Controlling molecular recognition with lipid/polymer domains in vesicle membranes.

Biological membranes and their synthetic analogues are formed by the self-assembly of amphiphilic molecules, either of low molecular weight (phospholipids) or high molecular weight (block copolymers). Both types of membranes (liposomes or polymersomes) can undergo lateral phase segregation processes, 6] which especially in lipid membranes comply with their biological functions in, for example, receptor clustering, viral recognition, and other specific protein binding in model as well as living cell membranes. So-called “lipid rafts” or domains are the consequence of the lateral phase segregation of complex lipid mixtures within membranes, which can be induced by molecular effects such as mismatches of the lengths of their lipid hydrophobic chains and by the interaction of cholesterol and sphingolipids (socalled “detergent-resistant membranes”, DRMs). In contrast, vesicles composed of block copolymers (“polymersomes”) can display either horizontal or lateral phase segregation through demixing processes caused by the immiscibility of their different polymer building blocks, or by the action of ions or nanoparticles. The recent trend in blending phospholipids and block copolymers to generate hybrid membranes, which combine the biofunctionality of liposomes with the remarkable mechanical stability and functional variability of polymersomal membranes, has further shown the formation of phase-separated membrane morphologies (domains), either as a result of immiscibility between the lipid and polymer component 16] or induced by a clustering agent as an external stimulus. It is of course tempting to speculate about the biological function of recognition processes in giant unilamellar vesicles (GUVs) composed of both lipids and polymers, in particular in addressing the location and assembly of glycosphingolipids (gangliosides) active in protein recognition processes. Can lateral domains within a hybrid vesicle prevent the recognition of a protein by blocking the function of a membrane receptor? Or is it possible to induce additional lateral phase separation by receptor–ligand interactions in a specially designed hybrid membrane? Herein we report our study of the binding of a biological protein to receptor-functionalized hybrid membranes, which consist of an amphiphilic block copolymer (BCP) and a natural lipid as the major components, and demonstrate the biofunctionality of such designed hybrid lipid/BCP membranes. In our current approach for studying receptor recognition on such a hybrid vesicle membrane we prepared hybrid membranes composed of the biocompatible polyisobutylene-block-polyethyleneoxide copolymer (PIB87-bPEO17; BCP 1) and 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC; 2) that were functionalized with gangliosideGM1 (3); we then studied the binding of cholera toxin B (CTB) to these hybrid membranes either in the laterally mixed or demixed phase state (Figure 1). When the designed amphiphilic block copolymer 1 is incorporated into gel-phase vesicles of DPPC, it induces phase heterogeneities within the “hybrid vesicle membrane” that can display a two-dimensional, lateral domain structure, as illustrated in Figure 1. The membrane morphology of these vesicles appears uniform at the resolution of confocal microscopy (roughly 200–300 nm) when prepared from mixtures containing less than 20 mol% and more than 28 mol% of BCP. In contrast, it is phase-separated into a patched membrane in the narrow compositional range between 20 and 28 mol% of BCP. Figure 1B shows a confocal microscopy image of such a phase-separated GUV (see also Figure 2), prepared from a mixture with 20 mol % of BCP (1) and 80 mol% of DPPC (2), where individual domains were visualized by the incorporation of the fluorescently labeled BCP 4, which was selectively excited at lexc = 488 nm (blue region in Figure 1B), and the lipid-analogue membrane dye DiDC18 (lexc = 633 nm; green region in Figure 1B). We found that the phase separation process leads to the formation of a less-ordered polymer-enriched phase (see Figure 2 and the Supporting Information) and a lipid-enriched domain (black patch in Figure 2A,C), the latter being depleted of polymer molecules. As reported in the literature, the lipid dye Rh-DHPE (lexc = 561 nm) is largely excluded from ordered phases in monoand bilayer membranes (e.g., liquid condensed phases). In Figure 2B, Rh-DHPE is preferentially incorporated into the polymer-enriched phase (compare with Figure 2A) which indicates a less-ordered phase state, [*] M. Schulz, Prof. Dr. W. H. Binder Martin-Luther University Halle-Wittenberg Faculty of Natural Sciences II Institute of Chemistry, Macromolecular Chemistry 06120 Halle (Saale) (Germany) E-mail: wolfgang.binder@chemie.uni-halle.de Homepage: http://www.macrochem. uni-halle.de