Manipulation and Biological Applications of Gold Nanorods
Rostro-Kohanloo, Betty Catalina
Doctor of Philosophy
This thesis compared anionic polyelectrolyte wrapping stabilization with poly(sodium 4-stryene-sulfonate), (PSS), polyelectrolyte and methoxy (polyethylene glycol)-thiol (mPEG (5000) -SH) strategies. From this data the critical gold nanorod (GNR) and cetyl-trimethylammonium bromide (CTAB) concentration ratio needed for GNR stabilization was determined using optical and chemical extraction methods. This was followed by functionalization with a heterobifunctional Polyethylene glycol (PEG) linker, such as a-thio-w-carboxy poly(ethylene glycol) termed t-PEG-c and carbodiimide chemistries for antibody linkage with Immunoglobulin G (IgG), and epidermal growth factor receptor (EGFR) based Human Epidermal growth factor Receptor 2 (Her2), and Cetuximab (C225) antibodies, for in vitro cancer cell targeting. Confocal, two-photon luminescence (TPL), and dark scattering microscopy, and fluorescence, zeta potential, and Nanoparticle Enzyme-linked immunosorbent assay (ELISA) were used to monitor changes to the GNR surface. An untreatable form of bladder cancer was then studied using the t-GNR-PEG-c-Ab bioconjugates with C225 antibody, which housed a glyceraldehyde-3-phosphate (GAPDH), Fluorescein isothiocyanate (FITC) labeled siRNA, termed GAPDH-siRNA-FITC, which was included within a Luciferase based plasmid. A salt based electrostatic heating method was used to trap the GAPDH-siRNA-FITC from the PEG layer by activating the PEG polymer pour point, while a laser based heating system was used for in vitro release inside cancer cells. The down regulation of the GAPDH gene was targeted by the siRNA. as GAPDH has been shown to be up-regulated in many cancers and down-regulated by chemotherapeutic drugs. Cell culture, and subsequent imaging by transmission electron microscopy (TEM), TPL and confocal microscopy were used to view the internalized conjugates, and reverse transcriptase polymerase chain reaction (RT-PCR) were used to determine if the release of the GAPDH-siRNA caused a reduction in the expression of GAPDH-mRNA. Plasmonic gene silencing of the gene by the GAPDH-siRNA was then compared to a lipid based Dharmafect control in terms of transfection ability. RT-PCR results evidenced gene silencing of the plasmonic-GAPDH-siRNA vector when compared to the Dharmafect control. Silencing likely resulted from the zwitterionic charges of the plasmonic vector and the encapsulated GAPDH-siRNA, which yielded near neutral charge tendencies. This differs significantly from the Dharmafect lipid vector, which is cationic in nature. Endosomal release of the plasmonic vector is further enhanced by the laser excitation of the GNR at the longitudinal surface plasmon resonance (LSPR), which allows for the endosomal release of the GAPDH-siRNA through pore formation leading to cytoplasmic transport and subsequent gene silencing. Near neutral charges were welcomed in this plasmonic gene therapy study as they tend to favor endosomal release, pore formation, and transport.