Stefan M. Schiller

Non-toxic catalysts for ring-opening polymerizations of biodegradable polymers at room temperature for biohybrid materials

Biodegradable biopolymers are versatile materials with a wide range of applications including suture, scaffolds, growth chambers and matrix materials for tissue-engineering and regenerative medicine. Their synthesis is usually based on tin-salts at high temperatures. Strategies to replace tin are interesting due to the toxicity of tin for aquatic organisms and potentially problematic effects in higher organisms. In the framework of this study we investigate nontoxic iron catalysts (iron chloride, bromide and perchlorate) and their potential to facilitate ring-opening polymerizations (ROP) at room temperature. To find the best combination of a catalyst and an initiator for the ROP of e-caprolactone, three iron-catalysts were analysed with four different initiators (water, isopropyl alcohol, benzyl alcohol and 2-allyl phenol). Functional entities such as the allyl group are interesting for bioconjugation and may allow functional access to functional biohybrid materials. The polymerizations were carried out solvent free in bulk and yielded polymers in the range 10–20 kDa for large monomer to catalyst/initiator ratios analysed by GPC, NMR and MALDI-TOF. We find that commercial iron salts can replace tin salts in many cases and allow for the ROP of e-caprolactone at room temperature while tin(II) 2-ethylhexanoate Sn(Oct)2 does not facilitate ROP at room temperature. The use of iron based ROP catalysts opens interesting access to biopolymers utilizing a nontoxic metal ion at low temperatures in a solvent free reaction scheme saving energy and the environment.

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.

Functional Tethered Bilayer Lipid Membranes

Tethered bilayer lipid membranes (tBLMs) constitute a novel experimental concept with very promising features for fundamental biophysical investigations of the general correlation between structural properties and functional processes in model membrane systems. Moreover, these architectures offer a robust platform for membrane-based bio-sensing principles that could eventually result in the design and fabrication of stable and cheap membrane chips. We first discuss a few synthetic strategies that lead to various types of architectures with unprecedented electrical properties: some of these tBLMs show an electrical resistance that exceeds even that of the best model membrane known so far, i.e., the bimolecular lipid membrane (BLM). The reconstitution of a synthetic ionophore, i.e., the carrier valinomycin, allows one to study the K+-selective and reversible increase of the membrane conductivity by more than 4 orders of magnitude. Next, the incorporation of various types of integrin receptors into the tethered bilayer membranes will be briefly discussed as an example of the versatility of this model system in membrane binding assays. And finally, the photopolymerization of polymerizable lipids will be introduced as a way to generate patterned bilayers that can serve as the basic structure for the construction of membrane chips for massive paralleled detection of membrane processes.

Biomimetic Lipoglycopolymer Membranes: Photochemical Surface Attachment of Supramolecular Architectures with Defined Orientation†

The assembly of defined supramolecular architectures on a molecular scale, such as biomimetic membranes, requires the synthesis of multifunctional building blocks and a sophisticated combination of nanotechnological surface preparation techniques. A drawback of current tethered bilayer lipid membrane (tBLM) systems is their limited submembrane decoupling distance from the solid support. The necessity for large (6 nm and more) cytoplasma analogue compartments originates from several biological and biophysical requirements, such as accommodation of cytoplasmatic subunits of membrane proteins, reduction of the F rster energy transfer for the use of fluorescent or photoaffinity probes in vicinity of metal surfaces, and compensation for surface roughness of sensor surfaces often preventing good electrical tBLMs properties. Current polymer-based tBLMs on gold do not achieve membrane– surface decoupling distances of more than 5 nm, nor are polymer based tBLMs able to maintain sufficient electrical properties (resistance of at least several MWcm). To overcome these limitations, we have devised a new strategy to expand the existing scope of tBLMs using lipoglycopolymers (LGP). Special emphasis was placed on increasing the cytoskeleton analogue compartment size (decoupling distance from the sensor surface) whilst retaining tightly sealing membranes, which requires the careful design of the lipid moiety, the tethering polymer, and the reactive self-assembling monolayer (SAM) to immobilize the LGP. Furthermore, we introduced a new covalent immobilization procedure that utilizes photochemical surface attachment to assemble complex supramolecular architectures of defined orientation from aqueous solution. This procedure may have interesting additional applications in the design of new protein nanoarrays and in bionanotechnology in general. The idea of using macromolecules as a cushion to mimic the cytsol/cytoskeleton of the cell to create a hydrophilic space between membrane and solid support was first introduced by Ringsdorf and Sackmann. Macromolecular tethers impose several challenging features. They adopt coiled conformations, which strongly depend on the solvent, and have a range of molecular weights and lengths, whilst interchain interactions determine shape and stability of thin films. Lipid-functionalized small macromolecular tether systems with less than 100 repeating units n that have been used to date reached no more than 40 % of their maximal theoretical thickness. Examples include poly(ethyloxazoline) (n = 50, length 3–3.5 nm) and PEG2000 (n = 45, length 4.9 nm). The requirements for a macromolecular tether can be summarized as follows: 1) There must be complete wetting between the surface and the hydrated polymer and between the membrane and the hydrated polymer; 12] 2) the interaction between membrane and surface needs to be repulsive to prevent dewetting; and 3) nonspecific contacts by van der Waals attraction (effective up to about 3 nm) between lipids and surface must be suppressed. Of all known systems, the glycocalix of the cell is one of the most efficient systems to exert the functions mentioned above. The carbohydrate units of the glycocalix maintain a relatively high osmotic pressure, allowing a stable cell–cell distance between 10–100 nm and a high cooperative stabilization within the glycocalix by hydrogen bonding. Therefore, a carbohydrate-modified or -based macromolecule tether can be envisioned that would enable suitable stabilization within and between tether chains. Theoretical calculations show that to realize a decoupling distance of at least 6 nm, a minimum of twelve b-1,4 linked monosaccharides is required. Apart from the fact that the synthesis of dodeca[*] Dr. S. M. Schiller Freiburg Institute for Advanced Studies (FRIAS), School of Soft Matter Research, Albert-Ludwigs-Universit t Freiburg Albertstrasse 19, 79104 Freiburg (Germany) Fax: (+ 49)761-203-97451 E-mail: Stefan.Schiller@FRIAS.Uni-Freiburg.de

Cell-free synthesis of functional human epidermal growth factor receptor: Investigation of ligand-independent dimerization in Sf21 microsomal membranes using non-canonical amino acids.

Cell-free protein synthesis systems represent versatile tools for the synthesis and modification of human membrane proteins. In particular, eukaryotic cell-free systems provide a promising platform for their structural and functional characterization. Here, we present the cell-free synthesis of functional human epidermal growth factor receptor and its vIII deletion mutant in a microsome-containing system derived from cultured Sf21 cells. We provide evidence for embedment of cell-free synthesized receptors into microsomal membranes and asparagine-linked glycosylation. Using the cricket paralysis virus internal ribosome entry site and a repetitive synthesis approach enrichment of receptors inside the microsomal fractions was facilitated thereby providing analytical amounts of functional protein. Receptor tyrosine kinase activation was demonstrated by monitoring receptor phosphorylation. Furthermore, an orthogonal cell-free translation system that provides the site-directed incorporation of p-azido-L-phenylalanine is characterized and applied to investigate receptor dimerization in the absence of a ligand by photo-affinity cross-linking. Finally, incorporated azides are used to generate stable covalently linked receptor dimers by strain-promoted cycloaddition using a novel linker system.

Hemolysin coregulated protein 1 as a molecular gluing unit for the assembly of nanoparticle hybrid structures

Summary Hybrid nanoparticle (NP) structures containing organic building units such as polymers, peptides, DNA and proteins have great potential in biosensor and electronic applications. The nearly free modification of the polymer chain, the variation of the protein and DNA sequence and the implementation of functional moieties provide a great platform to create inorganic structures of different morphology, resulting in different optical and magnetic properties. Nevertheless, the design and modification of a protein structure with functional groups or sequences for the assembly of biohybrid materials is not trivial. This is mainly due to the sensitivity of its secondary, tertiary and quaternary structure to the changes in the interaction (e.g., hydrophobic, hydrophilic, electrostatic, chemical groups) between the protein subunits and the inorganic material. Here, we use hemolysin coregulated protein 1 (Hcp1) from Pseudomonas aeruginosa as a building and gluing unit for the formation of biohybrid structures by implementing cysteine anchoring points at defined positions on the protein rim (Hcp1_cys3). We successfully apply the Hcp1_cys3 gluing unit for the assembly of often linear, hybrid structures of plasmonic gold (Au NP), magnetite (Fe3O4 NP), and cobalt ferrite nanoparticles (CoFe2O4 NP). Furthermore, the assembly of Au NPs into linear structures using Hcp1_cys3 is investigated by UV–vis spectroscopy, TEM and cryo-TEM. One key parameter for the formation of Au NP assembly is the specific ionic strength in the mixture. The resulting network-like structure of Au NPs is characterized by Raman spectroscopy, showing surface-enhanced Raman scattering (SERS) by a factor of 8·104 and a stable secondary structure of the Hcp1_cys3 unit. In order to prove the catalytic performance of the gold hybrid structures, they are used as a catalyst in the reduction reaction of 4-nitrophenol showing similar catalytic activity as the pure Au NPs. To further extend the functionality of the Hcp1_cys3 gluing unit, Fe3O4 and CoFe2O4 NPs are aligned in a magnetic field and connected by utilization of cysteine-modified Hcp1. After lyophilization, a fiber-like material of micrometer scale length can be observed. The Fe3O4 Hcp1_cys3 fibers show superparamagnetic behavior with a decreasing blocking temperature and an increasing remanent magnetization leading to a higher squareness value of the hysteresis curve. Thus the Hcp1_cys3 unit is shown to be very versatile in the formation of new biohybrid materials with enhanced magnetic, catalytic and optical properties.

Angiogenese und Vaskularisation beim Tissue Engineering von Fettgewebe

The current standard for the reconstruction of large soft tissue defects with exposed bone, nerves or blood vessels, for example after extensive tumor resections, complex injuries, severe burns or infections, is the local or free microsurgical tissue transfer. Despite the development of new surgical techniques and many synthetic materials, there are still a large number of limitations and complications at the donor and recipient site. Thus, in a subset of patients either complete treatment is not possible or poses problems. Therefore, there is a great need for the development of new methods and materials allowing for a permanent replacement with body own soft tissue. A promising therapeutic approach is the soft tissue replacement with autologous adipose tissue. Innovative research on the reconstruction of soft tissue by adipose tissue, and clinical and experimental studies on the long-term survival and transplantation of autologous adipose tissue showed that the free fat tissue graft without direct vascular connection come along with disappointing results. Often a loss of volume or a complete resorption of the graft because of insufficient tissue quality by lack of cell differentiation was observed. This fact points to the special role of the maintenance and development of the grafts blood supply (angiogenesis and vascularization) crucial for maintaining a constant volume of the tissue. The rapidly growing interdisciplinary field of tissue engineering offers alternative solutions to the existing treatment options with the aim to produce autologous adipose tissue, stable in volume in vitro as well as in vivo, which can be transplanted as a permanent tissue replacement to corresponding parts of the body. Numerous studies have demonstrated the important and most critical factor of vascularisation for quality, volume and long-term survival of transplanted newly generated adipose tissue constructs. Although our understanding of the regulatory mechanisms of adipogenesis is still limited, there are clear indications that the complex sequences of cell interactions in the differentiation and proliferation of adipocytes is directly related to angiogenesis.

De-novo Generierung von vaskularisiertem Gewebe mittels unterschiedlicher Gefässstielkonfigurationen in perforierten und geschlossenen Wachstumskammern

Growing three-dimensional tissue within a chamber requires vigorous angiogenesis initiated by, for example, an arteriovenous fistula or a ligated vascular pedicle. Growth may also be enhanced by contact with the external environment. In this study tissue growth in a rat model, vascularized via an arteriovenous loop (AV Loop) or ligated pedicle, was compared in chambers that were either closed or perforated. Chambers were harvested at 4 weeks and tissue volume and histology compared. In perforated chambers, more tissue were generated using the ligated pedicle (0.75 ml+/-0.04) than the AV Loop (0.59 ml+/-0.01). Perforated chambers generated larger volumes of tissue than closed chambers because they encouraged tissue ingrowth through the perforations. Both vessel configurations supported tissue growth but, interestingly, the ligated pedicle resulted in significantly more tissue in the perforated chambers.SummaryGrowing three-dimensional tissue within a chamber requires vigorous angiogenesis initiated by, for example, an arteriovenous fistula or a ligated vascular pedicle. Growth may also be enhanced by contact with the external environment. In this study tissue growth in a rat model, vascularized via an arteriovenous loop (AV Loop) or ligated pedicle, was compared in chambers that were either closed or perforated. Chambers were harvested at 4 weeks and tissue volume and histology compared. In perforated chambers, more tissue were generated using the ligated pedicle (0.75 ml±0.04) than the AV Loop (0.59 ml±0.01). Perforated chambers generated larger volumes of tissue than closed chambers because they encouraged tissue ingrowth through the perforations. Both vessel configurations supported tissue growth but, interestingly, the ligated pedicle resulted in significantly more tissue in the perforated chambers.ZusammenfassungDie Generierung dreidimensionaler Gewebekomplexe innerhalb einer Wachstumskammer setzt eine intensive Angiogenese voraus, die beispielsweise durch arteriovenöse Shunts oder einen ligierten Gefäßstiel induziert werden kann. Der Wachstumsprozess lässt sich weiter durch Kontakt mit dem umgebendem Gewebe stimulieren. In dieser Studie wurde das angiofibrinöse Gewebewachstum von arteriovenösen Shunts (AV-Shunts) und ligierten Gefäßstielen in perforierten sowie geschlossenen Kammern im Rattenmodell verglichen. Die Kammern wurden nach vier Wochen entfernt, das Volumen des Gewebes bestimmt und histomorphometrisch analysiert. In perforierten Kammern wurde mit ligierten Gefäßstielen (0,75 ml±0,04) mehr Gewebe als mit AV-Shunts (0,59 ml±0,01) generiert. Die perforierten Kammern produzierten größere Volumina als der geschlossene Typ, da sie eine zusätzliche Angiogenese durch die Perforationen erlaubten. Beide Gefäßstielkonfigurationen förderten das Wachstum von Gewebe, jedoch resultierte interessanterweise aus ligierten Gefäßstielen signifikant mehr Gewebe in den perforierten Modellen.

Steric and thermodynamic limits of design for the incorporation of large unnatural amino acids in aminoacyl-tRNA synthetase enzymes

Orthogonal aminoacyl‐tRNA synthetase/tRNA pairs from archaea have been evolved to facilitate site specific in vivo incorporation of unnatural amino acids into proteins in Escherichia coli. Using this approach, unnatural amino acids have been successfully incorporated with high translational efficiency and fidelity. In this study, CHARMM‐based molecular docking and free energy calculations were used to evaluate rational design of specific protein–ligand interactions for aminoacyl‐tRNA synthetases. A series of novel unnatural amino acid ligands were docked into the p‐benzoyl‐L‐phenylalanine tRNA synthetase, which revealed that the binding pocket of the enzyme does not provide sufficient space for significantly larger ligands. Specific binding site residues were mutated to alanine to create additional space to accommodate larger target ligands, and then mutations were introduced to improve binding free energy. This approach was used to redesign binding sites for several different target ligands, which were then tested against the standard 20 amino acids to verify target specificity. Only the synthetase designed to bind Man‐α‐O‐Tyr was predicted to be sufficiently selective for the target ligand and also thermodynamically stable. Our study suggests that extensive redesign of the tRNA synthatase binding pocket for large bulky ligands may be quite thermodynamically unfavorable. Proteins 2010.

Transient apoptosis inhibition in donor stem cells improves hematopoietic stem cell transplantation

During hematopoietic stem cell transplantation, a substantial number of donor cells are lost because of apoptotic cell death. Transplantation-associated apoptosis is mediated mainly by the proapoptotic BCL-2 family proteins BIM and BMF, and their proapoptotic function is conserved between mouse and human stem and progenitor cells. Permanent inhibition of apoptosis in donor cells caused by the loss of these BH3-only proteins improves transplantation outcome, but recipients might be exposed to increased risk of lymphomagenesis or autoimmunity. Here, we address whether transient inhibition of apoptosis can serve as a safe but efficient alternative to improve the outcome of stem cell transplantation. We show that transient apoptosis inhibition by short-term overexpression of prosurvival BCL-XL, known to block BIM and BMF, is not only sufficient to increase the viability of hematopoietic stem and progenitor cells during engraftment but also improves transplantation outcome without signs of adverse pathologies. Hence, this strategy represents a promising and novel therapeutic approach, particularly under conditions of limited donor stem cell availability.