Dokument

Summary:
This thesis deals with the application of polymer capsules for diagnostic and therapeutic purposes in mammalian cells. The capsules comprise a multilayer shell of oppositely charged polyelectrolytes surrounding a cavity and have a size of two to five microns. Concerning diagnostics, capsules were produced to monitor the dynamics of the lysosomal pH in cancer cells. The cavities of the capsules were filled with a fluorescent, pH-sensitive dye for optical readout of the signal. The cells were monitored under physiological conditions upon induced pH imbalances. The results showed that the capsules were appropriate for intracellular long-term measurements and could monitor changes of the pH. For therapy, biodegradable capsules filled with biologically active molecules were synthesized. Two strategies were employed. In one approach, the cavity was filled with polyplexes of DNA or RNA and polyethylenimine, which are used regularly for the delivery of foreign genetic material into host cells. This approach is an example for gene therapy. The results showed that delivery by the capsules was very efficient and the encapsulated polyplexes were less toxic for the cells than their free counterparts. The other strategy was to directly deliver functional enzymes into cells. For this approach, cell models representing lysosomal storage diseases were employed. One of these diseases is Fabry. Patients with Fabry disease are deficient of the enzyme α galactosidase A. The enzyme was encapsulated in biodegradable capsules and given to the cells. This therapy form is called enzyme replacement therapy. The intracellular enzyme activity was determined by quantification of the intracellular level of a fluorescently labeled substrate of α galactosidase A. As the products of the reaction were non-fluorescent, the intracellular fluorescence could be used to quantify the intracellular activity of the encapsulated enzyme. Finally, therapy and diagnostics were combined in a model of Krabbe disease, another lysosomal storage disorder. In Krabbe patients, sphingolipids and cerebrosides accumulate in the oligodendritic glia cells of the patients, as due to a gene defect the enzyme galactocerebrosidase usually converting these agents is not expressed. In the model, the cause of the disease was simulated by incubation of oligodendritic cells with psychosine, which belongs to the group of sphingolipids. Galactocerebrosidase was encapsulated in biodegradable capsules and delivered to the cells. The functionality was tested by a viability assay. Two types of cells were used, wild-type cells expressing galactocerebrosidase and knockout cells, which did not express the enzyme. The viability of the cells in the presence of psychosine was determined with and without addition of galactocerebrosidase-filled capsules. The results showed that the effect of the capsules on the viability of the two different cell types was contrary. Whereas knockout cells gained higher viability when capsules were administered, wild-type cells suffered a loss in viability. The diagnostic part was characterized by monitoring the lysosomal pH upon incubation with psychosine. The dynamics of the lysosomal pH of the two types of cells turned out to be different. Each of the cell types could therefore be identified with a specific pH profile and the decision to treat cells with the enzyme-filled capsules can be based on the measured pH profile. This is considered an in vitro-example of theranostics, the combination of therapy and diagnostics.

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