Recent electroporation-based systems for intracellular molecule delivery

Exploring molecular mechanisms in cells and manipulating the fate of dysregulated cells are both tasks that have been the focus of numerous biomedical studies.^1,2 Intracellular delivery of molecules such as DNA probes and plasmids is central to the successful decoding of cellular functions and reprogramming of aberrant cellular behavior.^3,4 However, the cell membrane acts as a defensive wall that resists the entrance of extraneous cargoes, forming an obstacle to intracellular molecule delivery.^5,6 A variety of methods have been developed to help cargoes break through this wall, such as nanomaterial-based carriers, direct penetration, and membrane permeabilization.^6–8 Among these, electroporation-based techniques have emerged as a robust tool able to deliver various types of molecules into cells, and these techniques now play a significant role in biomedical research as well as possessing great therapeutic potential.^9–11 

The systematic study of electroporation as a method for intracellular delivery can be traced back to the 1980s.¹¹ Neumann’s group reported that electroporation could deliver DNA plasmids into mouse lyoma cells.¹² This study played a pioneering role in explaining the structural rearrangements of the cell membrane that occur during electroporation. In the 1990s, Tsong and co-workers provided a detailed explanation of the process of electroporation, in which the cell membrane undergoes transient perforation under the action of a pulsed electric field, and this leads to the leakage of intracellular molecules and to increased uptake of exogenous drugs, molecular probes, DNA, etc.¹³ In recent years, benefiting in particular from the integration of micro- and nanodevices, there have been significant improvements in electroporation techniques in terms of delivery efficiency, dose uniformity, and cell safety.¹¹ The range of application of electroporation has also been expanded from in vitro cell research to in vivo targeted therapy.^14–16 

In this Review, we describe recently developed electroporation-based systems for delivering functional molecules into cells. First, we briefly introduce the principles of electroporation and describe the theoretical basis of membrane perforation and cargo delivery in bulk electroporation or micro-/nano-electroporation devices. To provide a comprehensive overview of recent applications of electroporation, we systematically summarize the current state of the art with regard to electroporation systems applied in biomedical areas (Fig. 1). For efficient delivery of molecules in vitro, we introduce electroporation devices based on nanochannel, nanostraw, and flow-through microfluidic chips. For in vivo applications of electroporation, we emphasize site-specific delivery for disease treatment according to the target organ, ranging from the skin to internal organs. The challenges faced by current electroporation systems are also described, with particular emphasis on clinical adoption. This Review will aid biomedical researchers in understanding the principles and applications of electroporation. We believe that electroporation-based delivery systems will become versatile tools both in the laboratory to facilitate the exploration of cell function and in clinical practice as innovative therapeutic techniques.

Electroporation uses an external electric field to perforate the cell membrane. The membrane is mainly composed of a phospholipid bilayer ∼5 nm in thickness. Because the conductivity of the membrane (about 3 × 10⁻⁷ S/m) is much lower than that of the cytoplasm (about 0.3 S/m) and extracellular medium (about 1.2 S/m), it can be regarded as a dielectric capacitor when subjected to an external electric field.¹⁷ The potential difference between the inside and outside of the cell membrane is called the transmembrane potential V_m. On application of an external voltage to the cell, when V_m reaches a threshold value of ∼0.2–1.0 V, the cell membrane will rearrange, with the formation of small hydrophilic openings [Figs. 2(a) and 2(b)].^18–20 

The value of V_m can be calculated from the Schwan equation:^11,17

where E is the external electric field, R is the cell radius, θ is the polar angle measured from the center of the cell relative to the direction of the field, t is the time elapsed since the onset of the field, and τ is the time constant of membrane charging, which varies with the conductivity of the medium.²¹ This equation can be used to estimate V_m for different cell types. Furthermore, V_m can be accurately predicted by establishing a mathematical model, such as through finite element analysis.^22,23 Numerical simulations can graphically reveal the distribution of the electric field around the cells. The values of V_m at different positions on the cell membrane can be obtained from calculations of the absolute value of the potential difference.

In the classical technique for electroporation, which is also called bulk electroporation (BEP), millions of cells are exposed to an external electric field between a pair of cuvette-style parallel electrodes [Figs. 2(c) and 2(d)].^11,24 The two electrodes are connected to a voltage generator to provide a pulsed voltage of over 100 V. During cell perforation, cargoes around the cells have the chance to diffuse through the cell membranes into the cellular interiors. However, in BEP, the cells are randomly distributed between the two electrodes, with the result that some cells close to the electrodes are exposed to an excessive electric field and suffer irreversible damage. At the same time, some cells far away from the electrodes do not undergo sufficient electroporation, leading to low delivery efficiency. Consequently, the dose delivered to each cell is nonuniform and cannot be controlled.^25,26

With the development of nanotechnology and microfabrication techniques, micro-/nano-electroporation (MEP/NEP) systems have emerged as challengers to conventional BEP.¹⁶ MEP/NEP systems take advantage of micro/nanodevices to realize controllable delivery of cargoes and significantly increase the precision of the electric field distribution.¹⁵ In general, the cells and cargoes are compartmentalized into adjacent chambers by an interface with micro/nanochannels.²⁶ Under a low external voltage, a concentrated electric field and biased voltage are formed around the micro/nanochannels [Figs. 2(e)–2(g)].²⁴ The regions of the cell membrane that tightly connected with the micro/nanochannels can be perforated instantaneously. Meanwhile, the charged cargoes are driven through the micro/nanochannels and into the cells by the electrophoretic force. Thanks to the focused electric field on the micro/nanochannels, the cell membrane can be perforated under a safe voltage (<10 V), which guarantees retention of cellular activity alongside efficient molecule delivery.²⁶ 

In the process of electroporation, the efficiency of intracellular delivery is influenced by the number and diameter of perforated pores on the cell membrane, as well as the size and electrical properties of the cargo that is to be delivered.^17,19,27 The number and diameter of the pores on the membrane can be controlled by the pulse intensity and duration, respectively.¹² In addition, large pores on the membrane are shrink rapidly during the pulse interval, and the smaller pores may remain open for minutes. Therefore, the delivery efficiency for macromolecules (e.g., plasmids) is lower than for small molecules (e.g., drugs and dyes). For the delivery of charged molecules, there will be an extra electrophoretic force to increase the delivery probability.¹⁷ Because the responses of different cells vary depending on their membrane composition and physiological properties, for specific electroporation tasks it is necessary to optimize the operating conditions, taking account of both delivery efficiency and cell safety.

To decipher intracellular molecular mechanisms, the introduction of exogenous molecules into cells is a crucial strategy.⁶ However, most exogenous molecular tools [e.g., plasmids, DNA probes, and small interfering RNAs (siRNAs)] are negatively charged, which prevents them from passing directly through the cell membrane, which has the same charge.²⁸ To efficiently deliver these molecules into cells without irreversible damage, various electroporation-based micro/nanodevices have been developed to control the electric field distribution (Table I).^15,29–31 According to their structural features, recent devices used for in vitro electroporation can be divided into three categories, namely, nanochannel-based devices, nanostraw-based devices, and flow-through microfluidic chips.

Nanochannel-based devices deploy nanoscale channels to concentrate the electric field for efficient cell electroporation. For example, Boukany et al.³² developed a nanochannel-based electroporation chip by etching a silicon wafer. An array of paired microchannels were horizontally connected by nanochannels (∼90 nm). The cells were placed in one microchannel and the materials for transfection in another. In this device, a through-channel electric field created tiny pores on the cell membrane and quickly drove propidium iodide (PI) dye into the cells (in <30 ms). Subsequently, to further increase cell throughput, the same team²⁴ developed a large-scale nanochannel array platform that enabled parallel delivery of ∼1000000 cells/cm². The cells were arranged on the chip by a simple “dewetting” approach. Biomolecules could be controllably delivered into cells under the action of an applied electric field. The device was used to investigate the dose effect of microRNAs in cardiomyocytes. In addition to “dewetting,” this team also realized cell manipulation by magnetic fields,²² dielectrophoresis,³³ and the application of a vacuum,²³ with increased loading efficiency for cell electroporation. These devices possess a resolution at the single-cell level, which is a significant development for research into cell heterogeneity. However, the fabrication of silicon-wafer-based chips requires high-precision equipment and considerable skill and experience. Commercial nanoporous polymer membranes have therefore become a more convenient choice to provide nanochannels for precise electroporation. Polycarbonate (PC)-membrane-based electroporation chips have realized intracellular delivery of PI dye, bovine serum albumin, and mCherry-encoding plasmid.^34,35 However, this approach lacks the ability to track specific cells, owing to the random cell location. Recently, Dong et al.²⁵ developed a microwell array on a nanoporous PC membrane to electro-deliver DNA probes into cells for in situ detection of tumor EGFR gene mutations and associated drug-resistant cellular behavior [Figs. 3(a)–3(c)]. This method enables single-cell gene analysis with high throughput and simple procedures, thus providing a versatile platform for cancer diagnosis and therapeutic guidance.

With nanostraw-based devices, the target cells are penetrated by nanostraws acting like hollow needles. Owing to their high aspect ratio and nanoscale tips, the nanostraws are in intimate contact with the cell membranes, as a result of which it is possible to significantly reduce the applied voltage and increase the uniformity of delivery.³⁶ For example, Cao et al.³⁷ used a nanostraw-based chip to quantitatively inject biomolecular cargoes (namely, mRNA, DNA, and proteins) into cells. The delivered dosage could be controlled precisely by adjusting the delivery duration, applied voltage, and reagent concentration. The nanostraw structures are usually fabricated by extending alumina from a track-etched membrane.^29,36 To simplify the complicated fabrication process of nanostraws, Wen et al.³⁸ developed an electrodeposition method for the controllable fabrication of conductive nanostraw arrays, expanding the range of processing materials. This nanostraw-based electroporation platform enabled the delivery of biomolecules (e.g., drugs, dyes, and DNA plasmids) and intracellular molecular detection with high efficiency and cell safety [Figs. 3(d) and 3(e)]. The same team has developed a branched nanostraw-based device that enables the effective capture of circulating tumor cells (CTCs), the delivery of biomolecules [namely, PI dyes and green fluorescent protein (GFP) plasmids] for intracellular molecular analysis, and the extraction of intracellular proteins (namely, caspase-3) for cell monitoring.³⁹ 

Flow-through microfluidic chips integrate the electrodes in microfluidic channels, and thus the cell membranes are perforated as the cells flow through the local electric field. Meanwhile, the functional molecules can permeate through the pores on the cell membranes and enter the cells. Owing to the continuous flow of cells, the throughput of these chips can be increased dramatically without the limitation of chip scales.⁴⁰ For example, Geng et al.⁴¹ presented a flow-through electroporation method for continuous transfection of cells [Figs. 3(f) and 3(g)]. Under a constant voltage, 10⁴–10⁸ cells were transfected per minute with high transfection efficiency and cell survival. To further improve cell viability, Wei et al.⁴² employed the dielectrophoretic force to sort viable cells. After electroporation and screening, plasmids were successfully delivered into vulnerable neuron cells and primary cells with high cell viability (∼90%). A droplet-based microfluidic chip has also been used to increase cell viability.⁴³ Single cells mixed with cargoes were isolated into microdroplets, and when these microdroplets successively passed through an electric field, the cells were perforated and molecular cargoes diffused into them. In this method, because the Joule heat generated by the electric field was rapidly cooled by the flowing solution, cell damage was significantly reduced.

With the development of electroporation-based systems, in vivo delivery of cargoes such as drugs and CRISPR gene-editing plasmids is considered to have clinical potential as a therapeutic approach.⁴⁴ Compared with traditional methods for drug delivery, such as oral uptake, electroporation-based delivery provides a more direct way to bring drugs into target organs with limited systemic damage.⁴⁵ The dosage delivered can also be accurately controlled by adjusting the applied electric field.⁴⁶ In this subsection, we describe applications of advanced electroporation-based systems to in vivo cargo delivery according to treatment location, including the skin and internal organs of the body.

Transdermal cargo delivery by electroporation is a less invasive mode for the therapy of skin diseases, such as melanoma, and it significantly minimizes the problems of drug degradation, low delivery efficiency, and systemic toxicity.^47–50 By applying electrodes to the surface of the skin or penetrating the deep skin tissue, the stratum corneum can be thoroughly penetrated under the action of an electric field. Interstitial fluid is forced to form transient conductive pathways for enhanced molecular permeation of the skin cells.^49,51,52 To reach the cells of the dermis, Huang et al.⁵³ developed a microneedle roller to produce deep cargo-loading microcavities [Fig. 4(a)]. An electric field was then applied by an electrode array, thereby generating uniform electroporation of the skin cells. By using this method, a red fluorescent protein (RFP) plasmid and siRNA were effectively delivered into normal mouse skin. Gallego-Perez et al.⁵⁴ used a nanochannel-based device for the electro-delivery of functionalized plasmids into neurons and endothelium [Figs. 4(b)–4(d)]. The limbs of murine models with ischemia were successfully rescued. Smartphone-powered electroporation for controlled transdermal delivery has been reported.⁵⁵ In this method, the integration of microneedle arrays and iontophoresis synergistically increased the delivery efficiency of insulin for diabetes treatment.

Internal tissues and organs are almost unreachable by the direct electroporation on the body surface. The common solution is to expose these deep locations surgically and then apply electroporation to the cells directly. Compared with traditional resection, the advantages of electroporation-based delivery are reflected mainly in the reprogramming of cells, which may change cellular behavior from that determined by gene expression levels.⁵⁶ In the process of cell reprogramming, plasmids are always employed as the tools for gene sequence modification and expression adjustment.⁵⁷ Despite their large molecular weight, plasmids can be delivered into cells by electroporation-based systems with high efficiency and controlled dosage, and without the risks of side effects that arise from the use of virus-based delivery methods.⁵⁸ For example, Latella et al.⁵⁹ used subretinal electroporation to deliver a CRISPR/Cas9 plasmid into mouse retina and successfully edit a mutant rhodopsin gene. An in vivo electroporation-based system has also been applied to intracranial delivery of plasmids.⁶⁰ Through simultaneous transfection of Etv2, Foxc2, and Fli1 (i.e., EFF) by nanochannel-based electroporation, fibroblasts in mouse brains were reprogrammed to accelerate the regeneration of blood vessels. Increased vascularity and neuronal cellularity, and reduced glial scar, indicated that this strategy provides a potential therapeutic approach for ischemic stroke. Cheng et al.⁶¹ developed an electronic blood vessel by integrating three layers of different blood-vessel cells on flexible electrodes [Fig. 4(e)]. This device enabled controlled electro-delivery of genes to the blood vessel. Its high biocompatibility was verified by three months of implantation in a rabbit model, showing the potential of this device for the treatment of cardiovascular diseases.

Electroporation opens new frontiers for research into intracellular mechanisms and treatment of disease by efficiently delivering functional molecules into cells. The controlled electroporation process enables molecule delivery with precise dosage control and high cell viability. In this Review, we have systematically described the principle of electroporation and recent applications of electroporation-based systems to intracellular delivery both in vitro and in vivo. For in vitro intracellular delivery, the micro/nanodevices used for electroporation enable cellular analysis with high throughput and single-cell precision. For in vivo intracellular delivery, electroporation techniques have great potential for cell reprogramming and disease treatment.

However, electroporation faces several challenges before it can become widely adopted. In the case of in vitro cell analysis, the inevitable cell damage caused by intracellular molecular leakage, cell contamination, etc. makes it impossible to analyze cells in an actual physiological state.⁶² Comprehensive optimization of electroporation devices and delivery conditions may effectively and fundamentally reduce cell damage. For example, compact nanochannels can increase the delivery efficiency with a reduced duration of cell membrane perforation, which may avoid interference with the cellular state. In addition, in the clinical application of electroporation to immunotherapy, there is a need for a dramatic increase in cell throughput to meet the requirement of simultaneous delivery of molecules to billions of single cells. Enlarging the scale of cell arrays on microwell chips or increasing the cell flow in flow-through microfluidic chips could be convenient ways to address this issue. For in vivo intracellular delivery, the limited spatial range of the effects of electroporation has been identified as a major issue that hinders its clinical applications. Although the delivered cells can release exosomes to transfer functional molecules to deeper-lying cells,⁵⁴ these effects will be minimal when it is desired to deliver drugs into large lesions. Integration of other methods, such as the use of long microneedles to expand the working area, may be helpful in solving this problem. In any case, it is clear that electroporation-based techniques have a crucial role to play in the field of intracellular delivery, although, as an emerging method, electroporation still needs improvements to allow its wider application in both biomedical research and clinical therapy.

This work was supported by the Beijing Natural Science Foundation (No. 7212204), Beihang University (JKE-YG-20-Z001), and the National Natural Science Foundation of China (Nos. 32071407 and 62003023).

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

 Dr. Zaizai Dong received her Ph.D. from the Institute of Chemistry, Chinese Academy of Sciences. She is currently a postdoctoral researcher at Beihang University, under the supervision of Professor Lingqian Chang. Her research interests are in the fields of biochip-based cell detection, multifunctional DNA probes for biomedical analysis, and electroporation micro- and nanodevices. She has published 12 journal papers and is the holder of three China Patents.

 Professor Lingqian Chang obtained his Ph.D. in biomedical engineering from Ohio State University, followed by postdoctoral training at the CCNE Nanoscale Center at Northwestern University with Professors Horacio Espinosa and Chad Mirkin. He then became an assistant professor at the University of North Texas. Currently, he is a full professor at Beihang University, and founded the Institute of Single Cell Engineering. His research is mainly focused on cellular micro- and nanotechnologies, with the aim of designing novel nanochips and nanosensors for gene detection and cell therapy in living cells. He has published more than 50 peer-reviewed papers, one book, and three book chapters and is the holder of five China Patents and three U.S. Patents.