Research Thrust 4:
Mechanism of Porous Substrate Electroporation for Electrokinetic Transport and Single Cell Intracellular Delivery
Intracellular delivery and extraction are critical aspects of biological study, with numerous applications ranging from fundamental biology to biomedical engineering. Many applications need an intracellular delivery method that is more highly repeatable and scalable than existing methods. Porous substrate electroporation (PSEP) is a promising new method with the potential to meet this need. During PSEP, cells are adhered to a porous substrate and an electric field is applied (Figure 4a-c). The channels in the porous substrate focus the electric field to discrete portions of the cell membrane, which temporarily permeabilizes the membrane and allows the intracellular delivery and extraction of proteins, drugs, genes, and other molecules of interest. Important aspects of the PSEP process, such as cargo delivery through the substrate channels and pore formation on the cell membrane, are determined by the voltage present at various points in the system when electric waveforms are applied. However, impedances of different components of the PSEP system had never been measured, making it impossible to understand what occurs between the application of a waveform and the delivery or extraction of cargo to or from cells. As a result, much of what is known about PSEP is based on simplified finite element models and PSEP remains poorly understood or optimized.
To better understand the processes that constitute PSEP, we developed the first circuit model of the PSEP system using experimental data (Figure 4d). Our model contains a combined faradaic and bulk electrolyte impedance, impedances for different porous substrates, and the impedance of a confluent cell monolayer. For the first time, we are able to show the influence of voltage, pulse frequency, and pulse duration on voltages present at different points in the PSEP system, including at the electrode-electrolyte interface, across the porous substrate channels, and the transmembrane potential (TMP) (Figure 4e-g). Faradaic impedances have never been included in simulations of PSEP, but they dominate the system when low voltage waveforms are applied. The voltage across the porous substrate channels governs the transport of cargo through the channels. Furthermore, the transmembrane potential determines pore formation. Using these impedance measurements and resulting voltages, we were also able to show for the first time that confluency significantly affects the voltage experienced by the cell, and that cargo transport through the channels may be influenced by electro-osmosis as well as electrophoresis (Figure 4h). The findings of our analysis were confirmed using electrokinetic transport through the substrate channels and intracellular protein delivery using PSEP (Figure 4i). Our novel circuit model of PSEP reveals numerous previously unknown aspects of PSEP, which will allow for greater understanding and optimization of PSEP in the future.
Porous substrate electroporation equivalent circuit model. a. Schematic of the electroporation setup. b. Magnified view of cells adherent to a porous substrate and subjected to an electric field. c. Image of the electroporation setup. d. Equivalent electrical circuit model with the impedances of the function generator, faradaic and bulk electrolyte, porous membrane, and cell monolayer. e. Simulated waveform applied to our model showing how the relative influences of the combined faradaic and bulk electrolytic impedance, PC membrane impedance, cell monolayer impedance, and TMP change with time. f. Percentage of applied voltage across each element as a function of voltage for a 10 ms, 20 Hz square wave. g. Percentage of applied voltage across each element as a function of pulse width for a 25 V, 20 Hz square wave. h. Electrophoretic mobility calculated through different membranes from delivered mass after subtracting diffusion. Error bars represent the standard error of the mean. i. Fluorescent microscope images of cells after electroporation when different seeding densities of cells are electroporated. The cells were stained with Hoechst to show all cells and calcein to show living cells, while Alexa Fluor 647 conjugated bovine serum albumin was delivered using PSEP.
Justin Brooks, Grayson Minnick, Prithvijit Mukherjee, Arian Jaberi, Lingqian Chang, Horacio D. Espinosa, and Ruiguo Yang. High Throughput and Highly Controllable Methods for In Vitro Intracellular Delivery. Small 16, no. 51 (2020): 2004917:1-20.