High Throughput and Highly Controllable Methods for In Vitro Intracellular Delivery

Abstract

In vitro and ex vivo intracellular delivery methods hold the key for releasing the full potential of tissue engineering, drug development, and many other applications. In recent years, there has been significant progress in the design and implementation of intracellular delivery systems capable of delivery at the same scale as viral transfection and bulk electroporation but offering fewer adverse outcomes. This review strives to examine a variety of methods for in vitro and ex vivo intracellular delivery such as flow-through microfluidics, engineered substrates, and automated probe-based systems from the perspective of throughput and control. Special attention is paid to a particularly promising method of electroporation using micro/nanochannel based porous substrates, which expose small patches of cell membrane to permeabilizing electric field. Porous substrate electroporation parameters discussed include system design, cells and cargos used, transfection efficiency and cell viability, and the electric field and its effects on molecular transport. The review concludes with discussion of potential new innovations which can arise from specific aspects of porous substrate-based electroporation platforms and high throughput, high control methods in general.

Keywords: electroporation; intracellular delivery; localized cell electroporation; porous substrates.

Figure 1.. Throughput and Control Classification of In Vitro/Ex Vivo Intracellular Delivery.

Tree structure of the three main categories of in vitro/ex vivo intracellular delivery, their subcategories, and specific methods. High throughput, low control methods are shown in yellow; low throughput, high control methods are shown in blue; and high throughput, high control methods are shown in green.

Figure 2.. High Throughput, High Control Methods.

Simplified depictions of high throughput, high control methods next to actual images of each method. The electroporation polarities shown are for delivering negative cargos. A. Automated probe-based injection. i. automated injection of zebrafish embryos ^[87] ii-iii. Different magnifications of an atomic force microscope tip with attached carbon nanotube needle (scale bar = 8 μm and 500 nm, respectively) ^[48] iv. cell held using a vacuum during injection ^[50] v-vi. real and simulated deformation during injection (needle diameter = 10 μm) ^[77] B. Automated probe-based electroporation. i-ii. image processing showing nuclear site in green and cytoplasmic site in red, followed by automated electrode positioning ^[90] iii. nanofountain probe electroporation (cell size ~ 10–20 μm) ^[58] iv. An improved version of nanofountain probe using silicon nitride for a soft touch (scale bar = 30 μm) ^[13]. C. Flow-through microfluidic electroporation. i. vortex microfluidic electroporation (scale bar ~ 720 μm) ^[97] ii. microfluidic electroporation device (scale bar = 6 mm) ^[96] iii. sawtooth microfluidic electroporation (scale bar = 40 μm) ^[95] D. Flow-through microfluidic mechanoporation, including cell squeezing and hydroporation. i. microfluidic constrictions for cell squeezing (scale bar ~ 250 μm) ^[92] ii-iii. microfluidic constrictions showing single and double deformation, respectively (scale bar = 10 μm) ^[103] iv. hydrodynamic shearing in hydroporation ^[105] v. spiral hydroporation ^[104] E. Nanostructures. i. nanoneedles (scale bar = 2 μm) ^[110] ii. cell adherent to nanostraws with false color added (scale bar = 10 μm) ^[121] iii. primary T cells on nanowires with false color added (scale bar = 10 μm) ^[115] iv. internalized nanowires with the cytoplasm dyed green and the cell membrane dyed red (scale bar = 10 μm) ^[111] v. neurons adherent to nanowires with false color added (scale bar = 10 μm) ^[114] vi-vii. silicon nanotubes used for biomolecular cargo delivery (scale bars = 1 um and 10 μm, respectively)^[118] G. Patterned electrode electroporation. i. electrode electroporation device with multiple inputs ^[149] ii. clover electrodes (scale bar = 5 mm) ^[143] iii. interdigited electrodes ^[136] iv. 3D interdigited electrodes (scale bar = 800 μm) ^[141] H. Porous substrate electroporation. i. anodic alumina membrane (scale bar = 1 μm) ^[155] ii. polycarbonate membrane microfluidic device (scale bar = 12 mm) ^[153] iii. porous array with nanostructure trapping mechanism (scale bar = 200 μm) ^[161] iv. DAPI stain showing cell seating on porous array (scale bar = 100 μm) ^[164]. Permission is needed.

Figure 3.. Porous Substrate Electroporation.

A. The 4 cell trapping mechanisms that have been demonstrated. From left: nanostructure, vacuum, magnetic tweezers, and dielectrophoresis. B. A cell adhered to a porous substrate and undergoing electroporation. Equivalent circuit elements are shown near their corresponding features. C. A magnified view of the cell-channel interface showing the voltage drop along the channel. D. The transition from hydrophobic pores to hydrophilic pores that occurs during electroporation. E. A further magnified view of the cell membrane showing the 3 nm radii pores that form at high voltage as predicted by Mukherjee et al. Under the current electrode configuration, electrophoresis extracts positive cargos and delivers negative cargos. From left, the cargos propidium iodide (PI), linear DNA, and bovine serum albumin (BSA) are shown to scale. F. An electroporation waveform consisting of two trains, each with three unipolar square bilevel pulses. The parameters high voltage (HV), low voltage (LV), high voltage duration (t₁), low voltage duration (t₂), and train interval (t₃) are shown. Single level and exponential decay pulses are also shown.