Nico Bruns

Biocompatible Functionalization of Polymersome Surfaces: A New Approach to Surface Immobilization and Cell Targeting Using Polymersomes

Vesicles assembled from amphiphilic block copolymers represent promising nanomaterials for applications that include drug delivery and surface functionalization. One essential requirement to guide such polymersomes to a desired site in vivo is conjugation of active, targeting ligands to the surface of preformed self-assemblies. Such conjugation chemistry must fulfill criteria of efficiency and selectivity, stability of the resulting bond, and biocompatibility. We have here developed a new system that achieves these criteria by simple conjugation of 4-formylbenzoate (4FB) functionalized polymersomes with 6-hydrazinonicotinate acetone hydrazone (HyNic) functionalized antibodies in aqueous buffer. The number of available amino groups on the surface of polymersomes composed of poly(dimethylsiloxane)-block-poly(2-methyloxazoline) diblock copolymers was investigated by reacting hydrophilic succinimidyl-activated fluorescent dye with polymersomes and evaluating the resulting emission intensity. To prove attachment of biomolecules to polymersomes, HyNic functionalized enhanced yellow fluorescent protein (eYFP) was attached to 4FB functionalized polymersomes, resulting in an average number of 5 eYFP molecules per polymersome. Two different polymersome-antibody conjugates were produced using either antibiotin IgG or trastuzumab. They showed specific targeting toward biotin-patterned surfaces and breast cancer cells. Overall, the polymersome-ligand platform appears promising for therapeutic and diagnostic use.

Horseradish Peroxidase as a Catalyst for Atom Transfer Radical Polymerization

The hemoprotein horseradish peroxidase (HRP) catalyzes the polymerization of N-isopropylacrylamide with an alkyl bromide initiator under conditions of activators regenerated by electron transfer atom transfer radical polymerization (ARGET ATRP) in the absence of any peroxide. This is a novel activity of HRP, which we propose to name ATRPase activity. Bromine-terminated polymers with polydispersity indices (PDIs) as low as 1.44 are obtained. The polymerization follows first order kinetics, but the evolution of molecular weight and the PDI upon increasing conversion deviate from the results expected for an ATRP mechanism. Conversion, M(n) and PDI depend on the pH and on the concentration of the reducing agent, sodium ascorbate. HRP is stable during the polymerization and does not unfold or form conjugates.

Mechanical nanosensor based on fret within a thermosome: Damage-reporting polymeric materials

Unter Spannung: Andert sich in einem Protein-Polymer-Hybridmaterial die mechanische Spannung der Polymermatrix, so lost dies eine Konformationsanderung des Proteinkomplexes aus: Das Material meldet eine strukturelle Schadigung (siehe Bild). Die Reporterkomponente ist ein Chaperonin, das ein Paar fluoreszierender Proteine kovalent bindet. Wird das Chaperonin deformiert, andert sich der Abstand zwischen den Fluorophoren und folglich auch das FRET-Signal. Under stress: Changes in stress of the polymer matrix in a protein-polymer hybrid material result in changes of conformation of the protein complex, thus resulting in a damage-reporting material (see picture). The reporter is an engineered chaperonin that covalently entraps a pair of fluorescent proteins. Deformation of the chaperonin leads to a change in fluorophore distance and a change in the fluorescene resonance energy transfer (FRET) signal.

pH-Responsive PDMS-b-PDMAEMA Micelles for Intracellular Anticancer Drug Delivery

A series of poly(dimethysiloxane)-b-poly(2-(dimethylamino)ethyl methacrylate) (PDMS-b-PDMAEMA) block copolymers were synthesized with atom transfer radical polymerization (ATRP). In aqueous solution the polymers self-assembled into micelles with diameters between 80 and 300 nm, with the ability to encapsulate DOX. The polymer with the shortest PDMAEMA block (5 units) displayed excellent cell viability, while micelles containing longer PDMAEMA block lengths (13 and 22 units) led to increased cytotoxicity. The carriers released DOX in response to a decrease in pH from 7.4 to 5.5. Confocal laser scanning microscopy (CLSM) revealed that nanoparticles were taken up by endocytosis into acidic cell compartments. Furthermore, DOX-loaded nanocarriers exhibited intracellular pH-response as changes in cell morphology and drug release were observed within 24 h.

Hemoglobin and red blood cells catalyze atom transfer radical polymerization.

Hemoglobin (Hb) is a promiscuous protein that not only transports oxygen, but also catalyzes several biotransformations. A novel in vitro catalytic activity of Hb is described. Bovine Hb and human erythrocytes were found to display ATRPase activity, i.e., they catalyzed the polymerization of vinyl monomers under conditions typical for atom transfer radical polymerization (ATRP). N-isopropylacrylamide (NIPAAm), poly(ethylene glycol) methyl ether acrylate (PEGA), and poly(ethylene glycol) methyl ether methacrylate (PEGMA) were polymerized using organobromine initiators and the reducing agent ascorbic acid in acidic aqueous solution. In order to avoid chain transfer from polymer radicals to Hbs cysteine residues, the accessible cysteines were blocked by a reaction with a maleimide. The formation of polymers with bromine chain ends, relatively low polydispersity indices (PDI), first order kinetics and an increase in the molecular weight of poly(PEGA) and poly(PEGMA) upon conversion indicate that control of the polymerization by Hb occurred via reversible atom transfer between the protein and the growing polymer chain. For poly(PEGA) and poly(PEGMA), the reactions proceeded with a good to moderate degree of control. Sodium dodecyl sulfate (SDS) gel electrophoresis, circular dichroism spectroscopy, and time-resolved ultraviolet-visible (UV-vis) spectroscopy revealed that the protein was stable during polymerization, and only underwent minor conformational changes. As Hb and erythrocytes are readily available, environmentally friendly, and nontoxic, their ATRPase activity is a useful tool for synthetic polymer chemistry. Moreover, this novel activity enhances the understanding of Hbs redox chemistry in the presence of organobromine compounds.

Real-time viscosity and mass density sensors requiring microliter sample volume based on nanomechanical resonators.

A microcantilever based method for fluid viscosity and mass density measurements with high temporal resolution and microliter sample consumption is presented. Nanomechanical cantilever vibration is driven by photothermal excitation and detected by an optical beam deflection system using two laser beams of different wavelengths. The theoretical framework relating cantilever response to the viscosity and mass density of the surrounding fluid was extended to consider higher flexural modes vibrating at high Reynolds numbers. The performance of the developed sensor and extended theory was validated over a viscosity range of 1-20 mPa·s and a corresponding mass density range of 998-1176 kg/m(3) using reference fluids. Separating sample plugs from the carrier fluid by a two-phase configuration in combination with a microfluidic flow cell, allowed samples of 5 μL to be sequentially measured under continuous flow, opening the method to fast and reliable screening applications. To demonstrate the study of dynamic processes, the viscosity and mass density changes occurring during the free radical polymerization of acrylamide were monitored and compared to published data. Shear-thinning was observed in the viscosity data at higher flexural modes, which vibrate at elevated frequencies. Rheokinetic models allowed the monomer-to-polymer conversion to be tracked in spite of the shear-thinning behavior, and could be applied to study the kinetics of unknown processes.

Protein cages and synthetic polymers: a fruitful symbiosis for drug delivery applications, bionanotechnology and materials science

Protein cages are hollow protein nanoparticles, such as viral capsids, virus-like particles, ferritin, heat-shock proteins and chaperonins. They have well-defined capsule-like structures with a monodisperse size. Their protein subunits can be modified by genetic engineering at predetermined positions, allowing for example site-selective introduction of attachment points for functional groups, catalysts or targeting ligands on their outer surface, in their interior and between subunits. Therefore, protein cages have been extensively explored as functional entities in bionanotechnology, as drug-delivery or gene-delivery vehicles, as nanoreactors or as templates for the synthesis of organic and inorganic nanomaterials. The scope of functionalities and applications of protein cages can be significantly broadened if they are combined with synthetic polymers on their surface or within their interior. For example, PEGylation reduces the immunogenicity of protein cage-based delivery systems and active targeting ligands can be attached via polymer chains to favour their accumulation in diseased tissue. Polymers within protein cages offer the possibility of increasing the loading density of drug molecules, nucleic acids, magnetic resonance imaging contrast agents or catalysts. Moreover, the interaction of protein cages and polymers can be used to modulate the size and shape of some viral capsids to generate structures that do not occur with native viruses. Another possibility is to use the interior of polymer cages as a confined reaction space for polymerization reactions such as atom transfer radical polymerization or rhodium-catalysed polymerization of phenylacetylene. The protein nanoreactors facilitate a higher degree of control over polymer synthesis. This review will summarize the hybrid structures that have been synthesized by polymerizing from protein cage-bound initiators, by conjugating polymers to protein cages, by embedding protein cages into bulk polymeric materials, by forming two- and three-dimensional crystals of protein cages and dendrimers, by adsorbing proteins to the surface of materials, by layer-by-layer deposition of proteins and polyelectrolytes and by encapsulating polymers into protein cages. The application of these hybrid materials in the biomedical context or as tools and building blocks for bionanotechnology, biosensing, memory devices and the synthesis of materials will be highlighted. The review aims to showcase recent developments in this field and to suggest possible future directions and opportunities for the symbiosis of protein cages and polymers.

Fluorescent Protein Senses and Reports Mechanical Damage in Glass-Fiber-Reinforced Polymer Composites

Yellow fluorescent protein (YFP) is used as a mechanoresponsive layer at the fiber/resin interface in glass-fiber-reinforced composites. The protein loses its fluorescence when subjected to mechanical stress. Within the material, it reports interfacial shear debonding and barely visible impact damage by a transition from a fluorescent to a non-fluorescent state.

Filling polymersomes with polymers by peroxidase-catalyzed atom transfer radical polymerization.

Polymersomes that encapsulate a hydrophilic polymer are prepared by conducting biocatalytic atom transfer radical polymerization (ATRP) in these hollow nanostructures. To this end, ATRPase horseradish peroxidase (HRP) is encapsulated into vesicles self-assembled from poly(dimethylsiloxane)-block-poly(2-methyl-2-oxazoline) (PDMS-b-PMOXA) diblock copolymers. The vesicles are turned into nanoreactors by UV-induced permeabilization with a hydroxyalkyl phenone and used to polymerize poly(ethylene glycol) methyl ether acrylate (PEGA) by enzyme-catalyzed ATRP. As the membrane of the polymersomes is only permeable for the reagents of ATRP but not for macromolecules, the polymerization occurs inside of the vesicles and fills the polymersomes with poly(PEGA), as evidenced by (1) H NMR. Dynamic and static light scattering show that the vesicles transform from hollow spheres to filled spheres during polymerization. Transmission electron microscopy (TEM) and cryo-TEM imaging reveal that the polymersomes are stable under the reaction conditions. The polymer-filled nanoreactors mimic the membrane and cytosol of cells and can be useful tools to study enzymatic behavior in crowded macromolecular environments.

Poly(N-vinylpyrrolidone)-poly(dimethylsiloxane)-based polymersome nanoreactors for laccase-catalyzed biotransformations.

Laccases (Lac) are oxidizing enzymes with a broad range of applications, for example, in soil remediation, as bleaching agent in the textile industry, and for cosmetics. Protecting the enzyme against degradation and inhibition is of great importance for many of these applications. Polymer vesicles (polymersomes) from poly(N-vinylpyrrolidone)-block-poly(dimethylsiloxane)-block-poly(N-vinylpyrrolidone) (PNVP-b-PDMS-b-PNVP) triblock copolymers were prepared and investigated as intrinsically semipermeable nanoreactors for Lac. The block copolymers allow oxygen to enter and reactive oxygen species (ROS) to leave the polymersomes. EPR spectroscopy proved that Lac can generate ROS. They could diffuse out of the polymersome and oxidize an aromatic substrate outside the vesicles. Michaelis-Menten constants Km between 60 and 143 μM and turn over numbers kcat of 0.11 to 0.18 s(-1) were determined for Lac in the nanoreactors. The molecular weight and the PDMS-to-PNVP ratio of the block copolymers influenced these apparent Michaelis-Menten parameters. Encapsulation of Lac in the polymersomes significantly protected the enzyme against enzymatic degradation and against small inhibitors: proteinase K caused 90% less degradation and the inhibitor sodium azide did not affect the enzymes activity. Therefore, these polymer nanoreactors are an effective means to stabilize laccase.