3. Laser-mediated cell poration for intracellular delivery

Optoporation, a physical delivery technique, utilizes a tightly focused laser beam to create a transient pore in the cell membrane [18, 19, 33, 34]. This technique offers high delivery efficiency, high cell viability and is versatile with respect to cargo and cell type. However, each cell has to be porated individually by focusing the laser beam directly onto the membrane, causing optoporation to have an extremely low throughput.Modifications, including the use of active flow in microfluidic channels and a nondiffracting beam, slightly increase the throughput but not to the scale necessary for applications such as cell therapy, which can require on the order of 108 cells [35, 36].

Figure 1. Schematic of gold nanoparticle-mediated intracellular delivery. (a) Gold nanoparticles adhere to the cell membrane. (b) The gold nanoparticles are illuminated by a pulsed laser system. (c) Laser illumination of the gold nanoparticles leads to the formation of a bubble around the gold nanoparticle. (d) The bubble creates a temporary pore in the cell membrane, through which membrane-impermeable cargo can enter. Reprinted with permission from [1]. Copyright 2013, Elsevier.

Laser-activated thermoplasmonic nanostructures improve the throughput of optoporation by efficiently absorbing the laser energy at multiple localized hotspots, generating a rise in temperature, and transferring the energy to the surrounding medium [2, 8–10, 31, 37–39]. This transfer of energy to the surrounding solution results in the creation of a bubble or pressure wave that can generate sufficient mechanical stress to create a transient pore in the cell membrane, through which membrane-impermeable cargo can diffuse into the cell [1, 3, 8–10, 40]. This process is shown briefly in Figure 1, and the physics of this process will be explained in greater detail in the following section of this thesis. Gold nanoparticles are the most commonly used plasmonic nanostructures for intracellular delivery and have been successfully used to porate cell membranes for a range of cell types [37, 38, 41–46]. Gold nanoparticles potentially outperform other physical techniques by offering high efficiency, viability, and throughput [1, 45]. However, the gold nanoparticles remain in the cell after delivery as metallic residue and can form aggregates, and the long-term toxicity of these gold nanoparticles is still not fully understood [47, 48].

Laser-activated nanostructured substrates bypass this potential toxicity problem, as cells can be cultured on the substrates, porated, and removed from the substrates (which remain intact) after intracellular delivery without leaving metallic particles within the cells [31, 39, 49–52]. In this thesis we explore the fabrication of various thermoplasmonic nanostructured substrates for intracellular delivery and use the fabricated substrates to deliver a wide range of membrane-impermeable cargoes (dyes, dextrans, proteins, etc.) to a wide range of cell types (HeLa CCL2 cells, induced pluripotent stem cells (iPSCs), etc.).
