Lipids and block copolymers can be individually assembled into unsupported, spherical membranes (liposomes or polymersomes), each having their own particular benefits and limitations. Here we demonstrate the preparation of microscale, hybrid "lipopolymersomes" composed of the common lipid POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine) and the commercially available copolymer PBd-b-PEO (polybutadiene-b-poly(ethylene oxide)) with the goal of incorporating the advantageous qualities of the unitary systems into mixed-membrane capsules. We investigate the lipopolymersomes using confocal fluorescence microscopy and demonstrate that these hybrid membranes are well mixed on nanoscopic length scales within the permittable compositional windows for hybrid vesicle formation. We measure the intramembrane dynamics and mechanical properties of these hybrid membranes by fluorescence recovery after photobleaching (FRAP) and micropipet aspiration, respectively. For the first time, we demonstrate the demixing of lipid-rich and polymer-rich membrane domains within the same vesicle membrane. This is achieved by the biotinylation of one of the constituent species and cross linking with the protein NeutrAvidin. The resultant domain patterning is dependent upon which component carries the biotin functionality: cross linking of the copolymer species results in domains that ripen into a single, large, copolymer-rich island, and cross linking of the lipids yields many small, "spot-like", lipid-rich domains within a copolymer-rich matrix. We discuss these morphological differences in terms of the fluidity and mechanical properties of the membrane phases and the possible resultant interdomain interactions within the membrane. These heterogeneous hybrid lipopolymersomes could find applications in fields such as targeted delivery, controlled release, and environmental detection assays where these capsules possess the characteristics of biocompatible lipid membranes combined with enhanced mechanical strength and stability from the copolymer matrix.

The release of cytochrome c from mitochondria is a key signaling mechanism in apoptosis. Although extramitochondrial proteins are thought to initiate this release, the exact mechanisms remain unclear. Cytochrome c (cyt c) binds to and penetrates lipid structures containing the inner mitochondrial membrane lipid cardiolipin (CL), leading to protein conformational changes and increased peroxidase activity. We describe here a direct visualization of a fluorescent cyt c crossing synthetic, CL-containing membranes in the absence of other proteins. We observed strong binding of cyt c to CL in phospholipid vesicles and bursts of cyt c leakage across the membrane. Passive fluorescent markers such as carboxyfluorescein and a 10-kDa dextran polymer crossed the membrane simultaneously with cyt c, although larger dextrans did not. The data show that these bursts result from the opening of lipid pores formed by the cyt c-CL conjugate. Pore formation and cyt c leakage were significantly reduced in the presence of ATP. We suggest a model, consistent with these findings, in which the formation of toroidal lipid pores is driven by initial cyt c-induced negative spontaneous membrane curvature and subsequent protein unfolding interactions. Our results suggest that the CL-cyt c interaction may be sufficient to allow cyt c permeation of mitochondrial membranes and that cyt c may contribute to its own escape from mitochondria during apoptosis.

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We demonstrate a method of heterogeneous vesicle binding using membrane-anchored, single-stranded DNA that can be used over several orders of magnitude in vesicle size, as demonstrated for large 100 nm vesicles and giant vesicles several microns in diameter. The aggregation behavior is studied for a range of DNA surface concentrations and solution ionic strengths. Three analogous states of aggregation are observed on both vesicle size scales. We explain the existence of these three regimes by a combination of DNA binding favorability, vesicle collision kinetics, and lateral diffusion of the DNA within the fluid membrane. The reversibility of the DNA hybridization allows dissociation of the structures formed and can be achieved either thermally or by a reduction in the ionic strength of the external aqueous environment. Difficulty is found in fully unbinding giant vesicles by thermal dehybridization, possibly frustrated by the attractive van der Waals minimum in the intermembrane potential when brought into close contact by DNA binding. This obstacle can be overcome by the isothermal reduction of the ionic strength of the solution: this reduces the Debye screening length, coupling the effects of DNA dehybridization and intermembrane repulsion due to the increased electrostatic repulsion between the highly charged DNA backbones.

Significant enhancement of membrane protein functional durability is demonstrated when reconstituted in hybrid lipid–block copolymer vesicles compared to conventional proteoliposomes.