SYNTHESE UND QUANTITATIVE ANALYSE VON STRUKTURIERTEN UND FUNKTIONELLEN HYDROGELSYSTEMEN ZUR REGULIERUNG DER ADHÄSIONSEIGENSCHAFTEN UNTERSCHIEDLICHER ZELLPHÄNOTYPEN AN GRENZFLÄCHEN

Der Fokus dieser Arbeit ist auf die Entwicklung eines künstlichen Modellsystems der extrazellulären Matrix (EZM) gerichtet, um die Reaktion diverser Zellphänotypen auf unterschiedliche Umweltbedingungen zu erfassen. Die Imitation der verschiedenen biochemischen sowie biomechanischen Eigenschaften von Gewebe durch das hier definierte System, diente dabei zur Untersuchung des Adhäsions- und Bewegungsverhaltens von Malaria Parasiten, speziell des Plasmodium Sporozoiten Stadiums, da diese nach Injektion durch die Anopheles Mücke zunächst Gewebe passieren müssen bevor sie in die Blutbahn gelangen. Die Basis des Modellsystems stellt ein proteinabweisendes Polyethylenglykol-Diacrylat (PEG-DA) Hydrogel dar, dessen Elastizität in einem Bereich von 0,6 kPa bis 6 MPa einstellbar ist. Die Hydrogeloberfläche wurde durch einen Transferprozess mit Goldnanopartikeln dekoriert, welche zuvor mittels Block-Copolymer-Nanolithographie in einer quasi-hexagonalen Ordnung mit gezielt justierbaren Partikelabständen von 40 nm bis 270 nm auf Glas generiert wurden. Diese Goldpartikel wurden über Thiolbindungen mit verschiedenen bioaktiven Molekülen funktionalisiert und fungierten hier als Adhäsionsanker, welche in definierten Abständen und somit unterschiedlicher Dichte angeboten werden können. Eine weitere Modifikation der Hydrogeloberfläche wurde durch Carbonsäure-Funktionen erzielt. Diese konnten in bestimmten Bereichen der Geloberfläche, beispielsweise in Form einer Mikrostruktur, erzeugt werden und anschließend über Peptidbindungen mit entsprechenden Molekülen weiter umgesetzt werden. Erstmals wurde eine Anbindung der unterschiedlich elastischen und funktionalisierten Hydrogele in Form dünner Schichten auf dünne Glassubstrate ermöglicht. Hierdurch konnten verschiedene Parameter, wie Elastizität, Abstände der Adhäsionsanker und Funktionalisierungen unabhängig voneinander in zahlreichen Kombinations-möglichkeiten auf einem Glassubstrat variiert werden. Diese Methode erbrachte große Vorteile für die Mikroskopie und Substrathandhabung. Die Untersuchung der Plasmodium Sporozoiten auf diesen Substraten zeigte die höchste Motilität dieser Parasiten auf Oberflächen mit Adhäsionsabständen von 55-100 nm und einer Elastizität entsprechend derer von Zellkulturplastik. Durch Zugabe eines Aktin-polymerisationsfördernden Wirkstoffes konnte die Motilität auf weicheren Substraten jedoch stimuliert werden. Das hier entwickelte System ermöglichte damit erstmals einen experimentellen Nachweis der Synergie zwischen Adhäsionsbildung und der Aktinpolymerisation und trägt durch die Möglichkeit der Untersuchung zahlreicher Faktoren entscheidend zum besseren Verständnis des Bewegungsmechanismus der Malariaerreger bei

De Laurentii Rhodomani vita et scriptis

von Theodor Wilhelm Heinrich Perschman

Tunable substrates unveil chemical complementation of a genetic cell migration defect

Cell migration is dependent on a number of physical and chemical parameters of the substrate that influence cellular signaling events as cell surface receptors interact with the substrate. These events can strengthen or loosen the contact of the cell with its environment and need to be orchestrated for efficient motility. A set of tunable substrates was used in combination with quantitative imaging to probe for potentially subtle differences in genetically modified and chemically treated rapidly migrating cells. As model cell, Plasmodium sporozoites were used, the forms of malaria parasites transmitted by the mosquito to the host. Sporozoites lacking a substrate-binding surface protein moved on different surfaces with consistently lower efficiency and were more sensitive to adhesion ligand spacing than wild type sporozoites. Addition of an actin filament stabilizing chemical agent temporarily increased sporozoite motility on soft but not on hard substrates. Defined conditions were found where the chemical completely compensates the reduced migration capacity of the genetically modified parasites. As the onset of motility was delayed for sporozoites on unfavourable gels it is suggested that the parasite can slowly adjust to environmental elasticity, possibly by adapting the interplay between surface adhesins and actin filament dynamics. This demonstrates the utility of tunable substrates to dissect molecular function in cell adhesion and motility

Induction of malaria parasite migration by synthetically tunable microenvironments

Interaction of Plasmodium sporozoites, the forms of the malaria parasite transmitted by the mosquito, with its microenvironment in form of adhesion and migration is essential for the successful establishment of infection. Myosin-based sporozoite migration relies on short and dynamic actin filaments. These are linked to transmembrane receptors, which in turn bind to the matrix microenvironment. In this work, we are able to define the characteristics that determine whether a matrix is favorable or adverse to sporozoite adhesion and motility using a specifically tunable hydrogel system decorated with gold nanostructures of defined interparticle spacing each equipped with molecules acting as receptor adhesion sites. We show that sporozoites migrate most efficiently on substrates with adhesion sites spaced between 55 and 100 nm apart. Sporozoites migrating on such substrates are more resilient toward disruption of the actin cytoskeleton than parasites moving on substrates with smaller and larger interparticle spacings. Plasmodium sporozoites adhesion and migration was also more efficient on stiff, bonelike interfaces than on soft, skinlike ones. Furthermore, in the absence of serum albumin, previously thought to be essential for motility, sporozoite movement was comparable on substrates functionalized with RGD- and RGE-peptides. This suggests that adhesion formation is sufficient for activating migration, and that modulation of adhesion formation and turnover during migration is efficiently controlled by the material parameters of the microenvironment, that is, adhesion site spacing and substrate stiffness. Our results and approaches provide the basis for a precise dissection of the mechanisms underlying Plasmodium sporozoites migration

Mimicking cellular environments by nanostructured soft interfaces

We present herein an innovative technique for decorating soft polymer surfaces with metallic nanostructures fabricated by diblock copolymer micelle nanolithography. Thus far, such nanolithography has been limited to plasma-resistant inorganic substrates such as glass. Our new development is based on the transfer of nanopatterns from glass to soft substrates. Special emphasis is given to hydrogel surfaces with respect to their properties for tailoring cell adhesion. Besides planar surfaces, periodic gold nanopatterns on curved surfaces have been fabricated, as demonstrated on the interior surface of a tubelike hydrogel, which potentially mimic situations of vessels in vivo

Protein repellent properties of covalently attached PEG coatings on nanostructured SiO₂ based interfaces

In this study, we report the systematic comparison of different poly(ethylene glycol) (PEG) self-assembled monolayers on glass with respect to their protein adsorption and cell adhesion resistance. Combining PEGylation with micellar nanolithography allowed the formation of gold nanoparticle arrays on glass and selective coverage of the free glass area by PEG. The gold nanoparticles serve as anchor points for the attachment of individual proteins and peptides such as the cell-matrix adhesion promoting cyclic RGDfK motif or the kinesin motor protein Eg5. The capability of the motor protein to bind microtubules remained unaffected by the immobilization. It was shown that the film thickness of a water swollen PEG layer is crucial to maximize the interaction between proteins and peptides with the nanostructures. Non-specific interaction between cells or microtubules and the surface was minimized. The optimum PEG layer thickness correlated with the size of gold nanoparticles which was approximately 5 nm

Investigation of early cell–surface interactions of human mesenchymal stem cells on nanopatterned β-type titanium–niobium alloy surfaces

Multi-potent adult mesenchymal stem cells (MSCs) derived from bone marrow have therapeutic potential for bone diseases and regenerative medicine. However, an intrinsic heterogeneity in their phenotype, which in turn results in various differentiation potentials, makes it difficult to predict the response of these cells. The aim of this study is to investigate initial cell-surface interactions of human MSCs on modified titanium alloys. Gold nanoparticles deposited on β-type Ti-40Nb alloys by block copolymer micelle nanolithography served as nanotopographical cues as well as specific binding sites for the immobilization of thiolated peptides present in several extracellular matrix proteins. MSC heterogeneity persists on polished and nanopatterned Ti-40Nb samples. However, cell heterogeneity and donor variability decreased upon functionalization of the gold nanoparticles with cyclic RGD peptides. In particular, the number of large cells significantly decreased after 24 h owing to the arrangement of cell anchorage sites, rather than peptide specificity. However, the size and number of integrin-mediated adhesion clusters increased in the presence of the integrin-binding peptide (cRGDfK) compared with the control peptide (cRADfK). These results suggest that the use of integrin ligands in defined patterns could improve MSC-material interactions, not only by regulating cell adhesion locally, but also by reducing population heterogeneity