Chemically Modified Substrates to Probe Cell Behaviour in Wound Healing

Abstract

Cell migration plays a major role in processes like wound healing, including lymphangiogenesis. It is especially influenced by the extracellular matrix. In this thesis chemical synthesis and material science are combined to address biological questions in a biological chemistry approach. This enables the analysis of the influence of different extracellular matrix modules on cell behaviour. On the one hand a concentration and density dependent effect of short hyaluronan is analysed. On the other hand a novel specific and unspecific binding model for the extracellular matrix is developed. Several chemical modification strategies are tested to immobilise hyaluronan on a surface. Therefore modifications at the reducing-end are introduced using cysteamine hydrochloride, propargylamine and dibenzocyclooctyne-amine and at the carboxy groups within the chain using again propargylamine. In case of the end-thiolated hyaluronan a degree of thiolation of 4.0 ± 0.5 % is achieved, and for the functionalisation within the chain a degree of alkynation of 16% is determined. All desired molecules could be synthesised and enable immobilisation of the hyaluronan species. After the chemical modification, the bioactivity is verified via the analysis of the interaction between the hyaluronan species and the hyaladherine aggrecan as well as LYVE-1. Especially the interaction between aggrecan and hyaluronan proves the conservation of the bioactivity during the modification. To determine the influence of short hyaluronan on lymphendothelial cells the AlamarBlue® and CyQuant® assay are established. A cell density of 131 mm-2, an incubation time of 2 h (AlamarBlue® assay) and 5min (CyQuant® assay) are determined. Compared are the influences of an enzymatically digested (10 kDa) and short hyaluronan (20 kDa) species which are applied to lymphendothelial cells in different concentrations and densities. To analyse the density related effect, the hyaluronan is immobilised on gold nanostructured surfaces. Between the two employed species no difference in cell behaviour is found. In the case of the application of hyaluronan in solution no difference in the relative metabolic activity is found. For the immobilised hyaluronan species a reduced ability to adhere to the surface is observed for the increasing nanoparticle density. Here, for both hyaluronan species a biphasic effect in relative metabolic activity is detected with maxima for a particle density of 540 μm-2. To investigate collective cell migration two novel photocleavable ligands are developed. Synthesised are an unspecific ligand 1-(4,5-dimethoxy-2-nitrophenyl) ethyl 11-mercaptoundecanoate and the specific, caged antagonist of the integrin α5β1 1-(4,5-dimethoxy-2-nitrophenyl)-ethyl (3(S))-3-(4-(3-(6-(3-mercaptopropanamido)- hexanamido)-propoxy)-benzamido)-4-(4-(3-((4-methoxypyridin-2-yl)-amino)-propoxy)phenyl)butanoate. 1-(4,5-dimethoxy-2-nitrophenyl)ethyl 11-mercaptoundecanoate can be synthesised with a yield of 18%. In QCM-D experiments, the adsorption happens with a specific decay time of (1899.0 ± 776.3) min at 21 ◦C, while the cleavage after UV irradiation takes (2.52 ± 0.64) min at 21 ◦C. The absorption spectrum of the molecule shows a band at 345 nm, which is blue shifted to 322 nm upon irradiation with UV light. Also, an isobestic point can be observed at 370 nm. An analysis of the toxicity of 4,5-dimethoxy-2-nitrophenyl ethanol shows an increasing metabolic activity of the used MDCK II cells for 4,5-dimethoxy- 2-nitrophenyl ethanol, but no effect for 1-(4,5-dimethoxy-2-nitrophenyl)ethyl 11-mercaptoundecanoate is observed. In summary, well-controlled chemical variations of surfaces enable novel approaches to study biological systems. Here a method is demonstrated to immobilise HA on a surface which allows the evaluation of the influence of HA on the basal side of lymphendothelial cells. Furthermore a novel intergrin-specific photocleavable ligand is developed to study collecitve cell migration.