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Current parenteral coronavirus disease 2019 (Covid-19) vaccines inadequately protect against infection of the upper respiratory tract. Additionally, antibodies generated by wild type (WT) spike-based vaccines poorly neutralize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. To address the need for a second-generation vaccine, we have initiated a preclinical program to produce and evaluate a potential candidate. Our vaccine consists of recombinant Beta spike protein coadministered with synthetic CpG adjuvant. Both components are encapsulated within artificial cell membrane (ACM) polymersomes, synthetic nanovesicles efficiently internalized by antigen presenting cells, including dendritic cells, enabling targeted delivery of cargo for enhanced immune responses. ACM vaccine is immunogenic in C57BL/6 mice and Golden Syrian hamsters, evoking high serum IgG and neutralizing responses. Compared to an ACM-WT spike vaccine that generates predominantly WT-neutralizing antibodies, the ACM-Beta spike vaccine induces antibodies that neutralize WT and Beta viruses equally. Intramuscular (IM)-immunized hamsters are strongly protected from weight loss and other clinical symptoms after the Beta challenge but show delayed viral clearance in the upper airway. With intranasal (IN) immunization, however, neutralizing antibodies are generated in the upper airway concomitant with rapid and potent reduction of viral load. Moreover, antibodies are cross-neutralizing and show good activity against Omicron. Safety is evaluated in New Zealand white rabbits in a repeated dose toxicological study under Good Laboratory Practice (GLP) conditions. Three doses, IM or IN, at two-week intervals do not induce an adverse effect or systemic toxicity. Cumulatively, these results support the application for a Phase 1 clinical trial of ACM-polymersome-based Covid-19 vaccine (ClinicalTrials.gov identifier: NCT05385991).

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of coronavirus disease 2019 (Covid-19), an ongoing global public health emergency. Despite the availability of safe and efficacious vaccines, achieving herd immunity remains a challenge due in part to rapid viral evolution. Multiple variants of concern (VOCs) have emerged, the latest being the heavily mutated Omicron, which exhibits the highest resistance to neutralizing antibodies from past vaccination or infection. Currently approved vaccines generate robust systemic immunity, yet poor immunity at the respiratory tract. We have demonstrated that a polymersome-based protein subunit vaccine with wild type (WT) spike protein and CpG adjuvant induces robust systemic immunity (humoral and T cell responses) in mice. Both antigen and adjuvant are encapsulated in artificial cell membrane (ACM) polymersomes – synthetic, nanoscale vesicles that substantially enhance the immune response through efficient delivery to dendritic cells. In the present study, we have formulated a vaccine candidate with the spike protein from Beta variant and assessed its immunogenicity in golden Syrian hamsters. Two doses of ACM-Beta spike vaccine administered via intramuscular (IM) injection evoke modest serum neutralizing titers that are equally efficacious towards WT and Beta viruses. In contrast, the ACM-WT spike vaccine induces a predominantly WT-specific serum neutralizing response with pronounced reduction in potency towards the Beta variant. Remarkably, immunogenicity of the ACM-Beta spike vaccine is greatly enhanced through intranasal (IN) administration. Following IN challenge with the Beta variant, IM-immunized hamsters are fully protected from disease but not infection, displaying similar peak viral RNA loads in oral swabs as non-vaccinated controls. In contrast, hamsters IN vaccinated with ACM-Beta spike vaccine are protected from disease and infection, exhibiting a ∼100-fold drop in total and subgenomic RNA load as early as day 2 post challenge. We further demonstrate that nasal washes from IN-but not IM-immunized animals possess virus neutralizing activity that is broadly efficacious towards Delta and Omicron variants. Altogether, our results show IN administration of ACM-Beta spike vaccine to evoke systemic and mucosal antibodies that cross-neutralize multiple SARS-CoV-2 VOCs. Our work supports IN administration of ACM-Beta spike vaccine for a next-generation vaccination strategy that not only protects against disease but also an infection of the respiratory tract, thus potentially preventing asymptomatic transmission.

Multiple successful vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are urgently needed to address the ongoing coronavirus disease 2019 (Covid-19) pandemic. In the present work, we describe a subunit vaccine based on the SARS-CoV-2 spike protein coadministered with CpG adjuvant. To enhance the immunogenicity of our formulation, both antigen and adjuvant were encapsulated with our proprietary artificial cell membrane (ACM) polymersome technology. Structurally, ACM polymersomes are self-assembling nanoscale vesicles made up of an amphiphilic block copolymer comprising poly(butadiene)-b-poly(ethylene glycol) and a cationic lipid, 1,2-dioleoyl-3-trimethylammonium-propane. Functionally, ACM polymersomes serve as delivery vehicles that are efficiently taken up by dendritic cells (DC1 and DC2), which are key initiators of the adaptive immune response. Two doses of our formulation elicit robust neutralizing antibody titers in C57BL/6 mice that persist at least 40 days. Furthermore, we confirm the presence of functional memory CD4+ and CD8+ T cells that produce T helper type 1 cytokines. This study is an important step toward the development of an efficacious vaccine in humans.

Multiple successful vaccines against SARS-CoV-2 are urgently needed to address the ongoing Covid-19 pandemic. In the present work, we describe a subunit vaccine based on the SARS-CoV-2 spike protein co-administered with CpG adjuvant. To enhance the immunogenicity of our formulation, both antigen and adjuvant were encapsulated with our proprietary artificial cell membrane (ACM) polymersome technology. Structurally, ACM polymersomes are self-assembling nanoscale vesicles made up of an amphiphilic block copolymer comprising of polybutadiene-b-polyethylene glycol and a cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane. Functionally, ACM polymersomes serve as delivery vehicles that are efficiently taken up by dendritic cells, which are key initiators of the adaptive immune response. Two doses of our formulation elicit robust neutralizing titers in C57BL/6 mice that persist at least 40 days. Furthermore, we confirm the presence of memory CD4+and CD8+T cells that produce Th1 cytokines. This study is an important step towards the development of an efficacious vaccine in humans.

In this work, we have used low-molecular-weight (PEG12-b-PCL6, PEG12-b-PCL9 or PEG16-b-PLA38, MW, 1.25&ndash, 3.45 kDa) biodegradable block co-polymers to construct nano- and micron-scaled hybrid (polymer/lipid) vesicles, by solvent dispersion and electroformation methods, respectively. The hybrid vesicles exhibit physical properties (size, bilayer thickness and small molecule encapsulation) of a vesicular boundary, confirmed by cryogenic transmission electron microscopy, calcein leakage assay and dynamic light scattering. Importantly, we find that these low MW polymers, on their own, do not self-assemble into polymersomes at nano and micron scales. Using giant unilamellar vesicles (GUVs) model, their surface topographies are homogeneous, independent of cholesterol, suggesting more energetically favorable mixing of lipid and polymer. Despite this mixed topography with a bilayer thickness similar to that of a lipid bilayer, variation in surface topology is demonstrated using the interfacial sensitive phospholipase A2 (sPLA2). The biodegradable hybrid vesicles are less sensitive to the phospholipase digestion, reminiscent of PEGylated vesicles, and the degree of sensitivity is polymer-dependent, implying that the nano-scale surface topology can further be tuned by its chemical composition. Our results reveal and emphasize the role of phospholipids in promoting low MW polymers for spontaneous vesicular self-assembly, generating a functional hybrid lipid-polymer interface.

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.

Identifying peptide substrates that are efficiently cleaved by proteases can give insights into the substrate recognition and specificity, guide the development of inhibitors and improve the sensitivity of assays. Peptide arrays and SAMDI mass spectrometry were used to identify a tetrapeptide substrate exhibiting high activity for the bacterial outer membrane protease (OmpT). Analysis of protease activity for the preferred residues at the cleavage site (P1, P1') and nearest neighbor positions (P2, P2') and their positional interdependence revealed FRRV as the optimal peptide with the highest OmpT activity. Substituting FRRV into a fragment of LL37, a natural substrate of OmpT, led to a >400 fold improvement in OmpT's catalytic efficiency with a kcat/Km value of 6.1 x 106 M-1 s-1. Interestingly, wild-type and mutant OmpT displayed significant differences in their substrate specificities, demonstrating that even modest mutants may not be suitable substitutes for the native enzyme.

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.

A plethora of polymer-based scaffolds have been designed to facilitate biochemical and biophysical investigation of membrane proteins, with a common goal to stabilize and present them in a functional format. In this review, an up-to-date account of such polymer-based supports and incorporation methodologies are presented. Furthermore, conceptual and imminent technological advances, with associated technical challenges are proposed.

Porous polymersomes based on block copolymers of isocyanopeptides and styrene have been used to anchor enzymes at three different locations, namely, in their lumen (glucose oxidase, GOx), in their bilayer membrane (Candida antarctica lipase B, CalB) and on their surface (horseradish peroxidase, HRP). The surface coupling was achieved by click chemistry between acetylene-functionalised anchors on the surface of the polymersomes and azido functions of HRP, which were introduced by using a direct diazo transfer reaction to lysine residues of the enzyme. To determine the encapsulation and conjugation efficiency of the enzymes, they were decorated with metal-ion labels and analysed by mass spectrometry. This revealed an almost quantitative immobilisation efficiency of HRP on the surface of the polymersomes and a more than statistical incorporation efficiency for CalB in the membrane and for GOx in the aqueous compartment. The enzyme-decorated polymersomes were studied as nanoreactors in which glucose acetate was converted by CalB to glucose, which was oxidised by GOx to gluconolactone in a second step. The hydrogen peroxide produced was used by HRP to oxidise 2,2′-azinobis(3-ethylbenzothiazoline- 6-sulfonic acid) (ABTS) to ABTS.+. Kinetic analysis revealed that the reaction step catalysed by HRP is the fastest in the cascade reaction.

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. Published version

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.

A block copolymer was designed to functionalise the surface of polystyrene-based polymersomes via coaggregation. An α,ω- diacetylene-functionalised poly(ethylene glycol) (PEG) was coupled to an azide-terminated polystyrene via a Cu(I)-catalysed cycloaddition to produce a PS-b-PEG polymer with an acetylene at its hydrophilic extremity. Incorporation of this 'anchor' compound in the bilayer of a polymersome places its bio-orthogonal group at the surface of this aggregate. Its accessibility was demonstrated using an azidofunctionalised Candida antarctica Lipase B (CalB), which retained its activity while immobilised on the polymersome.

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

Polystyrene40-b-poly(isocyanoalanine(2-thiophen-3-yl-ethyl) amide)50 (PS-PIAT) polymersomes have the unique property of being sufficiently porous to allow diffusion of small (organic) substrates while retaining large biomolecules such as enzymes inside. Herein we report on the encapsulation and protection of glucose oxidase (GOx) and horse radish peroxidase (HRP) in PS-PIAT polymersomes and the successful employment of these functionalised nanoreactors in a cascade reaction. The demonstrated concept allows for further application in other enzymatic cascade reactions, bio-organic hybrid systems and biosensing devices.

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.

A nanocompartment system composed of an ABA triblock copolymer, where A is poly(dimethylsiloxane) and B is poly(2-methyloxazoline), has been developed for selective recovery and detection of DNA. Translocation of TAMRA-labeled complementary primers into the nanocompartment system has been achieved through two deletion mutants (FhuA Delta1-129; FhuA Delta1-160) of the channel protein FhuA. Translocation was monitored by fluorescence resonance energy transfer through hybridization of the TAMRA-labeled primer to the complementary sequence of a nanophosphor-DNA-conjugate, which reduces its half-life (FhuA Delta1-129, 16.0% reduced; FhuA Delta1-160, 39.0% reduced).

Synthosomes are mechanically stable vesicles with a block copolymer membrane and an engineered transmembrane protein acting as selective gate. The polymer vesicles are nanometer-sized (50-1000 nm) and functionalized by loading them with enzymes for bioconversions or encapsulating charged macromolecules for selective compound recovery/release. The Synthosome system might become a novel technology platform for biocatalysis and selective product recovery. Progress in Synthosome research comprises employed block copolymers, transmembrane channel engineering, and functionalizations, which are discussed here in detail. The challenges in transmembrane protein engineering, as well as cost-effective production, in block copolymer design and the state of the art in Synthosome characterization comprising quantification of encapsulated protein, translocation efficiency, number of transmembrane channels per vesicle, and enzyme kinetics are also presented and discussed. An assessment of the Synthosome technology platform for prospective applications in industrial (white) biotechnology concludes this review.

A nanocompartment system based on two deletion mutants of the large channel protein FhuA (FhuA Delta1-129; FhuA Delta1-160) and an ABA triblock copolymer (PMOXA-PDMS-PMOXA) has been developed for putative biotechnological applications. FhuA is ideally suited for applications in biotechnology due to its monomeric structure, large pore diameter (39-46 A elliptical cross-section) that ensures rapid compound flux, and solved crystallographic structure. Two areas of application were targeted as proof of principle: (A) selective product recovery in nanocompartments and (B) enzymatic conversion in nanocompartments. Selective recovery of negatively charged compounds has been achieved on the example of sulforhodamine B by using positively charged polylysine molecules as trap inside the nanocompartment. Conversion in nanocompartments has been achieved by 3,3',5,5'-tetramethylbenzidine oxidation employing horseradish peroxidase (HRP).

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.

The pre-defined selective positioning of a controlled number of vesicles on a rigid substrate is crucial in many potential applications such as diagnostics, biosensors, lab-on-a chip, microanalyses and reaction chambers. In this paper, the vesicles made up of block copolymer using Poly [-(2-methyloxazoline) -poly- (dimethylsiloxane)-poly- (2-methyloxazoline)] (ABA) with dimensions of 100-200 nm are trapped by physisorption on hydrophilic surfaces. We discuss the protocols established for vesicle trapping. The optimum conditions obtained for physisorption is 15 minutes incubation followed by one cycle of DI water rinse. Trapping of 1-10 vesicles in lobe shape micro-wells fabricated by photo lithography using photoresist on UltraStick(™) slides was demonstrated. To overcome the issue of amalgamation of emitted light from optically sensitive photoresist and fluorescently tagged vesicles, an alternative approach of Si/SiO(2) microwell array coupled with APTES (3-AminoPropylTriEthoxySilane) treated bottom surfaces was developed.

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

We report the use of a novel microfluidics-based method to detect weak protein–protein interactions between membrane proteins. The tight junction protein, claudin-2, synthesised in vitro using a cell-free expression system in the presence of polymer vesicles as membrane scaffolds, was used as a model membrane protein. Individual claudin-2 molecules interact weakly, although the cumulative effect of these interactions is significant. This effect results in a transient decrease of average vesicle dispersivity and reduction in transport speed of claudin-2-functionalised vesicles. Polymer vesicles functionalised with claudin-2 were perfused through a microfluidic channel and the time taken to traverse a defined distance within the channel was measured. Functionalised vesicles took 1.19 to 1.69 times longer to traverse this distance than unfunctionalised ones. Coating the channel walls with protein A and incubating the vesicles with anti-claudin-2 antibodies prior to perfusion resulted in the functionalised vesicles taking 1.75 to 2.5 times longer to traverse this distance compared to the controls. The data show that our system is able to detect weak as well as strong protein–protein interactions. This system offers researchers a portable, easily operated and customizable platform for the study of weak protein–protein interactions, particularly between membrane proteins.

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.

To improve the stability of cell membrane mimics, there has been growing interest in the use of block copolymers. Here, we present an easy approach to create an array of planar polymeric matrices capable of hosting membrane proteins. The array of polymeric matrices was formed by the selective deposition of triblock copolymers onto an array of hydrophilic islands situated within a hydrophobic background. The thickness of these matrices corresponds to the length of a single polymer chain. These polymeric matrices were used to host cell-free expressed membrane proteins, and offers a prototype from which a membrane protein array can be created for diagnostics or drug discovery purposes.

The controlled introduction of azides in proteins provides targetable handles for selective protein manipulation. We present here an efficient diazo transfer protocol that can be applied in an aqueous solution, leading to the facile introduction of azides in the side chains of lysine residues and at the N-terminus of enzymes, e.g. horseradish peroxidase (HRP) and the red fluorescent protein DsRed. The effective introduction of azides was verified by mass spectrometry, after which the azido-proteins were used in Cu(I)-catalyzed [3 + 2] cycloaddition reactions. Azido-HRP retained its catalytic activity after conjugation of a small molecule. This modified protein could also be successfully immobilized on the surface of an acetylene-covered polymersome. Azido-DsRed was coupled to an acetylene-bearing protein allowing it to act as a fluorescent label, demonstrating the wide applicability of the diazo transfer procedure.

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.

Facile surface modification via the blending of lipids and block-co-polymers to assemble hybrid vesicles was investigated for improving cellular interaction and antigen delivery of poly(ethylene glycol) (PEG)-based polymersomes. Cationic lipids (DOTAP) incorporated into PEG-b-PBD polymersomes increased the binding and uptake of vesicles by antigen-presenting cells, while preserving the stability and biocompatibility of PEG-based polymersomes, resulting in the enhanced cellular delivery of a loaded lipid antigen sulfatide.

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.

The topic synthetic biology appears still as an 'empty basket to be filled'. However, there is already plenty of claims and visions, as well as convincing research strategies about the theme of synthetic biology. First of all, synthetic biology seems to be about the engineering of biology -about bottom-up and top-down approaches, compromising complexity versus stability of artificial architectures, relevant in biology. Synthetic biology accounts for heterogeneous approaches towards minimal and even artificial life, the engineering of biochemical pathways on the organismic level, the modelling of molecular processes and finally, the combination of synthetic with nature-derived materials and architectural concepts, such as a cellular membrane. Still, synthetic biology is a discipline, which embraces interdisciplinary attempts in order to have a profound, scientific base to enable the re-design of nature and to compose architectures and processes with man-made matter. We like to give an overview about the developments in the field of synthetic biology, regarding polymer-based analogs of cellular membranes and what questions can be answered by applying synthetic polymer science towards the smallest unit in life, namely a cell. (C) 2012 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

Compartmentalization as a design principle is 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 semipermeable outer compartment that allows passive diffusion of small molecules. The functionality of this multi compartment 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.

Cell, as a source of inspiration, is a key functional unit of living organism. Its interior consists of different structures composed of soft matter that are highly selective and functions with different roles. Compartmentalization is one of the main features of eukaryotic cells, giving organelles to achieve some essential functions, such as protein synthesis, replication and energy production within close proximity. With the purpose to mimic cellular compartments, lots of model systems based on lipids (liposomes) or polymers (polymersomes) have been developed. Polymersomes (also known as polymer vesicles) are spherical nanostructured entities with a hollow aqueous compartment enclosed by a bilayer membrane of synthetic amphiphilic block copolymers. Compared to the traditionally used liposomes, they have several properties which make them especially suited for lots of applications in biomedicine and biotechnology. Amongst others, their superior stability and excellent chemical diversity allowing their properties of its membranes to be tuned (e.g. selective permeability). Here, the thesis covers two parts of research work and the focus of the study is to use polymersomes system with specific functionality to build novel architectures for mimicking the complexity of living cells. DOCTOR OF PHILOSOPHY (MSE)

Liposome and polymersome are hollow bilayer vesicles nanostructures formed by self-assembly of phospholipids and synthetic amphiphilic block copolymers, respectively. Liposomes have been widely studied and developed for wide range of applications. On the other hand, polymersomes have several features which make them better suited than liposomes for certain technological applications, amongst which are their superior stability and excellent chemical versatility. Nevertheless, the self-assembly as well as the interaction of phospholipids and block copolymers, during or after assembly process, have not been well understood. Mixing phospholipids and block copolymers forming hybrid vesicle has been proposed to tune the membrane properties to combine the robustness and chemical tunability of polymersomes with the softness, fluidity and biocompatibility of liposomes. The self-assembly, phase behaviour, membrane properties, and parameters controlling these hybrid vesicles are not yet fully understood. In chapter 3, nanoscale hybrid phospholipid/block copolymer vesicles of various compositions were prepared by film-hydration-extrusion and the mixing behaviours, stability, and membrane properties of these nanostructures have been explored. It was found that the size, encapsulation efficiency and content-release behaviour of nanoscale polybutadiene-b-poly(ethylene oxide) (PB-PEO)/1-palmitoyl-2-oleoyl-sn-glycero-3-hosphatidylcholine (POPC) hybrid phospholipid/block copolymer vesicles can be tuned by the mixing ratio of the amphiphiles. It was generally believed that in binary mixtures of phospholipids and block copolymers, each forms spherical vesicles when present alone, and form spherical hybrid vesicles in aqueous solution. Detailed characterizations by Cryo-TEM reveal that, surprisingly, mixing POPC and PB-PEO results in spontaneous formation of tubular vesicles. This tendency for tubular vesicle formation is most pronounced when the amphiphile composition reaches equimolar proportion of the lipid and the polymer. The spontaneous stabilization of tubular vesicles, which requires a significant spontaneous bilayer curvature or energy, was proposed to arise from the unusually asymmetric lipid/polymer membrane composition. Membrane active polypeptides are of large interest for development of novel therapeutics including peptide-mediated drug delivery and release systems, transfection agents and antibiotics. Finding routes to tune specificity and activity of membrane active polypeptides is hence of large importance to improve their efficacy and minimize harmful side effects. Chapter 5 describes a de novo designed antimicrobial-like helix-loop-helix polypeptide (JR2KC) that partition into zwitterionic lipid membranes when specifically and covalently anchored to the membrane. Anchoring and subsequent membrane partitioning triggers a structural transition of the polypeptide from random coil to α-helical conformation and induces pore formation. The extent of pore formation can be dynamically tuned by varying the number of anchoring moieties or by introducing a complementary polypeptide (JR2E) that is designed to heterodimerize with JR2KC and fold into four-helix bundles. Dimerization competes with membrane partitioning of the polypeptides and reduces the pore forming capacity of JR2KC. This system thus enables a precise and very specific route for tuning the permeability of lipid membranes and demonstrates a novel strategy for development of recognition based membrane active polypeptides. In Chapter 6, a tetramethylrhodamine-labeled peptides binder was assembled on graphene oxide in solution to develop as a fluorescence turn-on sensor for Lipopolysaccharide (LPS)/endotoxin detection. The fluorescence of the dye-labeled peptide is quenched upon interaction with GO. Specific binding to LPS triggers release of the peptide-LPS complex from GO, resulting in fluorescence recovery. This fluorescent turn-on sensor offers a limit of detection of 130 pM, which is the lowest ever achieved among all synthetic LPS sensors to-date. Importantly, this sensor is applicable for detection of LPS in commonly used clinical injectable fluids, and it enables selective detection of LPS from different bacterial strains as well as LPS on the membrane of living E. coli. Finally, the applications of self-assembled membranes, liposomes, polymersomes, and polypeptides for biosensing will be also demonstrated in the latter part of the thesis. Doctor of Philosophy (MSE)