Background

Intracellular delivery of molecular cargoes allows for a wide variety of cell manipulation tasks with applications ranging from cell engineering and gene editing to cell therapy [1,2]. Given the wide implications of intracellular delivery for fundamental biology research and therapeutics, several technologies have emerged to achieve this task with high efficiency and precision that can be classified into two broad categories: i) direct delivery and ii) carrier-mediated delivery [3]. In direct-delivery methods, the cell membrane is permeated by subjecting it to physical disturbances such as mechanical forces [4–7], electric fields [8–10], or thermal shock [11]. Conversely, carrier-mediated delivery methods rely on chemical interactions between the cell membrane and the nanocarrier, which trigger endocytosis and encapsulation of the molecular cargo [3]. Examples of nanocarriers include lipid vesicles [12], spherical nucleic acids [13], and polymer nanoparticles [14]. Direct delivery methods offer more control and tunability over the membrane permeation process, which can result in tighter dosage precision compared with carrier-mediated methods [1,15].

Electroporation is one of the most widely used direct-delivery methods because of its ease of use and high throughput. In bulk electroporation systems, cells are placed in a cuvette with embedded electrodes that subject a suspension of cells to an electric field that induces the formation of pores across the cells when the electric potential across the membrane [transmembrane potential (TMP)] reaches a certain threshold. However, high applied voltages are required to achieve threshold TMP in bulk electroporation systems, which results in high cell mortality. Furthermore, the variability in the spatial distribution of the cells in the cuvette results in heterogeneous electric field conditions from cell to cell, which reduces dosage precision [1]. To circumvent these limitations, researchers developed electroporation systems that confine the electric field to a small fraction area of the cell membrane by interfacing cells with nanoscale orifices prior to the application of electric pulses [9,16,17]. The resulting highly localized electric fields enable the application of much lower voltages compared to bulk systems to achieve threshold TMPs, which results in higher viability. Examples of nanoscale features used in localized electroporation systems include nanochannel membranes [18,19], nanopipettes [20], nanostraws [21], and nanofountain probes [22].

Localized electroporation systems have been shown to be efficient in delivering various payloads (e.g., nucleic acids, plasmids, proteins, and nanoparticles) as well as sampling intracellular contents from cells [23,24]. The performance of localized electroporation systems depends on the electric field conditions, the mechanical properties of the cell membrane, the ionic environment in the surrounding fluid, and the physical properties of the molecular cargo (i.e., size and charge) [2,3]. While these systems offer significant advantages, they also pose challenges in scalability in the number of cells and require high cargo concentrations, which may limit their applicability in certain scenarios. Consequently, several parameters must be optimized to achieve the desired electroporation performance, including both delivery efficiency and cell health. To expedite the optimization process, we designed a well plate–based localized electroporation device (LEPD) equipped with multiplexing capabilities [9]. The multi-well LEPD system allows for tunability in the electric pulse conditions across the rows of the device and the molecular cargo or buffer conditions across columns. Furthermore, we integrated the LEPD with automated imaging and deep-learning enhanced segmentation to analyze the morphology of cells following delivery in addition to delivery efficiency and viability. The integrated approach provides a flexible platform suitable for adherent or suspended cell manipulation and analysis. In this protocol, we detail the experimental workflow of the LEPD spanning from device fabrication to optimization of plasmid delivery.