Kovtun, Anna:

Calcium phosphate nanoparticles as carriers of nucleic acids

Duisburg, Essen (2010), 149 S.
Dissertation / Fach: Chemie
Fakultät für Chemie » Anorganische Chemie
Epple, Matthias (Doktorvater, Betreuerin)
Schmuck, Carsten; Müller, Werner E. G. (GutachterIn)
Duisburg, Essen, Univ., Diss., 2010
Abstract:
The main purpose of this study was to apply calcium phosphate nanoparticles as carriers for nucleic acids into different cell systems to test their transfection properties and influence on cells. First, we performed an easy and straightforward synthesis of calcium phosphate nanoparticles partially or completely substituted by magnesium or aluminum. However, although these nanoparticles were much smaller than the pure calcium phosphate nanoparticles, they did not prove to be very efficient for the delivery of nucleic acids. In order to test the toxicity of the system we monitored the intracellular calcium level by using of radioactive 45Ca-marked nanoparticles and the calcium dye Fura-2. Our nanoparticulate delivery system proved to be nontoxic to the cells compared to other delivery systems (the commercial agent Polyfect® and the standard calcium-based method). Nanoparticles also did not lead to the disturbances of the intracellular calcium level which, otherwise, could be lethal for cells. In the next steps we functionalized the calcium phosphate nanoparticles with different oligonucleotides and tested them on cell culture for the gene silencing activity. The nanoparticles functionalized with siRNA were far more effective than Polyfect® and still nontoxic. The nanoparticles functionalized with shRNA effectively inhibited the synthesis of osteopontin and osteocalcin when also embedded into polyelectrolyte multilayers. This also allows the localized application of gene silencing. We functionalized the nanoparticles with the oligonucleotides which are able to mimic the microbial antigens, thus activating the immune system and breaking the innate tolerance of the organism. We used CpG oligonucleotides for the activation of the dendritic cells which leads to the synthesis of IL-12 and presumably other signaling molecules. We performed localized transfection experiments via deposition of the nanoparticles on metal substrates. In this case we could achieve high transfection efficiency and a local direct application. Thus, we showed that the synthesis of calcium phosphate nanoprecipitates and their subsequent functionalization is possible. Such system was effectively used as a carrier for DNA and RNAs into the different cell cultures and proved to be effective and nontoxic for cells.