Controlled Supramolecular Self-Assembly of Super-charged ß-Lactoglobulin A-PEG Conjugates into Nanocapsules.

The synthesis and characterization of a new protein-polymer conjugate composed of ß lactoglobulin A (ßLG A) and poly(ethylene glycol) PEG is described. ßLG A was selectively modified to self-assemble by super-charging via amination or succinylation followed by conjugation with PEG. An equimolar mixture of the oppositely charged protein-polymer conjugates self-assemble into spherical capsules of 80-100 nm in diameter. The self-assembly proceeds by taking simultaneous advantage of the amphiphilicity and polyelectrolyte nature of the protein-polymer conjugate. These protein-polymer capsules or proteinosomes are reminiscent of protein capsids, and are capable of encapsulating solutes in their interior. We envisage this approach to be applicable to other globular proteins.

Spontaneous formation of nanometer scale tubular vesicles in aqueous mixtures of lipid and block copolymer amphiphiles.

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 (Lt; 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.

Photo-induced conjugation of tetrazoles to modified and native proteins.

Bio-orthogonal chemistry has been widely used for conjugation of polymer molecules to proteins. Here, we demonstrate the conjugation of polyethylene glycol (PEG) to bovine beta-lactoglobulin (BLG) by photo-induced cyclo-addition of tetrazole-appended PEG and allyl-modified BLG. During the course of the investigation, a significant side-reaction was found to occur for the conjugation of PEG-tetrazole to native BLG. Further exploration of the underlying chemistry reveals that the presence of a tryptophan residue is sufficient for conjugation of tetrazole-modified molecules.

Conformational antibody binding to a native, cell-free expressed GPCR in block copolymer membranes.

G-protein coupled receptors (GPCRs) play a key role in physiological processes and are attractive drug targets. Their biophysical characterization is, however, highly challenging because of their innate instability outside a stabilizing membrane and the difficulty of finding a suitable expression system. We here show the cell-free expression of a GPCR, CXCR4, and its direct embedding in diblock copolymer membranes. The polymer-stabilized CXCR4 is readily immobilized onto biosensor chips for label-free binding analysis. Kinetic characterization using a conformationally sensitive antibody shows the receptor to exist in the correctly folded conformation, showing binding behaviour that is commensurate with heterologously expressed CXCR4.

Mixing, diffusion, and percolation in binary supported membranes containing mixtures of lipids and amphiphilic block copolymers.

Substrate-mediated fusion of small polymersomes, derived from mixtures of lipids and amphiphilic block copolymers, produces hybrid, supported planar bilayers at hydrophilic surfaces, monolayers at hydrophobic surfaces, and binary monolayer/bilayer patterns at amphiphilic surfaces, directly responding to local measures of (and variations in) surface free energy. Despite the large thickness mismatch in their hydrophobic cores, the hybrid membranes do not exhibit microscopic phase separation, reflecting irreversible adsorption and limited lateral reorganization of the polymer component. With increasing fluid-phase lipid fraction, these hybrid, supported membranes undergo a fluidity transition, producing a fully percolating fluid lipid phase beyond a critical area fraction, which matches the percolation threshold for the immobile point obstacles. This then suggests that polymer-lipid hybrid membranes might be useful models for studying obstructed diffusion, such as occurs in lipid membranes containing proteins.

An intercompartmental enzymatic cascade reaction in channel-equipped polymersome-in-polymersome architectures.

Compartmentalization, as a design principle, is a prerequisite for the functioning of eukaryotic cells. Although cell mimics in the form of single vesicular compartments such as liposomes or polymersomes have been tremendously successful, investigations of the corresponding higher-order architectures, in particular bilayer-based multicompartment vesicles, have only recently gained attention. We hereby demonstrate a multicompartment cell-mimetic nanocontainer, built-up from fully synthetic membranes, which features an inner compartment equipped with a channel protein and a semi-permeable outer compartment that allows passive diffusion of small molecules. The functionality of this multicompartment architecture is demonstrated by a cascade reaction between enzymes that are segregated in separate compartments. The unique architecture of polymersomes, which combines stability with a cell-membrane-mimetic environment, and their assembly into higher-order architectures could serve as a design principle for new generation drug-delivery vehicles, biosensors, and protocell models.

In vitro expressed GPCR inserted in polymersome membranes for ligand-binding studies.

The dopamine receptor D2 (DRD2), a G-protein coupled receptor is expressed into PBd(22)-PEO(13) and PMOXA(20)-PDMS(54)-PMOXA(20) block copolymer vesicles. The conformational integrity of the receptor is confirmed by antibody- and ligand-binding assays. Replacement of bound dopamine is demonstrated on surface-immobilized polymersomes, thus making this a promising platform for drug screening.

Multicompartmentalized polymersomes for selective encapsulation of biomacromolecules.

Multicompartmentalized polymersomes are formed using block co-polymers PMOXA-PDMS-PMOXA and PS-PIAT, and are subsequently proven to be capable of selective encapsulation of biomacromolecules. This architecture mimics the compartmentalization found in cells and may serve as a simple, albeit robust, model system.

A hydrogel-based enzyme-loaded polymersome reactor.

In this study we report the immobilization of enzyme-containing polymersomes into a macromolecular hydrogel. Whereas free enzyme shows progressive leakage from the hydrogel in a period of days, leakage of the polymersome-protected enzyme is virtually absent. The preparation of the hydrogel occurs under mild conditions and does not inhibit the activity of the encapsulated enzymes nor does it affect the structure of the polymersomes. The stability of the polymersome hydrogel architecture is demonstrated by the facile recycling of the polymersomes and their use in repeated reaction cycles. A 'continuous-flow polymersome reactor' is constructed in which substrate is added to the top of the reactor and product is collected at the bottom. This set-up allows the use of different enzymes and the processing of multiple substrates, as is demonstrated by the conversion of 2-methoxyphenyl acetate to tetraguaiacol in a reactor loaded with polymersome hydrogels containing the enzymes Candida antarctica lipase B (CALB) and glucose oxidase (GOx).

Tuning the properties of PS-block copolymers and their assembly into polymersomes.

The diblock copolymer polystyrene-b-polyisocyanoalanine(2-thiophene-3-yl-ethyl)amide (PS-PIAT) was prepared by reacting the isocyanide monomer (1) with a Ni(ii) initiator complex prepared from polystyrene amine (PS40NH2), either obtained by atom transfer radical polymerization (ATRP) or anionic polymerization (AP). It was found that polymerization of optically pure 1 followed first-order kinetics in monomer concentration and resulted in the formation of insoluble block copolymers, whereas the rate of polymerization of optical mixtures of 1 was retarded and yielded block copolymers that were better soluble. Furthermore, PS-PIAT polymersomes of which the PS-block was prepared by AP were more stable than polymersomes of which the PS-block was prepared by ATRP, as was indicated by combined turbidity and dynamic light scattering (DLS) measurements on the aggregate solutions.

Polymersome nanoreactors for enzymatic ring-opening polymerization.

Polystyrene-polyisocyanopeptide (PS-PIAT) polymersomes containing CALB in two different locations, one in the aqueous inner compartment and one in the bilayer, were investigated for enzymatic ring-opening polymerization of lactones in water. It is shown that the monomers 8-octanolactone and dodecalactone yield oligomers with this polymersome system. It is also observed that the polymerization activity is dependent on the position of the enzyme in the polymersome. SEM investigations show that the polymersome structures were destabilized during the polymerization. Further investigations show that the vesicular morphology of the polymersomes was destabilized only in the case of polymer product formation.