2. Currently available intracellular delivery techniques

The delivery of membrane-impermeable cargoes such as nanoparticles, genetic materials, or functional proteins directly into cells is a critical step for applications in biology and medicine [4]. For instance, the delivery of gene-editing tools could be used to manipulate cells and tissues for regenerative medicine or engineer cells for personalized cell therapies [4–7]. Intracellular delivery methods include biological vectors such as viruses, chemical modifications of delivery cargoes such as lipofection, and physical techniques such as microinjection, electroporation, and optoporation [11–24]. While research efforts have led to a continuous increase in efficiency and sophistication, each of the currently available approaches has its own advantages and disadvantages. To this point, no platform technology exists that combines highefficiency delivery, high-throughput processing, low-toxicity, versatility with respect to type of cell and cargo, and simple, cheap and affordable production. The research presented in this thesis is an attempt toward developing a solution to this problem.

Viral-based delivery is a popular biological technique that offers high-efficiency delivery at high throughput. However, it is limited in terms of cargo-carrying capacity, the ability to only deliver genetic material, the requirement to customize the virus for each cargo and cell type, and the potential for immunologic and oncogenic risks [11–15].

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

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,

Plasmonic Intracellular Delivery

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http://dx.doi.org/10.5772/intechopen.79384

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

Plasmonic structures have proven valuable in intracellular delivery as well as numerous other applications requiring the ability to generate electric fields in a highly localized manner [1]. These structures are capable of supporting plasmons, or quanta of plasma oscillations.

(HeLa CCL2 cells, induced pluripotent stem cells (iPSCs), etc.).

4. Physics of plasmonic intracellular delivery

4.1. Properties of localized surface plasmons

not fully understood [47, 48].

Elsevier.

Lipofection, a chemical method, offers high throughput but varies in efficiency depending on cell type, can require complex chemical customization depending on the cargo, and risks endosomal trapping of the cargo [7, 25, 26].

Electroporation, the most widely used physical delivery method, offers high-efficiency delivery and high throughput for a range of cargo types, but can lead to high cell death, particularly for sensitive cell types [27, 28]. Nucleofection, a variation of electroporation, offers improved viability but can require expensive customized reagents and can still be low viability for the most sensitive cell types. Ultrasound-mediated methods offer a low-cost high-throughput technique for delivering membrane-impermeable cargo into cells [12]. However, the cavitation dynamics are not spatially localized, which can lead to nonuniform results and high cell death. Other physical methods such as microinjection, nanowire-mediated delivery, and microfluidic squeezing are promising, but offer limited throughput and/or reproducibility [14, 29–32].
