Preparation and Characterization of Glycoacrylate-Based Polymer-Tethered Lipid Bilayers on Benzophenone-Modified Substrates

Abstract

Polymer-tethered lipid bilayers are promising models for biological membranes as they may provide a soft, lubricating environment with sufficient spacing between the substrate and bilayer for incorporating transmembrane proteins. We present such a system that uses a glycoacrylate-based telechelic lipopolymer in combination with a lipid analogue. Characterization of the mixed monolayers of lipopolymers and free lipids at the air-water interface is used to examine the molecular organization that dictates the final assembly properties. Isotherms indicate that the source of the dominating interactions, whether polymer interactions in the subphase or alkyl chain interactions, depends on both the tethering density and area per molecule. Moreover, a critical composition exists at which the alkyl chain interactions dominate the monolayer behavior regardless of the area per molecule. Isobaric creep and hysteresis experiments suggest that permanent states due to irreversible polymer-polymer interactions are not created as the monolayer is compressed. These data, combined with theoretical polymer predictions, are used to understand the organization of the monolayers at the air-water interface and, hence, the separation distance between the bottom of the bilayer and substrate in the water-swollen state of the final bilayer assembly. Atomic force microscopy is used to confirm that the measured separation distance of 11.2 nm is on the order of what would be predicted using a theoretical analysis for a representative 5 mol % lipopolymer-tethered bilayer. Next, the homogeneity of the final bilayer is probed at multiple scales. Fluorescence microscopy is used to demonstrate that homogeneous and continuous bilayers can be formed (within the optical resolution limit of 500 nm) with all polymer tethering densities used in this study. Atomic force microscopy studies demonstrate that homogeneity comparable to that of a solid-supported lipid bilayer can be achieved for a representative 5 mol % lipopolymer-tethered bilayer. Langmuir-Blodgett transfer conditions for depositing monolayers that can be used to create homogeneous, fluid bilayers are also discussed. Finally, the distal leaflet lateral mobility is measured using fluorescence recovery after photobleaching experiments and shown to be a function of the tethering density. A possible model for the mobility data is developed in which the tethered lipids in the proximal leaflet act as immobile lipid obstacles that couple to distal leaflet lipids.

α,ω‐Functionalized Poly(2‐Oxazoline)s Bearing Hydroxyl and Amino Functions

“…number of compounds has been found suitable for this purpose, in particular polymers [17,22] and oligomers [23][24][25][26] of ethyleneoxide but also oligopeptides bearing thiol groups [27] and acrylates. [28][29][30][31][32][33][34] In a series of contributions we and others demonstrated that tailored poly(2-oxazoline) (POx) lipopolymers are a highly versatile system for the construction of PTMs. [35][36][37][38][39][40][41][42][43][44] POx-based PTMs have been successfully used to determine lateral mobility and functionality of reconstituted membrane proteins, [17,23,41,42,45] to modulate the interactions between substrate and membrane [46] but also the PTM structure.…”

Effects of Fullerenes on Phospholipid Membranes: A Langmuir Monolayer Study

“…First, the small distance between the bilayer and the rigid substrate may be insufficient for foreign species to penetrate through the membrane. [16] Second, electrostatic interactions between the charged lipid head groups and the surface may modify the diffusion behavior of the transmembrane molecules. [17] The Langmuir balance is a simple but powerful tool for the construction and investigation of monolayers at gas/liquid interfaces with the ability to control surface pressure and composition.…”

Modulation of Substrate–Membrane Interactions by Linear Poly(2‐methyl‐2‐oxazoline) Spacers Revealed by X‐ray Reflectivity and Ellipsometry

“…Atomic force microscopy (AFM) operated in a contact mode can merely measure the height difference between the membrane surface and the area scratched under high forces. [18] An alternative way to evaluate the structures of such multilayered systems involves techniques based on reflectivity, such as ellipsometry [19][20][21] and specular X-ray and neutron reflectivity. [22][23][24][25][26][27] Taking the basic framework of Fresnel reflectivity, one can calculate the thickness and/or refractive index of the layered structures from the two measured ellipsometric angles.…”