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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.

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.

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.

Flow cytometry is a powerful technique for high-throughput, fluorescence-activated screening and sorting of cells (FACS). This methodology has been extended by Griffiths to the screening of water-in-oil microdroplets filled with an in vitro protein expression system.[1–6] The catalytic gene product was detected by the transformation of a co-encapsulated profluorescent substrate into a fluorescent product. Here we report a strategy that involves a versatile one-step preparation procedure of enzyme filled porous and stable polymeric capsules (polymersomes). Since the pores of the capsules are small enough to keep enzymes in, whereas these are sufficiently large to allow (profluorescent) substrates to enter, enzyme activity screening can be performed by the build-up of fluorescence, followed by FACS. To prevent the substrate from diffusing out of the capsules, a trapping agent was added inside the capsule. With this technology we were able to separate enzymatically active polymersomes from non-filled or non-active 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. Published version

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. None of the above

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.

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.

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.

Hybrid phospholipid/block copolymer vesicles, in which the polymeric membrane is blended with phospholipids, display interesting self-assembly behavior, incorporating the robustness and chemical versatility of polymersomes with the softness and biocompatibility of liposomes. Such structures can be conveniently characterized by preparing giant unilamellar vesicles (GUVs) via electroformation. Here, we are interested in exploring the self-assembly and properties of the analogous nanoscale hybrid vesicles (ca. 100 nm in diameter) of the same composition prepared by film-hydration and extrusion. We show that the self-assembly and content-release behavior of nanoscale polybutadiene-b-poly(ethylene oxide) (PB-PEO)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) hybrid phospholipid/block copolymer vesicles can be tuned by the mixing ratio of the amphiphiles. In brief, these hybrids may provide alternative tools for drug delivery purposes and molecular imaging/sensing applications and clearly open up new avenues for further investigation. Published version

G-protein-coupled receptors (GPCRs) constitute the largest gene family in the human genome (ca. 2 % of all genes) and play an indispensable role in cell communication, cell adhesion, and signal transduction. [1] Given their central role in diverse physiological processes, it comes as no surprise that these seven-transmembrane domain proteins are significantly involved in many diseases and, indeed, about 40 % of all marketed drugs or those in development target GPCRs. [2] For the majority of these receptors, however, the structure– function relationships remain elusive. Furthermore, there is a considerable number of orphan receptors with unknown endogenous ligands. [3] As such, substantial effort is directed towards the development of ligand-binding assays to identify either endogenous ligands or to screen for new drug leads. [4–9] A key determinant for the successful development of screening assays is the stable immobilization of GPCRs in an active conformation, preferably onto surfaces, to allow for a reliable micro-array-based screening format. Upon a screening hit such ligands can then be further tested in functional assays. Conventional methods of producing GPCRs involve overexpression in host cells. This approach typically exerts a strain

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.

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.

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 antarcticalipase B (CALB) and glucose oxidase (GOx). Accepted version

An overview of the wide range of polymer-based capsules that have been constructed from synthetic and biological building blocks or from biological building blocks that are taken out of their natural environment, using both hyperbranched and self-assembly approaches, was reviewed. The capsules that are discussed can be considered as the simplest mimics of an organelle or cell and contain a cavity in which chemical reactions can take place or cargo can be stored. The chemical tool box available for constructing polymer micelles and polymersomes is much larger, and natural motifs have been actively incorporated into their designs. Regarding LbL, polymersome, or polymeric micelle nanoreactors, it can be predicted that nature's biocatalysts will be increasingly used for encapsulation in these synthetic systems, holding promise for future nanoscale diagnostic devices. One of the main challenges in this field will be the effective stimulation of responsive polymersomes since most stimuli reported thus far cannot be applied in living organisms.

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.