3-D Scaffolds carrying Protein NanoAnchors (3DS-PNA)

The development of cellular environments, which are at the same time as defined and as close to nature as possible, is key to ex-vivo experiments on live cells where cell growth, activation, or differentiation are studied. In particular, three dimensional (3D) structuring inside microchannels, which can be used as models for vessels, remains a challenge. Current state of the art such as dip-pen lithography, nanoimprint lithography, or electron beam lithography is restricted to two dimensional structuring. High aspect ratios may give a "2.5-fold dimensionality" at the best. The only three dimensional technique which delivers well defined and non-periodic structures is two photon polymerization lithography (2PPL). Given its resolution in the micrometer range, it is well suited for the construction of three dimensional scaffolds (3DS) that allow for the adhesion and assembly of cells. However, no well-defined anchor points in the nanometer range can be produced so far using 2PPL. Such NanoAnchors are of interest for example, to mimic ultrafine lesions of vessels, or to serve as presentation sites for single proteins such as antibodies or activation factors. In the group of the proposer T. Klar, an extension of 2PPL was established which now allows for the creation of three dimensional structures with nanoscopic resolution. This extension is called stimulated emission depletion (STED) lithography, a close relative of STED microscopy which was co- invented by the applicant T. Klar and allows for resolution far below the Abbe-limit. It is the aim of the proposed project, to create nanoscopic anchors for antigens, antibodies, or activation factors using STED-2PPL on top of a microscopic scaffold produced with classical 2PPL. This yields 3-D Scaffolds carrying Protein Nano Anchors (3DS-PNA). A combination of the 3DS-PNA with a microfluidic system will be used to mimic blood vessels and to tackle questions on thrombocyte activation under various conditions (e.g. presentation of different proteins, activation factors and flow parameters). The project consortium covers all research fields which are crucial for the success of the project. It consists of the Institute of Applied Physics (one out of only five institutes in the world where STED-2PPL can be carried out right now), and the Red Cross Blood Transfusion Service of Upper Austria (BTS) providing the biomedical background in platelets biology. Further synergies will arise via international and national cooperations, for instance with Prof. Stefan Hell (MPI Göttingen, Germany), Dr. Lrnd Kelemen (Univ. Szeget, Hungary), Prof. Peter Bettelheim (Elisabethinen Hospital, Linz), or Dr. Heinz Redl (Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Linz). These groups could apply a 3DS-PNA platform in their own biophysical or medical research with great profit.

The development of cellular environments, which are at the same time as defined and as close to nature as possible, is key to ex-vivo experiments on live cells where cell growth, activation, or differentiation are studied. In particular, three dimensional (3D) structuring inside microchannels, which can be used as models for vessels, has been a challenge. In this project, we managed to write such structures in flow cells and to attach the important factor for blood clothing, the von-Willebrand-factor, at predefined locations in three dimensional space. This allows for realistic studies on the activation of blood platelets and the early stage development of a thrombosis as a function of several important parameters. Such parameters are, for instance, the density of the von-Willebrand-factors on the scale of some microns (size of a platelet) or on the scale of some tens of nanometres (scale of an adhesion site on a platelet), or to vary the flow velocity. With this, we achieved an important progress, as previously applied methods for nanostructuring such as dip-pen lithography, nanoimprint lithography, or electron beam lithography are restricted to two dimensional structuring. The progress became possible by STED lithography, a further development of two photon lithography, however with a structuring and resolution power which is unbound to diffraction, very similar to STED microscopy which was co-developed by Prof. Klar and allows for resolution far below the Abbe-limit. Besides the construction of three dimensional scaffolds for cells and proteins, it was an important challenge to find a way to specifically adhere proteins, antigens, antibodies or factors to nanoscale spots in three dimensions, ideally using covalent bonds. Several strategies were followed within the project and a number of well working solutions were found. As an additional example of possible applications, a three-dimensional immunoassay was developed. With this, it was possible to fish specific particles in a flow cell such as high density lipoprotein particles which play an important role in the transport of cholesterol in the human body. The results of the project have been published in 4 scientific articles in renowned Journals, one more is submitted and another one is in preparation. Further, the results were disseminated in more than 20 oral or poster presentations.