Chemical modification strategies for the preparation of bioactive interphases

Abstract

Biomimetic systems and interfaces allow to understand and control cellular behavior in a well defined and reproducible manner. In this study three different strategies are developed to prepare such simplified, well-defined biomimetic materials. Firstly, a combination of click chemistry and gold thiol interactions allows the presentation of two distinct signaling molecules at controlled density and arrangement to investigate the cross-talk between two signaling molecules in cell culture. Secondly, the commonly used Ni2+-NTA interaction with His6- tagged proteins is substantially improved in its stability and inertness for protein immobilization on SAMs by replacing the Ni2+ ions with Co3+ in the complex. Thirdly, His6-tagged proteins are stably tethered on TiO2 nanoparticles for targeted delivery. To produce dual functionalized gold nanostructured interfaces, first the presentation of a ligand of interest with azide functionality on glass substrates at controlled density is established. For this pirpose, alkyne terminated poly(ethylene glycol) (PEG) is covalently bound to glass through a silanization reaction and subsequently modified through copper catalyzed azide alkyne cycloaddition (CuAAC). The functionalization density can be statistically tuned through the coimmobilization of a methoxy-terminated PEG. The surface coating and its modification with the CuAAC is analyzed using fluorescence microscopy, XPS, an enzymatic digestion assay for the determination of the ligand density, QCM-D and in cell adhesion studies. This PEG coating is used in combination with the established gold nanostructured surfaces to generate orthogonally dual functionalized biomimetic interfaces where one of the ligands is attached to the PEG coating between the gold nanoparticles using the CuAAC and the second ligand is attached to the gold nanoparticles using the gold thiol interaction. These interfaces, which present two distinct ligands at controlled density and arrangement, are suitable to investigate the mutual influence of two signaling molecules on cell behavior. Exemplarily, the combined effect of the adhesion peptide cRGD and the synergy site PHSRN on REF fibroblast adhesion is investigated. While on neither of the monofunctionalized substrates the cells can attach, the cells adhere on the dual functionalized cRGD and PHSRN presenting interfaces. The second part of this study deals with the stable immobilization of His-tagged proteins on NTA presenting surfaces using the cobalt(III) mediated interaction. The cobalt(III) complex is generated by first preforming the well established cobalt(II) complex between NTA and His6-tagged proteins and the subsequent chemical oxidation of Co2+ to Co3+ with hydrogen peroxide. A comparison of the Ni(II) and Co(III) mediated interaction between NTA moieties and His6-GFP reveals the lability of the Ni(II) and stability of the Co(III) complexes against high concentrations of competing ligands and washing off overtime. Further, also the resistance of the Co(III) mediated interaction against reducing agents is demonstrated. The oxidation step in this immobilization strategy can potentially harm the protein’s activity and this has to be investigated case by case. To illustrate that this method can be used to immobilize functional protein, the His6-tagged protein A is immobilized through the Co(III) mediated interaction and it is shown that the oxidation step dosen’t influence the immunoglobulin binding activity. In the third part the Co(III) mediated stable immobilization of His-tagged proteins is used to biofunctionalize TiO2 nanoparticles. Here, the photocatalytic activity of TiO2 is taken advantage of to perform the oxidation of Co(II) complexes between the chelating TETT surface coating on the TiO2 nanoparticles and a His-tagged protein. The Co2+ ion loading capacity of the nanoparticles and their photocatalytic activity is characterized with a colorimetric assay, fluorescence studies using terephtahlic acid as radical detection reagent, absorbance measurements, DLS and zeta potential measurements proving the photo-mediated oxidation of coordinated Co2+ ions to Co3+. Exemplarily, the stable immobilization of the model protein His6-GFP and of the glycoprotein transferrin-His6 is studied.