Recent electroporation-based systems for intracellular molecule delivery

Zaizai Dong and Lingqian Chang

ABSTRACT Intracellular delivery of functional molecules,such as DNA probes and plasmids,is an important method for investigating cellular mechanisms and changing cell fates in biomedicine.Among various delivery methods,recent years have seen the emergence of electroporation-based techniques that provide versatile platforms for molecule delivery,with high efficienc and controlled dosage.In this Review,we describe recent electroporation-based systems for intracellular molecule delivery.The principles of electroporation for cell membrane perforation and cargo delivery are briefl summarized.Focusing on various scenarios for the application of electroporation,we review electroporation devices that variously employ structures based on nanochannels,nanostraws,and flow-throug microfluidi channels for in vitro intracellular molecule delivery.We also consider in vivo targeted therapies based on delivery of active molecules by electroporation according to the lesion locations.Finally,we discuss the current challenges facing electroporation-based techniques,as well as opportunities for their future development,which may lead to innovations in intracellular molecule delivery both for cellular analysis in the laboratory and treatment in the clinic.

KEYWORDS Electroporation,Intracellular delivery,Micro/nanodevice,Targeted therapy

I.INTRODUCTION

Exploring molecular mechanisms in cells and manipulating the fate of dysregulated cells are both tasks that have been the focus of numerous biomedical studies.1,2Intracellular delivery of molecules such as DNA probes and plasmids is central to the successful decoding of cellular functions and reprogramming of aberrant cellular behavior.3,4However,the cell membrane acts as a defensive wall that resists the entrance of extraneous cargoes,forming an obstacle to intracellular molecule delivery.5,6A variety of methods have been developed to help cargoes break through this wall,such as nanomaterial-based carriers,direct penetration,and membrane permeabilization.6–8Among 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 significan 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.11Neumann’s group reported that electroporation could deliver DNA plasmids into mouse lyoma cells.12This 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.13In recent years,benefitin in particular from the integration of micro-and nanodevices,there have been significan improvements in electroporation techniques in terms of delivery efficiency dose uniformity,and cell safety.11The range of application of electroporation has also been expanded fromin vitrocell research toin vivotargeted therapy.14–16

FIG.1. Electroporation-based systems for intracellular molecule delivery.As described in this Review,in vitro intracellular delivery mainly includes electroporation devices based on nanochannels,nanostraws,and flow-through microfluidic chips.In vivo intracellular delivery includes the applications of electroporation for the treatment of lesions on the skin,retina,blood vessels,etc.

In this Review,we describe recently developed electroporationbased systems for delivering functional molecules into cells.First,we briefl 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 efficien delivery of moleculesin vitro,we introduce electroporation devices based on nanochannel,nanostraw,and flow-throug microfluidi chips.Forin vivoapplications of electroporation,we emphasize site-specifi 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.

II.PRINCIPLES OF ELECTROPORATION FOR INTRACELLULAR DELIVERY

A.Theoretical basis of electroporation

Electroporation uses an external electric fiel 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 3×10−7S/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 field17The potential difference between the inside and outside of the cell membrane is called the transmembrane potentialVm.On application of an external voltage to the cell,whenVmreaches 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 ofVmcan be calculated from the Schwan equation:11,17

whereEis the external electric fieldRis the cell radius,θis the polar angle measured from the center of the cell relative to the direction of the fieldtis the time elapsed since the onset of the field andτis the time constant of membrane charging,which varies with the conductivity of the medium.21This equation can be used to estimateVmfor different cell types.Furthermore,Vmcan be accurately predicted by establishing a mathematical model,such as through finit element analysis.22,23Numerical simulations can graphically reveal the distribution of the electric fiel around the cells.The values ofVmat different positions on the cell membrane can be obtained from calculations of the absolute value of the potential difference.

B.Basic electroporation device for intracellular delivery

In the classical technique for electroporation,which is also called bulk electroporation (BEP),millions of cells are exposed to an external electric fiel between a pair of cuvette-style parallel electrodes[Figs.2(c)and 2(d)].11,24The 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 fiel and suffer irreversible damage.At the same time,some cells far away from the electrodes do not undergo sufficien 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.16MEP/NEP systems take advantage of micro/nanodevices to realize controllable delivery of cargoes and significantl increase the precision of the electric fiel distribution.15In general,the cells and cargoes are compartmentalized into adjacent chambers by an interface with micro/nanochannels.26Under a low external voltage,a concentrated electric fiel and biased voltage are formed around the micro/nanochannels [Figs.2(e)–2(g)].24The 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 fiel on the micro/nanochannels,the cell membrane can be perforated under a safe voltage(<10 V),which guarantees retention of cellular activity alongside efficien molecule delivery.26

FIG.2. Principles of electroporation-based intracellular delivery.(a) Rearrangement of the cell membrane under an electric field.19 (b) Micrograph of a cell membrane(60 000×)after application of a 40 ms electrical pulse.20 Reprinted with permission from D.C.Chang and T.S.Reese,Biophys.J.58,1–12(1990).Copyright 1990 The Biophysical Society.(c)Schematic of a bulk electroporation(BEP)device.The cells are randomly distributed between the two electrodes.(d)Physical simulation of the electric field distribution in the cuvette of a BEP device.24 (e)Distribution of biased voltage in a nanochannel-based device.24 (f)There is a high potential concentrated around the nanochannel.24 (g)There is a low potential drop(∼4 V)over the cell membrane on the nanochannel.24 (d)–(g)Reprinted with permission from L.Chang et al.,Small 12,5971–5980(2016).Copyright 2016 John Wiley &Sons,Inc.

In the process of electroporation,the efficienc of intracellular delivery is influence 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.17,19,27The number and diameter of the pores on the membrane can be controlled by the pulse intensity and duration,respectively.12In 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 efficienc 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.17Because the responses of different cells vary depending on their membrane composition and physiological properties,for specifi electroporation tasks it is necessary to optimize the operating conditions,taking account of both delivery efficienc and cell safety.

III.APPLICATION OF ELECTROPORATION TO INTRACELLULAR DELIVERY

A. In vitro electroporation-based systems for intracellular delivery

To decipher intracellular molecular mechanisms,the introduction of exogenous molecules into cells is a crucial strategy.6However,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.28To efficientl deliver these molecules into cells without irreversible damage,various electroporation-based micro/nanodevices have been developed to control the electric fiel distribution (Table I).15,29–31According to their structural features,recent devices used forin vitroelectroporation can be divided into three categories,namely,nanochannel-based devices,nanostrawbased devices,and flow-throug microfluidi chips.

Nanochannel-based devices deploy nanoscale channels to concentrate the electric fiel for efficien cell electroporation.For example,Boukanyet al.32developed 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 fiel 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 team24developed a large-scale nanochannel array platform that enabled parallel delivery of ∼1 000 000 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 realizedcell manipulation by magnetic fields22dielectrophoresis,33and the application of a vacuum,23with increased loading efficienc for cell electroporation.These devices possess a resolution at the single-cell level,which is a significan 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.34,35However,this approach lacks the ability to track specifi cells,owing to the random cell location.Recently,Donget al.25developed a microwell array on a nanoporous PC membrane to electrodeliver DNA probes into cells forin situdetection of tumorEGFRgene 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.

TABLE I.Summary of electroporation-based systems for intracellular delivery.

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 significantl reduce the applied voltage and increase the uniformity of delivery.36For example,Caoet al.37used 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.29,36To simplify the complicated fabrication process of nanostraws,Wenet al.38developed 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 efficienc 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 fluorescen protein(GFP)plasmids]for intracellular molecular analysis,and the extraction of intracellular proteins(namely,caspase-3)for cell monitoring.39

Flow-through microfluidi chips integrate the electrodes in microfluidi channels,and thus the cell membranes are perforated as the cells flo 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 flo of cells,the throughput of these chips can be increased dramatically without the limitation of chip scales.40For example,Genget al.41presented a flow-throug electroporation method for continuous transfection of cells[Figs.3(f)and 3(g)].Under a constant voltage,104–108cells were transfected per minute with high transfection efficienc and cell survival.To further improve cell viability,Weiet al.42employed 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 microfluidi chip has also been used to increase cell viability.43Single 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 fiel was rapidly cooled by the flowin solution,cell damage was significantl reduced.

B. In vivo electroporation-based systems for intracellular delivery

FIG.3.In vitro electroporation-based systems for intracellular delivery.(a)Cross-sectional view of a single-living-cell analysis nanoplatform for intracellular RNA detection and cell behavior analysis by electric delivery of the Domino-probe into living cells.25 (b)Layered assembly schematic of a microwell array-based nanochip for electroporationbased probe delivery and single-cell analysis.25(c)Images of mutant cells detected by the Domino-probe on the microwell array.The scale bar represents 50 μm.25(a)–(c)Reprinted with permission from Z.Dong et al.,Nano Lett. 21,4878–4886 (2021).Copyright 2021 American Chemical Society.(d) Illustration of a nanostraw-integrated electroporation system for intracellular cargo delivery.38 (e) Scanning electron microscope (SEM) image of a HeLa cell cultured on platinum nanostraws.38 (d) and (e)Reprinted with permission from R.Wen et al.,ACS Appl.Mater.Interfaces 11,43936–43948 (2019).Copyright 2019 American Chemical Society.(f) Apparatus for cell transfection using flow-through electroporation based on a constant voltage.41(g)Illustration of a flow-through microfluidic chip for electroporation-based molecule delivery.PDMS,polydimethylsiloxane.41(f)and(g)Reprinted with permission from T.Geng et al.,Nat.Protoc.6,1192–1208(2011).Copyright 2011 Nature Publishing Group.

With the development of electroporation-based systems,in vivodelivery of cargoes such as drugs and CRISPR gene-editing plasmids is considered to have clinical potential as a therapeutic approach.44Compared 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.45The dosage delivered can also be accurately controlled by adjusting the applied electric field46In this subsection,we describe applications of advanced electroporation-based systems toin vivocargo 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 significantl minimizes the problems of drug degradation,low delivery efficiency and systemic toxicity.47–50By 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 flui is forced to form transient conductive pathways for enhanced molecular permeation of the skin cells.49,51,52To reach the cells of the dermis,Huanget al.53developed a microneedle roller to produce deep cargo-loading microcavities [Fig.4(a)].An electric fiel was then applied by an electrode array,thereby generating uniform electroporation of the skin cells.By using this method,a red fluorescen protein (RFP)plasmid and siRNA were effectively delivered into normal mouse skin.Gallego-Perezet al.54used 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.55In this method,the integration of microneedle arrays and iontophoresis synergistically increased the delivery efficienc of insulin for diabetes treatment.

FIG.4.In vivo electroporation-based systems for intracellular delivery.(a)Main processes of the electroporation-based strategy for the delivery of drugs into mouse skin using a microneedle roller and flexible interdigitated electroporation array(FIEA).53 Reprinted with permission from D.Huang et al.,Theranostics 8,2361–2376(2018).Copyright 2018 Ivyspring International Publisher,licensed under a Creative Commons Attribution(CC BY-NC)License.(b)Schematic of the nano-transfection(TNT)process on skin.Applied electric field condition:250 V;10 ms/pulse;10 pulses.54 (c)Ischemic limbs with and without delivering of EFF plasmids(day 14).EFF:reprogramming factors Etv2,Foxc2,and Fli1.54 (d)Enhanced angiogenesis obtained after delivery of EFF by electroporation.54 (b)–(d)Reprinted with permission from D.Gallego-Perez et al.,Nat.Nanothechnol. 12,974–979 (2017).Copyright 2017 Nature Publishing Group.(e) Schematic of electronic blood vessel in the carotid artery of a rabbit.The fluorescence image shows the distribution of three types of blood-vessel cells,namely,human umbilical vein endothelial cells(HUVECs,CellTracker Violet),smooth muscle cells(SMCs,CellTracker Green)and human artery-derived fibroblasts(HAFs,CellTracker Deep Red)on the electronic blood vessel.61 Reprinted with permission from S.Cheng et al.,Matter 3,1664–1684(2020).Copyright 2020 Elsevier,Inc.

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 reflecte mainly in the reprogramming of cells,which may change cellular behavior from that determined by gene expression levels.56In the process of cell reprogramming,plasmids are always employed as the tools for gene sequence modificatio and expression adjustment.57Despite their large molecular weight,plasmids can be delivered into cells by electroporation-based systems with high efficienc and controlled dosage,and without the risks of side effects that arise from the use of virus-based delivery methods.58For example,Latellaet al.59used subretinal electroporation to deliver a CRISPR/Cas9 plasmid into mouse retina and successfully edit a mutant rhodopsin gene.Anin vivoelectroporation-based system has also been applied to intracranial delivery of plasmids.60Through simultaneous transfection ofEtv2,Foxc2,andFli1(i.e.,EFF) by nanochannel-based electroporation,fibroblast 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.Chenget al.61developed an electronic blood vessel by integrating three layers of different blood-vessel cells on flexibl electrodes [Fig.4(e)].This device enabled controlled electro-delivery of genes to the blood vessel.Its high biocompatibility was verifie by three months of implantation in a rabbit model,showing the potential of this device for the treatment of cardiovascular diseases.

IV.CONCLUSION AND OUTLOOK

Electroporation opens new frontiers for research into intracellular mechanisms and treatment of disease by efficientl 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 bothin vitroandin vivo.Forin vitrointracellular delivery,the micro/nanodevices used for electroporation enable cellular analysis with high throughput and single-cell precision.Forin vivointracellular 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 ofin vitrocell analysis,the inevitable cell damage caused by intracellular molecular leakage,cell contamination,etc.makes it impossible to analyze cells in an actual physiological state.62Comprehensive optimization of electroporation devices and delivery conditions may effectively and fundamentally reduce cell damage.For example,compact nanochannels can increase the delivery efficienc 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 flo in flow through microfluidi chips could be convenient ways to address this issue.Forin vivointracellular delivery,the limited spatial range of the effects of electroporation has been identifie as a major issue that hinders its clinical applications.Although the delivered cells can release exosomes to transfer functional molecules to deeper-lying cells,54these 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 electroporationbased techniques have a crucial role to play in the fiel of intracellular delivery,although,as an emerging method,electroporation still needs improvements to allow its wider application in both biomedical research and clinical therapy.

ACKNOWLEDGMENTS

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 finan cial interests or personal relationships that could have appeared to influenc the work reported in this paper.