Hans-Peter M. de Hoog

Self-assembled architectures with multiple aqueous compartments

A vital organizational feature of living cells is that of compartmentalization. This allows cells to run concurrently incompatible metabolic processes and to regulate these processes by selective trans-membrane transport. Although strategies that effectively mimic cell function in simple architectures have been researched extensively, soft matter systems with membranes that delineate distinct and multiple aqueous environments have only recently caught attention. We highlight a range of multi-compartmentalized soft matter systems including vesosomes, capsosomes, polymersomes, double emulsions, and their combinations, and demonstrate that the unique properties of the multi-compartmentalized architectures have the potential to add value to application areas such as drug-delivery and multi-enzyme biosynthesis.

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.

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.

A facile and fast method for the functionalization of polymersomes by photoinduced cycloaddition chemistry

Polymersomes are promising platforms for use in biosensing, where their stability may be crucial over that of liposomes. For the introduction of the desired functionality multiple strategies have been reported for functionalization of polymersomes. However, none of them have combined readily available starting materials, facility and in situ quantification. We show a simple 4-step method for functionalization of polymersomes starting from commercially available materials. For the key conjugation step a recently explored light induced cycloaddition was used which is relatively fast (15 min) and allows in situ quantification by the intrinsic fluorescence of the conjugate. The facility of the protocol, the ease of preparation and quantification make this ‘click’-type conjugation method a promising alternative to the established strained cycloadditions.

Third-Party ATP Sensing in Polymersomes: A Label-Free Assay of Enzyme Reactions in Vesicular Compartments

Polymersomes are encapsulated with a fluorescent reporter and a non-labeled enzyme for sensing of adenosine triphosphate (ATP). As B. Liedberg and co-workers report on page 442, passive diffusion of exogenously added ATP through the membrane is sensed by monitoring the ATPinduced fluorescence quenching of the reporter polymer followed by partial recovery of its emission due to hydrolysis of reporter-bound ATP by alkaline phosphatase.

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.

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.

Controlled Supramolecular Self-Assembly of Super-charged β-Lactoglobulin A–PEG Conjugates into Nanocapsules

The synthesis and characterization of a new protein-polymer conjugate composed of β lactoglobulin A (βLG A) and poly(ethylene glycol) PEG is described. βLG 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.