Evaluation Of In Vitro Biological Noble Metal Nanoparticles And Research Iological Effect Of Nanoparticles And Related

With the rapid development of nanotechnology and the wide application of its products, nanomaterials will be released into the environment and into the human body through various pathways. However, due to a lack of a clear understanding of the interaction between nanomaterials and biological systems, its biological safety has not yet been fully analyzed and evaluated. So far, the evaluations of nanomaterial biosafety are almost directly obtained through traditional biological methods to describe data. In view of the special physical and chemical properties of nanomaterials may interfere with these biological test methods which not be excluded, In fact, it might not guaranteed that these methods can work equally well for nanomaterials. For accurate and comprehensive assessment of the biological safety of nanoparticles, confirming the nanoparticles’ interference with the test method itself is necessary the system. In addition, the biological effects of nanomaterials also result from the interaction between nanomaterials and biomolecules.Once contacting with biological fluids(such as blood, cell fluid), the surface of nanoparticles will be rapidly adsorbed layer or layers of protein molecules to form protein corona. Properties of nanoparticles in the organism are mainly determined by the surface protein shell. The type and quantity of biological molecules adsorbed on the surface of the nanoparticles give new biological properties of nanomaterials in a biological environment. Therefore, the study of protein corona provides the most basic information to predict the physiochemical behavior of nanoparticles in biological systems and the design of specific features nanoparticles.Focus on the in vitro biological effects of nanomaterials and biological molecules and nanomaterials interaction expand research, this thesis studied nanoparticles’ interference in the evaluation of in vitro toxicity of silver nanoparticles, and the interactions between nanomaterials and protein by fluorescence correlation spectroscopy. Specific research results are summarized as follows:Firstly, from three aspects including light absorbing, surface adsorption and reaction with chemical reagents, a systematic and quantitative interference study of silver nanoparticle with different size and surface functional groups(AgNP-PVP-20, AgNP-CIT-20 and AgNP-CIT-110) on several common cytotoxicity assessment test, such as LDH release, MTS assay, NO generation and ROS formation. Results indicate that AgNPs does cause interference on the detection, especially LDH release, 42.8 μg mL-1 AgNP-PVP-20 with 0.5 U mL-1 LDH standard solution affected LDH detection by about 50%, while with a concentration of 42.8 μg mL-1 AgNP-CIT-20 affected LDH detection by about 70%. The results imply that prior to assess the biological safety effects of AgNPs or other nanomaterials, it is required to confirm whether nanoparticles will interfere with the detection of experimental methods.Secondly, to explore different surface functional groups of silver nanopaticle(AgNP-PVP-20, AgNP-CIT-20) produced cytotoxicity, ROS production levels, as well as distribution of silver nanoparticle on Hep G2. MTS, LDH, NO and ROS assay showed cytotoxicity of AgNP-CIT-20 significantly higher than AgNP-PVP-20, and the greater the concentration, the stronger its cytotoxicity. Experimental analysis AgNPs can cause cell membrane rupture, LDH leakage, cell proliferation inhibited, oxidative stress, and different degree of cell necrosis or apoptosis.Thirdly, based on the deconvolution fluorescence lifetime correlation spectroscopy model, utilizing metal-enhanced fluorescence effect to change fluorescence lifetime, and research equilibrium dynamics between gold nanoparticles and protein molecules. During the progress, synthesized 65 nm AuNPs, selected flexible HS-PEG-NH2 as its functional group, controlled the distance between the metal and fluorophores, and labeled with Alexa Fluo 594 NHS ester. Results indicate that HS-PEG-NH2 with different molecular weight have little effect on the fluorescence lifetime of Alexa Fluo 594 NHS ester in our pre-conditions.