Pulsed laser-activated thermoplasmonic substrates for intracellular delivery

Abstract

This thesis describes advancements made in the field of laser-activated thermoplasmonic-substrate based intracellular delivery. The second chapter describes background knowledge relevant to this work. In the third chapter, we present a fabrication process for generating large-area thermoplasmonic substrates patterned with gold-coated micropyramids. Temperature simulations of ns-pulsed laser illumination of these substrates in aqueous surroundings show the surrounding liquid reaches temperatures high enough to generate a bubble. We therefore propose an intracellular delivery mechanism in which a heat-mediated bubble generates sufficient thermomechanical stress to porate the cell membrane. We show results for delivery of cargoes ranging in size from 0.6 to 2000 kDa to HeLa CCL2 cells using these substrates. Delivery efficiencies and cell viabilities are quantified by flourescence microscopy and flow cytometry. In Chapter 4, we explore the utility of thermoplasmonic substrates for translational research by delivering CRISPR-Cas9 ribonucleoprotein (RNP) complexes to induced pluripotent stem cells (iPSCs) to knockout green-fluorescent protein (GFP). We verify GFP-knockout with fluorescence microscopy and flow cytometry, and verify maintenance of pluripotency markers after laser-treatment with immunocytochemistry (ICC) staining and quantitative polymerase chain reaction (qPCR). In the fifth chapter, we show spatially-selective delivery by delivering fluorescent cargo to a specific region of a differentiated C2C12 myotube cell. We also measure and calculate the approximate radius and lifetime of the pore by loading a C2C12 myoblast cell with fluorescent dye and recording the drop in fluorescence after poration. In the sixth chapter, we present an affordable and simple fabrication technique for generating thermoplasmonic nanocavity substrates that is based on self-assembly and colloidal templating. Previously, it was assumed that plasma-mediated bubbles generated from ps- or fs-pulsed laser sources were necessary for high-viability cell poration. A significant finding from this work is that heat-mediated bubbles generated from a ns-pulsed laser source are sufficient. The combination of simple substrate fabrication and the ability to use a ns-pulsed laser source makes thermoplasmonic intracellular delivery more widely accessible. In addition, this work presents the first instance of thermoplasmonic substrates being used to deliver CRISPR-Cas9 to iPSCs for successful gene-editing, advancing the potential utility of this technique for translational research.