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.

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.

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.

Porphyrin-polymer nanocompartments: singlet oxygen generation and antimicrobial activity.

A new water-soluble photocatalyst for singlet oxygen generation is presented. Its absorption extends to the red part of the spectrum, showing activity up to irradiation at 660 nm. Its efficiency has been compared to that of a commercial analogue (Rose Bengal) for the oxidation of L-methionine. The quantitative and selective oxidation was promising enough to encapsulate the photocatalyst in polymersomes. The singlet oxygen generated in this way can diffuse and remain active for the oxidation of L-methionine outside the polymeric compartment. These results made us consider the use of these polymersomes for antimicrobial applications. E. coli colonies were subjected to oxidative stress using the photocatalyst-polymersome conjugates and nearly all the colonies were damaged upon extensive irradiation while under the same red LED light irradiation, liquid cultures in the absence of porphyrin or porphyrin-loaded polymersomes were unharmed.

Self-assembled structures from amphiphilic peptides.

Nanotechnology and its applications are strongly influenced by structures self-assembled from a variety of different materials. This review covers nanostructures, including micelles, rod-like micelles, fibers and peptide beads, self-assembled from de novo designed amphiphilic peptides. The latter are promising candidates for the development of nanoscale carrier systems because they are completely composed of amino acids. In addition to designing primary sequences, secondary structure and external parameters are also discussed with respect to their impact on self-assembly. Moreover, the assembly process itself is examined. Potential applications range from gene and drug delivery devices to diagnostics, thereby highlighting the versatility of the system.

High-density reconstitution of functional water channels into vesicular and planar block copolymer membranes.

The exquisite selectivity and unique transport properties of membrane proteins can be harnessed for a variety of engineering and biomedical applications if suitable membranes can be produced. Amphiphilic block copolymers (BCPs), developed as stable lipid analogs, form membranes that functionally incorporate membrane proteins and are ideal for such applications. While high protein density and planar membrane morphology are most desirable, BCP-membrane protein aggregates have so far been limited to low protein densities in either vesicular or bilayer morphologies. Here, we used dialysis to reproducibly form planar and vesicular BCP membranes with a high density of reconstituted aquaporin-0 (AQP0) water channels. We show that AQP0 retains its biological activity when incorporated at high density in BCP membranes, and that the morphology of the BCP-protein aggregates can be controlled by adjusting the amount of incorporated AQP0. We also show that BCPs can be used to form two-dimensional crystals of AQP0.

Inverting glucuronidation of hymecromone in situ by catalytic nanocompartments.

Glucuronidation is a metabolic pathway that inactivates many drugs including hymecromone. Adverse effects of glucuronide metabolites include a reduction of half-life circulation times and rapid elimination from the body. Herein, we developed synthetic catalytic nanocompartments able to cleave the glucuronide moiety from the metabolized form of hymecromone in order to convert it to the active drug. By shielding enzymes from their surroundings, catalytic nanocompartments favor prolonged activity and lower immunogenicity as key aspects to improve the therapeutic solution. The catalytic nanocompartments (CNCs) consist of self-assembled poly(dimethylsiloxane)-block-poly(2-methyl-2-oxazoline) diblock copolymer polymersomes encapsulating ß-glucuronidase. Insertion of melittin in the synthetic membrane of these polymersomes provided pores for the diffusion of the hydrophilic hymecromone-glucuronide conjugate to the compartment inside where the encapsulated ß-glucuronidase catalyzed its conversion to hymecromone. Our system successfully produced hymecromone from its glucuronide conjugate in both phosphate buffered solution and cell culture medium. CNCs were non-cytotoxic when incubated with HepG2 cells. After being taken up by cells, CNCs produced the drug in situ over 24 hours. Such catalytic platforms, which locally revert a drug metabolite into its active form, open new avenues in the design of therapeutics that aim at prolonging the residence time of a drug.

From spherical compartments to polymer films: exploiting vesicle fusion to generate solid supported thin polymer membranes.

Solid supported polymer membranes as scaffold for the insertion of functional biomolecules provide the basis for mimicking natural membranes. They also provide the means for unraveling biomolecule-membrane interactions and engineering platforms for biosensing. Vesicle fusion is an established procedure to obtain solid supported lipid bilayers but the more robust polymer vesicles tend to resist fusion and planar membranes rarely form. Here, we build on vesicle fusion to develop a refined and efficient way to produce solid supported membranes based on poly(dimethylsiloxane)-poly(2-methyl-2-oxazoline) (PMOXA-b-PDMS-b-PMOXA) amphiphilic triblock copolymers. We first create thiol-bearing polymer vesicles (polymersomes) and anchor them on a gold substrate. An osmotic shock then provokes polymersome rupture and drives planar film formation. Prerequisite for a uniform amphiphilic planar membrane is the proper combination of immobilized polymersomes and osmotic shock conditions. Thus, we explored the impact of the hydrophobic PDMS block length of the polymersome on the formation and the characteristics of the resulting solid supported polymer assemblies by quarz crystal microbalance with dissipation monitoring (QCM-D), atomic force microscopy (AFM) and spectroscopic ellipsometry (SE). When the PDMS block is short enough, attached polymersomes restructure in response to osmotic shock, resulting in a uniform planar membrane. Our approach to rapidly form planar polymer membranes by vesicle fusion brings many advantages to the development of synthetic planar membranes for bio-sensing and biotechnological applications.

Supramolecular and nanochemistry.

The following contributions describe various research activities of the Department of Chemistry, University of Basel in the area of nanochemistry and supramolecular chemistry.

Porphyrin Containing Polymersomes with Enhanced ROS Generation Efficiency: In Vitro Evaluation.

Porphyrins are molecules possessing unique photophysical properties making them suitable for application in photodynamic therapy. The incorporation of porphyrins into natural or synthetic nano-assemblies such as polymersomes is a strategy to improve and prolong their therapeutic capacities and to overcome their limitations as therapeutic and diagnostic agents. Here, 5,10,15,20-tetrakis(1-(6-ethoxy-6-oxohexyl)-4-pyridin-1-io)-21H,23H-porphyrin tetrabromide porphyrin is inserted into polymersomes in order to demonstrate that the encapsulation enhances its ability to generate highly reactive singlet oxygen (1 O2 ) upon irradiation in vitro. The photoactivation of the free and polymersome-encapsulated porphyrin is evaluated by electron spin resonance and cell viability assays on three different mammalian cell lines. The results indicate that by encapsulating the porphyrin, a controlled ROS delivery within the cells is achieved, at the same time avoiding side effects such as dark toxicity, non-specific porphyrin release and over time decreased activity in vitro. This work focuses on showing a not-toxic model system for modern therapeutic nanomedicine, which works under mild irradiation and dosage conditions.

Probing membrane asymmetry of ABC polymersomes.

We report the sensitivity of the membrane asymmetry of ABC (PEO-b-PCL-b-PMOXA) polymersomes towards the end-group modification of a shorter C block. While a non-modified ABC polymer formed polymersomes with the A block outside and the C block inside, a mixture of ABC and ABC-biotin formed polymersomes with the C block outside.

Biomolecules Turn Self-Assembling Amphiphilic Block Co-polymer Platforms Into Biomimetic Interfaces.

Biological membranes constitute an interface between cells and their surroundings and form distinct compartments within the cell. They also host a variety of biomolecules that carry out vital functions including selective transport, signal transduction and cell-cell communication. Due to the vast complexity and versatility of the different membranes, there is a critical need for simplified and specific model membrane platforms to explore the behaviors of individual biomolecules while preserving their intrinsic function. Information obtained from model membrane platforms should make invaluable contributions to current and emerging technologies in biotechnology, nanotechnology and medicine. Amphiphilic block co-polymers are ideal building blocks to create model membrane platforms with enhanced stability and robustness. They form various supramolecular assemblies, ranging from three-dimensional structures (e.g., micelles, nanoparticles, or vesicles) in aqueous solution to planar polymer membranes on solid supports (e.g., polymer cushioned/tethered membranes,) and membrane-like polymer brushes. Furthermore, polymer micelles and polymersomes can also be immobilized on solid supports to take advantage of a wide range of surface sensitive analytical tools. In this review article, we focus on self-assembled amphiphilic block copolymer platforms that are hosting biomolecules. We present different strategies for harnessing polymer platforms with biomolecules either by integrating proteins or peptides into assemblies or by attaching proteins or DNA to their surface. We will discuss how to obtain synthetic structures on solid supports and their characterization using different surface sensitive analytical tools. Finally, we highlight present and future perspectives of polymer micelles and polymersomes for biomedical applications and those of solid-supported polymer membranes for biosensing.

Nanoparticle-based highly sensitive MRI contrast agents with enhanced relaxivity in reductive milieu.

Current magnetic resonance imaging (MRI) contrast agents often produce insufficient contrast for diagnosis of early disease stages, and do not sense their biochemical environments. Herein, we report a highly sensitive nanoparticle-based MRI probe with r1 relaxivity up to 51.7 ± 1.2 mM(-1) s(-1) (3T). Nanoparticles were co-assembled from Gd(3+) complexed to heparin-poly(dimethylsiloxane) copolymer, and a reduction-sensitive amphiphilic peptide serving to induce responsiveness to environmental changes. The release of the peptide components leads to a r1 relaxivity increase under reducing conditions and increases the MRI contrast. In addition, this MRI probe has several advantages, such as a low cellular uptake, no apparent cellular toxicity (tested up to 1 mM Gd(3+)), absence of an anticoagulation property, and a high shelf stability (no increase in free Gd(3+) over 7 months). Thus, this highly sensitive T1 MRI contrast nanoparticle system represents a promising probe for early diagnosis through possible accumulation and contrast enhancement within reductive extracellular tumour tissue.

Functional surface engineering by nucleotide-modulated potassium channel insertion into polymer membranes attached to solid supports.

Planar solid-supported membranes based on amphiphilic block copolymers represent promising systems for the artificial creation of structural surfaces. Here we introduce a method for engineering functional planar solid-supported membranes through insertion of active biomolecules. We show that membranes based on poly(dimethylsiloxane)-block-poly(2-methyl-2-oxazoline) (PDMS-b-PMOXA) amphiphilic diblock copolymers, which mimic natural membranes, are suitable for hosting biomolecules. Our strategy allows preparation of large-area, well-ordered polymer bilayers via Langmuir-Blodgett and Langmuir-Schaefer transfers, and insertion of biomolecules by using Bio-Beads. We demonstrate that a model membrane protein, the potassium channel from the bacterium Mesorhizobium loti, remains functional after insertion into the planar solid-supported polymer membrane. This approach can be easily extended to generate a platform of functional solid-supported membranes by insertion of different hydrophobic biomolecules, and employing different types of solid substrates for desired applications.

Self-assembled peptide beads used as a template for ordered gold nanoparticle superstructures.

Using peptide-based materials to tailor self-assembled, nano-scaled hybrid materials with potentially high biocompatibility/biodegradability is gaining importance in developing a broad range of new applications, in areas such as diagnostics and medicine. Here, we investigated how the self-assembly ability of amphiphilic peptides can be used to create organized inorganic materials, i.e. gold nanoparticles. A bead-forming, purely peptidic amphiphile Ac-[K(Ac)]3-[W-l]3-W-NH2, containing acetylated (Ac) l-lysine (K), l-tryptophan (W) and d-leucine (l), was C-terminally modified with a l-cysteine (C) and linked to gold nanoparticles. Subsequent peptide-driven self-assembly of the peptide-coated gold nanoparticles with increasing water content led to controlled aggregation of the gold-core micelles, forming composite peptide-gold superstructures. The individual gold nanoparticles did not agglomerate but were separated from each other by a peptide film within the composite material, as revealed by electron microscopy studies. Structural investigation on 2D template-stripped gold demonstrated the ability of the peptides to form self-assembled monolayers. Structural elements of ß-turns and weak hydrogen bonding of the hydrophobic moiety of the peptide were evident, thereby suggesting that the secondary structure remains intact.

Effects of silver nanoparticles on primary mixed neural cell cultures: uptake, oxidative stress and acute calcium responses.

In the body, nanoparticles can be systemically distributed and then may affect secondary target organs, such as the central nervous system (CNS). Putative adverse effects on the CNS are rarely investigated to date. Here, we used a mixed primary cell model consisting mainly of neurons and astrocytes and a minor proportion of oligodendrocytes to analyze the effects of well-characterized 20 and 40 nm silver nanoparticles (SNP). Similar gold nanoparticles served as control and proved inert for all endpoints tested. SNP induced a strong size-dependent cytotoxicity. Additionally, in the low concentration range (up to 10 µg/ml of SNP), the further differentiated cultures were more sensitive to SNP treatment. For detailed studies, we used low/medium dose concentrations (up to 20 µg/ml) and found strong oxidative stress responses. Reactive oxygen species (ROS) were detected along with the formation of protein carbonyls and the induction of heme oxygenase-1. We observed an acute calcium response, which clearly preceded oxidative stress responses. ROS formation was reduced by antioxidants, whereas the calcium response could not be alleviated by antioxidants. Finally, we looked into the responses of neurons and astrocytes separately. Astrocytes were much more vulnerable to SNP treatment compared with neurons. Consistently, SNP were mainly taken up by astrocytes and not by neurons. Immunofluorescence studies of mixed cell cultures indicated stronger effects on astrocyte morphology. Altogether, we can demonstrate strong effects of SNP associated with calcium dysregulation and ROS formation in primary neural cells, which were detectable already at moderate dosages.