with interesting parallels to those found in plasma membranes. In that sense, the described multi-bilayer architecture represents an attractive model membrane platform for a variety of different biophysical studies.

Physisorbed polymer-tethered lipid bilayers consisting of phospholipids and lipopolymers represent an attractive planar model membrane platform, in which bilayer fluidity and membrane elastic properties can be regulated through lipopolymer molar concentration. Herein we report a method for the fabrication of such a planar model membrane system with a lateral gradient of lipopolymer density. In addition, a procedure is described, which leads to a sharp boundary between regions of low and high lipopolymer molar concentrations. Resulting gradients and sharp boundaries are visualized on the basis of membrane buckling structures at elevated lipopolymer concentrations using epifluorescence microscopy and atomic force microscopy. Furthermore, results from spot photobleaching experiments are presented, which provide insight into the lipid lateral fluidity in these model membrane architectures. The presented experimental data highlight a planar, solid-supported membrane characterized by fascinating length scale-dependent dynamics and elastic properties with remarkable parallels to those observed in cellular membranes.