Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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    Cephalopod-Inspired High Dynamic Range Mechano-Imaging in Polymeric Materials
    Clough, Jess M. ; Gucht, Jasper van der; Kodger, Thomas E. ; Sprakel, Joris - \ 2020
    Advanced Functional Materials (2020). - ISSN 1616-301X
    colloids - mechanochemistry - mechanochromism - photonics - polymers

    Cephalopods, such as squid, cuttlefish, and octopuses, use an array of responsive absorptive and photonic dermal structures to achieve rapid and reversible color changes for spectacular camouflage and signaling displays. Challenges remain in designing synthetic soft materials with similar multiple and dynamic responsivity for the development of optical sensors for the sensitive detection of mechanical stresses and strains. Here, a high dynamic range mechano-imaging (HDR-MI) polymeric material integrating physical and chemical mechanochromism is designed providing a continuous optical read-out of strain upon mechanical deformation. By combining a colloidal photonic array with a mechanically responsive dye, the material architecture significantly improves the mechanochromic sensitivity, which is moreover readily tuned, and expands the range of detectable strains and stresses at both microscopic and nanoscopic length scales. This multi-functional material is highlighted by creating detailed HDR mechanographs of membrane deformation and around defects using a low-cost hyperspectral camera, which is found to be in excellent agreement with the results of finite element simulations. This multi-scale approach to mechano-sensing and -imaging provides a platform to develop mechanochromic composites with high sensitivity and high dynamic mechanical range.

    Electron Storage in Electroactive Biofilms
    Heijne, A. ter; Pereira, M.A. ; Pereira, J. ; Sleutels, T. - \ 2020
    Trends in Biotechnology (2020). - ISSN 0167-7799 - 9 p.
    electroactive biofilms - electron storage - microbial electrochemical technologies - polymers

    Microbial electrochemical technologies (METs) are promising for sustainable applications. Recently, electron storage during intermittent operation of electroactive biofilms (EABs) has been shown to play an important role in power output and electron efficiencies. Insights into electron storage mechanisms, and the conditions under which these occur, are essential to improve microbial electrochemical conversions and to optimize biotechnological processes. Here, we discuss the two main mechanisms for electron storage in EABs: storage in the form of reduced redox active components in the electron transport chain and in the form of polymers. We review electron storage in EABs and in other microorganisms and will discuss how the mechanisms of electron storage can be influenced.

    Melamine-Based Microporous Organic Framework Thin Films on an Alumina Membrane for High-Flux Organic Solvent Nanofiltration
    Amirilargani, Mohammad ; Yokota, Giovana N. ; Vermeij, Gijs H. ; Merlet, Renaud B. ; Delen, Guusje ; Mandemaker, Laurens D.B. ; Weckhuysen, Bert M. ; Winnubst, Louis ; Nijmeijer, Arian ; Smet, Louis C.P.M. de; Sudhölter, Ernst J.R. - \ 2020
    ChemSusChem 13 (2020)1. - ISSN 1864-5631 - p. 136 - 140.
    membranes - microporous materials - organic solvent nanofiltration - polymers - porous organic frameworks

    Microporous polymer frameworks have attracted considerable attention to make novel separation layers owing to their highly porous structure, high permeability, and excellent molecular separation. This study concerns the fabrication and properties of thin melamine-based microporous polymer networks with a layer thickness of around 400 nm, supported on an α-alumina support and their potential use in organic solvent nanofiltration. The modified membranes show excellent solvent purification performances, such as n-heptane permeability as high as 9.2 L m−2 h−1 bar −1 in combination with a very high rejection of approximately 99 % for organic dyes with molecular weight of ≥457 Da. These values are higher than for the majority of the state-of-the-art membranes. The membranes further exhibit outstanding long-term operation stability. This work significantly expands the possibilities of using ceramic membranes in organic solvent nanofiltration.

    ‘We’ve still got a way to go’
    Thoden van Velzen, E.U. ; Molenveld, K. - \ 2018
    recycling - biobased economy - polymers - residual streams - organic wastes - biofuels - bioenergy - renewable energy
    Nonlinear shear and dilatational rheology of viscoelastic interfacial layers of cellulose nanocrystals
    Berg, Merel van den; Kuster, Simon ; Windhab, E.J. ; Sagis, L.M.C. ; Fischer, P. - \ 2018
    Physics of Fluids 30 (2018)7. - ISSN 1070-6631 - 11 p.
    compressibility - contact angle - Hydrophobicity - nanomechanics - nanoparticles - polymers - Rheology - softening - viscoelasticity - work hardening
    We present a nonlinear rheological investigation of model rod-like particles at the air/water interface in dilatation and shear. Cellulose nanocrystals were modified to vary their surface hydrophobicity, creating a range of surface-active particles with varying contact angle. The interfacial rheological properties were studied using a series of frequency sweeps in small amplitude oscillatory shear as well as strain sweeps under large amplitude oscillatory shear (LAOS) and large amplitude oscillatory dilatation (LAOD) to include the nonlinear behavior. A multi-mode Maxwell model was used to fit the frequency sweeps that were obtained during formation of the interfacial layer. A shift toward longer
    relaxation times was found, more pronounced for particles with higher hydrophobicity. Lissajous plots in LAOS revealed strain stiffening, yielding, and unconstrained flow of the interfacial layers.
    Lissajous plots in LAOD revealed strain hardening in compression and strain softening in expansion, increasing with surface pressure and with particle hydrophobicity. While interfacial layers commonly show gel or solid-like behavior, our findings imply a weakly aggregated system. The rheological
    behavior indicates the formation of larger clusters for particles with high hydrophobicity compared to smaller clusters for particles with low hydrophobicity. The particle-particle interactions therefore vary with hydrophobicity, suggesting that capillary interactions are important for the formation of these microstructures.
    Christiaan Bolck (Biobased Performance Materials): ‘We doen onderzoek dat impact heeft’
    Bolck, C.H. - \ 2018
    biobased economy - biobased materials - biomass - polymers - bioplastics - dyes - pigments - paints - plant extracts
    Laccase-Mediated Grafting on Biopolymers and Synthetic Polymers : A Critical Review
    Slagman, Sjoerd ; Zuilhof, Han ; Franssen, Maurice C.R. - \ 2018
    ChemBioChem 19 (2018)4. - ISSN 1439-4227 - p. 288 - 311.
    biomass - enzyme catalysis - grafting - polymers - surface chemistry
    Laccase-mediated grafting on lignocelluloses has gained considerable attention as an environmentally benign method to covalently modify wood, paper and cork. In recent decades this technique has also been employed to modify fibres with a polysaccharide backbone, such as cellulose or chitosan, to infer colouration, antimicrobial activity or antioxidant activity to the material. The scope of this approach has been further widened by researchers, who apply mediators or high redox potential laccases and those that modify synthetic polymers and proteins. In all cases, the methodology relies on one- or two-electron oxidation of the surface functional groups or of the graftable molecule in solution. However, similar results can very often be achieved through simple deposition, even after extensive washing. This unintended adsorption of the active substance could have an adverse effect on the durability of the applied coating. Differentiating between actual covalent binding and adsorption is therefore essential, but proves to be challenging. This review not only covers excellent research on the topic of laccase-mediated grafting over the last five to ten years, but also provides a critical comparison to highlight either the lack or presence of compelling evidence for covalent grafting.
    Production of protein‐based polymers in Pichia pastoris
    Werten, Marc W.T. - \ 2017
    Wageningen University. Promotor(en): M.A. Cohen Stuart; G. Eggink, co-promotor(en): F.A. de Wolf. - Wageningen : Wageningen University - ISBN 9789463436069 - 241
    proteins - polymers - pichia pastoris - gelatin - proteolysis - biosynthesis - eiwitten - polymeren - pichia pastoris - gelatine - proteolyse - biosynthese

    From a chemistry perspective, proteins can be thought of as polymers of amino acids, linked by amide bonds. They feature unsurpassed control over monomer sequence and molecular size. The amino acid sequence of proteins determines their three-dimensional folded structure, resulting in unique properties. Proteins such as collagen, elastin, and silk play a crucial structure-forming role in various tissues and animal architecture such as spider webs. These proteins are characterized by highly repetitive amino acid sequences, and can reversibly self-assemble into supramolecular structures through the formation of noncovalent bonds. These unique properties have sparked the interest of material scientists, and mimics of these proteins have been designed and produced as heterologous proteins in suitable expression systems.

    The most commonly employed host for these so-called protein-based polymers, or protein polymers for short, is the bacterium Escherichia coli. In this thesis, we explored the use of an alternative platform, namely the methylotrophic yeast Pichia pastoris (Komagataella phafii). This organism is well-known for its often relatively high yields, and offers a choice between intracellular and secretory production. Secretion of the polymer into the medium provides a highly effective first purification step, and precludes the need for cell disruption procedures that are cost-prohibitive at an industrial scale.

    We evaluated the secretory production in P. pastoris of various protein polymers: murine collagen fragments (gelatins), a de novo-designed highly hydrophilic gelatin, silk-like proteins, hydrogel-forming triblock copolymers with collagen-inspired end blocks, block copolymers with heterodimer-forming modules, and silk-inspired triblock copolymers that feature integrin-binding or proteoglycan-binding cell-adhesive motifs. All of these protein polymers were produced at g/L levels, and various bioprocessing and strain engineering strategies were employed to address problems such as proteolytic degradation and other undesired posttranslational modifications. The basic physicochemical properties of the polymers produced were studied, which revealed interesting characteristics. Some of these polymers show promise for further development towards biomedical applications such as tissue engineering and controlled drug release.

    Harvesting and cell disruption of microalgae
    Lam, Gerard Pieter 't - \ 2017
    Wageningen University. Promotor(en): R.H. Wijffels; M.H.M. Eppink, co-promotor(en): M.H. Vermuë. - Wageningen : Wageningen University - ISBN 9789463431736 - 206
    algae - harvesting - flocculation - polymers - chlorella vulgaris - biorefinery - electric field - organelles - algen - oogsten - uitvlokking - polymeren - chlorella vulgaris - bioraffinage - elektrisch veld - organellen

    Microalgae are a potential feedstock for various products. At the moment, they are already used as feedstock for high-valuable products (e.g. aquaculture and pigments).

    Microalgae pre-dominantly consist out of proteins, lipids and carbohydrates. This makes algae an interesting feedstock for various bulk-commodities. To successfully produce bulk-commodities, a multi-product biorefinery should be adopted that aims on production of both bulk- and high value co-products. In the downstream process, however, harvesting- and cell disruption are technological hurdles for cost effective multi-product biorefinery.

    Flocculation is considered as a low-cost harvesting process. Flocculating microalgae at high salinities used to be not feasible We demonstrated that marine microalgae can successfully be flocculated and harvested by using cationic polymers.

    In the second part of this thesis we studied Pulsed Electric Field (PEF) as potential cheap and non-disruptive technology to open microalgae. PEF-treatment evokes openings/’holes’ in micro-organisms. PEF in combination with a pre-treatment to weaken the cell wall resulted in release of proteins from microalgae at low energy consumption.

    Recent advances in technology development learned that harvesting of micro-algae is no longer a bottleneck. Future research and development should focus on cell disruption and mild extraction technologies. Costs for the biorefinery will decrease by process simplification. For that unit operations for cell disruption and extraction need to be integrated.

    This project was part of a large public private partnership program AlgaePARC biorefinery (www.AlgaePARC.com). Objective of this program is to develop a more sustainable and economically feasible microalgae production process. For that all biomass components (e.g. proteins, lipids, carbohydrates) should be used at minimal energy requirements and minimal costs while keeping the functionality of the different biomass components. Biorefining of microalgae is very important for the selective separation and use of the different functional biomass components.

    Thermo-responsive block copolymers : synthesis, self-assembly and membrane development
    Mocan Cetintas, Merve - \ 2017
    Wageningen University. Promotor(en): F.A.M. Leermakers, co-promotor(en): M.M.G. Kamperman. - Wageningen : Wageningen University - ISBN 9789463431583 - 177
    polymer chemistry - polymers - membranes - synthesis - self assembly - thermal properties - polymeerchemie - polymeren - membranen - synthese - zelf-assemblage - thermische eigenschappen

    Block copolymers (BCPs) are remarkable materials because of their self-assembly behavior into nano-sized regular structures and high tunable properties. BCPs are in used various applications such as surfactants, nanolithography, biomedicine and nanoporous membranes. In these thesis, we aimed to fabricate thermo-responsive iso- and nanoporous membranes from BCPs.

    First, we optimized the synthesis of a thermo-responsive BCP, i.e. polystyrene-poly(N-isopropyl acrylamide) (PS-PNIPAM) with desired properties using controlled/living polymerization methods. We fabricated membranes using self-assembly and non-solvent induced phase separation (SNIPS) method. The membranes were nanoporous, thermo-responsive and exhibited an interconnected worm-like surface.

    We investigated the self-assembly behavior of BCPs using both theoretical and experimental approaches. The theoretical investigation involves self-consistent field modelling of Scheutjens and Fleer (SF-SCF) which is used for the first time for BCP self-assembly phenomena. Using SF-SCF, first, we found a chain length dependence on the critical point of BCP phase diagram which confirms well with the reported literature. Second, we worked on the stability of the common mesophases (e.g. single and double gyroids, double diamond, hexagonally perforated lamellae) that is observed between hexagonally ordered cylindrical (HEX) and lamellar (LAM) phases; at chain length, =300 and at intermediate segregation regime, =30. Among the mentioned mesophases double gyroid was the only phase dominant over HEX and LAM phases. At strong segregation regime of =120 with the same chain length, double gyroid was found as a metastable phase.

    The experimental approach of the BCP self-assembly was performed by solvent annealing of BCP thin films. For annealing, common laboratory solvents e.g. methanol, tetrahydrofuran, toluene were used with various ratios to tune the selectivity of the solvent mixtures to the blocks in the copolymer. A lamellar forming triblock copolymer using the solvent mixtures methanol: THF (v:v) 1:2 or methanol: toluene (v:v) 1:1 resulted in HEX phase. In contrast, no sustained long-range order was found when only one type of solvent was used.

    Next, we optimized the membrane fabrication parameters to obtain membranes with an isoporous surface. We investigated the effect of solvent selectivity, evaporation time and polymer concentration. For PS selective solvents, membranes exhibited a disordered surface whereas PNIPAM selective solvents resulted in membranes with an isoporous surface. For a large parameter space, isoporous membranes were attained which is not common for SNIPS method. Permeability tests at various temperatures proved fully reversible thermo-responsive behavior of these membranes.

    Finally, we concluded our work with future recommendations to obtain block copolymer membranes that have improved properties and suggested tests that will prove membranes’ suitability for industrial applications.

    Dynamic covalent polymers
    Garcia Melo, Fatima ; Smulders, Maarten M.J. - \ 2016
    Journal of Polymer Science. Part A, Polymer Chemistry 54 (2016)22. - ISSN 0887-624X - p. 3551 - 3577.
    dynamic covalent chemistry - gels - nanoparticles - polymers - responsive materials - self-healing materials

    This Highlight presents an overview of the rapidly growing field of dynamic covalent polymers. This class of polymers combines intrinsic reversibility with the robustness of covalent bonds, thus enabling formation of mechanically stable, polymer-based materials that are responsive to external stimuli. It will be discussed how the inherent dynamic nature of the dynamic covalent bonds on the molecular level can be translated to the macroscopic level of the polymer, giving access to a range of applications, such as stimuli-responsive or self-healing materials. A primary distinction will be made based on the type of dynamic covalent bond employed, while a secondary distinction will be based on the consideration whether the dynamic covalent bond is used in the main chain of the polymer or whether it is used to allow side chain modification of the polymer. Emphasis will be on the chemistry of the dynamic covalent bonds present in the polymer, in particular in relation to how the specific (dynamic) features of the bond impart functionality to the polymer material, and to the conditions under which this dynamic behavior is manifested.

    Controlling the self-assembly of protein polymers via heterodimer-forming modules
    Domeradzka, Natalia Eliza - \ 2016
    Wageningen University. Promotor(en): Frans Leermakers, co-promotor(en): Renko de Vries; Frits de Wolf. - Wageningen : Wageningen University - ISBN 9789462578661 - 166
    polymers - nanotechnology - pichia pastoris - modules - mass spectrometry - microscopy - sds-page - rheology - fluorescence emission spectroscopy - protein purification - fermentation - chromatography - polymeren - nanotechnologie - pichia pastoris - modules - massaspectrometrie - microscopie - sds-page - reologie - fluorescentie-emissiespectroscopie - eiwitzuivering - fermentatie - chromatografie

    Supramolecular assemblies formed by protein polymers are attractive candidates for future biomaterials. Ideally, one would like to be able to define the nanostructure, in which the protein polymers should self-assemble, and then design protein polymer sequences that assemble exactly into such nanostructures. Despite progress towards ‘programmability’ of protein polymer self-assembly, we do not yet have such control. This holds especially for hierarchical structures such as self-assembled fibril bundles, where one would like to have independent control over the structures at the different length-scales. In this thesis we explore the use of heterodimerization as a strategy to control self-assembly of protein polymers at multiple length-scales. We tested a selected set of heterodimer-forming peptide modules. The heterodimer-forming modules are genetically incorporated at the C-terminus of protein polymers with a previously characterized self-assembly behavior. Several newly constructed protein polymers were biosynthesized in the yeast Pichia pastoris and, for these new protein polymers we investigated whether the inclusion of the heterodimer-forming blocks improved the control over the assembly of nanostructures.

    The incorporation of heterodimer-forming modules into protein polymers is not the only tool that can be used for improving programmability of assembly. In Chapter 2 we present an overview of several tools that can be use, and we highlighted their advantages and disadvantages.

    In Chapter 3 we test de novo designed heterodimerizing coiled coils DA = LEIRAAFLRQRNTALRTEVAELEQEVQRLENEVSQYETRYGPLGGGK and DB = LEIEAAFLERENTALETRVAELRQRVQRLRNRVSQYRTRYGPLGGGK. These peptides were fused to hydrophilic random coil protein polymer (CP4) and homotrimer forming protein polymer (T9-CP4). We present data on the production, characterization and functionality for four new protein polymers: CP4-DA, CP4-DB, T9-CP4-DA and T9-CP4-DB. When the new protein polymers were produced using the fermentation process established previously for other protein polymers such as CP4 (i.e. standard fermentation), we found the new protein polymers to be partly degraded. The use of a protease deficient strain, as well as changes in aeration or pH were found ineffective in preventing degradation, but nearly intact products were obtained from a fermentation in which the induction was done at 20 ˚C and in which the medium was supplemented with casamino acids. With respect to the physical properties of the new protein polymers, size exclusion chromatography (SEC) showed that an equimolar mixture of CP4-DA and CP4-DB contained mostly dimers, whereas unmixed CP4-DA and CP4-DB contained only monomers. However, we also found that CP4-DB forms homooligomers at concentrations ≥100 µM. A mixture of T9-CP4-DA and T9-CP4-DB forms a hydrogel, most probably due to both homotypic and heterotypic DA/DB associations. We conclude that when used at low concentration, this pair of coiled coils seems to be suitable to control self-assembly of protein polymers produced in Pichia Pastoris.

    Next, in Chapter 4 we test another pair of de novo designed coiled coils. These are much shorter and have lower reported values of the association constant as compared to the DA/DB coiled coils. The systems consist of a peptide DE = (EIAALEK)3 and a peptide DK = (KIAALKE)3. The two peptides were C-terminally fused to protein polymers CP4 and T9-CP4. The standard fermentations resulted in intact CP4-DE and T9-CP4-DE, but protein polymers CP4-DK and T9-CP4-DK were found to be partly degraded. The degradation of variants with DK module could not be readily resolved by fermentation at higher pH or using proteases deficient strain. For CP4-DK, ion exchange chromatography showed that about 40% of protein polymer (by mass) was intact. We find that for this pair of coiled-coils, homotypic interactions are so strong that they can drive gel formation in the case of T9-CP4-DE, and a strong increase in viscosity for T9-CP4-DK. Mixtures of the complimentary triblocks also form hydrogels, but it is not yet clear to what extent this is due to homotypic DE/ DE and DK/ DK associations, and to what extent it is due to DE/ DK heterodimer formation.

    A very different type of heterodimer-forming block is the so-called WW domain that is found in many natural proteins, and which forms heterodimers with proline-rich peptides PPxY. In Chapter 5 we test the interaction between a naturally occurring WW domain (DWW) and its proline-rich ligand (DPPxY). Both were C-terminally fused to the hydrophilic random coil protein polymer CP4. The new protein polymers CP4-DWW and CP4-DPPxY were produced intact during standard fermentations, but CP4-DPPxY was shown to be glycosylated. Using genetic engineering, we mutated the CP4-DPPxY protein polymer sequence by the substitution Ser12→Ala. A standard fermentation resulted in an intact and non-glycosylated protein polymer CP4-DPPxY*. Interaction studies (ITC and steady state tryptophan fluorescence quenching), showed that both CP4-DPPxY and CP4-DPPxY* bind to CP4-DWW with an equilibrium dissociation constant on the order of mM.

    Finally, to demonstrate that heterodimer-forming blocks can be used to independently control protein polymer self-assembly at multiple length-scales, we selected the heterodimer-forming modules DA and DB to control the lateral interactions of fibrils self-assembled from the previously designed triblock protein polymer C2-SH48-C2. In Chapter 6 we construct the protein polymers C2-SH48-C2-DA and C2-SH48-C2-DB. The C2-SH48-C2 protein polymers assemble into long and stiff fibrils at neutral pH. The aim of the C-terminal attachment of the DA/DB blocks was to be able to control subsequent physical cross-linking and bundling of the fibrils. Both protein polymers C2-SH48-C2-DA and C2-SH48-C2-DB were produced intact and with high yield during fermentation at optimal conditions as discussed in Chapter 3. Using Atomic Force Microscopy (AFM) we show that at neutral pH, fibrils consisting of 100% C2-SH48-C2-DA or C2-SH48-C2-DB protein polymers bundle up and cross-link via homotypic DA/DA and DB/DB associations. Control over the degree of cross-linking and bundling can be obtained by using mixed fibrils consisting of C2-SH48-C2 with controlled amounts of the newly developed protein polymers C2-SH48-C2-DA and C2-SH48-C2-DB. While the effect of the heterodimers on the structure of the fibril network as judged from AFM is very strong, oscillation rheology shows that the inclusion of the heterodimer forming blocks merely leads to a moderate increase in gel stiffness.

    In order to place the research discussed in this thesis into the broader perspective, in Chapter 7 we provide a General Discussion. We discuss several general strategies that can be used to control protein polymer self-assembly and discuss why and when there is a need for using heterodimer forming blocks. After providing an overview over results obtained in this thesis, we highlight the most urgent questions that need to be answered next. This is followed by a discussion on the benefits that heterodimer-driven self-assembly may bring to possible future applications of protein polymers as biomaterials. We also discuss the possible risks for human health end environment that might arise from the use of protein polymers technology. Finally we present some speculations about the future of the field of self-assembling protein polymers.

    Co-assembled DNA-protein polymer bottlebrushes : main-chain stiffening & liquid crystallinity
    Storm, I.M. - \ 2016
    Wageningen University. Promotor(en): Martien Cohen Stuart; Frans Leermakers, co-promotor(en): Renko de Vries. - Wageningen : Wageningen University - ISBN 9789462577466 - 161
    polymers - liquid crystals - dna - proteins - polymeren - vloeibare kristallen - dna - eiwitten

    Bottlebrushes are macromolecules consisting of a backbone polymer onto which side chains are either physically or chemically grafted. Early theories suggested that attaching side chains to a (flexible) backbone molecule would induce the so-called main-chain stiffening effect. This newly formed bottlebrush molecule should therefore behave as a semi-flexible polymer rather than a flexible polymer. Due to this semi-flexible behaviour bottlebrushes should also be able to show liquid crystalline behaviour. However, there are very few examples of bottlebrush systems that are able to make liquid crystalline phases. In this thesis, we present a co-assembled bottlebrush system that consist of DNA as the backbone molecule and genetically engineered protein polymers as side chains. This co-assembled system is one of the few bottlebrush systems that actually does show liquid crystalline behaviour. This ability makes this bottlebrush system a perfect system to explain why it is so very difficult to make liquid crystalline phases with bottlebrushes. We have shown that attaching side chains will, at first, result in an effectively more flexible bottlebrush system. Only for systems with very densely packed and long side chains is the stiffness of the bottlebrush molecule increasing. Moreover, with osmotic stress experiments we have shown that the presence of free polymers also has a negative influence on the stiffness of bottlebrush molecules and hence this reduces the tendency for the system to form liquid crystals.

    Nanoscale force sensors to study supramolecular systems
    Cingil, E.H. - \ 2016
    Wageningen University. Promotor(en): Martien Cohen Stuart, co-promotor(en): Joris Sprakel. - Wageningen : Wageningen University - ISBN 9789462576971 - 136
    sensors - supramolecular chemistry - molecules - biopolymers - polymers - methodology - rheology - sensors - supramoleculaire chemie - moleculen - biopolymeren - polymeren - methodologie - reologie

    Supramolecular systems are solutions, suspensions or solids, formed by physical and non-covalent interactions. These weak and dynamic bonds drive molecular self-assembly in nature, leading to formation of complex ordered structures in high precision. Understanding self-assembly and co-assembly is crucial to unravel and mimic many processes occurring in nature. However, the challenge cannot be easily addressed especially in biological systems as it involves many dynamic interactions which may cooperatively, noncooperatively or competitively generate a complex manifold of interaction pathways. In this thesis, we employed two techniques to understand these complex interactions in various supramolecular systems at the nanoscale 1) multiple particle tracking microrheology to study thermoreversible assembly of triple helices in a collagen-inspired recombinant polypeptide in the form of a triblock copolymer gel former; and 2) polyfluorene-based conjugated polyelctrolyte mechosensors to monitor electrostatic co-assembly dynamics of (i) a recombinant diblock copolypeptide which encapsulates the conjugated polyelectrolyte like a protein capsid and (ii) various synthetic diblock copolymers which forms complex coacervate micelles; and finally the orthogonal self-assembly dynamics of (iii) a recombinant viral coat protein which mimics natural rod-like viruses. These novel polymeric mechanosensors work as versatile, non-invasive tools to detect even low degrees of analyte binding or complex formation due to the stress applied on their conjugated backbone. This mechanical stress causes the polymeric backbone to stretch which can be detected by a shift in its fluorescence spectra.

    Silky gels for cells : Self-assembling protein-based polymers for use in tissue engineering
    Wlodarczyk, M.K. - \ 2016
    Wageningen University. Promotor(en): Martien Cohen Stuart; Marleen Kamperman; S.C.G. Leeuwenburgh. - Wageningen : Wageningen University - ISBN 9789462576230 - 194
    polymers - proteins - biomedical engineering - biomaterials - recombinant dna - transplantation - compatibility - encapsulation - heparin - biodegradation - physical properties - polymeren - eiwitten - biomedische techniek - biomaterialen - recombinant dna - transplantatie - compatibiliteit - inkapselen - heparine - biodegradatie - fysische eigenschappen

    Tissue engineering is a relatively new, but actively developing field of biomedical science. It aims at organ or tissue regeneration by use of scaffolds, which are usually seeded with cells prior to implantation, and stimulated by bioactive cues or growth factors. It is a promising and valuable alternative to the use of transplants, for which the demand is greater than the supply, and for which application is connected with high risk of rejection and infection due to immunosuppressant medication. One of the main challenges of tissue engineering, that we tried to address in this thesis, is the design of biocompatible and functional biomaterials that could serve as cell scaffold. We investigated, if protein-based polymers, more specifically, if the de novo designed, C2SH48C2 copolymer, which self-assembles into fibers upon a pH-trigger, is a suitable material for cell scaffolds.

    In Chapter 2 we described the design and production, by means of recombinant DNA technology, of C2SH48C2. The protein was efficiently secreted by Pichia pastoris at high yields of g/l levels and we proposed an effective purification method. We showed that fibers and gels form by self-assembly upon pH adjustment, and that rheological properties of the obtained hydrogels depend on the total protein concentration. In view of potential biomedical applications, erosion studies were performed, which indicated that the gels exhibited long term stability in conditions mimicking those in body fluid. The biocompatibility of the gel scaffolds was demonstrated in a 2D cell culture study. However, despite the cell viability, a low proliferation rate was observed.

    To improve cell performance in contact with C2SH48C2 hydrogels (Chapter 3) we incorporated active domains in the C2SH48C2 protein by recombinant functionalization. We described the synthesis of two protein variants: (1) BRGDC2SH48C2, N-terminally enriched in integrin-binding domains (RGD) and (2) BKRSRC2SH48C2, N-terminally enriched in heparin binding domains (KRSR). We showed precise control over the amount of active domains in the final gels, by simply mixing the variants of the proteins in the desired molar ratio before inducing gelation. A 23-day cell culture study, performed using MG-63 cells, revealed that the presence of RGD and KRSR domains positively influenced cell attachment, spreading and activity. A synergistic effect was observed, i.e. scaffolds containing both bioactive domains yielded fully confluent layers of cells at an earlier stage during cell culture than the other gels. We concluded that cell behavior can be controlled by tuning the content of functional domains.

    In Chapter 4, we tested the suitability of the C2SH48C2 protein, enriched in RGD domains, for cell encapsulation, as the conditions of 3D cell culturing are more similar to the environment of cells in the body. We independently varied gel stiffness (by means of protein concentration) and functional motif (RGD) density, and analyzed the influence of these parameters on the cellular response. The viability and proliferation of MG-63 cells, encapsulated in the gels at different protein concentrations and RGD densities, was investigated with a cell activity assay, and by quantitative analysis of confocal pictures of nuclei (DAPI stain) and F-actin (phalloidin). We showed that optimal cell behavior is obtained in the presence of RGD domains and at low protein concentrations. The results indicated that RGD functionality is not the sole requirement; the gel matrix needs to exhibit the right mechanical properties and architecture to allow for cell growth, cytoplasmic extension and migration.

    Finally, in Chapter 5, we showed that active domains (here KRSR) can serve multiple functions in the material. We demonstrated the cross-linking ability of KRSR domains in the presence of heparin, leading to structural and mechanical changes in the scaffolds. In dilute systems (0.1 % (w/v)), heparin increases the rate of fiber growth, and induces fiber bundling. At higher protein concentrations, leading to the hydrogel formation (2 % (w/v)), the gelation rate and final storage modulus can be tuned by the amount of heparin and KRSR domain density. We concluded that with this approach, the material properties of C2SH48C2 protein gels can be effectively and simply controlled in a straightforward and biocompatible way.

    In Chapter 6 we described the main requirements for biomaterials and discussed to what extent they are fulfilled by protein-based polymers, and in particular, by the presented C2SH48C2 protein. The main advantages over alternative materials, and the challenges that need to be addressed before application in tissue engineering becomes a reality, were discussed. We ended with suggestions to improve the properties of C2SH48C2 protein for use as a biomaterial, especially its biodegradability, and its structural and mechanical properties.

    Composite hydrogels of bio-inspired protein polymers : mechanical and structural characterization
    Rombouts, W.H. - \ 2015
    Wageningen University. Promotor(en): Jasper van der Gucht. - Wageningen : Wageningen University - ISBN 9789462575721 - 172
    gels - formation - proteins - polymers - networks - mechanical properties - gels - formatie - eiwitten - polymeren - netwerken - mechanische eigenschappen

    In this thesis we presented various combinations of custom-designed protein polymers that formed composite hydrogels. In chapter 2, composite hydrogels were prepared by mixing silk-like block copolymers (CP2SE48CP2) with collagen-like block copolymers (T9CR4T9). We found that by adding the collagen-like protein polymer the storage modulus, the critical stress and critical strain values of the composite hydrogels were significantly improved in comparison to the single networks. With cryo-transmission electron microscopy (cryo-TEM) we observed that the silk-like fibers were bundled in the presence of the collagen-like protein polymer, probably due to depletion attraction interactions. In follow-up research on these composite hydrogels in chapter 3, we tried to get more insight into the exact toughening mechanism and self-healing capabilities of the composite network by performing cyclic loading/unloading tests. We found that mechanical hysteresis occurred in these composite hydrogels. The energy that was dissipated could be split into two contributions: a part belonging to the permanent rupture of silk-like fibers, and a viscoelastic part belonging to the assembly and disassembly of collagen-like triple helices. Both these contributions increased as the concentration of the collagen-like protein polymer in the composite network was increased, resulting in toughening of the composite network. Furthermore, it was observed that the silk-like fiber network was not able to recover, while the composites could recover up to 70% of the original storage modulus after failure. In chapter 4 we studied composite networks of silk-like block copolymers (CP2SE48CP2) and a FMOC-functionalized dipeptide (FMOC-LG) which could both form fibers. With cryo-TEM and atomic force microscopy (AFM) we found that two different types of fibers were formed, indicating that orthogonal self-assembly occurred in this system. We found with rheology that the storage moduli of the composite fiber networks were significantly higher (75 kPa vs. 400 kPa) than that of the single networks. Strain-hardening present in the FMOC-LG fiber network disappeared when the silk-like protein polymer was present. In chapter 5 hydrogels with both physical and chemical crosslinks were prepared from collagen-like protein polymers (T9CRT9). The chemical crosslinks were introduced by crosslinking lysine residues present in the random-coil middle blocks with glutaraldehyde. We found with rheology that the order in which the physical and chemical networks were formed did not influence the final storage modulus of the hydrogel. Depending on the amount of glutaraldehyde added we found an increase of up to an order of magnitude in the storage modulus for the collagen-like hydrogels. To investigate effects on the nonlinear rheological properties cyclic loading/unloading tests were performed. It was observed that before hydrogel failure occurred no hysteresis was observed between consecutive cycles. Both physical and chemical crosslinks ruptured when the hydrogel was fractured. In chapter 6 we studied hydrogels formed by the co- assembly of an asymmetric silk-collagen-like protein polymer (SH8CR4T9) with a symmetric oppositely charged silk-like protein polymer (CP2SE48CP2). This was done in a step-wise approach: (1) the S blocks were co-assembled into silk-like fibers. (2) the T blocks were assembled into triple helical nodes by reducing the temperature. We confirmed with confocal laser scanning microscopy and NMR that both monomers were present in the same fibers. With rheology we found that these composite hydrogels did respond in a reversible manner to temperature changes, with which the mechanical strength of the hydrogel can be tuned. In chapter 7 hydrogel formation of a modified silk-like protein polymer with a cysteine-residue attached to the C-terminal side (CP2SH48CP2-Cys) was studied. With rheology we showed that hydrogels that were formed in oxidizing conditions, where disulfide-bridges could form, were much stronger than those formed in reducing conditions. Both hydrogels formed in oxidizing and reducing conditions showed a scaling of modulus versus concentration consistent with entangled semi-flexible networks. This result implied that the disulfide-bridges formed between cysteine-residues formed loops in the coronae of the fibers. The increase in mechanical strength of the fibers was related to the increase in persistence length of the fibers in oxidizing conditions observed with AFM. With self-consistent field theory-simulations it was shown that the formation of loops in the corona resulted in a slight reduction of the lateral pressure in the corona of the fibers. However, this effect is by itself not sufficient to cause a significant change in persistence length. Due to the reduction in lateral pressure, the stacking of monomers into fibers is probably influenced: fibers with a more crystalline structure and with less detects are formed, resulting in improved mechanical properties of the hydrogels. In the general discussion in chapter 8, we reflect on our work, discuss about future directions of research, and possible applications of protein polymers.

    Reversible Temperature-Switching of Hydrogel Stiffness of Coassembled, Silk-Collagen-Like Hydrogels
    Rombouts, W.H. ; Kort, D.W. de; Pham, T.T.H. ; Mierlo, C.P.M. van; Werten, M.W.T. ; Wolf, F.A. de; Gucht, J. van der - \ 2015
    Biomacromolecules 16 (2015)8. - ISSN 1525-7797 - p. 2506 - 2513.
    block-copolymers - gels - ph - gelatin - elastin - biosynthesis - proteins - polymers
    Recombinant protein polymers, which can combine different bioinspired self-assembly motifs in a well-defined block sequence, have large potential as building blocks for making complex, hierarchically structured materials. In this paper we demonstrate the stepwise formation of thermosensitive hydrogels by combination of two distinct, orthogonal self-assembly mechanisms. In the first step, fibers are coassembled from two recombinant protein polymers: (a) a symmetric silk-like block copolymer consisting of a central silk-like block flanked by two soluble random coil blocks and (b) an asymmetric silk-collagen-like block copolymer consisting of a central random-coil block flanked on one side by a silk-like block and on the other side a collagen-like block. In the second step, induced by cooling, the collagen-like blocks form triple helices and thereby cross-link the fibers, leading to hydrogels with a thermo-reversibly switchable stiffness. Our work demonstrates how complex self-assembled materials can be formed through careful control of the self-assembly pathway.
    Structure of multiresponsive brush-decorated nanoparticles: A combined electrokinetic, DLS, and SANS study
    Martin, J.R.S. ; Bihannic, J. ; Santos, C. ; Farinha, J.P. ; Deme, B. ; Leermakers, F.A.M. ; Pinheiro, J.P. - \ 2015
    Langmuir 31 (2015)16. - ISSN 0743-7463 - p. 4779 - 4790.
    functional-group distributions - soft particles - temperature - water - poly(n-isopropylacrylamide) - electrophoresis - microgels - polymers - core
    Particles consisting of a glassy poly(methyl methacrylate) core (ca. 40 nm in radius) decorated with a poly(N-isopropylacrylamide) anionic corona are synthesized using either methacrylic acid (MA) or acrylic acid (AA) as reactive comonomers in the shell. The different reactivity ratios of MA and AA toward N-isopropylacrylamide originates p(MA-N) and p(N-AA) particles with carboxylate charges supposedly located, preferentially, in the close vicinity of the core and at the shell periphery, respectively. The corresponding swelling features of these nanoparticles are addressed over a broad range of pH values (4 to 7.5), NaNO3 concentrations (3 to 200 mM), and temperatures (15 to 45 °C) by dynamic light scattering (DLS) and small angle neutron scattering (SANS). DLS shows that the swelling of the particle shells increases their thickness from ~10 to 90 nm with decreasing temperature, ionic strength, or increasing pH, with the effect being more pronounced for p(N-AA) whose lower critical solution temperature is shifted to higher values compared to that of p(MA-N). Potentiometric titration and electrokinetic results further reflect the easier dissociation of carboxyl groups in p(N-AA) and a marked heterogeneous interfacial swelling of the latter with decreasing solution salt content. The DLS response of both particles is attributed to the multiresponsive nature of a peripheral dilute shell, while SANS only probes the presence of a quasi-solvent-free dense polymer layer, condensed on the core surface. The thickness of that layer slightly increases from ~6 to 9.5 nm with increasing temperature from 15 to 45 °C (at 15 mM NaNO3 and pH 5) due to the collapse of the outer dilute shell layer. Overall, results evidence a nonideal brush behavior of p(MA-N) and p(N-AA) and their microphase segregated shell structure, which supports some of the conclusions recently formulated from approximate self-consistent mean-field computations.
    Detection of sunflower oil in extra virgin olive oil by fast differential scanning calorimetry
    Wetten, I.A. ; Herwaarden, A.W. ; Splinter, R. ; Boerrigter-Eenling, R. ; Ruth, S.M. van - \ 2015
    Thermochimica Acta 603 (2015). - ISSN 0040-6031 - p. 237 - 243.
    crystallization - discrimination - adulteration - polymers - tool - dsc
    Extra virgin olive oil (EVOO) is an economically valuable product, due to its high quality and premium price. Therefore it is vulnerable for adulteration by means of the addition of cheaper vegetable oils. Differential scanning calorimetry (DSC) has been suggested as a fast technique for the detection of adulteration. However, measurements still take several hours. Fast DSC measurements take several minutes. Therefore this study investigates the applicability of fast DSC for the detection of sunflower oil (SFO) in EVOO. Nine EVOOs, five SFOs and three mixtures were analysed. Cooling curves of EVOO and SFO show one major exothermic peak. Because the cooling curves of EVOO and SFO are very similar they cannot be used for the detection of adulteration. Heating curves of EVOOs show two major endothermic peaks after slow cooling (-2 °C/s), heating curves of SFOs only one. Addition of SFO to EVOO caused a rapid decrease in the coldest endothermic peak and can therefore be used in the detection of adulteration of EVOO by SFO. Depending on the type of olive oil, the presence of 2–10% SFO can already be detected.
    Dilute self-healing hydrogels of silk-collagen-like block copolypeptides at neutral pH
    Golinska, M.D. ; Wlodarczyk-Biegun, M.K. ; Werten, M.W.T. ; Cohen Stuart, M.A. ; Wolf, F.A. de; Vries, R.J. de - \ 2014
    Biomacromolecules 15 (2014)3. - ISSN 1525-7797 - p. 699 - 706.
    spider dragline silk - drug-delivery - fibrous biomaterials - actin solutions - contact-lenses - scaffolds - gels - peptides - polymers - elasticity
    We report on self-healing, pH-responsive hydrogels that are entirely protein-based. The protein is a denovo designed recombinant triblock polypeptide of 66 kg/mol consisting of a silk-like middle block (GAGAGAGH)48, flanked by two long collagen-inspired hydrophilic random coil side blocks. The pH-dependent charge on the histidines in the silk block controls folding and stacking of the silk block. At low pH the protein exists as monomers, but above pH 6 it readily self-assembles into long fibers. At higher concentrations the fibers form self-healing physical gels. Optimal gel strength and self-healing are found at a pH of around 7. The modulus of a 2 wt % gel at pH 7 is G' = 1700 Pa. Being protein-based, and amenable to further sequence engineering, we expect that these proteins are promising scaffold materials to be developed for a broad range of biomedical applications.
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