The aim of the present work is the establishment of a synthesis of azide-functionalized ultrasmall gold nanoparticles that can be modified by the click reaction with alkynes in such a way that these can be tailored to specific biomedical questions. The work can be divided into four milestones, describing each milestone chronologically as follows: 1.) Establishment of a synthesis of azide-functionalized ultrasmall gold nanoparticles and characterization of the system. 2.) Establishment and verification of the click reaction of alkynes on the azide-tipped surface of the ultrasmall gold nanoparticles. 3) Clicking of fluorophores to enable intercellular tracking of the ultrasmall gold nanoparticles and the resulting qualification testing of the particles for applications in biomedicine and protein control. 4) Demonstration of the specific application and avidity of the system by clicking of supramolecular ligands.
The first milestone was the establishment of a novel synthesis route for the synthesis of azid-functionalized ultrasmall gold nanoparticles. Beyond the synthesis described in literature, a novel method for the preparation of clickable ultrasmall gold nanoparticles has been established, because the azide-terminated ultrasmall gold nanoparticles were intrinsically produced by using the azide-modified peptide in a one-pot synthesis. The binding of the tripeptide to the nanoparticle surface was achieved by the incorporation of a cysteine following the model of a glutathione structure. After the removal of the free peptide by ultrafiltration, IR and NMR spectroscopic investigations showed that the peptide is not only bound to the gold nanoparticle surface by the sulfur but is also further acidified. The characteristic azide vibration at 2100 cm-1 was detected after synthesis. Further, the NMR spectroscopic experiment TOCSY showed an intact peptide after gold nanoparticle synthesis by correlating the 1H nuclei. Using 1H-DOSY, it was demonstrated clearly that the signals of the peptide have the same diffusion coefficients and the hydrodynamic diameter of the peptide increases after formation of the particles. The quantification of the particle-bound ligand was performed using the ERETIC method. A surface loading of the synthetic peptide of ~ 70% was observed. The HRTEM images of the azide functionalized ultrasmall gold nanoparticles confirmed a spherical gold core with a mean diameter in the ~ 2 nm dimension range.
The second milestone was to show evidence of a successful click reaction onto the nanoparticle surface. The 13C labelled propargyl alcohol was clicked onto the azide-functionalized nanoparticles as a model alkyne. Using the NMR method 13C-SOFAST-HMQC, it was shown that the signal at ~ 8 ppm after the click reaction is a triazole proton. It was also determined that with the inclusion of a coupled 1H-NMR spectrum, the signal of the triazole proton splits into a doublet resulting from the 13C-atoms of the alcohol. The 1H-DOSY of the click product showed that the signals of the clicked propargyl alcohol diffuse as fast as the signals of the particle bound peptide. Using HRTEM images, it has been demonstrated that the click reaction has no significant effect on the size and shape of the gold core.
The third milestone of this work was the clicking of alkyne-modified fluorophores. By clicking functional molecules to the ultrasmall gold nanoparticles, an applied aspect of the system was obtained. The fluorophores enabled the tracking of the particles in various cell culture studies. It was found that in comparison to the dissolved fluorophore, the particles managed the cellular entry down to the cell nucleus. In addition, clicking of fluorophores with different excitation wavelengths demonstrated the modular character of the system and the easy adjustment to cell biological investigations. The UV-vis bands of the clicked fluorophores allowed the spectroscopic quantification of the clicked ligands on the nanoparticle surface and between 10 and 5 fluorophores per nanoparticle were covalently bound.
The fourth milestone of this work was the clicking of supramolecular ligands. By clicking these ligands, the possibility of targeting biomolecules and optimizing the avidity of supramolecular ligands was investigated. The arginine and lysine-binding tweezers were clicked onto the ultrasmall gold nanoparticles. After the characterization of the colloidal-chemical parameters, NMR titrations were carried out with this system. Titration with 13C-15N lysine showed that the clicked tweezers were intact and still acted as lysine binders. NMR titration with the 13C-15N model protein showed that the tweezer bound to the gold surface addressed lysines and arginines. The binding affinity of the system was investigated with a protein that is relevant for cancer research. The binding constant of the functionalized particles was almost identical to that of the dissolved tweezers. A significant increase in the affinity of the tweezers by binding it to the ultrasmall gold nanoparticles was not observed.
The results of this work indicate that the surface functionalization of ultrasmall gold nanoparticles provides a tailor-made transport vehicle in the field of biomedicine. After the click reaction, a colloidally stable system is obtained that is characterized by ultrasmall gold cores and a functional ligand shell.
Besides the surface functionalization of ultrasmall gold nanoparticles by the click chemistry, the peptide mimetic oligo(amidoamine) of Hartmann group was investigated as a ligand class for ultrasmall gold nanoparticles. In addition to the hydrophilic EDS building blocks, the oligo(amidoamine)s are characterized by several cysteines. The free thiol groups of the cysteines are responsible for binding the precision macromolecules to the nanoparticle surface. SAXS and HRTEM showed that bi- and hexavalent macromolecules stabilize ultrasmall gold nanoparticles. The obtained particle size distributions are very narrow and indicate monodisperse samples. NMR spectroscopic investigations showed that the particle-bound macromolecules are intact after synthesis and the 1H resonances of the cysteines become so broad that they are no longer traceable as signals in the spectra. The strong enhancement of the binding building blocks indicates that the multi-thiol ligands bind multivalently to the nanoparticle surface. 1H-DOSY experiments clearly show that the particle-bound macromolecules are coordinated around the gold core, with significant increases in hydrodynamic diameters of over ~ 50%. However, it also clarifies that the multivalence of the ligands does not generate cross-linking to other gold nuclei. The quantification of the particle-bound macromolecules confirmed the assumption that the functionalization state of the particles can be controlled by the multi-thiol ligands described here.
The results are promising and suggest, that multi-thiol oligo(amidoamine) present an interesting and advanced class of molecules for the functionalization of ultrasmall gold nanoparticles. Especially a mono-functionalization of the particles with a multi-thiol macromolecule would simplify the handling of the particles enormously, because a resulting nanoparticle system with a defined size, shape and one ligand per particle can be assumed.
|Advisor:||Epple , Matthias|
|School:||Universitaet Duisburg-Essen (Germany)|
|Source:||DAI-C 82/4(E), Dissertation Abstracts International|
|Subjects:||Inorganic chemistry, Biomedical engineering, Nanotechnology|
|Keywords:||gold nanoparticles, click reaction|
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