Development of colloids for cell and tissue targeting: bisphosphonate-functionalized gold nanoparticles for the investigation of bone targeting

Gold nanoparticles (GNPs) possess a great potential as model systems for the investigation of nanoparticulate drug delivery due to their ease of production, low toxicity and also the high stability of the noble metal in biological fluids. Due to their unique surface chemistry, they can be used as building platform for stable self assembled monolayers of thioalkylated poly(ethylene glycol)s with different surface functionalities, allowing the investigation of different drug targeting principles. In this thesis gold nanoparticles, also for suitable for intravenous in vivo applications, were prepared in aqueous media by citrate reduction of gold chloride, yielding particles in the size range of 40 to 150 nm with appropriate size distributions. Adjustment of the particle size was achieved by varying the ratio of citrate to gold salt and subsequently the obtained particles were investigated via photon correlation spectroscopy and transmission electron microscopy for sufficient size determination. For a subsequent stabilization and functionalization, heterobifunctional poly(ethylene glycol) derivatives [PEG] were synthesized, carrying a thioalkyl chain for stable binding to gold and a hydrophilic PEG chain as hydrophilic coating. The synthesis of the different thioalkylated poly(ethylene glycol)s was further improved applying a new synthetic strategy, in order to avoid non binding polymer components interfering with the subsequent particle coating. For the additional functionalization with targeting ligands, the polymers were furthermore modified to provide a functional amine group, which can be used to attach hydrophilic molecules necessary for specific binding of the colloids to their target sites. In this thesis two different polymers [methoxy poly(ethylene glycol)-undecyl mercaptane and amino poly(ethylene glycol)-undecyl mercaptane] were synthesized and conjugated to a bone targeting ligand, namely a bisphosphonate, using standard DCC/NHS chemistry. The obtained polymers were characterized by 1H-NMR, HPLC and mass spectroscopy and demonstrated a high degree of purity, suitable for further immobilization on gold surfaces. In order to functionalize the prepared gold particles, bisphosphonate and methoxy terminated poly(ethylene glycol)-undecyl mercaptane (BP-PEG-AlkSH and mPEG-AlkSH) were mixed in different ratios and used to coat the nanoparticles. The investigations demonstrated that the polymer coated nanoparticles slightly increased in size as determined by PCS, but no significant changes were observed with TEM due to the low contrast of the polymer layer. The surface charge and UV-Vis absorption spectra of the particles were also only slightly changed, indicating well separated particles and a successful replacement of the citrate coating by the different polymers. The polymer-coated nanoparticles proved to be stable in different media (5M NaCl, 200 µg/ml BSA and 10% (V/V) serum), indicating a strong binding and a well established steric stabilization of the uncharged polymers. Bisphosphonate coated particles were furthermore incubated with porous hydroxyapatite ceramics (Endobon®, Biomet) and it was observed that their adsorption to apatite was strongly dependent on the content of bisphosphonate in the particle surface and the adsorption was not affected by calcium ions or proteins added to the media. The conducted in vivo investigations using radioactively labeled particles furthermore indicated that the functionalized and non-functionalized gold nanoparticles showed only a minimum uptake by the reticuloendothelial system (liver and spleen) and therefore circulated for very long time in the bloodstream as compared to other nanoparticulate systems. On the other hand the particles also showed a strong tendency to extravasate and distribute into other organs after intravenous injection into mice, but the largest accumulation was observed in the kidneys. Besides that a steady accumulation of the bisphosphonate functionalized nanoparticles in bone was observed in comparison to control nanoparticles, but further studies have to be conducted in order to investigate especially the long term fate in bone and the accurate location of the particles in the kidney. Due to the beneficial in vivo behavior of the developed particles, the here presented drug delivery system may be furthermore a suitable model system for the investigation also of other active targeting strategies in many different fields