Hierarchical Nanopatterns for Cell Adhesion Studies

Hierarchical nanopatterned interfaces are an intriguing tool to study clustering processes of proteins like for example integrins that mediate cell adhesion. The aim of this work is the development of innovative methods for the fabrication of hierarchical micro-nanopatterned surfaces and the use of such systems as platforms to study cell adhesion. In the first part of this work different approaches are presented which are suitable for preparing micro-nanopatterned interfaces at a large scale and high sample throughput as required for biological studies. Nanopatterning is achieved by employing diblock copolymer lithography, a method previously reported to be suitable for the fabrication of extended arrays of noble metal nanoparticles by pure selfassembly. The particles are thereby embedded in a micellar shell built up by the polymer, which can be transferred to solid interfaces. Within this work the method has been combined with conventional lithographic techniques to control the particle orientation on discrete areas on the substrate material with single particle precision. Electron beam lithography was used to immobilize gold particles by cross-linking the polymeric matrix with a focused electron beam. The benefits of high precision, single particle deposition and arbitrary pattern design of this technique are opposed by the lack of ability to cover areas larger than a square millimeter in one day exposure time. To overcome this drawback, nanopatterned silicon chips were completely coated with an electron sensitive resist that covered all particles on the substrate. After illuminating the resist by electron beam lithography in desired areas, the unexposed parts including the underlying particles could be removed. Washing off the protecting resist in exposed parts revealed the gold particles pattern. With this technique exposed areas could be increased to square centimeter areas within one day exposure time. As a further approach a new method was developed by exposing the substrate through a metal grid to electrons emitted by an electron flood gun rather than scanning the substrate by a focused beam. Micropatterned areas of several square centimeters could be prepared within minutes, even on non-conductive glass substrates. The three different approaches now provide a toolbox out of which a method can be chosen that suits the respective scientific purpose. Possibilities range from single particle deposition to larger scale arbitrary patterns, that can even be transferred to non-conductive and transparent substrates. The second part of the work, the cellular interactions of rat embryonic fibroblasts (REF) and dendritic cells (DC) with the biofunctionalized micro - nanopatterns produced were studied. Biofunctionalization included linkage of a cell receptor addressing peptide to the nano-particles and a protein repellent layer in between to avoid unspecific interaction. Fluorescent and electron microscopy images revealed, that cellular anchor points are confined to the underlying micro-nanopattern of gold particles. Intracellular actin networks connect to these protein anchor points, forming so called focal adhesions, and thereby mediate mechanical stress. At sizes of the squared adhesive patches of larger than or equal to 1 µm actin fibers connected to one adhesive patch. Whereas, if patterns consisted of squared patches smaller than or equal to 500 nm side length the actin fibers bridged these pattern gaps over several adhesion domains. Patterns with edge lengths of 100 nm comprising 6 ± 1 particles per patch were found to be the minimum number of adhesion receptors that need to cluster in order to induce adhesion. Cell-surface interactions have also been studied with dendritic cells, that play a key role in the immune system since they capture antigens in peripheral tissues and migrate to lymph nodes to present the processed antigen to T-cells and trigger an immune response. In contrast to fibroblasts, DCs were also able to adhere to gold particles that were functionalized with a control peptide that does not address integrins and to particles that were not functionalized at all. Additionally DC adhesion could be induced even on homogeneous patterns with spacings of up to 130 nm. Dendritic anchor points were confined to squared adhesive patches if the pattern comprised edge lengths of 5 µm or 10 µm, but were able to bridge pattern gaps if hierarchical structures consisted of 1 µm and 500 nm adhesive areas