146. Golden Cells: Gold Conjugate Labeling as a Potential Enhancer of Electro-Gene Transfer

Abstract

When cells undergo electroporation, electric pulses cause a reversible breakdown of the membrane, allowing the entry of DNA. Electroporation can be performed on quiescent cells and there is litter or no risk of insertional mutagenesis. However, mechanical damage to cells makes the process less efficient at non-toxic conditions. Colloidal gold particles are electron-dense, negatively charged spheres that exist as monodisperse suspensions due to hydrostatic repulsions. They are further stabilized when halos of protein are non-covalently adsorbed to the particles, thus a receptor-specific gold conjugate can be formed. Since the gold particles are conductive metals, the electric field strength will be more dense in the region surrounding the gold particles. Theoretically, electro-gene transfer should be enhanced in cells labeled with sufficiently large gold particles in close proximity to the cell membrane. To investigate this hypothesis, Hela cells were labeled with gold particles conjugated to transferrin receptor (TfR)-targeting proteins. First, a mouse monoclonal anti-human TfR and a sheep anti-mouse IgG conjugated to FITC were bound to Hela cells. Fluorescence of cell surface confirmed the presence of the transferrin receptor. Then, the primary TfR-recognizing antibody was bound to a 6nm immunogold particle coated with goat anti-mouse IgG. Luciferase plasmid was introduced into the cells using a T820 ElectroSquarePoratorTM. The presence of gold particles on the cells was confirmed using an Aurion Silver Enhancement Reagent. Under these conditions, no significant difference was noted between the control and gold labeled cells. Assuming the gold particle was too small or too far away from the membrane to effect electro-gene transfer, we made two adjustments to the protocol. First, the experiment was conducted with 25nm immunogold particles with goat anti-mouse IgG. Second, the electroporation was conducted with 25nm gold particles conjugated directly to holo-transferrin protein, thus decreasing the distance between cell and gold particle. The indirect conjugation using the 25nm gold particle did not significantly increase transfection efficiency. However, the cells with the gold-holo-transferrin conjugate showed significantly higher luciferase activity relative to the control group after 24 hours (95%CI, P<0.015). These results indicate that direct gold particle attachment is a promising method for increasing the efficiency of electro-gene transfer.