Gold Nanoparticles Prepared From Amino Acid, Peptide And Protein

Gold nanoparticles have attracted tremendous scientific interests in biomedical area due to their facile synthesis, chemical stability, and unique optical properties. Recently, scientists have paid more attention to the cytotoxicity of nanoparticles. However, by now, how cytotoxic effects are related with key physicochemical parameters of nanoparticles, such as colloidal stability, size, composition, surface chemistry, and their ability to adsorb environmental compounds such as proteins, is still not very clear. It is important to develop new approaches to produce gold nanoparticles with good biocompatibility.Amino acid, peptide, and protein are suitable materials to produce gold nanoparticles due to their natural source, low cost, good biocompatibility and abundant functional groups. In this thesis, amino acid, peptide and protein are used as both reducing and capping agents to produce biocompatible gold nanoparticles. The biocompatibility of the produced gold nanoparticles was investigated. This thesis contains the following four parts.1. Gold nanoparticles with aspartate, glycine, leucine, lysine, and serine surfaces (Au@Asp, Au@Gly, Au@Leu, Au@Lys and Au@Ser) were produced from the mixed solutions of HAuCl4 and respective amino acids via UV irradiation at pH 10. UV irradiation was proved to be a controllable and effective method to produce gold nanoparticles. The amino acids bind on the nanoparticle surfaces via amine groups and their carboxylic groups extend outside to stabilize the nanoparticles. The nanoparticles have hydrodynamic diameters of 15-47 nm in pH 7.4 aqueous solution and have the diameters of 62-73 nm after 48 h incubation in cell culture containing 10% fetal bovine serum. The nanoparticles adsorb human and bovine serum albumins on their surfaces by specific interactions characterized by the intrinsic fluorescence quenching of the albumins. The albumin adsorption decreases the aggregation of the nanoparticles in cell culture and also decreases the intracellular uptake. There is no coincident correlativity between the intracellular uptake of the gold nanoparticles and cytotoxicity. The gold nanoparticles produced from leucine and lysine, which have amphiphilic surfaces, present much more biocompatibility than the other gold nanoparticles.2. An effective, green, and facile approach to synthesize gold nanoparticle-loaded protein-polysaccharide nanogels was developed. Biocompatible gold nanoparticle-loaded lysozyme-dextran (Au@Lyso-Dex) nanogels were produced using lysozyme-dextran nanogels as reducing and stabilizing agents without any chemicals and separation process. Lysozyme-dextran nanogels have a size about 200 nm and a structure of lysozyme core and dextran shell. At pH around 4, AuCl4 ions are attracted and locally enriched by lysozyme due to the electrostatic and coordination interactions. When the solution under UV irradiation, the AuCl4 ions are reduced to gold nanoparticles in situ by solvated electrons and reactive radicals produced from aromatic amino acid residues in the lysozyme. The produced gold nanoparticles with a size about 8 nm are trapped inside the nanogels and the Au@Lyso-Dex nanogels are stable in aqueous solution by virtue of the dextran shell. Antitumor drug, doxorubicin, can be loaded effectively inside Au@Lyso-Dex nanogels via diffusion. In vitro study demonstrates the doxorubicin loaded Au@Lyso-Dex nanogels have the same antitumor activity as free doxorubicin. The nanogels can be used as a contrast agent in optical cell imaging, in which direct visual images of the subcellular distributions of the gold nanoparticles and the released doxorubicin are presented synchronously. The dual functional drug loaded Au@Lyso-Dex nanogels are a promising system for simultaneous drug delivery and biomedical imaging.3. A preparative reversed phase-high performance liquid chromatography-electrospray ionization mass spectrometry (RP-HPLC/ESI-MS) method was developed to obtain low-cost casein peptides. Casein was hydrolyzed by trypsin and the hydrolysate was analyzed by RP-HPLC/ESI-MS firstly. The solvent gradient performed in analytical column was optimized to achieve a better separation. Then, the optimal analytical condition was applied in preparative column directly. In each loading of the hydrolysate, several pure peptide fractions were collected automatically by the inductions of UV absorbance and mass spectrometry signals together. The process is simple and effective. The effect of solvent pH on the separation and the influence of loading amount in preparative column were investigated intensively. For the collection consisting of hydrophilic peptides that were poorly separated in the primary process, secondary separation was performed. By changing the solvent gradient, several different pure peptides were obtained.4. YR10 peptide (YLGYLEQLLR) with amphiphilic molecular structure was chosen from casein hydrolysate to prepare gold nanoparticles and its self-assembly behavior was investigated. At pH 2, YR10 peptide forms nanofibers with 10 nm in diameter and several micrometers in length. The nanofibers have β-sheet secondary structure and are composed of several smaller fibrils. At neutral and basic pH, gold nanoparticles smaller than 10 nm were produced using YR10 peptide as both reducing and capping agents via UV-irradiation or heating. However, at acid pH and room temperature, self-assembly phenomenon occurred shortly after YR10 peptide was mixed with HAuCl4. Spherical aggregates with a size about 400 nm formed after reaction. The aggregates contain many gold nanoparticles of about 10 nm. Raising temperature can decrease the size of the aggregates. The process of self-assembly was traced by transmission electron microscopy, dynamic light scattering,ξ-potential and UV-Vis spectroscopy. It was found that spherical aggregates formed first as mixing YR10 peptide with HAuCl4, which followed by the reduction of HAuCl4 to form gold nanoparticles.