Natural channel protein inserts and functions in a completely artificial, solid-supported bilayer membrane

Reconstitution of membrane proteins in artificial membrane systems creates a platform for exploring their potential for pharmacological or biotechnological applications. Previously, we demonstrated amphiphilic block copolymers as promising building blocks for artificial membranes with long-term stability and tailorable structural parameters. However, the insertion of membrane proteins has not previously been realized in a large-area, stable, and solid-supported artificial membrane. Here, we show the first, preliminary model of a channel membrane protein that is functionally incorporated in a completely artificial polymer, tethered, solid-supported bilayer membrane (TSSBM). Unprecedented ionic transport characteristics that differ from previous results on protein insertion into planar, free-standing membranes, are identified. Our findings mark a change in understanding protein insertion and ion flow within natural channel proteins when inserted in an artificial TSSBM, thus holding great potential for numerous applications such as drug screening, trace analyzing, and biosensing

Block Copolymer Giant Unilamellar Vesicles for High-Throughput Screening

Bottom-up synthetic cells offer the potential to study cellular processes with reduced complexity. Giant unilamellar vesicles (GUVs) can mimic cells in their morphological characteristics because their architecture is precisely controllable. We propose a block copolymer-based GUV system that can be used for high-throughput screening. Through droplet microfluidic methods, we produce double emulsions that then serve as templates for GUVs with adjustable inner, polymer membrane, and outer composition. Using flow cytometry, we are able to analyze tens of thousands of GUVs in a short amount of time, enabling their use for screening assays

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

Clusters of polymersomes and Janus nanoparticles hierarchically self-organized and controlled by DNA hybridization

The combination of "hard", structurally well-defined particles with "soft", functional compartments bears great potential to produce structurally intricate hybrid nanomaterials that promote a multitude of applications that require multimodal agents and that permit the production of molecular factories. However, the co-assembly of "hard" and "soft" components in a programmable and directional manner is challenging due to the strongly differing mechanical properties of such disparate entities. Here, a versatile strategy to generate clusters by the directional and controlled self-organization of "hard" Janus nanoparticles (JNPs) with "soft" polymersomes is described. The hybridization of complementary ssDNA strands bound to the components drives cluster formation, while the asymmetry of the JNPs governs the directionality of the self-organization. Various factors have been explored to simultaneously preserve the integrity of the polymersomes and program the cluster formation. Differently loaded polymersomes on each lobe of the JNPs preserved their architecture in the clusters which, were shown to be non-toxic when interacting with cell lines. The architecture of the clusters, as a molecular factory where each component can be separately controlled bears great promise for use in advanced medical applications, including theranostics and correlative imaging

Surfaces with Dual Functionality through Specific Coimmobilization of Self-Assembled Polymeric Nanostructures

Coimmobilization of functional, nanosized assemblies broadens the possibility to engineer dually functionalized active surfaces with a nanostructured texture. Surfaces decorated with different nanoassemblies, such as micelles, polymersomes, or nanoparticles are in high demand for various applications ranging from catalysis, biosensing up to antimicrobial surfaces. Here, we present a combination of bio-orthogonal and catalyst-free strain-promoted azide–alkyne click (SPAAC) and thiol–ene reactions to simultaneously coimmobilize various nanoassemblies; we selected polymersome–polymersome and polymersome–micelle assemblies. For the first time, the immobilization method using SPAAC reaction was studied in detail to attach soft, polymeric assemblies on a solid support. Together, the SPAAC and thiol–ene reactions successfully coimmobilized two unique self-assembled structures on the surfaces. Additionally, poly(dimethylsiloxane) (PDMS)-based polymersomes were used as “ink” for direct immobilization from a PDMS-based microstamp onto a surface creating locally defined patterns. Combining immobilization reactions has the advantage to attach any kind of nanoassembly pairs, resulting in surfaces with “desired” interfacial properties. Different nanoassemblies that encapsulate multiple active compounds coimmobilized on a surface will pave the way for the development of multifunctional surfaces with controlled properties and efficiency

Selective ion-permeable membranes by insertion of biopores into polymersomes

In nature there are various specific reactions for which highly selective detection or support is required to preserve their bio-specificity or/and functionality. In this respect, mimics of cell membranes and bio-compartments are essential for developing tailored applications in therapeutic diagnostics. Being inspired by nature, we present here biomimetic nanocompartments with ion-selective membrane permeability engineered by insertion of ionomycin into polymersomes with sizes less than 250 nm. As a marker to assess the proper insertion and functionality of ionomycin inside the synthetic membrane, we used a Ca2+-sensitive dye encapsulated inside the polymersome cavity prior to inserting the biopore. The calcium sensitive dye, ionomycin, and Ca2+ did not influence the architecture and the size of polymersomes. Successful ionomycin functionality inside the synthetic membrane with a thickness of 10.7 nm was established by a combination of fluorescence spectroscopy and stopped-flow spectroscopy. Polymersomes equipped with ion selective membranes are ideal candidates for the development of medical applications, such as cellular ion nanosensors or nanoreactors in which ion exchange is required to support in situ reactions

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

Asymetric Triblock Copolymer Nanocarriers for Controlled Localization and pH-Sensitive Release of Proteins

Designing nanocarriers to release proteins under specific conditions is required to improve therapeutic approaches, especially in treating cancer and protein deficiency diseases. We present here supramolecular assemblies based on asymmetric poly(ethylene glycol)-b-poly(methylcaprolactone)-b-poly(2-(N,Ndiethylamino)ethyl methacrylate) (PEG-b-PMCL-b-PDMAEMA) copolymers for controlled localization and pH-sensitive release of proteins. Copolymers self-assembled in soft nanoparticles with a core domain formed by PMCL, and a hydrophilic domain based on PEG mainly embedded inside, and the branched PDMAEMA exposed at the particle surface. We selected as model proteins to be attached to the nanoparticles bovine serum albumin (BSA) and acid sphingomyelinase (ASM), the latter being an ideal candidate for protein replacement therapy. The hydrophilic/hydrophobic ratio, nanoparticle size, and the nature of biomolecules are key factors for modulating protein localization and attachment efficiency. The predominant outer shell of PDMAEMA allows efficient pH-triggered release of BSA and ASM, and in acidic conditions >70% of the bound proteins were released. Uptake of protein-attached nanoparticles by HELA cells, together with low toxicity and pH-responsive release, supports such protein-bound nanoparticles as efficient stimuli-responsive candidates for protein therapy

Sistema de control embebido conectable a PC : diseño y aplicaciones prácticas

Ladarescu Palivan, I. (2010). Sistema de control embebido conectable a PC : diseño y aplicaciones prácticas. http://hdl.handle.net/10251/9118.Archivo delegad

Biomolecule-polymer hybrid compartments: combining the best of both worlds

Compartmentalization is a fundamental principle in biology that is needed for the temporal and spatial separation of chemically incompatible reactions and biomolecules. Nano- or micro-sized compartments made of synthetic polymers are used to mimick this principle. The self-assembly of these polymers into vesicular objects is highly compatible with the integration of biomolecules, either into the lumen, the membrane or onto the surface of the vesicles. Thus, a great variety of biohybrid nano- and microscaled compartments has been developed exploiting the specific function and properties of targeting peptides, antibodies, enzymes, nucleic acids or lipids. Such biohybrid compartments have moved from simple systems encapsulating e.g. a model protein into complex multicompartmentalized structures that are able to combine the activity of different biomolecular cargos getting closer to the realization of artifical organelles or cells. Encapsulation of medically relevant cargos combined with careful design of the polymeric scaffold and specific surface functionalization have led to a significant progress in therapeutical applications such as targeted drug delivery or enzyme replacement therapy