Synthesis, characterization and application of calcium phosphate nanoparticles for the transfection of cells
In this work chemical, biological and biochemical investigations were carried out in the cooperation with the Chair of Molecular Neurobiochemistry at the University of Bochum and with the Institute of Physico-Chemical Biology, Moscow State University. In this cooperation the main purpose was to find optimal conditions for the preparation of stable monodisperse calcium phosphate colloids and their applications in biological investigations on living cells. This interdisciplinary cooperation was necessary to understand major barriers in the gene delivery system and to develop a new system which can overcome these barriers or at least improve the trafficking of nucleic acids into the cells. We have demonstrated how by a comparatively easy and well reproducible process nanodisperse calcium phosphate/DNA nanoparticles with a size below 100 nm can be prepared, especially if calcium is partially substituted by magnesium or aluminum. These ions obviously have an inhibitory effect on the particle growth of calcium phosphate. Multi-shell calcium phosphate/DNA-nanoparticles with a calcium phosphate core and DNA/calcium phosphate shells help to protect DNA from the degradation by enzymes (nucleases) inside the cytoplasm of cultured endothelial cells, reaching the efficiency of Polyfect®. Calcium phosphates should be advantageous due to their high biocompatibility and good biodegradability compared to other types of nanoparticles used for cell transfection such as iron oxide (magnetite), gold or silica. In contrast to the classical calcium phosphate method, the particle/DNA dispersions can be stored for weeks without loss of their transfection efficiency. We have developed a delivery system for gene silencing in which these nanoparticles are used for down-regulation of gene expression by targeting the mRNA resulting in effective gene silencing. The preparation of stable calcium phosphate/oligonucleotide dispersions and their successful biological application in vitro to HeLa-EGFP cells was shown. The most challenging work in non-viral gene therapy is a successful delivery of DNA into the nuclei overcoming all the extracellular and intracellular physicochemical barriers. To get information about the targeting of calcium phosphate nanoparticles into T-HUVEC, fluorescence microscopy was used. The routing of nanoparticles inside the cells was studied. It was shown that the transfection efficiency correlates with the distribution of the nanoparticles in the cells. DNA/BSA aggregates alone were not able to penetrate the cell membrane, i.e. the inorganic nanoparticles (calcium phosphate) were necessary as carriers to enter the cell. We also performed the preparation of calcium phosphate samples with ubiquitin by the co-precipitation method. It was shown that ubiquitin can be adsorbed as well as incorporated into the calcium phosphate particles. This method shows opportunities in the preparation of calcium phosphate ceramics with proteins similar to ubiquitin, i.e. proteins of the BMP family. An implantation of such biofunctionalized calcium phosphate ceramics (e.g. cold-isostatically pressed into an appropriate shape) in bone defects can stimulate the development of new bone at the implantation site.