Submicrometer, porous polymeric vesicles, composed of an amphiphilic di‐block copolymer, polystyrene‐b‐polyisocyanoalanine (2‐thiophene‐3‐yl‐ethyl) amide, are used to encapsulate an enzyme, alkaline phosphatase, and a fluorescent reporter polymer poly 1(3((4methylthiophen‐3‐yl)oxy) propyl) quinuclidin‐1‐ium. Passive diffusion of exogenously added adenosine triphosphate (ATP) through the membrane was sensed by monitoring the ATP‐induced fluorescence quenching of the reporter polymer followed by partial recovery of its emission due to hydrolysis of reporter‐bound ATP by alkaline phosphatase.

Many common amphiphiles self-assemble in water to produce heterogeneous populations of discrete and symmetric but polydisperse and multilamellar vesicles isolating the encapsulated aqueous core from the surrounding bulk. But when mixtures of amphiphiles of vastly different elastic properties co-assemble, their non-uniform molecular organization can stabilize lower symmetries and produce novel shapes. Here, using high resolution electron cryomicroscopy and tomography, we identify the spontaneous formation of a membrane morphology consisting of unilamellar tubular vesicles in dilute aqueous solutions of binary mixtures of two different amphiphiles of vastly different origins. Our results show that aqueous phase mixtures of a fluid-phase phospholipid and an amphiphilic block copolymer spontaneously assume a bimodal polymorphic character in a composition dependent manner: over a broad range of compositions (15-85 mol% polymer component), a tubular morphology co-exists with spherical vesicles. Strikingly, in the vicinity of equimolar compositions, an exclusively tubular morphology (L; diameter, ∼15 nm; length, >1 μm; core, ∼2.0 nm; wall, ∼5-6 nm) emerges in an apparent steady state. Theory suggests that the spontaneous stabilization of cylindrical vesicles, unaided by extraneous forces, requires a significant spontaneous bilayer curvature, which in turn necessitates a strongly asymmetric membrane composition. We confirm that such dramatic compositional asymmetry is indeed produced spontaneously in aqueous mixtures of a lipid and polymer through two independent biochemical assays - (1) reduction in the quenching of fluorophore-labeled lipids and (2) inhibition in the activity of externally added lipid-hydrolyzing phospholipase A2, resulting in a significant enrichment of the polymer component in the outer leaflet. Taken together, these results illustrate the coupling of the membrane shape with local composition through spontaneous curvature generation under conditions of asymmetric distribution of mixtures of disparate amphiphiles.

The encapsulation of chloroperoxidase from Caldariomyces fumago (CPO) in block copolymer polymersomes is reported. Fluorescence and electron microscopy show that when the encapsulating conditions favour self-assembly of the block copolymer, the enzyme is incorporated with concentrations that are 50 times higher than the enzyme concentration before encapsulation. The oxidation of two substrates by the encapsulated enzyme was studied: i) pyrogallol, a common substrate used to assay CPO enzymatic activity and ii) thioanisole, of which the product, (R)-methyl phenyl sulfoxide, is an important pharmaceutical intermediate. The CPO-loaded polymersomes showed distinct reactivity towards these substrates. While the oxidation of pyrogallol was limited by diffusion of the substrate into the polymersome, the rate-limiting step for the oxidation of thioansiole was the turnover by the enzyme.

. The methods described are suitable for preparing the pure compounds in quantities of 1 kg or more. For each of the hydrocarbons and also for n-heptane and 2.2,4-trimethylpentane we have determined accurate values for the boiling point, melting point, densities at 15" and 20" or at 20" and 25", refractive indices and molecular refractions at the same temperatures for the wave lengths 6563. 5876, 4861, 4341 A. W e have also recorded accurate values for the surface tension at 20" and for the parachor. The critical solution temperatures with aniline have also been determined.

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