Patterned self-assembled fibrillar networks for specific cell immobilization
M. Humenik¹* C. Heinritz¹, Z. Lamberger¹, K. Kocourková², A. Minařík<sup>2

1</sup> Department of Biomaterials, Faculty of Engineering Science, Universität Bayreuth, Prof.-Rüdiger-Bormann.Str. 1, 95447 Bayreuth, Germany, ² Department of Physics and Materials Engineering, Tomas Bata University in Zlín, Vavrečkova 275, 760 01 Zlín, Czech Republic
*martin.humenik@bm.uni.bayreuth.de


 We established DNA-spider silk conjugates using “click” coupling of the recombinant protein eADF4(C16) with short DNA strands to create hybrid materials. Whereas the spider silk moiety enabled self-assembly into nanofibrils controlled by phosphate ions in aqueous buffers, the DNA part enabled DNA specific fibril labeling [1] or sequence specific immobilization of DNA-spider silk conjugates [2]. Recently, we developed surface nucleated self-assembly of the DNA-modified recombinant spider silk proteins into immobilized fibril-based networks, which revealed swelling and softening properties of nanohydrogels [3]. The DNA-functionalization in the highly hydrated, cell repellent networks could be used in a specific cell binding. For this, a mild lipid-DNA incorporation into cell membrane was employed, which showed high labelling efficiency as well as negligible cytotoxicity. Based on the complementarity of the nucleic acids, highly specific DNA-assisted immobilization of the cells on the nanohydrogels with tuneable cell densities was possible. Designed competitor DNA probes enabled the cell’s lift off on demand.  We employed also modification of the nanohydrogels with DNA-aptamers capable of binding to cell markers to immobilize different types of cancer cells specifically.  The principle of surface nucleated nanohydrogel self-assembly was further combined with photolithography. Using a positive-tone resist on an amino-reactive substrate, arbitrarily shaped microwells were patterned, which bottom served to define the binding of spider silk nucleation sites and formation of the fibrillar networks. After stripping of the sacrificial photoresists, nanohydrogel micro-structured pattern were achieved enabling either the DNA-assisted immobilization of DNA-labelled cells or the aptamer-cancer cell marker interactions with high spatial fidelity [4].

References
[1] M. Humenik & T. Scheibel, ACS Nano 2014, 8, 1342-1349
[2] M. Humenik, A. Molina & T. Scheibel, Biomacromolecules 2019, 20, 347–352
[3] M. Humenik, T. Preiß, S. Gödrich, G. Papastavrou and T. Scheibel, Materials Today Bio 2020, 6, 100045
[4] C. Heinritz, Z. Lamberger, K. Kocourková, A. Minařík; M. Humenik submitted