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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    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.

    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.

    Groepsgedrag op de nanoschaal
    Gucht, J. van der - \ 2014
    Wageningen : Wageningen University, Wageningen UR - ISBN 9789461739711 - 23
    nanotechnologie - colloïden - groepsgedrag - polymeren - nanotechnology - colloids - group behaviour - polymers
    Organic monolayers and fluoropolymer brushes : functionalization, stability and tribology
    Bhairamadgi, N.S. - \ 2014
    Wageningen University. Promotor(en): Han Zuilhof; Cees van Rijn. - Wageningen : Wageningen University - ISBN 9789461739636 - 178
    unimoleculaire films - organische verbindingen - organische fluorverbindingen - colloïden - polymeren - adhesie - frictie - oppervlakteverschijnselen - unimolecular films - organic compounds - organofluorine compounds - colloids - polymers - adhesion - friction - surface phenomena

    This thesis deals with the adhesion and friction properties of densely grafted and covalently bound fluoropolymer brushes on silicon surfaces with varying thickness and fluorine content. A novel surface-functionalizing method is described using the thiol-yne click (TYC) reaction. The TYC reaction is highly useful for the attachment of functional (bio-)molecules and immobilization of radical initiators onto a surface with high density. Next, the hydrolytic and thermal stability of 24 different types of monolayers on Si(111), Si(100), SiC, SiN, SiO2, CrN, ITO, PAO, Au and stainless steel surfaces was evaluated. Subsequently, based on this outcome, highly stable fluorinated polymers are described as obtained using surface-initiated atom transfer radical polymerization (SI-ATRP) reactions. The effects of thickness and fluorine content on tribological properties of these layers were studied. The adhesion and friction properties were investigated using colloidal probe atomic force microscopy under dry and ambient conditions. The solvent-free lubricating properties of obtained fluoropolymer brushes have been characterized in detail, and demonstrate their potential for e.g., MEMS/NEMS devices.

    Protein-based polymers that bond to DNA : design of virus-like particles and supramolecular nanostructures
    Hernandez Garcia, A. - \ 2014
    Wageningen University. Promotor(en): Martien Cohen Stuart, co-promotor(en): Renko de Vries; P. Schoot. - Wageningen : Wageningen University - ISBN 9789461738233 - 242
    polymeren - polymeerchemie - eiwitten - dna - dna-bindende eiwitten - virussen - nanotechnologie - virusreplicatie - virusachtige deeltjes - polymers - polymer chemistry - proteins - dna - dna binding proteins - viruses - nanotechnology - viral replication - virus-like particles

    In this thesis it is demonstrated that it is possible to use Protein-based Polymers (PbPs) as synthetic binders of DNA (or any other negatively charged polyelectrolyte). The PbPs co-assemble with their DNA templates to form highly organized virus-like particles and supramolecular structures. A range of PbPs have been developed over the last decades that can be used as precision functional polymers, and which integrate the unique properties of both proteins and polymers. Many PbPs are based on nature-inspired simple repetitive amino acid sequences. In this thesis, different kinds of such sequences have been combined into PbPs that mimic complex natural functionalities. Being intermediate between proteins and polymers, it has been able to mimic complex functionalities typically found for folded proteins, while retaining the tunability and ease of control that is more characteristic for (synthetic) polymers. Indeed, using clear design rules, biosynthetic PbPs sequences have been obtained and produced that co-assemble with nucleic acids to form true artificial viruses, which mimic their natural counterparts in many respects.

    The motivation for developing artificial viruses derives among others from the growing interest in exploiting natural self-assembled virus structures to develop nanostructured materials. In addition, natural viruses are being used as scaffolds for delivering DNA in the context of gene therapy, to serve as vaccines (by displaying antigens), to template diverse materials, to produce energy, to catch light, to catalyze reactions, to serve as contrasting agents, etc. Developing artificial viruses would serve not only to advance our capabilities to understand and control the co-assembly of nanostructures, but would also generate useful synthetic biomaterials that are even more suited than natural viruses to be used as building blocks for nanostructured materials. In short, the successful development of artificial viruses may be expected to give rise not only to new insights on templated self-assembly, but will also be very important for a range of applications.

    The main part of the thesis is divided into three parts. In part I, “Complexation of DNA into virus-like particles”, we describe details of the molecular biomimetic strategy to design and produce PbPs with functionalities that mimic those of natural viruses. Part II, “Applications of protein-DNA complexes”, deals with the development of diblock PbP that coat DNA, and with their applications in gene delivery and optical mapping of long DNA. Finally, in part III: “Supramolecular nanostructures beyond DNA” we consider the co-assembly of our PbPs with templates other than DNA, and also consider their self-assembly in the absence of DNA.

    Dual responsive physical networks from asymmetric biosynthetic triblock copolymers
    Pham, T.H.T. - \ 2013
    Wageningen University. Promotor(en): Martien Cohen Stuart; Jasper van der Gucht, co-promotor(en): Frits de Wolf. - Wageningen : Wageningen UR - ISBN 9789461737359 - 163
    polymeren - gels - biopolymeren - biosynthese - elastine - collageen - polymers - gels - biopolymers - biosynthesis - elastin - collagen

    The aim of the project is to develop biosynthetically produced amino acid polymers which are composed of three distinct blocks A-C-B, each with a separate function. A is a first self-assembling block capable of ‘recognizing’ (upon a trigger) other A blocks; C is an inert, random coil-like connector, and B is a second self-assembling block. A and B have to be chosen such that they do not cross-assemble. With these molecules it should be possible to fabricate hydrogels in which direct ‘loops’ are excluded. We exploited genetic engineering to design proper genes encoding asymmetric triblock protein polymer and fermentation to produce monodisperse protein polymers. There different asymmetric triblock protein polymers were produced and characterized.

    The first molecule, silk-elastin hybrid molecule (SCE), was inspired by natural silk and elastin. The silk-like block (S) forms a pH-sensitive beta-roll (beta-sheet like) structure that further stacks into long fibrils. The elastin-like block(E) has thermo-responsive properties; above the lower critical solution temperature (LCST), it forms aggregates. We find that polymers that have both silk and elastin-like domains show temperature dependent fibril formation. At high temperature, the elastin blocks irreversibly induce bundling and aggregation of fibrils. The presence of the elastin-like block also changes the kinetics of fibril formation. Whereas silk-like protein without elastin forms monodisperse fibrils, the presence of elastin results in polydisperse fibrils due to homogenous nucleation.

    The self-assembly of silk-elastin hybrid molecule is further analysed in the presence of NaCl. We find that the thermo-responsive behaviors of elastin-like block are strongly dependent on salt concentration. At high salt concentration, the aggregation transition is much more pronounced. At high pH, where the S block does not self-assemble, the polymer forms micellar aggregates upon heating in the presence of NaCl. At low temperature, lowering the pH leads to fibril formation. When both blocks are induced to self-assemble, the final structure reveals a pathway-dependence. Heating the solution of fibrils formed at low temperature results in fibril aggregates which do not dissociate upon cooling. The pH-triggered fibril formation of preheated protein solutions leads to the formation of large objects, which likely cause sedimentation. The structural difference is also demonstrated clearly in the morphology of gels formed at high protein concentration: whereas the gel formed in the first pathway (first lower the pH, then increase the temperature) is transparent, the gel formed in the latter pathway (first increase the temperature, then lower the pH) is milky and has a higher elastic modulus.

    The second type of asymmetric triblock copolymer (TR4H or TR4K) has a collagen-like, triple-helix-forming motif at one end, and a poly cationic block at the other. The collagen-like end-block T consists of 9 (PGP) repeats and forms thermo-responsive triple helices upon cooling. Such helices are reversibly disrupted when the temperature is raised above the melting temperature. The other end-block has 6 positively charged amino acids (histidine-H or lysine-K) and forms micelles when a negatively charged polymer is added. The charge-driven complexation of this block depends on its degree of deprotonation, which is determined by the pKa and the pH. The additives used in this study are a flexible polyanion (polystyrene sulfonate, PSS) and a semi-flexible polyanion (xanthan). We find micelle-to-network transition of the triblock TR4H in complexation with PSS. First, the self-assembly of each end-block is studied separately. As expected, the collagen-like block reversibly forms triple helices upon cooling. The cationic H block forms charge-driven complexes upon adding PSS, leading to micelles with an aggregation number that depends on ionic strength. At high concentration, the micellar TR4H/PSS solutions form a viscoelastic gel upon cooling, which melts at high temperature, indicating the formation of helical junctions between the micelles. Liquid-liquid phase separation is observed when the concentration is below the gelation point (around 90 g/L). This leads to a dilute phase on top of a concentrated gel phase. The phase separation is driven by the attraction between charge-driven micelles caused by the triple helices. It disappears when the solution is heated or when the ionic strength is increased.

    The charge-driven complexation of TR4K with xanthan, a negatively charged polysaccharide is also studied. At high temperature and at very low xanthan concentration, the TR4K binds to the xanthan backbone via the charged block K, leading to charge-driven bottle brushes, as indicated by a significant increase in light scattering intensity due to the increased mass. This interaction is dependent on the pH, due to protonation of the cationic K block. The xanthan/TR4K complex shows thermo-sensitivity due to the helical interaction of the collagen-like blocks. At a xanthan concentration around the overlap concentration (~7 g/L), the presence of the triblock results in an increase in elastic modulus of xanthan gels. At high temperature, the elastic modulus increases by 3 times after adding the triblock. As triple helices do not form, this must be due to changes in the entanglement of the bottle brushes. Also the non-linear rheology of the xanthan/TR4K gels differs significantly from that of xanthan alone. At low temperatures when the helical junctions are formed, the elastic modulus increases even further approximately two times compared with the corresponding value at high temperature. This is ascribed to the formation of crosslinks induced by the proteins between the xanthan molecules. The triblock protein modifies the properties of the xanthan hydrogels in three ways: (1) a significant increase in storage modulus, (2) thermo-sensitivity and (3) a two-step strain softening, where the first step is probably due to unbinding of the proteins from the xanthan backbones.

    The third molecule is an asymmetric triblock copolymer (TR4T-Cys), which has two triple helix forming end-blocks (T), with a cysteine residue (Cys) added to one of these. Under oxidizing conditions, the cysteine residues can form disulfide bonds between two polymers whereas reducing conditions restore the thiol groups. Since cysteine can form only one S-S bridge, intramolecular loops are prevented. The presence of S-S bonds significantly enhances the thermal stability of the triple helical network. This results in the appearance of two melting temperatures, of which the higher one is due to the S-S stabilized triple helices. The elastic modulus of the physical gels in the presence of S-S bonds is almost 2 times higher than that of the physical gels in the absence of S-S bonds. The relaxation time also triples under oxidizing conditions, which indicates that triple helical knots are also kinetically stabilized by S-S bonds.

    In summary, the design of S-C-S (S: functional end-block, C: connector) network-forming components might meet the increasing demands of high performance biomaterials that must be able to build a physical gel under narrowly defined conditions. Such class of telechelic polymer might display various interesting dynamic behaviors including shear banding, self-assembly, rheochaos, and phase-separation. Another aspect is the functionality of the end-block which self-assembles upon triggering. However, connectors often return to the same nodes, resulting in loop formation. Loop formation is a structural imperfection that weakens network connectivity and lowers the material’s elasticity. The asymmetric triblock with two different end-blocks is designed in order to: (1) prevent unimolecular loops and improve mechanical properties (2) achieve multi-responsiveness: this allows us to observe different assembling pathways. In this work, with respect to (1), we indeed observed the decrease in loop formation in physical gels formed by TR4T-Cys due to the formation of S-S bridges. With respect to (2), we indeed obtained multi-responsive hydrogels with all three asymmetric triblock proteins. However, we have only scratched the surface as understanding kinetics of self-assembly and pathway dependent processes. Further investigations are needed to get more insights into how to manipulate various parameters in controlling the final structures.

    Conductive polymers for carbon dioxide sensing
    Doan, T.C.D. - \ 2012
    Wageningen University. Promotor(en): Cees van Rijn. - S.l. : s.n. - ISBN 9789461734105 - 194
    polymeren - geleidingsvermogen - aftasten - kooldioxide - polymers - conductivity - sensing - carbon dioxide

    Augmented levels of carbon dioxide (CO2) in greenhouses stimulate plant growth through photosynthesis. Wireless sensor networks monitoring CO2 levels in greenhouses covering large areas require preferably low power sensors to minimize energy consumption. Therefore, the main objective of this research is to develop CO2 sensors using conductive polymer/polyelectrolyte blends as low power sensing layers operating at room temperature. The transduction principle is based on a relative change in conductivity of the polymer/blend film with regard to variation in CO2 concentration. Conductive polymers including emeraldine base polyaniline (EB-PANI), sodium salt of sulfonated polyaniline(SPAN-Na) and their blends with poly(vinyl alcohol) (PVA) were investigated for CO2 sensing. Conductivity of EB-PANI did not vary in the required pH range for CO2 sensing (pH4 - pH7), however a sulfonated derivative (SPAN-Na) showed an appropriate conductivity change in this pH range. Frequency-dependent impedance of the polymer films casted on interdigitated platinum electrodes were measured. A significant decrease in impedance of the SPAN-Na:PVA blend films was observed at high CO2 concentrations (above 20,000 ppm) under high humidity. The effect of humidity on intrinsic and ionic conductivity of the polymerswas investigated by electrochemical impedance spectroscopy. In addition, polyethyleneimine (PEI) and its blends with other polyelectrolytes including SPAN-Na, poly(sodium 4-styrenesulfonate) (PSS-Na) and Nafion sodium salt (Nafion-Na) exhibited a better sensitivity over a wide range of CO2 concentrations (from 400 ppm to 10,000 ppm). Both dc resistance and ac impedance increased when the films were exposed to CO2 at high humidity. The relative change in impedance of the PEI films was about 6-12%. The response time was 4-5 min but recovery time was quite long from 20 to 60 min. A novel solution to reduce the recovery time was achieved with PEI blends. The blend of PEI:SPAN-Na exhibited a fast response (1.5-4 min) and a short recovery time (1.5-10 min) but a reduced sensitivity in comparison with pure PEI. Furthermore, blends of PEI with PSS-Na, Nafion-Na gave a good sensitivity (up to 2-3 order improvement) and relatively short recovery time (10-20 min). The interactions between sulfonate groups with amine groups of PEI might explain the higher CO2 sensitivity of this PEI blend. Some perspectives are sketched for polymer sensors to be applied in wireless sensor network for greenhouses and other potential applications.

    Structure of binary mixed polymer Langmuir layers
    Bernardini, C. - \ 2012
    Wageningen University. Promotor(en): Martien Cohen Stuart; Frans Leermakers. - S.l. : s.n. - ISBN 9789461732149 - 200
    polymeren - colloïden - colloïdale eigenschappen - oppervlakteverschijnselen - polymers - colloids - colloidal properties - surface phenomena

    The possibility of preparing 2D stable emulsions through mixing of homopolymers in a Langmuir monolayer is the core topic of this thesis. While colloid science has achieved well established results in the study of bulk dispersed systems, accounts on properties of mixed monomolecular films are fewer, and seldom systematic. The aim of this investigation is to contribute to a deeper understanding of the subject, in order to explore opportunities to apply the acquired knowledge to the fabrication of technologically relevant materials. In particular, this study focused on a possibly applicable, innovative strategy for the manipulation of the morphology and the patterning of mixed Langmuir monolayers: the possibility to stabilize and control a dispersion of homopolymers through the addition of a lineactant (the equivalent of a surfactant in three dimensional systems), able to adsorb preferentially at the interfacial contact line of polymer domains, thereby lowering the interfacial energy (line tension) in the system and favoring an effective dispersion of one component into the other.

    The state of the art of the preparation and investigation of 2D colloids is the subject of Chapter 2, which is a comprehensive review on several systems able to yield phase–separated Langmuir monolayers, and includes a general definition of the concept of a 2D colloid, the most relevant instrumental techniques and experimental tools available, a summary of several systems suitable for preparing 2D colloid dispersions, an introduction to the concept of lineactant, and several examples, both experimental and theoretical, in which compounds acting as lineactants have been investigated. This review clearly shows that the polymer–based mixtures are a poorly explored subject, when compared to amphiphiles of natural origin, and so the rest of the thesis has been devoted to the investigation of polymer–based Langmuir monolayers.

    This investigation has been carried out with two parallel approaches: classical experiments at the Langmuir trough and morphological characterization of the Langmuir monolayers with the Brewster Angle Microscope have been performed, along with Self–Consistent Field modeling of the same systems. The setup of the SCF model and comparison of SCF calculation with experimental data from the reference experiments are dealt with in Chapter 3. Surface pressure isotherms at the air/water interface were reproduced for four different polymers, poly–l–lactic acid (PLLA), poly (dimethylsiloxane) (PDMS), poly (methyl methacrylate) (PMMA), and poly (isobutylene) (PiB). The polymers are all insoluble in water, but display a different degree of amphiphilicity; therefore the four isotherms differed strongly. The polymers were described through a SCF model on a united atom level, taking the side groups on the monomer level into account. In line with experiments, the model shown that PiB spread in a monolayer which smoothly thickened at a very low surface pressure and area/monomer value. The monolayer made of PMMA had an autophobic behavior: a PMMA liquid did not spread on top of the monolayer of PMMA at the air/water interface. A thicker PMMA layer only formed after the collapse of the film at a relatively high pressure. The isotherm of PDMS had regions with extreme compressibility which were linked to a layering transition. Finally, PLLA wetted the water surface and spread homogeneously at larger areas per monomer. The classical SCF approach features only short–range, nearest–neighbor interactions. For the correct positioning of the layering and for the thickening of the polymer films, a power–law van der Waals contribution was taken into account in this model. Two–gradient SCF computations were performed to model the interface between two coexistent PDMS films at the layering transition, and an estimation of the length of their interfacial contact was obtained, together with the associated line tension value. The SF–SCF molecularly detailed modeling of PLLA, PDMS, PMMA, and PiB monolayers, spread at the air/water surface, has proven to be consistent with experimental data: the incorporation in the model of a detailed molecular description of the monomeric features of the four compounds examined has been crucial to reproducing the features of the adsorption and pressure/area isotherms.

    In Chapter 4, the same approach was applied to the description of polymer mixtures spread at the air/water interface. The aim of this chapter was to analyze topics such as 2D phase separation and partitioning in mixed polymeric Langmuir monolayers. Two of the four polymers studied in Chapter 3 were selected in order to obtain a mixed Langmuir monolayer. A system consisting of water–insoluble, spreadable, fluid–like polymers was prepared. The polymers were polydimethylsiloxane (PDMS) and polymethylmethacrylate (PMMA), combined, in some cases, with a minority of PDMS–b–PMMA copolymer. Both Langmuir trough pressure/area isotherm measurements and Brewster angle microscopy (BAM) observations were performed, and complemented with molecularly detailed self–consistent field (SCF) calculations. It was shown that PDMS undergoes a layering transition that is difficult to detect by BAM. Addition of PMMA enhanced contrast in BAM, showing a two–phase system: if this consisted of separate two–dimensional (2D) PMMA and PDMS phases, a PDMS–PMMA diblock should accumulate at the phase boundary. However, the diblock copolymer of PDMS–PMMA failed to show the expected “lineactant” behavior, i.e., failed to accumulate at the phase boundary. The calculations pointed to a non-trivial arrangement of the polymer chains at the interface: in mixtures of the two homopolymers, in a rather wide composition ratio, a vertical (with respect to the air/water interfacial plane) configuration was found, with PMMA sitting preferably at the PDMS/water interface of the thicker PDMS film, during the PDMS layering phase transition. This also explained why the diblock copolymer was not a lineactant. Both PMMA and PDMS–b–PMMA were depleted from the thin–thick PDMS film interface, and the line tension between the phases consequently increased in the binary mixtures, as well as in the ternary ones. The results shown in this chapter proved that gaining an accurate control over thin film structures at the microscopic level is a far from trivial task, and the acquisition of fundamental knowledge is necessary in order to interpret experimental data in an appropriate way.

    As a consequence, in Chapter 5 an investigation based solely on SCF modeling was carried out, in order to analyze which polymer blends could have the possibility to undergo lateral phase separation in two dimensions. Specifically, the model system investigated consisted of water–supported Langmuir monolayers, obtained from binary polyalkyl methacrylate mixtures (PXMA, where X stands for any of the type of ester side groups used: M, methyl–; E, ethyl–; B, butyl–; H, hexyl–; O, octyl–; L, lauryl–methacrylate). In particular, the conditions which determined demixing and phase separation in the two–dimensional system were addressed, showing that a sufficient chain length mismatch in the ester side group moieties is able to drive the polymer demixing. When the difference in length of the alkyl chain of the ester moieties on the two types of polymers was progressively reduced, from 11 carbon atoms (PMMA/PLMA) to 4 carbons only (POMA/PLMA), the demixing tendency was also reduced; it vanished, indeed, for POMA/PLMA. In the latter case the polymer/subphase interactions affected more the distribution of the polymer coils in the blend monolayer: mixing of the two polymers was observed, but also a partial layering along the vertical direction.

    Lineactancy was also considered, by selecting the mixture in which phase separation was best achieved: a third component, namely a symmetrical diblock copolymer of the type PLMA–b–PMMA, was added to a PMMA/PLMA blended monolayer. Adsorption of the diblock copolymer was observed exclusively at the contact line between the two homopolymer domains, together with a concomitant lowering of the line tension. The line tension varied with chemical potential of the diblock copolymer according to the Gibbs’ law, which demonstrated that PLMA–b–PMMA indeed acted as a lineactant (the two–dimensional analog of a surfactant) in the model system made of a binary demixed PMMA/PLMA Langmuir monolayer.

    In conclusion, the requirements needed to achieve polymer blend demixing in a Langmuir monolayer are the following: spreadable, insoluble polymers, with the same amphiphilicity degree, combined to a certain chemical mismatch of the side moieties are necessary in order to cause lateral demixing at the air/water interface. The polyalkyl methacrylate example investigated in the chapter represented a suitable model system, since the methacrylate backbone guarantees that the different polymers have the same affinity towards the water subphase, while the different ester moieties drive the occurrence of lateral demixing. The dependency of the lateral demixing on the difference in length between the two ester side groups chosen was demonstrated. A rather complex interplay of forces regulates the distribution of the polymer coils in the monolayer: subtle alterations of this complex balance might favor the dewetting of the mixture in a single domain, together with the layering of the blended polymers along the direction normal to the air/water interface, as well as accumulation of one polymer at the domain edge, instead of the occurrence of the lateral phase separation. Furthermore, the possibility to control emulsification of two–dimensional demixed polymer blends was proven. This was achieved by use of a diblock copolymer, which acted as a lineactant by adsorbing at the contact line of the polymer domains. The calculations demonstrated the possibility to extend the lineactant concept, first elaborated in the context of lipid membrane investigations, to the field of study of polymer thin films.

    Natuurlijke vezelversterkte composieten
    Oever, M.J.A. van den - \ 2010
    natuurlijke vezels - polymeren - stijven - sterkte - biobased economy - composieten - natural fibres - polymers - sizing - strength - biobased economy - composite materials
    De specifieke eigenschappen van natuurlijke vezels maakt ze geschikt voor toepassing in vezelversterkte polymeren (composieten) met een hoge sterkte-stijfheid en een lage dichtheid. Momenteel bestaan er op commerciële schaal vier combinaties van verwerkingsmethoden en toepassingen van natuurlijke vezelcomposieten.
    Collagen-like block copolymers with tunable design : production in yeast and functional characterisation
    Teles, H.M. - \ 2010
    Wageningen University. Promotor(en): Gerrit Eggink, co-promotor(en): Frits de Wolf. - [S.l.] : S.n. - ISBN 9789085857082 - 152
    collageen - polymeren - gels - gelering - biologische productie - industriële microbiologie - gelatine - pichia pastoris - collagen - polymers - gels - gelation - biological production - industrial microbiology - gelatin - pichia pastoris
    Animal-derived collagen and gelatin have been extensively used in the past decades for several pharmaceutical and biomedical applications. However, there is need for collagen-based materials with predictable and tailorable properties.
    The aim of this thesis is the design and microbial production of gel forming non-hydroxylated collagen-like proteins. Recombinant protein expression and protein engineering are used to develop collagen-like polymers with defined composition, structure, and tunable physical-chemical properties. The possibility of using these proteins as controlled release systems is also explored, as well as the set-up of efficient and scalable production procedures using P. pastorisas a microbial factory.

    In chapter 2 we describe the genetic design, recombinant production and preliminary characterisation of a new class of ABA triblock copolymers forming thermosensitive gels with highly controllable and predictable properties. Gel formation is obtained by combining proline-rich collagen-inspired (Pro-Gly-Pro)9 end-blocks (T), which have triple helix-forming ability, with highly hydrophilic random coil blocks (Pn or Rn) defining the distance between the trimer forming end-blocks. We report the secreted production in yeast at several g/l of two such non-hydroxylated ~42 kDa triblock copolymers, TP4T and TR4T. The dynamic elasticity (storage modulus) of the gels from these collagen-inspired triblock copolymers was comparable to animal gelatin with a similar content of triple helices. In favourable contrast to traditional gelatin, the dynamic elasticity of the new material, in which only one single (well-defined) type of cross links is formed, is independent of the thermal history of the gel. The novel hydrogels have a ~37 °C melting temperature. However, the thermostability of the hydrogels formed by these polymers can be tailored by changing the number of (Pro-Gly-Pro) repeats. The concept allows to produce custom-made precision gels for biomedical applications.

    In chapter 3 it was shown that small, but tailored changes in the length of the mid-block of the collagen-inspired triblock copolymers results in significant changes in the viscoelastic properties of the hydrogels. We compared 4 different triblock copolymers, differing only in their mid-block size or mid-block amino sequence. The shorter versions, i.e. TP4T and TR4T, had mid-blocks made of ~400 amino acids, and their longer counterparts, i.e. TP8T and TR8T, ~800 amino acids. These results obtained indicate that the elastic properties of the network are not only a function of concentration and temperature but also of polymer length. The experimental results were well described by an analytical model that was based on classical gel theory and accounted for the particular molecular structure of the gels, and the presence of loops and dangling ends. These results suggest that, by controlling the structure of the present type of hydrogel-forming polymers through genetic engineering their physical-chemical properties can be predicted, and tailored in order to match a specific application

    In chapter 4 we explored the potential of hydrogels from collagen-inspired triblock co-polymers as drug delivery systems. We studied the erosion and protein release kinetics of two of these hydrogel-forming polymers, i.e. TR4T and TR8T, differing only in their mid-block length (mid-block molecular weights ~37 kDa and ~73 kDa). By varying polymer length and concentration, the elastic properties of the hydrogels as well as their mesh size, swelling and erosion behaviour can be tuned. We show that the hydrogel networks are highly dense and that the decrease of gel volume is mainly the result of surface erosion, which in turn depends on both temperature and initial polymer concentration. In addition, we show that the release kinetics of an entrapped protein is governed by a combined mechanism of erosion and diffusion. The prevalence of one or the other is strongly dependent on polymer concentration. Most importantly, the encapsulated protein was quantitatively released demonstrating that these hydrogels offer great potential as drug delivery systems.

    The development of efficient large-scale production processes can be a critical factor in whether or not a relevant pharmaceutical material is available in sufficient amounts to be used for application studies and eventually enter human clinical trials and the marketplace. In chapter 5 we describe the development of a pilot-scale process for the fermentation and purification of five collagen-inspired triblock copolymers (TP4T, TR4T, TP8T, TR8T and TP12T) with molecular weights ranging from ~42 kDa to ~114 kDa. P. pastoris strains were grown in a 140 liter bioreactor using a three-phase fermentation process. The fermentation culture reached high cell densities, and all proteins were efficiently expressed and secreted into the fermentation medium at a concentration of ~700-800 mg/l of cell free broth. The downstream processing principles elaborated previously at lab-scale were successfully adapted to the larger scale and resulted in 80-95 % recovery. The purified proteins were intact and showed a similar performance to those obtained using lab-scale procedures. The good productivity and efficient downstream processing (DSP) shown in this study provides a promising perspective towards a potential further scale-up to industrial production of these proteins.

    In chapter 6 some of the results obtained in the thesis are highlighted and suggestions for further research are given.
    The contents of this thesis provide a good starting point for future development of this novel class of hydrogel forming collagen-like proteins.


    Productie van platformchemicaliën door planten
    Meer, I.M. van der; Koops, A.J. - \ 2010
    chemische industrie - lysine - polymeren - transgene planten - aardappelen - biobased economy - monomeren - chemicaliën uit biologische grondstoffen - chemical industry - lysine - polymers - transgenic plants - potatoes - biobased economy - monomers - biobased chemicals
    Info sheet over de productie van platformchemicaliën door planten. Chemicaliën die stikstofatomen bevatten en als uitgangsmateriaal dienen voor de petrochemische industrie zijn chemisch moeilijk te maken. Productie van dergelijke chemicaliën is vaak kostbaar en vervuilend. Sommige van deze stikstofbevattende chemicaliën komen echter ook in planten voor. Ook andere bouwsteenmoleculen uit aardolie worden door planten gemaakt, zoals organische zuren. Deze infosheet geeft een voorbeeld van de ontwikkeling van platformchemicaliën in planten.
    Self-organization of polymers in bulk and at interfaces
    Charlaganov, M. - \ 2009
    Wageningen University. Promotor(en): Frans Leermakers; Martien Cohen Stuart, co-promotor(en): O.V. Borisov. - [S.l. : S.n. - ISBN 9789085855026 - 129
    polymeren - moleculaire structuur - colloïdale eigenschappen - polymers - molecular conformation - colloidal properties
    Fully atomistic analysis of polymeric systems is computationally very demanding because the time and length scales involved span over several orders of magnitude. At the same time many properties of polymers are universal in the sense that they do not depend on the chemical nature of the comprising monomers. This makes coarse-grained methods, such as self-consistent field (SCF) modeling, an ideal tool for studying them. In this thesis we employ SCF modeling to study intra- and intermolecular self-organization organization of polymers and ordering of polymers near interfaces. Where possible, the results are compared to experiments and predictions of analytical theories.
    Block copolymer self-assembly and co-assembly : shape function and application
    Li, F. - \ 2009
    Wageningen University. Promotor(en): Frans Leermakers; Ernst Sudhölter, co-promotor(en): Ton Marcelis. - [S.l. : S.n. - ISBN 9789085854739 - 197
    polymeren - micellen - blaasjes - zelf-assemblage - nanotechnologie - polymers - micelles - vesicles - self assembly - nanotechnology
    Amphiphilic block copolymers can, in selective solvents such as water, assemble into various shapes and architectures. Among those, polymer vesicles, polymer micelles and polymer fibers are very popular structures in current nanotechnology. These objects each have their own particular properties and can serve as containers or templates for different nanotechnological applications. Polymer vesicles, for example, can encapsulate both hydrophobic and hydrophilic molecules, and are therefore considered as attractive candidates for drug delivery, nanoreactors and also microtemplates. Polymeric micelles are usually significantly smaller. Their hydrophobic cores are typically used to solubilize hydrophobic drugs or carry biomolecules. Polymer fibers by construction are long continuous entities. Because of this, they easily form gels at relatively low concentrations and may find functions as templates/substrates in biological applications. In some cases the can carry or conduct charges which may trigger their use in electronic devices.
    In reality, many applications of these objects require stimuli-responsive properties, and very importantly for biomedical applications, the block copolymer building blocks need to have zero or very low toxicity and preferably biocompatibility. It is a big challenge to assemble functional molecules into stable nanostructures with desired size and shape.
    To achieve these goals, (bio)functional polymers chains have to be internalized in the objects. For this purpose two distinct routes are proposed. One of them is self-assembly of functional block copolymers into a defined nanoparticle, which is a direct approach. In this case some preliminary work is required because the (bio)functional groups have to be chemically incorporated in the block copolymer. Alternatively, more than one type of copolymer can be co-assembled into large objects with fixed stoichiometry, each with their own functionalities. In these coassembled objects, we combine functionalities of its constituents in a controlled way.
    Both approaches are applied in this dissertation and results from both routes are promising. Pluronic polymers are an interesting class of materials that in water self-assemble in association colloids of various sizes and shapes. These polymers are highly temperature sensitive. One member of the Pluronics family forms unilamellar vesicles, namely L121, but these vesicles are extremely hard to work with. This is because they are extremely fragile and termally unstable. Much work that is reported in this dissertation has resulted from attempts to improve the stability of L121 vesicles. It was found that these vesicles now can also act as a template for the assembly of stimuli-responsive polypeptide block copolymers. These co-assembled vesicles can serve as multifunctional containers for transport of several biomolecules in vivo. The assembly rules apply to several other systems as well. This dissertation is divided into three parts, and a short overview for each part is given below.
    Part 1: Self assembly
    In Chapter 2 it is described how Pluronic L121 vesicles can be stabilized by means of a polymerized network of pentaerythritol tetraacrylate (PETA). The permanent interpenetrating network allows the Pluronic L121 copolymers to reversibly associate with the vesicles. The size of the vesicles can easily be varied from at least 100 nm to 1 μm, depending on the pore size of the extrusion membrane. The stabilized vesicles can retain their sizes for more than one month at room temperature.
    In Chapter 3 it was shown that such Pluronic L121 vesicles, stabilized by means of a network of polymerized PETA can become negatively charged upon mixing with polyacrylic acid (PAA). This can be used to immobilized these vesicles onto glass as well as onto mica surfaces in combination with Mg2+ ions that are used as bridge between the two negatively charged components, polyelectrolyte and the surface. This permits relatively easy observations with CLSM. It was found that the vesicles can change their shape to tubular vesicles upon addition of another micelle-forming Pluronic block copolymer. The latter shape transition also occurs for corresponding conditions in the bulk.
    Part 2: Co-assembly
    In Chapter 4 is described how small unilamellar vesicles spontaneously form in simple mixtures of a lamellae-forming block copolymer PB10PE10 and a micelleforming block copolymer PB10PE18. The co-assembled vesicles are rather monodisperse with a core radius down to 30 nm. Furthermore, it was found that under these conditions the polymersomes coexist with mixed spherical micelles. The polydispersity of the vesicles as well as their sizes are well controlled both by the mixing ratio of the components as by the polymer concentrations used.
    In Chapter 5, a molecularly detailed self-consistent field theory is used to study the assembly characteristics of binary polymer mixtures in a selective solvent. We mimic the binary mixture of the lamellae-forming copolymer PB10PE10 mixed with the micelle-forming PB10PE18 species. It is possible to arrive at a mixing ratio where small unilamellar vesicles coexist with many spherical micelles. We argue that the vesicle size can be tuned by the copolymer mixing ratio. For still modest PE18/PE10 ratios, one enters a regime where the micelles are the dominant species and these micelles buffer the chemical potential of the copolymers in the system. In such a situation one can store more PE18 chains in smaller vesicles than in larger ones. Hence, upon an increase of the amount of PB10PE18 in the system, the vesicles evolves towards small sizes. In addition the SCF model explains many other salient properties of these systems. Hence, the SCF analysis gives strong support for the expectation that in binary polymer mixtures it is possible to have thermodynamically stable small unilamellar vesicles.
    In Chapter 6, a new type of co-assembled, highly-ordered conjugated nanotape, made from either positively or negatively charged triblock peptide copolymers, and a conjugated zwitterionic polythiophene derivative (PTT) is described. The interaction of these compounds results in a change of the conformation and the electronic structure of the polythiophene. Morphological studies show that the co-assembled nanotapes are longer at a higher concentration and a higher PTT/triblock peptide copolymer ratio.
    In Chapter 7, a similar species was prepared from this conjugated zwitterionic polythiophene (PTT) derivative and a low molecular weight gelator. The inter action of these compounds results in complex assemblies with very large aspect ratios, wherein the polythiophenes molecules are in an extended, planar conformation. Morphologic studies have shown that the co-assembled nanowires adopt a helical structure that forms well-defined nanowires with lengths up to several μm long. These nanowires can be rather simply and reversibly manipulated by external stimuli, such as pH.
    Part 3: Applications
    In an attempt to further improve the working window of L121 vesicles, in Chapter 8, Pluronic L121 vesicles were stabilized by both PETA and Pluronic P85 micelles. The double-stabilized Pluronic L121 vesicles are stable to at least 33 ºC. We can routinely control the size of these vesicles by extruding them through an appropriately sized filter before the interpenetrating polymer network is formed. Furthermore, we can covalently polymerize fluorescent molecules in the interpenetrating network, and these stabilized fluorescent vesicles can be easily taken up by cells. This system is potentially useful for molecular imaging, biological assays, as biomarker, for cell-labeling, etc.
    In Chapter 9, a study on the co-assembly of a genetically designed peptide block copolymer with Pluronic vesicles to form templated protein vesicles is described. Now, several oppositely charged biological polyelectrolytes can interact with the peptide in the vesicle and become incorporated into the protein polymer. The structural features of the protein polymersome are investigated in detail, and the functionalized protein vesicles can be simply internalized in human cells. This method provides an intelligent approach to incorporate multifunctional materials for drug delivery and gene delivery applications.
    In Chapter 10, Pluronic micelles are stabilized by an interpenetrating network, which keeps them stable for months. Furthermore, fluorescent molecules are covalently polymerized in the interpenetrating network, and these stabilized fluorescent micelles can be easily taken up by HeLa cells and remain integrated in the cytoplasm. FCS studies of these micelles in living cells indicate that the diffusion properties are significantly different from those of micelles dissolved in aqueous solution. These results may be used to probe the local viscosity in the cell, because there is sufficient evidence that the micelles do not change their size and shape after internalisation.
    In Chapter 11, it is shown that similar nanoparticles can act as nanometersized thermal sensors. Within a short temperature range, the emission of the nanoparticles changes dramatically and reversibly as a function of temperature. These stabilized fluorescent micelles can be easily taken up by HeLa cells, where they can be used as intracellular nanosized thermometers.
    Brushes and particles
    Vos, W.M. de - \ 2009
    Wageningen University. Promotor(en): Martien Cohen Stuart, co-promotor(en): Arie de Keizer; Mieke Kleijn. - [S.l. : S.n. - ISBN 9789085854524 - 264
    polymeren - oppervlaktespanningsverlagende stoffen - oppervlakte-interacties - fysische chemie - polymers - surfactants - surface interactions - physical chemistry
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