23 publications

Ultrathin Poly(glycidyl ether) Coatings on Polystyrene for Temperature-Triggered Human Dermal Fibroblast Sheet Fabrication

Daniel David Stöbener, Melanie Uckert, José Luis Cuellar Camacho, Anke Hoppensack, Marie Weinhart

ACS | 2017-07-18

ACS Biomater. Sci. Eng., Just Accepted Manuscript

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The fabrication of cell sheets is a major requirement for bottom-up tissue engineering purposes (e.g. cell sheet engineering) and regenerative medicine. Employing thermoresponsive polymer coatings as tissue culture substrates allows for the mild, temperature-triggered detachment of intact cell sheets along with their extracellular matrix (ECM). It has been shown before that biocompatible, thermoresponsive poly(glycidyl ether) monolayers on gold substrates can be utilized to harvest confluent cell sheets by simply reducing the temperature to 20 °C. Herein, we report on the first poly(glycidyl ether)-based coating on an application-relevant tissue culture plastic substrate. We devised a simple, substrate geometry-independent method to functionalize polystyrene (PS) surfaces from dilute ethanolic solution via the physical adsorption process of a thermoresponsive poly(glycidyl ether) block-copolymer (PGE) bearing a short, hydrophobic, and photoreactive benzophenone (BP) anchor block. Subsequently, the PGE-coated PS is UV irradiated for covalent photo-immobilization of the polymer on the PS substrate. Online monitoring of the adsorption process via QCM-D measurements and detailed characterization of the resulting coatings via AFM, ellipsometry, and water contact angle (CA) measurements revealed the formation of an ultrathin PGE layer with an average dry thickness of 0.7 ± 0.1 nm. Adhesion and proliferation of human dermal fibroblasts on PGE-coated PS and TCPS were comparable. For temperature-triggered detachment, fibroblasts were cultured in PGE-coated PS culture dishes at 37 °C for 24 h until they reached confluency. Intact cell sheets could be harvested from the thermoresponsive substrates within 51 ± 17 min upon cooling to 20 °C, whereas sheets could not be harvested from uncoated PS and tissue culture PS (TCPS) control dishes. Live/dead staining and flow cytometry affirmed a high viability of the fibroblasts within the cell sheets. Hence, ultrathin layers of thermoresponsive poly(glycidyl ether)s on hydrophobic PS substrates are functional coatings for cell sheet fabrication.

Thermoresponsive Poly(glycidyl ether) Brushes on Gold: Surface Engineering Parameters and their Implication for Cell Sheet Fabrication

Silke Heinen, José Luis Cuéllar-Camacho, Marie Weinhart

Elsevier | 2017-06-20

Acta Biomaterialia Article ASAP

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Thermoresponsive polymer coatings, optimized for cell adhesion and thermally-triggered cell detachment, allow the fabrication of confluent cell sheets with intact extracellular matrix. However, rational design guidelines for such coatings are rare, since temperature-triggered cell adhesion and detachment from thermoresponsive surfaces are mechanistically not well understood. Herein, we investigated the impact of molecular weight (2, 9, 24 kDa), grafting density (0.04 – 1.4 chains nm-2), morphology, and roughness of well-characterized thermoresponsive poly(glycidyl ether) brushes on the cell response at 37 and 20 °C. NIH 3T3 mouse fibroblasts served as a model cell line for adhesion, proliferation, and cell sheet detachment. The cell response was correlated with serum protein adsorption from cell culture medium containing 10% fetal bovine serum. Intact cell sheets could be harvested from all the studied poly(glycidyl ether) coated surfaces, irrespective of the molecular weight, provided that the morphology of the coating was homogenous and the surface was fully shielded by the hydrated brush. The degree of chain overlap was estimated by the ratio of twice the polymer’s Flory radius in a theta solvent to its interchain distance, which should be located in the strongly overlapping brush regime (2 Rf/l > 1.4). In contrast, dense PNIPAM (2.5 kDa) control monolayers did not induce protein adsorption from cell culture medium at 37 °C and, as a result, did not allow a significant cell adhesion. These structural design parameters of functional poly(glycidyl ether) coatings on gold will contribute to future engineering of these thermoresponsive coatings on more common, cell culture relevant substrates.

Poly(glycidyl ether)-Based Monolayers on Gold Surfaces: Control of Grafting Density and Chain Conformation by Grafting Procedure, Surface Anchor, and Molecular Weight

Silke Heinen, Marie Weinhart

ACS | 2017-02-13

Langmuir, 2017, 33 (9), 2076–2086

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For a meaningful correlation of surface coatings with their respective biological response reproducible coating procedures, well-defined surface coatings, and thorough surface characterization with respect to layer thickness and grafting density are indispensable. The same applies to polymeric monolayer coatings which are intended to be used for, e.g., fundamental studies on the volume phase transition of surface end-tethered thermoresponsive polymer chains. Planar gold surfaces are frequently used as model substrates, since they allow a variety of straightforward surface characterization methods. Herein we present reproducible grafting-to procedures performed with thermoresponsive poly(glycidyl ether) copolymers composed of glycidyl methyl ether (GME) and ethyl glycidyl ether (EGE). The copolymers feature different molecular weights (2 kDa, 9 kDa, 24 kDa) and are equipped with varying sulfur-containing anchor groups in order to achieve adjustable grafting densities on gold surfaces and hence control the tethered polymers’ chain conformation. We determined “wet” and “dry” thicknesses of these coatings by QCM-D and ellipsometry measurements and deduced anchor distances and degrees of chain overlap of the polymer chains assembled on gold. Grafting under cloud point conditions allowed for higher degrees of chain overlap compared to grafting from a good solvent like ethanol, independent of the used sulfur-containing anchor group for polymers with low (2 kDa) and medium (9 kDa) molecular weights. By contrast, the achieved grafting densities and thus chain overlaps of surface-tethered polymers with high (24 kDa) molecular weights were identical for both grafting methods. Monolayers prepared from an ethanolic solution of poly(glycidyl ether)s equipped with sterically demanding disulfide-containing anchors revealed the lowest degrees of chain overlap. The ratio of the radius of gyration to the anchor distance (2 Rg/l) of the latter coating was found to be lower than 1.4, indicating that the assembly was rather in the mushroom-like than in the brush regime. Polymer chains with thiol-containing anchors of different alkyl chain lengths (C11SH vs C4SH) formed assemblies with comparable degrees of chain overlap with 2 Rg/l values above 1.4 and are thus in the brush regime. Molecular weights influenced the achievable degree of chain overlap on the surface. Coatings prepared with the medium molecular weight polymer (9 kDa) resulted in the highest chain packing density. Control of grafting density and thus chain overlap in different regimes (brush vs mushroom) on planar gold substrates are attainable for monolayer coatings with poly(GME-ran-EGE) by adjusting the polymer’s molecular weight and anchor group as well as the conditions for the grafting-to procedure.

A Perfect Match: Fast and Truly Random Copolymerization of Glycidyl Ether Monomers to Thermoresponsive Copolymers

Silke Heinen, Simon Rackow, Andreas Schäfer, Marie Weinhart

ACS | 2016-12-23

Macromolecules, 2017, 50 (1), 44–53.

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Thermoresponsive and highly biocompatible poly(glycidyl ether) copolymers of glycidyl methyl ether (GME) and ethyl glycidyl ether (EGE) with adjustable molecular weight and defined end groups are synthesized by a monomer-activated anionic ring-opening polymerization with NOct4Br as initiator and i-Bu3Al as activator. In contrast to a conventional oxyanionic (nonactivated) copolymerization, higher molecular weights and a truly random incorporation of the monomers are accomplished. The monomer reactivity ratios were determined by the Kelen–Tüdõs approach to be rGME = 0.98 and rEGE = 0.95. The thermoresponsive properties of these copolymers with varying molecular weight were characterized by UV–vis transmittance and dynamic light scattering. Conformational changes of the copolymer during the phase transition on the molecular level were studied by 1H and 13C NMR spectroscopy in D2O and revealed only a partial dehydration during the collapse of the copolymer affecting both side chains and polymer backbone.

Reversible hemostatic properties of sulfabetaine/quaternary ammonium modified hyperbranched polyglycerol

Jiying Wen, Marie Weinhart, Benjamin F. Lai, Jayachandran Kizhakkedathu, Donald E. Brooks

Elsevier | 2016-02-02

Biomaterials, 2016, 86, 42–55.

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A library of hyperbranched polyglycerols (HPGs) functionalized with different mole fractions of zwitterionic sulfabetaine and cationic quaternary ammonium ligands was synthesized and characterized. A post-polymerization method was employed that utilized double bond moieties on the dendritic HPG for the coupling of thiol-terminated ligands via UV initiated thiol-ene “click” chemistry. The proportions of different ligands were precisely controlled by varying the ligand concentration during the irradiation process. The effect of the polymer library on hemostasis was investigated using whole human blood. It was found that polymer with ≥40% of alkenes converted to positive charges and the remainder to sulfabetaines caused hemagglutination at ≥1 mg/mL, without causing red blood cell lysis. The quaternary ammonium groups can interact with the negative charged sites on the membranes of erythrocytes, which provides the bioadhesion. The zwitterionic sulfabetaine evidently provides a hydration layer to partially mask the adverse effects that are likely to be caused by cationic moieties. The polymer was also found able to enhance platelet aggregation and activation in a concentration and positive charge density-dependent manner, which would contribute to initiating hemostasis. In a variety of other assays the material was found to be largely biocompatible. The polymer-induced hemostasis is obtained by a process independent of the normal blood clotting cascade but dependent on red blood cell agglutination, where the polymers promote hemostasis by linking erythrocytes together to form a lattice to entrap the cells.

Biocompatible fluorinated polyglycerols for droplet microfluidics as an alternative to PEG-based copolymer surfactants

Olaf Wagner, Julian Thiele, Marie Weinhart, Linas Mazutis, David A. Weitz, Wilhelm T. S. Huck, Rainer Haag

RSC | 2015-11-24

Lab Chip, 2016,16, 65-69

Full TextSupporting InformationSupplementary Movie

In droplet-based microfluidics, non-ionic, high-molecular weight surfactants are required to stabilize droplet interfaces. One of the most common structures that imparts stability as well as biocompatibility to water-in-oil droplets is a triblock copolymer surfactant composed of perfluoropolyether (PFPE) and polyethylene glycol (PEG) blocks. However, the fast growing applications of microdroplets in biology would benefit from a larger choice of specialized surfactants. PEG as a hydrophilic moiety, however, is a very limited tool in surfactant modification as one can only vary the molecular weight and chain-end functionalization. In contrast, linear polyglycerol offers further side-chain functionalization to create custom-tailored, biocompatible droplet interfaces. Herein, we describe the synthesis and characterization of polyglycerol-based triblock surfactants with tailored side-chain composition, and exemplify their application in cell encapsulation and in vitro gene expression studies in droplet-based microfluidics.

Dendritic polyglycerol sulfate inhibits microglial activation and reduces hippocampal CA1 dendritic spine morphology deficits

Dusica Maysinger, Dominic Gröger, Andrew Lake, Kai Licha, Marie Weinhart, Philip K.-Y. Chang, Rose Mulvey, Rainer Haag, R. Anne McKinney

ACS | 2015-07-28

Biomacromolecules, 2015, 16 (9), 3073–3082.

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Hyperactivity of microglia and loss of functional circuitry is a common feature of many neurological disorders including those induced or exacerbated by inflammation. Herein, we investigate the response of microglia and changes in hippocampal dendritic postsynaptic spines by dendritic polyglycerol sulfate (dPGS) treatment. Mouse microglia and organotypic hippocampal slices were exposed to dPGS and an inflammogen (lipopolysaccharides). Measurements of intracellular fluorescence and confocal microscopic analyses revealed that dPGS is avidly internalized by microglia but not CA1 pyramidal neurons. Concentration and time-dependent response studies consistently showed no obvious toxicity of dPGS. The adverse effects induced by proinflammogen LPS exposure were reduced and dendritic spine morphology was normalized with the addition of dPGS. This was accompanied by a significant reduction in nitrite and proinflammatory cytokines (TNF-α and IL-6) from hyperactive microglia suggesting normalized circuitry function with dPGS treatment. Collectively, these results suggest that dPGS acts anti-inflammatory, inhibits inflammation-induced degenerative changes in microglia phenotype and rescues dendritic spine morphology.

In-Depth Analysis of Switchable Glycerol Based Polymeric Coatings for Cell Sheet Engineering

Tobias Becherer, Silke Heinen, Qiang Wei, Rainer Haag, Marie Weinhart

Elsevier | 2015-06-30

Acta Biomaterialia, 2015, 25, 43-55.

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Scaffold-free cell sheet engineering using thermoresponsive substrates provides a promising alternative to conventional tissue engineering which in general employs biodegradable scaffold materials. We have previously developed a thermoresponsive coating with glycerol based linear copolymers that enables gentle harvesting of entire cell sheets. In this article we present an in-depth analysis of these thermoresponsive linear polyglycidyl ethers and their performance as coating for substrates in cell culture in comparison with commercially available poly(N-isopropylacrylamide) (PNIPAM) coated culture dishes. A series of copolymers of glycidyl methyl ether (GME) and glycidyl ethyl ether (EGE) was prepared in order to study their thermoresponsive properties in solution and on the surface with respect to the comonomer ratio. In both cases, when grafted to planar surfaces or spherical nanoparticles, the applied thermoresponsive polyglycerol coatings render the respective surfaces switchable. Protein adsorption experiments on copolymer coated planar surfaces with surface plasmon resonance (SPR) spectroscopy reveal the ability of the tested thermoresponsive coatings to be switched between highly protein resistant and adsorptive states. Cell culture experiments demonstrate that these thermoresponsive coatings allow for adhesion and proliferation of NIH 3T3 fibroblasts comparable to TCPS and faster than on PNIPAM substrates. Temperature triggered detachment of complete cell sheets from copolymer coated substrates was accomplished within minutes while maintaining high viability of the harvested cells. Thus such glycerol based copolymers present a promising alternative to PNIPAM as a thermoresponsive coating of cell culture substrates.

Affinity-based Design and Discovery of a Synthetic Universal Reversal Agent for Clinically Used Parenteral Heparin Anticoagulants

Rajesh A. Shenoi, Benjamin F. Lai, Marie Weinhart, Kai Yu, Cedric .J. Carter, Donald E. Brooks, Jay N. Kizhakkedathu

American Association for the Advancement of Science | 2014-10-29

Science Translational Medicine, 2014, 6 (260), 260ra150.

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Heparin-based anticoagulant drugs have been widely used for the prevention of blood clotting during surgical procedures and for the treatment of thromboembolic events. However, bleeding risks associated with these anticoagulants demand continuous monitoring and neutralization with suitable antidotes. Protamine, the only clinically approved antidote to heparin, has shown adverse effects and ineffectiveness against low–molecular weight heparins and fondaparinux, a heparin-related medication. Alternative approaches based on cationic molecules and recombinant proteins have several drawbacks including limited efficacy, toxicity, immunogenicity, and high cost. Thus, there is an unmet clinical need for safer, rapid, predictable, and cost-effective anticoagulant-reversal agents for all clinically used heparins. We report a design strategy for a fully synthetic dendritic polymer–based universal heparin reversal agent (UHRA) that makes use of multivalent presentation of branched cationic heparin binding groups (HBGs). Optimization of the UHRA design was aided by isothermal titration calorimetry studies, biocompatibility evaluation, and heparin neutralization analysis. By controlling the scaffold’s molecular weight, the nature of the protective shell, and the presentation of HBGs on the polymer scaffold, we arrived at lead UHRA molecules that completely neutralized the activity of all clinically used heparins. The optimized UHRA molecules demonstrated superior efficacy and safety profiles and mitigated heparin-induced bleeding in animal models. This new polymer therapeutic may benefit patients undergoing high-risk surgical procedures and has potential for the treatment of anticoagulant-related bleeding problems.

A crucial role of L-selectin in C protein-induced experimental polymyositis in mice

Kyosuke Oishi, Yasuhito Hamaguchi, Takashi Matsushita, Minoru Hasegawa, Naoko Okiyama, Jens Dernedde, Marie Weinhart, Rainer Haag, Thomas F Tedder, Kazuhiko Takehara, Hitoshi Kohsaka, Manabu Fujimoto

Wiley | 2014-06-27

Arthritis & Rheumatology , 2014, 66 (7), 1864–1871.

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Objective: To investigate the role of adhesion molecules in C protein–induced myositis (CIM), a murine model of polymyositis (PM). Methods: CIM was induced in wild-type mice, L-selectin–deficient (L-selectin−/−) mice, intercellular adhesion molecule 1 (ICAM-1)–deficient (ICAM-1−/−) mice, and mice deficient in both L-selectin and ICAM-1 (L-selectin−/−ICAM-1−/− mice). Myositis severity, inflammatory cell infiltration, and messenger RNA expression in the inflamed muscles were analyzed. The effect of dendritic polyglycerol sulfate, a synthetic inhibitor that suppresses the function of L-selectin and endothelial P-selectin, was also examined. Results: L-selectin−/− mice and L-selectin−/−ICAM-1−/− mice developed significantly less severe myositis compared to wild-type mice, while ICAM-1 deficiency did not inhibit the development of myositis. L-selectin−/− mice that received wild-type T cells developed myositis. Treatment with dendritic polyglycerol sulfate significantly diminished the severity of myositis in wild-type mice compared to treatment with control. Conclusion: These data indicate that L-selectin plays a major role in the development of CIM, whereas ICAM-1 plays a lesser role, if any, in the development of CIM. L-selectin–targeted therapy may be a candidate for the treatment of PM.

Selectivity in Bone Targeting with Multivalent Dendritic Polyanion Dye Conjugates

Dominic Gröger, Michael Kerschnitzki, Marie Weinhart, Sabine Reimann, Tobias Schneider, Benjamin Kohl, Wolfgang Wagermaier, Gundula Schulze-Tanzil, Peter Fratzl, Rainer Haag

Wiley | 2013-08-29

Advanced Healthcare Materials, 2014, 3 (3), 375–385.

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Targeting bone with anionic macromolecules is a potent approach for the development of novel diagnostics and therapeutics for bone related diseases. A highly efficient modular synthesis of dendritic polyglycerol (dPG) polyanion dye conjugates, namely, sulfates, sulfonates, carboxylates, phosphates, phosphonates, and bisphosphonates via click chemistry is presented. By investigating the microarchitecture of stained bone sections with confocal laser scanning microscopy, the bisphosphonate, phosphonate, and phosphate functionalized polymers are identified as strongly penetrating compounds, whereas sulfates, sulfonates, and carboxylates reveal a weaker binding to hydroxyapatite (HA) but a more pronounced affinity toward collagen. In a quantitative HA binding assay, the affinity of the dPG sulfonate, sulfate, and carboxylate toward collagen and the exceptional high HA affinity of the phosphorous containing polyelectrolytes are validated. This shows the potential of dendritic polyphosphates and phosphonates as alternatives to the commonly employed bisphosphonate modification. In cytotoxicity studies with murine fibroblasts, the conjugates have no significant effect on the cell viability at 10-5m. All polyanions are taken up into the cells within 24 h. The presented synthetic approach allows versatile extensions for preparing conjugates for selective bone imaging applications, tissue engineering, and drug delivery.

Subject: bisphosphonates;bone targeting;conjugates;hydroxyapatite;inflammation

Synthesis and Biological Evaluation of Radio and Dye Labeled Amino Functionalized Dendritic Polyglycerol Sulfates as Multivalent Antiinflammatory Compounds

Dominic Gröger, Florian Paulus, Kai Lich, Pia Welker, Marie Weinhart, Cornelia Holzhausen, Lars Mundhenk, Achim D. Gruber, Ulrich Abram, Rainer Haag

ACS | 2013-08-07

Bioconjugate Chemistry, 2013, 24 (9), 1507–1514

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Herein we describe a platform technology for the synthesis and characterization of partially aminated, 35S-labeled, dendritic polyglycerol sulfate (dPG35S amine) and fluorescent dPGS indocarbocyanine (ICC) dye conjugates. These polymer conjugates, based on a biocompatible dendritic polyglycerol scaffold, exhibit a high affinity to inflamed tissue in vivo and represent promising candidates for therapeutic and diagnostic applications. By utilizing a one-step sequential copolymerization approach, dendritic polyglycerol (Mn ≈ 4.5 kDa) containing 9.4% N-phthalimide protected amine functionalities was prepared on a large scale. Sulfation and simultaneous radio labeling with 35SO3 pyridine complex, followed by cleavage of the N-phthalimide protecting groups, yielded dPG35S amine as a beta emitting, inflammation specific probe with free amino functionalities for conjugation. Furthermore, efficient labeling procedures with ICC via iminothiolane modification and subsequent “Michael” addition of the maleimide functionalized ICC dye, as well as by amide formation via NHS derivatized ICC on a dPGS amine scaffold, are described. The dPGS-ICC conjugates were investigated with respect to their photophysical properties, and both the radiolabeled and fluorescent compounds were comparatively visualized in histological tissue sections (radio detection and fluorescence microscopy) of animals treated with dPGS. Furthermore, cellular uptake of dPGS-ICC was found in endothelial cord blood (HUVEC) and the epithelial lung cells (A549). The presented synthetic routes allow a reproducible, controlled synthesis of dPGS amine on kilogram scale applying a one-pot batch reaction process. dPGS amine can be used for analysis via radioactivity or fluorescence, thereby creating a new platform for inflammation specific, multimodal imaging purposes using other attachable probes or contrast agents.

Surface Functionalization of Poly(ether imide) Membranes with Linear, Methylated Oligoglycerols for Reducing Thrombogenicity

Maik Lange, Steffen Braune, Karola Luetzow, Klaus Richau, Nico Scharnagl, Marie Weinhart, Axel T. Neffe, Friedrich Jung, Rainer Haag, Andreas Lendlein

Wiley | 2012-07-23

Macromolecular Rapid Communications, 2012, 33(17), 1487–1492.

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Materials for biomedical applications are often chosen for their bulk properties. Other requirements such as a hemocompatible surface shall be fulfilled by suitable chemical functionalization. Here we show, that linear, side-chain methylated oligoglycerols (OGMe) are more stable to oxidation than oligo(ethylene glycol) (OEG). Poly(ether imide) (PEI) membranes functionalized with OGMes perform at least as good as, and partially better than, OEG functionalized PEI membranes in view of protein resistance as well as thrombocyte adhesion and activation. Therefore, OGMes are highly potent surface functionalizing molecules for improving the hemocompatibility of polymers.

Subject: hemocompatibility;poly(ethylene glycol);polyglycerol;polyimides;surface chemistry

Fluorescence Imaging with Multifunctional Polyglycerol Sulfates: Novel Polymeric near-IR Probes Targeting Inflammation

Kai Licha, Pia Welker, Marie Weinhart, Nicole Wegner, Sylvia Kern, Stefanie Reichert, Ines Gemeinhardt, Carmen Weissbach, Bernd Ebert, Rainer Haag, Michael Schirner

ACS | 2011-11-17

Bioconjugate Chemistry, 2011, 22 (12), 2453–2460.

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We present a highly selective approach for the targeting of inflammation with a multivalent polymeric probe. Dendritic polyglycerol was employed to synthesize a polyanionic macromolecular conjugate with a near-infrared fluorescent dye related to Indocyanine Green (ICG). On the basis of the dense assembly of sulfate groups which were generated from the polyol core, the resulting polyglycerol sulfate (molecular weight 12 kD with ∼70 sulfate groups) targets factors of inflammation (IC50 of 3–6 nM for inhibition of L-selectin binding) and is specifically transported into inflammatory cells. The in vivo accumulation studied by near-IR fluorescence imaging in an animal model of rheumatoid arthritis demonstrated fast and selective uptake which enabled the differentiation of diseased joints (score 1–3) with a 3.5-fold higher fluorescence level and a signal maximum at 60 min post injection. Localization in tissues using fluorescence histology showed that the conjugates are deposited in the inflammatory infiltrate in the synovial membrane, whereas nonsulfated control was not detected in association with disease. Hence, this type of polymeric imaging probe is an alternative to current bioconjugates and provides future options for targeted imaging and drug delivery.

Linear and Hyperbranched Polyglycerol Derivatives as Excellent Bioinert Coating Materials

Marie Weinhart, Tobias Becherer, Nicolai Schnurbusch, Karin Schwibbert, Hans-Jörg Kunte, Rainer Haag

Wiley | 2011-09-21

Advanced Engineering Materials, 2011, 13(12), B501–B510.

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The non-specific adsorption of proteins to surfaces in contact with biofluids constitutes a major problem in the biomedical and biotechnological field, due to the initiation of biofilm formation and the resulting improper function of devices. Therefore, non-fouling surfaces modified with poly(ethylene glycol) (PEG) are usually applied. In this study, we report the synthesis of triethoxysilane modified glycerol based polymers of linear and branched architecture for the preparation of covalently attached monolayers on glass. Evaluation of the biocompatibility of these surfaces was performed in comparison to bare non-coated glass, hydrophobic hexadecane modified glass, and mPEG modified glass as the controls. Protein adsorption of BSA and fibrinogen (1 mg · mL−1 in PBS) after 4 and 24 h immersion was reduced by more than 96 and 90%, respectively, compared to the adsorption on bare glass substrates. In addition, mouse NIH-3T3 fibroblast cells showed only marginal adhesion on the polyglycerol and mPEG coated slides after 3 and 7 d incubation in cell suspension, which demonstrates the long-term stability of the applied glass coatings. The non-adhesive properties of these coatings were further reflected in bacterial adhesion tests of Escherichia coli K12 and three clinically relevant Gram-positive and negative strains (Staphylococcus aureus, Pseudomonas aeruginosa, and Aeromonas hydrophila), since linear polyglycerol (LPG(OH)), linear poly(methyl glycerol) (LPG(OMe)), and hyperbranched polyglycerol (HPG) reduced the adhesion for all tested strains by more than 99% compared to bare glass. Therefore, polyglycerol derivatives present an excellent non-fouling surface coating as an alternative to PEG with feasibility for surface modification of various substrates.

Effect of Polymer Brush Architecture on Antibiofouling Properties

Gesine Gunkel, Marie Weinhart, Tobias Becherer, Rainer Haag, Wilhelm T. S. Huck

ACS | 2011-09-20

Biomacromolecules, 2011, 12 (11), 4169–4172.

Full TextSupporting Information

Polymer brushes show great promise in next-generation antibiofouling surfaces. Here, we have studied the influence of polymer brush architecture on protein resistance. By carefully optimizing reaction conditions, we were able to polymerize oligoglycerol-based brushes with sterically demanding linear or dendronized side chains on gold surfaces. Protein adsorption from serum and plasma was analyzed by surface plasmon resonance. Our findings reveal a pronounced dependence of biofouling on brush architecture. Bulky yet flexible side chains as in dendronized brushes provide an ideal environment to repel protein—possibly through formation of a hydration layer, which can be further enhanced by presenting free hydroxyl groups on the polymer brushes. A deeper understanding of how brush architecture influences protein resistance will ultimately enable fabrication of surface coatings tailored to specific requirements in biomedical applications.

The Role of Dimension in Multivalent Binding Events: Structure-Activity Relationship of Dendritic Polyglycerol Sulfate Binding to L-selectin in Correlation with Size and Surface Charge Density

Cover Macromol Biosci 08/2011

Marie Weinhart, Dominic Gröger, Sven Enders, Sebastian B. Riese, Jens Dernedde, Rajesh K. Kainthan, Donald E. Brooks, Rainer Haag

Wiley | 2011-07-03

Macromolecular Bioscience, 2011, 11(8), 1088–1098.

Full TextSupporting Information

L-, P-, and E-Selectin are cell adhesion molecules that play a crucial role in leukocyte recruitment from the blood stream to the afflicted tissue in an acute and chronic inflammatory setting. Since selectins mediate the initial contact of leukocytes to the vascular endothelium, they have evolved as a valuable therapeutic target in diseases related to inflammation by inhibition of the physiological selectin–ligand interactions. In a previous study, it was demonstrated that dPGS, a fully synthetic heparin analogue, works as an efficient inhibitor towards L- and P-selectin in vitro as well as in vivo. Herein, the focus is directed towards the effect of size and charge density of the polyanion. The efficiency of L-selectin inhibition via an SPR-based in vitro assay and a cell-based flow chamber assay is investigated with dPGS ranging from approximately 4 to 2000 kDa. SPR measurements show that the inhibitory potential of highly sulfated dPGS increases with size and charge density. Thereby, IC50 values from the micromolar to the low picomolar range are determined. The same tendency could be observed in a cell-based flow chamber assay with three representative dPGS samples. This structure–affinity relationship of dPGS suggests that the strong inhibitory potential of dPGS is not only based on the strong electrostatic interaction with areas of cationic surface potential on L-selectin but is also due to a steric shielding of the carbohydrate binding site by large, flexible dPGS particles.

Subject: hyperbranched polymers, multivalency, polyelectrolytes, selectin inhibition

Synthesis of Dendritic Polyglycerol Anions and Their Efficiency Toward L-Selectin Inhibition

Marie Weinhart, Dominic Gröger, Sven Enders, Jens Dernedde, Rainer Haag

ACS | 2011-05-20

Biomacromolecules, 2011, 12 (7), 2502–2511.

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A versatile route for the synthesis of highly functionalized, polyanionic macromolecules based on dendritic polyglycerol was applied by means of the Huisgen–Sharpless–Meldal 1,3-dipolar cycloaddition (“click-reaction”) of polyglycerolazide precursors and alkyne-functionalized anions such as sulfonates, carboxylates, phosphonates, and bisphosphonates. In addition, the corresponding polyglycerol phosphate has been synthesized via direct hydroxyl interconversion of polyglycerol to the corresponding phosphate with a degree of functionalization >80% by analogy to the synthesis of previously reported polyglycerol sulfates (dPGS). On the basis of the finding that dPGS exhibits high affinity for L- and P-selectin, the potential of these novel polyanionic, multivalent macromolecules of varying anionic nature as L-selectin inhibitors has been evaluated in vitro by means of a competitive concentration dependent binding assay. Affinity of all polyanions toward L-selectin was demonstrated with distinct IC50 values ranging from the low nanomolar to the high micromolar range. The efficiency of L-selectin inhibition increases in the order carboxylate < phosphate < phosphonate ≈ sulfonate < bisphosphonate < sulfate. Additional DLS and ζ-potential measurements of these polyanions were performed to correlate their binding affinity toward L-selectin with their anionic nature. However, a direct correlation of effective charge and particle size with the determined IC50 values turned out to require further in-depth studies on the microstructure of the polyanions but clearly indicate an exceptional position of dPGS among the studied dendritic polyelectrolytes.

Computational entropy estimation of linear polyether modified surfaces and correlation with protein resistant properties of such surfaces

Marcus Weber, Alexander Bujotzek, Karsten Andrae, Marie Weinhart, Rainer Haag

Taylor & Francis Group | 2011-02-23

Molecular Simulation, 2011, 37(11), 899-906. ...

The non-specific adsorption of proteins on surfaces is a well-known and mostly undesirable phenomena, which is reduced by a surface coating with the linear polyether poly(ethylene glycol) (PEG) as the current benchmark material. However, the molecular mechanism of protein-resistant surfaces is still not fully understood. Two main hypotheses are generally applied. The first one is steric repulsion of the highly flexible tethered polymer chains, leading to an entropic penalty by adsorption of proteins due to the reduction in polymer chain mobility. The second one argues with well-hydrated polymer chains generating a repulsive interfacial water layer. In this article, we compare the three different protein-resistant polyether structures PEG, linear polyglycerol (LPG(OH)) and linear poly(methyl glycerol) (LPG(OMe)) to get new insights into the molecular mechanism behind protein resistance. In a theoretical approach, we apply an entropy estimator that assesses the conformational states of the tethered polyethers from MD simulations. It reveals the entropy differences between these polyethers to be in the order PEG>LPG(OH) > LPG(OMe). Moreover, experiments on fibrinogen adsorption of these surfaces via surface plasmon resonance spectroscopy are performed and correlated with the theoretical studies. We find that protein resistant properties of surfaces are likely to arise from an interplay of different factors.

Subject: protein-resistant surfaces, polyethylene glycol, MD simulation, SPR, entropy estimation

Switchable, biocompatible surfaces based on glycerol copolymers, Chemical Communications

Marie Weinhart, Tobias Becherer, Rainer Haag

RSC | 2010-11-11

Chemical Communications, 2011, 47, 1553-1555

Full TextSupporting Information

Two thermoresponsive copolymers based on glycerol were synthesized and tethered onto gold surfaces via SAM formation. Below the LCST protein resistant surfaces were obtained while fibrinogen adsorbed above the LCST. It was demonstrated that these switchable properties can be applied to control fibroblast cell adhesion and detachment.

Dendritic polyglycerol sulfates as multivalent inhibitors of inflammation

Jens Dernedde, Alexandra Rausch, Marie Weinhart,Sven Enders, Rudolf Tauber, Kai Licha, Michael Schirner, Ulrich Zuegel, Arne von Bonin, Rainer Haag

United States National Academy of Sciences | 2010-11-01

PNAS, 2010, 107(46), 19679-19684.

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Adhesive interactions of leukocytes and endothelial cells initiate leukocyte migration to inflamed tissue and are important for immune surveillance. Acute and chronic inflammatory diseases show a dysregulated immune response and result in a massive efflux of leukocytes that contributes to further tissue damage. Therefore, targeting leukocyte trafficking may provide a potent form of anti-inflammatory therapy. Leukocyte migration is initiated by interactions of the cell adhesion molecules E-, L-, and P-selectin and their corresponding carbohydrate ligands. Compounds that efficiently address these interactions are therefore of high therapeutic interest. Based on this rationale we investigated synthetic dendritic polyglycerol sulfates (dPGS) as macromolecular inhibitors that operate via a multivalent binding mechanism mimicking naturally occurring ligands. dPGS inhibited both leukocytic L-selectin and endothelial P-selectin with high efficacy. Size and degree of sulfation of the polymer core determined selectin binding affinity. Administration of dPGS in a contact dermatitis mouse model dampened leukocyte extravasation as effectively as glucocorticoids did and edema formation was significantly reduced. In addition, dPGS interacted with the complement factors C3 and C5 as was shown in vitro and reduced C5a levels in a mouse model of complement activation. Thus, dPGS represent an innovative class of a fully synthetic polymer therapeutics that may be used for the treatment of inflammatory diseases.

Subject: anti-inflammatory drug, complement inhibition, multiple target binding, multivalent selectin inhibitor, synthetic polymer

Linear Poly(methyl glycerol) and Linear Polyglycerol as Potent Protein and Cell Resistant Alternatives to Poly(ethylene glycol)

Marie Weinhart, Ingo Grunwald, Monika Wyszogrodzka, Linda Gaetjen, Andreas Hartwig, Rainer Haag

Wiley | 2010-09-03

Chemistry - An Asian Journal, 2010, 5(9), 1992-2000

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The nonspecific interaction of proteins with surfaces in contact with biofluids leads to adverse problems and is prevented by a biocompatible surface coating. The current benchmark material among such coatings is poly(ethylene glycol) (PEG). Herein, we report on the synthesis of linear polyglycerol derivatives as promising alternatives to PEG. Therefore, gold surfaces as a model system are functionalized with a self-assembled monolayer (SAM) by a two-step anhydride coupling and a direct thiol immobilization of linear poly(methyl glycerol) and polyglycerol. Surface plasmon resonance (SPR) spectroscopy reveals both types of functionalized surfaces to be as resistant as PEG towards the adsorption of the test proteins fibrinogen, pepsin, albumin, and lysozyme. Moreover, linear polyglycerols adsorb even less proteins from human plasma than a PEG-modified surface. Additional cell adhesion experiments on linear poly(methyl glycerol) and polyglycerol-modified surfaces show comparable cell resistance as for a PEG-modified surface. Also, in the case of long-term stability, high cell resistance is observed for all samples in medium. Additional in vitro cell-toxicity tests add to the argument that linear poly(methyl glycerol) and polyglycerol are strong candidates for promising alternatives to PEG, which can easily be modified for biocompatible functionalization of other surfaces.

Patterning microfluidic device wettability using flow confinement

Adam R. Abate, Julian Thiele, Marie Weinhart, David A. Weitz

RSC | 2010-05-05

Lab Chip, 2010,10, 1774-1776.

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We present a simple method to spatially pattern the surface properties of microfluidic devices using flow confinement. Our technique allows surface patterning with micron-scale resolution. To demonstrate its effectiveness, we use it to pattern wettability to form W/O/W and O/W/O double emulsions.