Eva-Kathrin Sinner

Photocontrol of Cell Adhesion Processes: Model Studies with Cyclic Azobenzene-RGD Peptides

A photoresponsive integrin ligand was synthesized by backbone-cyclization of a heptapeptide containing the integrin binding motif Arg-Gly-Asp (RGD) with 4-(aminomethyl)phenylazobenzoic acid (AMPB). Surface plasmon enhanced fluorescence spectroscopy showed that binding of the azobenzene peptide to alpha(v)beta(3) integrin depends on the photoisomeric state of the peptide chromophore. The higher affinity of the trans isomer could be rationalized by comparing the NMR conformations of the cis and trans isomers with the recently solved X-ray structure of a cyclic RGD-pentapeptide bound to integrin.

Proteopolymersomes: In vitro production of a membrane protein in polymersome membranes

Polymersomes are stable self-assembled architectures which mimic cell membranes. For characterization, membrane proteins can be incorporated into such bio-mimetic membranes by reconstitution methods, leading to so-called proteopolymersomes. In this work, we demonstrate the direct incorporation of a membrane protein into polymersome membranes by a cell-free expression system. Firstly, we demonstrate pore formation in the preformed polymersome membrane using α-hemolysin. Secondly, we use claudin-2, a protein involved in cell-cell interactions, to demonstrate the in vitro expression of a membrane protein into these polymersomes. Surface plasmon resonance (Biacore) binding studies with the claudin-2 proteopolymersomes and claudin-2 specific antibodies are performed to show the presence of the in vitro expressed protein in polymersome membranes.

Biomimetic supported membranes from amphiphilic block copolymers

A unique combination of surface chemistry and self-assembly of amphiphilic block copolymers was employed to obtain—for the first time—solid-supported biomimetic polymer bilayers. An organized monolayer from sulfur-functionalized poly(butadiene)-b-poly(ethylene oxide) was covalently attached to ultrasmooth gold upon Langmuir-Blodgett transfer. Hydrophobic interactions, on the other hand, were exploited to attach the second monolayer. As a result, we obtained a homogeneous hydrophilic-hydrophobic-hydrophilic structure, similar to supported lipid bilayers by architecture, stability and fluidity. Our polymer bilayers, however, outperform such lipid membranes with regard to tunability of thickness and stability in gaseous environments. As characterized by surface analysis tools (AFM, SPR), solid-supported polymer membranes are smooth with a thickness of ca. 11 nm, resistant to rinsing with aqueous solutions and stable upon drying and rehydration. These properties could be attractive for nanotechnological applications, such as immobilization of functional molecules or nanoparticles, sensor development or preparation of chemically responsive functional surfaces.

Binding and Docking of Synthetic Heterotrimeric Collagen Type IV Peptides with α1β1 Integrin

Collagen type IV, whose major and ubiquitous form consists of one 2 and two 1 chains, 2] forms a network that determines the biomechanical stability and macromolecular organization of the basement membrane and provides a scaffold into which other constituents of the tissue are incorporated . This collagen benzotriazol (HOAT) as activating reagents and the coupling times were extended to 3 ±4 h to avoid incomplete peptide bond formation. After the entire biotin-tagged and farnesylated peptide 23 had been assembled on the polymeric support, the seven Aloc groups present were removed simultaneously by treatment with Pd[PPh3]4 in the presence of piperidine for four hours. Removal of the catalyst was achieved by simple washing, which rendered the troublesome purification of the unmasked oligolysine peptide unnecessary. Finally, fully unmasked lipidated K-Ras peptide 2 was released from the solid support by treatment with 1% TFA in the presence of 2% TES. Under these conditions both O-trityl groups present in 24 were removed as well and the farnesyl group remained unattacked. Purification of the target peptide was readily achieved by means of HPLC on an RP-C18 column to yield the desired biotin-tagged and lipidated oligolysine peptide 2 (Figure 1) in high purity and with 11% overall yield.

Planar Block Copolymer Membranes by Vesicle Spreading

An easy route to planar solid-supported polymer membranes by vesicle spreading is described. Pre-organized poly(butadiene)-block-poly(ethylene oxide)(PB-PEO) assemblies were spread on two different supports, i.e. strongly hydrophilic glass surfaces and ultrasmooth gold substrates. Polymer membranes were produced on a hydrophilic support by spreading hydroxyl-functionalized polymer vesicles, while covalently immobilized polymer membranes were obtained by spreading LA-functionalized polymer vesicles on gold substrates. Covalently bound membranes were further incubated with the peptide polymyxin B. Interactions with the polymer membrane were detected by EIS. These systems are of great interest to fundamental membrane science and have potential in technological applications, such as drug screening and (bio)sensing.

Current limitations and challenges in nanowaste detection, characterisation and monitoring.

Engineered nanomaterials (ENMs) are already extensively used in diverse consumer products. Along the life cycle of a nano-enabled product, ENMs can be released and subsequently accumulate in the environment. Material flow models also indicate that a variety of ENMs may accumulate in waste streams. Therefore, a new type of waste, so-called nanowaste, is generated when end-of-life ENMs and nano-enabled products are disposed of. In terms of the precautionary principle, environmental monitoring of end-of-life ENMs is crucial to allow assessment of the potential impact of nanowaste on our ecosystem. Trace analysis and quantification of nanoparticulate species is very challenging because of the variety of ENM types that are used in products and low concentrations of nanowaste expected in complex environmental media. In the framework of this paper, challenges in nanowaste characterisation and appropriate analytical techniques which can be applied to nanowaste analysis are summarised. Recent case studies focussing on the characterisation of ENMs in waste streams are discussed. Most studies aim to investigate the fate of nanowaste during incineration, particularly considering aerosol measurements; whereas, detailed studies focusing on the potential release of nanowaste during waste recycling processes are currently not available. In terms of suitable analytical methods, separation techniques coupled to spectrometry-based methods are promising tools to detect nanowaste and determine particle size distribution in liquid waste samples. Standardised leaching protocols can be applied to generate soluble fractions stemming from solid wastes, while micro- and ultrafiltration can be used to enrich nanoparticulate species. Imaging techniques combined with X-ray-based methods are powerful tools for determining particle size, morphology and screening elemental composition. However, quantification of nanowaste is currently hampered due to the problem to differentiate engineered from naturally-occurring nanoparticles. A promising approach to face these challenges in nanowaste characterisation might be the application of nanotracers with unique optical properties, elemental or isotopic fingerprints. At present, there is also a need to develop and standardise analytical protocols regarding nanowaste sampling, separation and quantification. In general, more experimental studies are needed to examine the fate and transport of ENMs in waste streams and to deduce transfer coefficients, respectively to develop reliable material flow models.

In vitro expressed GPCR inserted in polymersome membranes for ligand-binding studies

The dopamine receptor D2 (DRD2), a G-protein coupled receptor is expressed into PBd(22)-PEO(13) and PMOXA(20)-PDMS(54)-PMOXA(20) block copolymer vesicles. The conformational integrity of the receptor is confirmed by antibody- and ligand-binding assays. Replacement of bound dopamine is demonstrated on surface-immobilized polymersomes, thus making this a promising platform for drug screening.

Synthetic biology, inspired by synthetic chemistry

We like to give an overview about the developments in the field of synthetic biology, regarding polymer‐based analogs of cellular membranes and what questions can be answered by applying synthetic polymer science towards the smallest unit in life, namely a cell.

Polymer-Tethered Bimolecular Lipid Membranes

This contribution describes the assembly and structural and functional characterization of various types of polymer-supported lipid bilayer membranes.We start with the description of the polymer-cushioned membrane that can be prepared by first attaching (covalently) polymer coils (as tethers or cushions) from solution to a reactive solid support, followed by the covalent coupling of a lipid monolayer containing reactive anchor lipids. Alternatively, a lipopolymer monolayer (if needed mixed with “normal” lipids) is pre-organized at the water-air interface in a Langmuir trough and then transferred to a solid substrate which is again pre-functionalized by a reactive coating. A special case discussed is the use of glycolipopolymers for the assembly of the proximal tethered monolayer. From all these interfacial architectures the final structure, the supported bilayer, is obtained by the fusion of vesicles forming the distal monolayer of the membrane.

Molecularly controlled functional architectures

This paper summarizes some of our efforts in designing and synthesizing bio-functional layers at solid/solution interfaces, characterizing their structure and dynamics, and optimizing their functional properties. We explore different materials and architectures, focusing here on hydrogels and lipid bilayer membranes.