Bridging between bioactive and biomimicking materials : cascade reactions in catalytic compartments

Based on the theme of this thesis, Chapter 1 introduces the concept cells as the paramount example of compartmentalization in nature and the use of polymeric assemblies encapsulating enzymes as mimics. It then proceeds to discuss the principles behind self-assembly of polymers and applications of such systems. Building on that, Chapter 2 states the aim of the thesis, delineating its background and the vision that lead to a coherent research process. For this thesis, vesicular polymeric compartments composed of the triblock copolymer PMOXA-b-PDMS-b-PMOXA were produced, harbouring various proteins in their lumen and membranes, for catalysis and membrane permeabilization. In a first step, I contributed to the development of multicompartment cell mimics, micrometer-sized polymeric vesicles that behave like cells in their internal organization and segregation, triggered environmental responses and architectural plasticity. In Chapter 3, such assemblies are able to sense the redox potential of the exterior and, with a cascade resembling receptor-mediated pathways in cells, activate responses ranging from enzymatic activity to selective permeability and cytoskeleton reorganization. In Chapter 4 and 5, the polymeric vesicles were “shrunk” to diameters of 200 nm and less, to work on biological settings, using sizes smaller than cells for future biomedical applications, with binary mixture of vesicles encapsulating a single type of enzyme. They lost their internal compartmentalization but gained a more intimate relationship with living matter, acting first as cell models, then as symbionts to detoxify the cell medium from uric acid (Chapter4.1) and finally as artificial organelles to study the effect of the overproduction of the signaling molecule cGMP through an already-present cascade (5.1). These two studies shed light not only on the general behavior of binary cascades at the nanoscale, but also on technological limitations of such system, that is the difficult transmembrane diffusion through the porin OmpF, and the effect of distance. To solve the first matter, we studied melittin as a replacement for OmpF. The pore-forming peptide was studied in its interaction with PMOXA-b-PDMS-b-PMOXA membranes (Chapter 6), and we determine the parameters governing their interaction, both from the polymer (stiffness, length, chain dispersity, roughness), from the geometry of the assembly (curvature) and its stability when it interacts with the peptide. A kind of catalytically active polymeric vesicles was produced to prove melittin’s functionality. To solve the problem of substrate diffusion, we designed clusters of catalytic vesicles, tethered via complementary DNA strands, and permeabilized by melittin. Enzymes part of the same cascade were in close proximity, below 20 nm, leading to a net gain in reaction efficiency when compared to the same unclustered conditions. Additionally, the DNA clusters adhered to the surface of lung cells, suggesting a future as targeted delivery. The conclusions of Chapter 8 summarize the results of this work and suggest the future outlook for research in this field, whereas Chapter 9 lists all the materials and methods used.

Bioactive Catalytic Nanocompartments Integrated into Cell Physiology and Their Amplification of a Native Signaling Cascade

Bioactive nanomaterials have the potential to overcome the limitations of classical pharmacological approaches by taking advantage of native pathways to influence cell behavior, interacting with them and eliciting responses. Herein, we propose a cascade system mediated by two catalytic nanocompartments (CNC) with biological activity. Activated by nitric oxide (NO) produced by inducible nitric oxidase synthase (iNOS), soluble guanylyl cyclase (sGC) produces cyclic guanosine monophosphate (cGMP), a second messenger that modulates a broad range of physiological functions. As alterations in cGMP signaling are implicated in a multitude of pathologies, its signaling cascade represents a viable target for therapeutic intervention. Following along this line, we encapsulated iNOS and sGC in two separate polymeric compartments that function in unison to produce NO and cGMP. Their action was tested in vitro by monitoring the derived changes in cytoplasmic calcium concentrations of HeLa and differentiated C2C12 myocytes, where the produced second messenger influenced the cellular homeostasis

Nanoscale Enzymatic Compartments in Tandem Support Cascade Reactions in Vitro

Compartmentalization at the nanoscale is fundamental in nature, where the spatial segregation of biochemical reactions within cells ensures optimal conditions for regulating metabolic pathways. Here, we present a nature-inspired approach to engineer enzymatic cascade reactions taking place between separate vesicular nanocompartments (polymersomes), each containing one enzyme type. We propose, by the selected combination of enzymes, an efficient solution to detoxify the harmful effect of uric acid and prevent the accumulation of the derived H2O2, both being associated with various pathological conditions (e.g., gout and oxidative stress). Fungal uricase and horseradish peroxidase combined to act in tandem, and they were separately encapsulated within nanocompartments that were equipped with channel porins as gates to allow passage of substrates and products from each step of the reaction. We established the molecular factors affecting the efficiency of the overall reaction, and the protective role of the compartments. Interestingly, the cascade reaction between separate nanocompartments was as efficient as for free enzymes in complex media, such as human serum. The nanocompartments were nontoxic toward cells, and more importantly, addition of the tandem catalytic nanocompartments to cells exposed to uric acid provided simultaneous detoxification of uric acid and the H2O2. Such catalytic nanocompartments can be used as a platform for understanding fundamental factors affecting intracellular communication and can introduce non-native metabolic reactions into living systems for therapeutic applications

An outer membrane‐inspired polymer coating protects and endows Escherichia coli with novel functionalities

A bio‐inspired membrane made of Pluronic L‐121 is produced around Escherichia coli thanks to the simple co‐extrusion of bacteria and polymer vesicles. The block copolymer‐coated bacteria can withstand various harsh shocks, for example, temperature, pressure, osmolarity, and chemical agents. The polymer membrane also makes the bacteria resistant to enzymatic digestion and enables them to degrade toxic compounds, improving their performance as whole‐cell biocatalysts. Moreover, the polymer membrane acts as an anchor layer for the surface modification of the bacteria. Being decorated with α‐amylase or lysozyme, the cells are endowed with the ability to digest starch or self‐predatory bacteria are created. Thus, without any genetic engineering, the phenotype of encapsulated bacteria is changed as they become sturdier and gain novel metabolic functionalities

Polymer-Lipid Hybrid Membranes as a Model Platform to Drive Membrane-Cytochrome c Interaction and Peroxidase-like Activity

Controllable attachment of proteins to material surfaces is very attractive for many applications including biosensors, bioengineered scaffolds or drug screening. Especially, redox proteins have received considerable attention as a model system not only to understand the mechanism of electron transfer in biological systems, but also the development of novel biosensors. However, current research attempts suffer from denaturation of the protein after its attachment to solid substrates. Here, we present how lipid, polymer and hybrid membranes based on mixtures of lipids and copolymers on a solid support provide a more favorable environment to drive selective and functional attachment of a model redox protein, cytochrome c (cyt c). Polymer membranes provided chemical versatility to support covalent attachment of cyt c, whereas lipid membranes provided flexibility and biocompatibility to support insertion of cyt c through its hydrophobic part. Hybrid membranes combine the most promising characteristics of both lipids and polymers and allowed attachment of cyt c with both covalent attachment and insertion driven by hydrophobic interactions. We then investigated the effect of different attachment strategies on the accessibility and peroxidase-like activity of cyt c, in the presence of different membranes. The real-time combination of cyt c with the planar membranes was investigated by quartz crystal microbalance with dissipation. It was possible to selectively drive the insertion of cyt c into a specific lipid domain of hybrid membranes. In addition, protein accessibility and its functionality were dependent on the specificity of the combination strategy: covalent conjugation of cyt c to polymer and hybrid membranes promoted higher accessibility and supported higher peroxidase-like activity. Taking together, the combination of biomolecules with planar membranes can be modulated in such a way to improve the accessibility of the biomolecules and their resulting functionality for the development of efficient âEuro�active surfacesâEuro�

Multicompartment Polymer Vesicles with Artificial Organelles for Signal-Triggered Cascade Reactions Including Cytoskeleton Formation

Abstract Organelles, i.e., internal subcompartments of cells, are fundamental to spatially separate cellular processes, while controlled intercompartment communication is essential for signal transduction. Furthermore, dynamic remodeling of the cytoskeleton provides the mechanical basis for cell shape transformations and mobility. In a quest to develop cell-like smart synthetic materials, exhibiting functional flexibility, a self-assembled vesicular multicompartment system, comprised of a polymeric membrane (giant unilamellar vesicle, GUV) enveloping polymeric artificial organelles (vesicles, nanoparticles), is herein presented. Such multicompartment assemblies respond to an external stimulus that is transduced through a precise sequence. Stimuli-triggered communication between two types of internal artificial organelles induces and localizes an enzymatic reaction and allows ion-channel mediated release from storage vacuoles. Moreover, cytoskeleton formation in the GUVs` lumen can be triggered by addition of ionophores and ions. An additional level of control is achieved by signal-triggered ionophore translocation from organelles to the outer membrane, triggering cytoskeleton formation. This system is further used to study the diffusion of various cytoskeletal drugs across the synthetic outer membrane, demonstrating potential applicability, e.g., anticancer drug screening. Such multicompartment assemblies represent a robust system harboring many different functionalities and are a considerable leap in the application of cell logics to reactive and smart synthetic materials

Mimicking Cellular Signaling Pathways within Synthetic Multicompartment Vesicles with Triggered Enzyme Activity and Induced Ion Channel Recruitmen

Subcellular compartmentalization of cells, a defining characteristic of eukaryotes, is fundamental for the fine tuning of internal processes and the responding to external stimuli. Reproducing and controlling such compartmentalized hierarchical organization, responsiveness, and communication is important toward understanding biological systems and applying them to smart materials. Herein, a cellular signal transduction strategy (triggered release from subcompartments) is leveraged to develop responsive, purely artificial, polymeric multicompartment assemblies. Incorporation of responsive nanoparticles-loaded with enzymatic substrate or ion channels-as subcompartments inside micrometer-sized polymeric vesicles (polymersomes) allowed to conduct bioinspired signaling cascades. Response of these subcompartments to an externally added stimulus is achieved and studied by using confocal laser scanning microscopy (CLSM) coupled with in situ fluorescence correlation spectroscopy (FCS). Signal triggered activity of an enzymatic reaction is demonstrated in multicompartments through recombination of compartmentalized substrate and enzyme. In parallel, a two-step signaling cascade is achieved by triggering the recruitment of ion channels from inner subcompartments to the vesicles' membrane, inducing ion permeability, mimicking endosome-mediated insertion of internally stored channels. This design shows remarkable versatility, robustness, and controllability, demonstrating that multicompartment polymer-based assemblies offer an ideal scaffold for the development of complex cell-inspired responsive systems for applications in biosensing, catalysis, and medicine

Clustering of catalytic nanocompartments for enhancing an extracellular non-native cascade reaction

Compartmentalization is fundamental in nature, where the spatial segregation of biochemical reactions within and between cells ensures optimal conditions for the regulation of cascade reactions. While the distance between compartments or their interaction are essential parameters supporting the efficiency of bio-reactions, so far they have not been exploited to regulate cascade reactions between bioinspired catalytic nanocompartments. Here, we generate individual catalytic nanocompartments (CNCs) by encapsulating within polymersomes or attaching to their surface enzymes involved in a cascade reaction and then, tether the polymersomes together into clusters. By conjugating complementary DNA strands to the polymersomes' surface, DNA hybridization drove the clusterization process of enzyme-loaded polymersomes and controlled the distance between the respective catalytic nanocompartments. Owing to the close proximity of CNCs within clusters and the overall stability of the cluster architecture, the cascade reaction between spatially segregated enzymes was significantly more efficient than when the catalytic nanocompartments were not linked together by DNA duplexes. Additionally, residual DNA single strands that were not engaged in clustering, allowed for an interaction of the clusters with the cell surface as evidenced by A549 cells, where clusters decorating the surface endowed the cells with a non-native enzymatic cascade. The self-organization into clusters of catalytic nanocompartments confining different enzymes of a cascade reaction allows for a distance control of the reaction spaces which opens new avenues for highly efficient applications in domains such as catalysis or nanomedicine