Study On Inhibition Mechanism Of Gold Nanoparticles On Drug-metabolizing Cytochrome P450Enzymes

Today, gold nanoparticles (AuNPs) are emerging as promising therapeutic agents for treatment of AIDS, tumor and Parkinson’s disease. Additionally, more and more nanogold-containing products become readily available on the market, thus increasing the chance of exposure to and the health risks of engineered nanogold in food, cosmetics and other consumer products through the skin, lungs, or gastro-intestinal tract. As a special kind of exogenous materials exposed in bio-tissues, nanoparticles may be able to disrupt the biological functions of drug-metabolizing enzymes by direct contact with CYPs. So, changes in NP-CYP interactions and NP-associated effects on CYP activities are to be expected. There is a few data available on the induction and inhibition of certain CYPs by several types of NPs, including metallic and polymeric nanomaterials. The potential influence of nanoparticles on main human hepatic cytochrome P450(CYP) isozymes mediated Phase I biotransformation and relevant mechanism were investigated in this thesis, the main work is as follows:(1) To evaluate the activity of CYP isozymes, we established a LC-MS/MS analytical method, which can quantify6metabolites at the same time. Accuracy and precision of the method were investigated. Meanwhile, we applied a new and green salt-induced phase segregation and nanoprecipitation method to remove water-soluble nanoparticles and to extract analytes from complex AuNP-containing bio-samples into an organic phase for direct liquid chromatographic quantification.(2) Six drugs, which served as probe substrate for different CYP isozymes, were separately incubated with different sized AuNPs (5-100nm) in vitro. AuNPs demonstrated a strong inhibition of CYP2C9,2C19,2D6and3A4depending on the concentration and size of NPs.(3) Under microsomal incubation conditions, UV-vis spectroscopy, dynamic light scattering and zeta-potential measurement were used to examined the changes in surface properties of AuNPs during the incubation period. As expected, the red-shift of the surface plasmon resonance peak, as well as the drop of surface charge and the increase of the hydrodynamic diameter indicated the conjugation of proteins to NPs. Remarkably, a critical concentration of human liver microsomes (HLM) made the biggest growth of diameter and red-shift was observed for7and70nm AuNPs, respectively. This study suggest that membrane structural HLM may form micelle around AuNPs and hence affecting the integrity of membrane, resulting in alteration of CYPs activity.