Study On Controlled-Biosynthesis Of Au Nanoparticles And Their Cell Bioeffects

Nanomaterials with some excellent characterizations in physical and chemical aspects, including of large specific surface area, high catalytic activity and biological compatibility, have gotten some important applications in catalysis, energy sources, biotechnologies and analytics. As the development of the physical and chemical synthesis processes, the biosynthesis technology attracts the increasing attention as the clean, nontoxic and environmentally property. The biosynthesized nanoparticles capped some biological active molecules are endowed with the unique properties including good monodispersity and stabilization, even biological activity. Newly, a large member of organisms have been used in the biosynthesis of nanoparticles, including prokaryotes (bacteria and actinomycetes), and eukaryotes (yeasts, fungi and plant). And a range of nanoparticles have been biosynthesized, such as metal nanoparticles of Au, Ag, Au-Ag, Magnetite, Palladium, Barium Titanate, and semiconductor nanomaterials of CdSe, CdS, ZnS and amorphous silica nanoprticles. The controlled biosynthesis of nanoparticles, however, is still the advanced research in the new synthesis nanotechnology. On the basis of our correlative researches, the dissertation focuses on the following studies of the biochemical synthesis mechanism of Au nanoparticles by different fungi, size-controlled biosynthesis of Au nanoparticles intra-and entracellular using Penicillium sp., the biological effects and cytotoxicity to human cells by biogeneic Au nanoparticles using Penicillium sp.,, Fusarium sp., Fusarium oxysporum (F. oxysporum) and the plant of Dolichomitriopsis diversiformis (D. diver sifor mis) as the biological materials. The main content of each chapter are as follows.1. Biochemical synthesis mechanism aspects of Au nanoparticles by different fungiThe intracellular gold nanoparticles were biosynthesized using three fungi including A. pullulans, Fusarium sp. and F. oxysporum after immersion in AuCl4-ions solution. The active biomolecules, such as reducing sugar of A. pullulans, proteins in both fungi of Fusarium sp. and F. oxysporum, were positive by providing the function of the reduction and the formation of the gold nanoparticles. SDS-PAGE results showed three proteins with molecular weight (WM) about100kDa,25kDa and19kDa in the gold nanoparticles by Fusarium sp. and two proteins with WM about25kDa and19kDa in the gold nanoparticles by F. oxysporum. Further, the indentification of proteins by LC-MS/MS indicated that three fungal proteins with WM about100kDa,25kDa and19kDa from Fusarium sp. were the plasma membrane ATPase,3-glucan binding protein and glyceraldehyde-3-phosphate dehydrogenase, respectively.The ultrathin sections images of the Au nano-fungal cells by TEM showed that the gold nanoparticles were mainly in intracellular vacuoles of Fusarium sp. and F. oxysporum. The growth of gold nanoparticles intracellular indicated the reducing sugar led to the gold nanoparticles in spherical morphology and proteins benefited to form the gold aggregates.2. Based temperature controlled on biosynthesis of different size Au nanoparticles intracellular using fungal Penicillium sp.The biosynthesis of different size gold nanoparticles using fungal Penicillium sp. was developed. Fungus Penicillium sp. could successfully reduct and nucleate AuCl4-ions after exposure to reaction solution, and leading to the assembly and formation of intracellular Au nanoparticles in spherical morphology and with good monodispersity. The result of biological compositions analysis indicated the intracellular reducing sugar of fungus Penicillium sp. could play an important role in the occurrence of reduction of AuCl4-ions and the formation of gold nanoparticles. Reaction temperature, as an important physiological parameter for the growth of fungus Penicillium sp., could significantly control the size of the intracellular Au nanoparticles. By control of the temperature at4,28and20-30℃, Penicillium sp. could biosynthsed the intracellular gold nanoparticles in size of8±2nm,30±5nm and75±35nm, respectively. Furthermore, the intracellular gold nanoparticles could be easily separated from the fungal cell lysate by ultrasonication and centrifugation.3. Based pH-inductive on plant manipulated-biosynthesis of constrained shape Au nanoparticles extracellularA pH-inductive protein-scaffold biosynthesis of shape-tunable gold nanoparticles at room temperature has been developed. By simple manipulation of the reaction solution’s pH, anisotropic gold nanoparticles including spheres, triangles and cubes biosynthesized by D. diversiformis biomasses incubating in an aqueous solution of sodium tetrachloroaurate. A moss protein with molecular weight of about71kDa and pI of4.9was involved in the biosynthesis of gold nanoparticles as the primary biomolecule. The analysis of CD spectrum implied that the moss protein displayed the different secondary configuration at the experimental pH solution, including random coil, a-helix and intermediate conformations between random coil and a-helix. Further the growth process of gold nanoparticles showed that the moss protein with different configurations provided the function of template scaffold for the shape-controlled biosynthesis of gold nanoparticles. The constrained shape of the gold nanoparticles, however, disappeared in boiled moss extract. The gold nanoparticles with designed morphology could be reconstructed using purified moss protein at the experimental pH solution. Structural characterizations by SEM, TEM and SAED showed that the triangular and cubic gold nanoparticles were single crystalline.4. Biological effects of Au nanoparticles capped different biomolecules biosynthsed by fungiSpherical Au nanoparticles capped different biological molecules were biosynthesized by three fungi of Penicillium sp., Fusarium sp. and F. oxysporum, respectively. Biological effects of BEL7404and L-02human cells were tested including the cell morphological change, surface adhesion, and viability of cell after incubating with Au nanoparticles biosynthesized by Penicillium sp., Fusarium sp. and F. oxysporum, respectively. The results suggested that both of BEL7404and L-02cells were more sensitive to the reduce sugar-Au nanoparitlces by Penicillium sp.. And the IC50values reached to511μg. In contrast, the gold nanoparticles capped protein by Fusarium sp. and F. oxysporum were nontoxic to the both BEL7404and L-02cells at some concentration. The biological effects of BEL7404and L-02cells showed the characteristic of biomolecules dependent. The Au nanoparticles capped different biological molecules is expected to develop the potential nanoparticle drugs to apply in the biomedical field.5. Cytotoxicity mechanism of protein Au nanoparticles to human tumor cellSpherical protein Au nanoparticles were biosynthesized using moss plant D. diver si for mis. The cellular toxicity, biological effects and possible mechanism of internalization by protein Au nanoparticles were evaluated. The results suggested that protein Au nanoparitcles could efficiently deliver into the experimental cells. After treatmented with the protein Au nanoparticle for24h, the normal morphology of BEL7404cell were disappearing. The L-02cell, however, changed smaller in morphology than BEL7404cell. The analysis by FCM (Flow Cytometry) showed same cytotoxicity to MCF-7and Hela cells. Futher the results by FCM suggested that the possible mechanism of toxicity to tumor cells was caused by the protein capped on gold nanoparticles. The cytotoxicity of protein Au nanoparticles to human tumor cells could be expected to apply in the cancer therapy.