Synthetic Biology: Bottom-Up Assembly of Molecular Systems.

The bottom-up assembly of biological and chemical components opens exciting opportunities to engineer artificial vesicular systems for applications with previously unmet requirements. The modular combination of scaffolds and functional building blocks enables the engineering of complex systems with biomimetic or new-to-nature functionalities. Inspired by the compartmentalized organization of cells and organelles, lipid or polymer vesicles are widely used as model membrane systems to investigate the translocation of solutes and the transduction of signals by membrane proteins. The bottom-up assembly and functionalization of such artificial compartments enables full control over their composition and can thus provide specifically optimized environments for synthetic biological processes. This review aims to inspire future endeavors by providing a diverse toolbox of molecular modules, engineering methodologies, and different approaches to assemble artificial vesicular systems. Important technical and practical aspects are addressed and selected applications are presented, highlighting particular achievements and limitations of the bottom-up approach. Complementing the cutting-edge technological achievements, fundamental aspects are also discussed to cater to the inherently diverse background of the target audience, which results from the interdisciplinary nature of synthetic biology. The engineering of proteins as functional modules and the use of lipids and block copolymers as scaffold modules for the assembly of functionalized vesicular systems are explored in detail. Particular emphasis is placed on ensuring the controlled assembly of these components into increasingly complex vesicular systems. Finally, all descriptions are presented in the greater context of engineering valuable synthetic biological systems for applications in biocatalysis, biosensing, bioremediation, or targeted drug delivery.

Multicomponent Copolymer Planar Membranes with Nanoscale Domain Separation.

Domain separation is crucial for proper cellular function and numerous biomedical technologies, especially artificial cells. While phase separation in hybrid membranes containing lipids and copolymers is well-known, the membranes' overall stability, limited by the lipid part, is hindering the technological applications. Here, we introduce a fully synthetic planar membrane undergoing phase separation into domains embedded within a continuous phase. The mono- and bilayer membranes are composed of two amphiphilic diblock copolymers (PEO45-b-PEHOx20 and PMOXA10-b-PDMS25) with distinct properties and mixed at various concentrations. The molar ratio of the copolymers in the mixture and the nature of the solid support were the key parameters inducing nanoscale phase separation of the planar membranes. The size of the domains and resulting morphology of the nanopatterned surfaces were tailored by adjusting the molar ratios of the copolymers and transfer conditions. Our approach opens new avenues for the development of biomimetic planar membranes with a nanoscale texture.

Block Length-Dependent Protein Fouling on Poly(2-oxazoline)-Based Polymersomes: Influence on Macrophage Association and Circulation Behavior.

Polymersomes are vesicular structures self-assembled from amphiphilic block copolymers and are considered an alternative to liposomes for applications in drug delivery, immunotherapy, biosensing, and as nanoreactors and artificial organelles. However, the limited availability of systematic stability, protein fouling (protein corona formation), and blood circulation studies hampers their clinical translation. Poly(2-oxazoline)s (POx) are valuable antifouling hydrophilic polymers that can replace the current gold-standard, poly(ethylene glycol) (PEG), yet investigations of POx functionality on nanoparticles are relatively sparse. Herein, a systematic study is reported of the structural, dynamic and antifouling properties of polymersomes made of poly(2-methyl-2-oxazoline)-block-poly(dimethylsiloxane)-block-poly(2-methyl-2-oxazoline) (PMOXA-b-PDMS-b-PMOXA). The study relates in vitro antifouling performance of the polymersomes to atomistic molecular dynamics simulations of polymersome membrane hydration behavior. These observations support the experimentally demonstrated benefit of maximizing the length of PMOXA (degree of polymerization (DP) > 6) while keeping PDMS at a minimal length that still provides sufficient membrane stability (DP > 19). In vitro macrophage association and in vivo blood circulation evaluation of polymersomes in zebrafish embryos corroborate these findings. They further suggest that single copolymer presentation on polymersomes is outperformed by blends of varied copolymer lengths. This study helps to rationalize design rules for stable and low-fouling polymersomes for future medical applications.

Tailoring a Solvent-Assisted Method for Solid-Supported Hybrid Lipid-Polymer Membranes.

Combining amphiphilic block copolymers and phospholipids opens new opportunities for the preparation of artificial membranes. The chemical versatility and mechanical robustness of polymers together with the fluidity and biocompatibility of lipids afford hybrid membranes with unique properties that are of great interest in the field of bioengineering. Owing to its straightforwardness, the solvent-assisted method (SA) is particularly attractive for obtaining solid-supported membranes. While the SA method was first developed for lipids and very recently extended to amphiphilic block copolymers, its potential to develop hybrid membranes has not yet been explored. Here, we tailor the SA method to prepare solid-supported polymer-lipid hybrid membranes by combining a small library of amphiphilic diblock copolymers poly(dimethyl siloxane)-poly(2-methyl-2-oxazoline) and poly(butylene oxide)-block-poly(glycidol) with phospholipids commonly found in cell membranes including 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, sphingomyelin, and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(glutaryl). The optimization of the conditions under which the SA method was applied allowed for the formation of hybrid polymer-lipid solid-supported membranes. The real-time formation and morphology of these hybrid membranes were evaluated using a combination of quartz crystal microbalance and atomic force microscopy. Depending on the type of polymer-lipid combination, significant differences in membrane coverage, formation of domains, and quality of membranes were obtained. The use of the SA method for a rapid and controlled formation of solid-supported hybrid membranes provides the basis for developing customized artificial hybrid membranes.

Stabilizing Enzymes within Polymersomes by Coencapsulation of Trehalose.

Enzymes are essential biocatalysts and very attractive as therapeutics. However, their functionality is strictly related to their stability, which is significantly affected by the environmental changes occurring during their usage or long-term storage. Therefore, maintaining the activity of enzymes is essential when they are exposed to high temperature during usage or when they are stored for extended periods of time. Here, we stabilize and protect enzymes by coencapsulating them with trehalose into polymersomes. The anhydrobiotic disaccharide preserved up to about 81% of the enzyme's original activity when laccase/trehalose-loaded nanoreactors were kept desiccated for 2 months at room temperature and 75% of its activity when heated at 50 °C for 3 weeks. Moreover, the applicability of laccase/trehalose-loaded nanoreactors as catalysts for bleaching of the textile dyes orange G, toluidine blue O, and indigo was proven. Our results demonstrate the advantages of coencapsulating trehalose within polymersomes to stabilize enzymes in dehydrated state for extended periods of time, preserving their activity even when heated to elevated temperature.

Expanding the Potential of the Solvent-Assisted Method to Create Bio-Interfaces from Amphiphilic Block Copolymers.

Artificial membranes, as materials with biomimetic properties, can be applied in various fields, such as drug screening or bio-sensing. The solvent-assisted method (SA) represents a straightforward method to prepare lipid solid-supported membranes. It overcomes the main limitations of established membrane preparation methods, such as Langmuir-Blodgett (LB) or vesicle fusion. However, it has not yet been applied to create artificial membranes based on amphiphilic block copolymers, despite their enhanced mechanical stability compared to lipid-based membranes and bio-compatible properties. Here, we applied the SA method on different amphiphilic di- and triblock poly(dimethylsiloxane)-block-poly(2-methyl-2-oxazoline) (PDMS-b-PMOXA) copolymers and optimized the conditions to prepare artificial membranes on a solid support. The real-time membrane formation, the morphology, and the mechanical properties have been evaluated by a combination of atomic force microscopy and quartz crystal microbalance. Then, selected biomolecules including complementary DNA strands and an artificial deallylase metalloenzyme (ADAse) were incorporated into these membranes relying on the biotin-streptavidin technology. DNA strands served to establish the capability of these synthetic membranes to interact with biomolecules by preserving their correct conformation. The catalytic activity of the ADAse following its membrane anchoring induced the functionality of the biomimetic platform. Polymer membranes on solid support as prepared by the SA method open new opportunities for the creation of artificial membranes with tailored biomimetic properties and functionality.

Metal cation responsive anionic microgels: behaviour towards biologically relevant divalent and trivalent ions.

Anionic poly(vinylcaprolactam-co-itaconicacid-co-dimethylitaconate) microgels were synthesized via dispersion polymerization and their responsiveness towards cations, namely Mg2+, Sr2+, Cu2+ and Fe3+, was investigated. The itaconic moieties chelate the metal ions which act as a crosslinker and decrease the electrostatic repulsion within the network, leading to a decrease in the gel size. The responsiveness towards the metal ion concentration has been studied via dynamic light scattering (DLS) and the number of ions bonded within the network has been quantified with ion chromatography. Through the protonation of the carboxylate groups in the gel network, their interaction with the cations is significantly lowered, and the metals are consequently released back in solution. The number of ions released was assessed also via ion chromatography for all four ions, whilst Mg2+ was also used as a model ion to display the reversibility of the system. The microgels can bond and release divalent cations over multiple cycles without undergoing any loss of functionality. Moreover, these gels also selectively entrap Fe3+ with respect to the remaining divalent cations, opening the possibility of using the proposed gels in the digestive tract as biocompatible chelating agents to fight iron overaccumulation.

How Do the Properties of Amphiphilic Polymer Membranes Influence the Functional Insertion of Peptide Pores?

Pore-forming peptides are of high biological relevance particularly as cytotoxic agents, but their properties are also applicable for the permeabilization of lipid membranes for biotechnological applications, which can then be translated to the more stable and versatile polymeric membranes. However, their interactions with synthetic membranes leading to pore formation are still poorly understood, hampering the development of peptide-based nanotechnological applications, such as biosensors or catalytic compartments. To elucidate these interactions, we chose the model peptide melittin, the main component of bee venom. Here, we present our systematic investigation on how melittin interacts with and inserts into synthetic membranes, based on amphiphilic block copolymers, to induce pore formation in three different setups (planar membranes and micrometric and nanometric vesicles). By varying selected molecular properties of block copolymers and resulting membranes (e.g., hydrophilic to hydrophobic block ratio, membrane thickness, surface roughness, and membrane curvature) and the stage of melittin addition to the synthetic membranes, we gained a deeper understanding of melittin insertion requirements. In the case of solid-supported planar membranes, melittin interaction was favored by membrane roughness and thickness, but its insertion and pore formation were hindered when the membrane was excessively thick. The additional property provided by micrometric vesicles, curvature, increased the functional insertion of melittin, which was evidenced by the even more curved nanometric vesicles. Using nanometric vesicles allowed us to estimate the pore size and density, and by changing the stage of melittin addition, we overcame the limitations of peptide-polymer membrane interaction. Mirroring the functionality assay of planar membranes, we produced glucose-sensing vesicles. The design of synthetic membranes permeabilized with melittin opens a new path toward the development of biosensors and catalytic compartments based on pore-forming peptides functionally inserted in synthetic planar or three-dimensional membranes.

Directed Insertion of Light-Activated Proteorhodopsin into Asymmetric Polymersomes from an ABC Block Copolymer.

Nanoscopic artificial vesicles containing functional protein transporters are fundamental for synthetic biology. Energy-providing modules, such as proton pumps, are a basis for simple nanoreactors. We report on the first insertion of a functional transmembrane protein into asymmetric polymersomes from an ABC triblock copolymer. The polymer with the composition poly(ethylene glycol)-poly(diisopropylaminoethyl methacrylate)-poly(styrenesulfonate) (PEG-PDPA-PSS) was synthesized by sequential controlled radical polymerization. PEG and PSS are two distinctively different hydrophilic blocks, allowing for a specific orientation of our protein, the light-activated proton pump proteorhodopsin (PR), into the final proteopolymersome. A very interesting aspect of the PEG-PDPA-PSS triblock copolymers is that it allowed for simultaneous vesicle formation and oriented insertion of PR simply by adjusting the pH. The intrinsic positive charge of PR's intracellular surface was enhanced by a His-tag, which aligns readily with the negative charges of the PSS on the outside of the polymersomes. The directed insertion of PR was confirmed by a light-dependent pH change of the proteopolymersome solution, indicating the intended orientation. We have hereby demonstrated the first successful oriented insertion of a proton pump into an artificial asymmetric membrane.

Effect of Divalent Cation on Swelling Behavior of Anionic Microgels: Quantification and Dynamics of Ion Uptake and Release.

Poly(N-vinylcaprolactam-co-itaconate) (P(VCL-co-IADME) microgels were synthesized varying the molar ratio between VCL and IADME via free radical precipitation polymerization in the presence of quaternary ammonium surfactant. In order to determine the effect of the divalent metal ions on the structure and the swelling behavior of the microgel systems, both neutral and charged forms of the hydrogels after hydrolysis were investigated. The triggered gel collapse caused by the divalent metal ion together with the quantification of the metal ion uptake was studied in detail by titration and ion chromatography methods and revealed the minimum concentration around 0.1 mM to trigger gel collapse on the treated gels. Uptake and release dynamics of the gels were followed by turbidity measurements and were in the time-range of 2 and 17 s, depending on the composition and the concentrations.