Bioelectrochemistry 156 (2024) 108629

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Bioelectrochemistry
journal homepage: www.journals.elsevier.com/bioelectrochemistry

Therapeutic perspectives of high pulse repetition rate electroporation

Alexia de Caro, Franck Talmont , Marie-Pierre Rols , Muriel Golzio *, Jelena Kolosnjaj-Tabi *
Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, CNRS, UPS, Toulouse, France

A R T I C L E I N F O A B S T R A C T

Keywords: Electroporation, a technique that uses electrical pulses to temporarily or permanently destabilize cell mem­
Electric pulses branes, is increasingly used in cancer treatment, gene therapy, and cardiac tissue ablation. Although the tech­
High repetition rate nique is efficient, patients report discomfort and pain. Current strategies that aim to minimize pain and muscle
High frequency
contraction rely on the use of pharmacological agents. Nevertheless, technical improvements might be a valuable
Electroporation
Bipolar pulse
tool to minimize adverse events, which occur during the application of standard electroporation protocols. One
Monopolar pulse recent technological strategy involves the use of high pulse repetition rate. The emerging technique, also referred
Muscle contraction as “high frequency” electroporation, employs short (micro to nanosecond) mono or bipolar pulses at repetition
Bipolar cancellation rate ranging from a few kHz to a few MHz. This review provides an overview of the historical background of
electric field use and its development in therapies over time. With the aim to understand the rationale for novel
electroporation protocols development, we briefly describe the physiological background of neuromuscular
stimulation and pain caused by exposure to pulsed electric fields. Then, we summarize the current knowledge on
electroporation protocols based on high pulse repetition rates. The advantages and limitations of these protocols
are described from the perspective of their therapeutic application.

1. Introduction the application of short pulses (typically tens of microseconds down to

Electroporation, a physical method relying on electric pulses to
transiently or permanently destabilize cell membranes, is gaining mo­
mentum in different therapeutic fields. Numerous preclinical studies
have shown its considerable potential for cancer treatment, gene ther­
apy and cardiac tissue ablation, and its growing use in clinics attests to
its efficacy and safety [1]. Yet, electroporation-based procedures might
generate discomfort in patients, as they can induce muscle contraction
and pain. Strategies are thus being developed to alleviate these in­
conveniences: pharmacological approaches include the administration
of (local) anesthetics or muscle relaxing agents [2–4], and technical
approaches involve the development of different pulse parameters,
namely, the application of electric pulses delivered at high pulse repe­
tition rates [5,6]. The latter can reduce muscle contractions and asso­
ciated pain [7,8]. When using high repetition rate, protocol adjustments
are required to attain permeabilization efficacy comparable to protocols
with low repetition rate electroporation. Indeed, with high frequency
protocols, cancellation effect may occur, which is caused by short time
delay during microsecond or nanosecond bipolar pulses, resulting in the
cancellation of the positive pulse by the negative one [9,10].
Technically, “high frequency” electroporation protocols consist of
nanoseconds) or pulse bursts, applied at repetition rate ranging from
kHz to MHz, with field intensities ranging from hundreds of V/cm to
tens of kV/cm, respectively. The term “pulse repetition rate” is defined
as the number of electric pulses delivered per unit of time, expressed in
pulses per second (s− 1 or Hz). While the term “high frequency” is often
used in the literature to refer to a high pulse repetition rate, the wording
is somewhat inaccurate, because pulse frequency is a spectral charac­
teristic of a particular waveform, which is related to pulses width/length
and the rate at which the waveform changes polarity or oscillates. In this
review, we will use both terms, high repetition rate and “high fre­
quency”, keeping in mind these specificities. Since the literature does
not provide a formal boundary for “high frequency” or high repetition
rate electroporation protocols, we have elected to set this boundary at
repetition rates of 5 kHz and above. This value was chosen taking into
account previously published electroporation protocols that aimed at
reducing muscle contractions and repetition rates that most commer­
cially available generators used in electroporation procedures in clinical
practice are able to deliver [11].
With the prospect of “high frequency” electroporation protocols
being used in clinical practice, this review aims to summarize the cur­
rent state of knowledge on the subject. At first, we briefly overview the

* Corresponding authors.
E-mail addresses: muriel.golzio@ipbs.fr (M. Golzio), jelena.kolosnjaj-tabi@ipbs.fr (J. Kolosnjaj-Tabi).

https://doi.org/10.1016/j.bioelechem.2023.108629
Received 28 April 2023; Received in revised form 15 December 2023; Accepted 16 December 2023
Available online 21 December 2023
1567-5394/© 2023 Elsevier B.V. All rights reserved.
A. de Caro et al.
historical context of electric field use for therapies. We first describe
some physiological background regarding neuro and muscular stimu­
lation and tissue-induced inflammation, which occur upon exposure to
electric pulses, and may have an impact on pain. Then, after a short
description of technical progress on electroporators and electrode
design, we provide an overview of emerging “high frequency” electro­
poration protocols, applied together with anticancer agents (cisplatin,
bleomycin, doxorubicin, calcium) for electrochemotherapy (ECT), or
nucleic acids (such as DNA) for gene electrotransfer (GET), or electric
pulses applied as standalone treatment for tissue ablation that is known
as irreversible electroporation (IRE). Finally, we discuss the advantages
and limitations of these protocols for their respective therapeutic
applications.
2. Historical context
After the discovery of electricity, its effects on biological systems
intrigued numerous scientists. To mention just a few, Jean-Antoine
Nollet, an 18th century French physicist explored electrical phenom­
ena at the interface between physics and the living, and described,
among other phenomena, his “Electric boy” experiment. A boy was
suspended to the ceiling using insulating cords and was electrified,
causing the body to accumulate charge and thus attract objects or
generate sparks when a person approached [12]. In addition, Nollet
described that electric spark applied to human skin can induce the for­
mation of red spots, an effect which, according to our current knowl­
edge, could be caused either by irreversible electroporation or by an
electro-thermal burn, or a combination of both. At about the same
time, Luigi Galvani showed that electricity applied to a dead frog’s
spinal cord led to muscle twitching in the frog’s leg and published
several papers, including his major work “De viribus electricitatis in motu
Scheme 1. Timeline summarizing experiments at the interface of electric fields’ use and biological applications.
2
Bioelectrochemistry 156 (2024) 108629
musculari commentarius” (“Notes on the electrical forces in the move­
ment of muscles” [13]). Alessandro Volta’s invention of the electric
battery can be also mentioned, allowing the generation of a steady
electric current, which opened up new possibilities for electrical stim­
ulation of biological tissues. Later, Carlo Matteucci’s experiments
showed that electric fields could stimulate the heart and other muscles
[14] and Emil Du Bois-Reymond’s studies investigated the electrical
properties of nerves and showed that nerve impulses were electrical in
nature [15]. In 1872, Gabriel Lippmann’s invention of the capillary
electrometer allowed the measurement of very small electrical poten­
tials. In 1968, Sale and Hamilton showed that the lytic effects of electric
pulses on erythrocytes were not due to thermal effects but to an increase
of the transmembrane potential of the cells [16]. This process was later
described by Neumann and Rosenheck in 1972, showing that the per­
meabilization of the plasma membrane led to non-selective exchange
between intra- and extracellular media [17]. In 1982 Neumann et al.
then demonstrated that the application of an electric-field not only
destabilized the cell membrane, but could also introduce DNA into cells
[18]. Since then, electroporation has been finely optimized and widely
used for medical applications to deliver therapeutic agents and transfer
genes into cells or to ablate tissues.
Scheme 1 recapitulates the experiments conducted in the 18th and
19th centuries, which detailed macroscopic effects of electric fields on
the body and its components. The intimate interactions between the cell
membranes and the electric field were revealed in the second half of the
20th century, when electroporation flourished and ECT, GET and IRE
procedures were then developed (Schematic 1). The trends of the 21st
century converge towards minimizing the muscle stimulation in elec­
troporation procedures, for which high frequency electroporation pro­
tocols seem particularly promising for the reduction of the associated
pain.
A. de Caro et al.
2.1. Current electroporation protocols for medical applications
2.1.1. Electrochemotherapy
Electroporation-enhanced drug delivery has been extensively stud­
ied in the field of oncology for cutaneous and subcutaneous cancer
treatments [19,20] as well as for the treatment of deep-seated tumors
[21–23]. To induce reversible electroporation, which transiently de­
stabilizes cell membranes and allows a massive entry of hydrophilic
drugs, increasing their cytotoxicity, eight electric pulses are generally
applied (with electric field strength of 1000 V/cm, and pulse frequency
< 5 kHz). This corresponds to the electrochemotherapy procedure,
which was standardized in the framework of the European Standard
Operating Procedure on Electrochemotherapy (ESOPE) multicenter
trial. This approach is known as electrochemotherapy (ECT). Molecules
such as bleomycin [24], cisplatin [25], and calcium [26] are generally
used (Scheme 2.A).
2.1.2. Gene electrotherapy
Electroporation led to the successful transfer of non-viral genes for
gene therapies, vaccination or immunotherapies [27]. Electroporation-
enhanced gene transfer is more complex than the transfer of therapeu­
tic molecules. Indeed, due to the negative charge of the DNA, an elec­
trophoretic force must be engaged. In order to induce sufficient
electrophoretic force to anchor DNA to cell membranes, pulses of the
order of milliseconds and electric field intensities of the order of hun­
dreds of volts per centimeter were reported as the most efficient
[28–30]. To affect the nuclear membrane and the membrane of the
cellular organelles, the use of shorter pulses was proposed, of the order
of nanoseconds. The first studies enabled DNA electrotransfer using low
pulse repetition rate (1 or 10 pulses per second), with monopolar mil­
liseconds pulses [31,32] lasting hundreds of microseconds [33] or
nanopulses followed by one millisecond pulse [34]. In addition, the use
of bipolar pulses was studied for gene transfer, for which it was shown
that the inversion of polarity led to the interaction of DNA on both sides
of the cells facing the electrodes [35], when pulses were delivered at a
frequency of 1 Hz. To deepen the understanding of the key step of DNA/
membrane complex formation, our group investigated the dependence
of DNA/membrane interaction and gene expression on electric pulse
polarity and repetition frequency [36]. The results obtained [36]
revealed the existence of two classes of DNA/membrane interactions: (i)
a metastable DNA/membrane complex formation, from which DNA can
dislodge and return to the external medium and (ii) a stable DNA/
membrane complex formation, where DNA cannot be removed, even by
applying electric pulses of inversed polarity. Only DNA belonging to the
second class leads to effective gene expression. The life-time of DNA/
membrane complex formation is of the order of 1 s. This has to be taken
into account to improve protocols of electro-mediated gene delivery
[36] (Schematic 2A).
2.1.3. Irreversible electroporation
When electroporation is used to ablate tumors, about one hundred
pulses are used to permanently alter the cell membrane, leading to cell
death induced with the so-called irreversible electroporation (IRE). The
pulses used are in the kV/cm range, also referred to as high intensity
electric pulses [37–41]. This method is used in the clinic to ablate deep-
seated tumors, such as prostate tumors [41], pancreatic cancers [2] or
liver tumors [42]. In addition, irreversible electroporation can also be
used for ablation procedures of specific cardiac regions such as those
involved in arrhythmia [43,44] (Schematic 2A).
Generally, the pulses applied in standard ECT, gene electrotransfer or
in IRE are delivered with repetition rates of about 1 Hz [19,20,37,38]
(Schematic 2A). While very efficient, side effects such as pain sensation
or discomfort have been reported by patients (Table 1) leading to the
need for new protocol developments.
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Bioelectrochemistry 156 (2024) 108629
2.2. Emerging electroporation protocols
Classical electroporation protocols generate muscle contractions and
are associated with pain during and after electrical stimulation [45].
Muscle contractions may also cause electrodes displacement during the
treatment, and thus jeopardize the treatment’s outcome.
New protocols have been established, which limit or avoid pain
sensation and discomfort. Among these new protocols, some use
microsecond electrical pulses, applied at high pulse repetition rate (high
PRR), also called “high frequency” electroporation protocols (Schematic
2B). They can be used to deliver chemotherapeutics or to ablate tissues.
In ablation procedures, the term “high frequency irreversible electro­
poration” (H-FIRE) is often used [6,46].
Beside microsecond high PRR protocols, thanks to specific genera­
tors able to deliver high electric field intensities in the nanosecond time
range, it has been possible to push forward the exploration of the pa­
rameters with nanosecond pulsed electric field (nsPEF) [46–48]. These
nsPEF can lead to plasma membrane and intracellular cellular organelles
effects [47] and nsPEF protocols are being increasingly studied (as
recently reviewed by Ruiz-Fernandez et al. [48]), and have been trans­
lated to clinical trials for cancer treatments [49–51] and cardiac ablation
[43,44].
All these different electroporation protocols may involve different
types of cell death pathways (comprehensively reviewed by Batista
Napotnik et al. [52]). The necessity to optimize the electroporation
protocols and to understand the underlying mechanisms of excitation
and electroporation has thus emerged for the rational use of
electroporation-based approaches. “High frequency” electroporation
protocols therapeutic applications are further detailed in section 5.
3. Effects of pulsed electric fields on nerve and muscle: pain,
stimulation and electroporation
Pain is a very complex phenomenon involving sensitive and
emotional elements. The direct application of electric fields on the skin
may induce pain perception and pain sensation. Pain perception (noci­
ception) is a sensory phenomenon transmitted to the spinal cord and
brain by neurons in the form of an electrical signal. This is different from
pain sensation, which involves the interpretation of the electric signal by
the brain. As each human or animal is unique, signal processing may be
different from one to the other. In humans, pain perception is usually
assessed using a scale graduated from zero to ten where zero is no pain
and ten is an intolerable pain.
Some of the studies reporting pain sensation in patients treated by
electroporation are summarized in Table 1. Pain sensations were
recorded in patients treated with electrochemotherapy and irreversible
electroporation under local and regional anesthesia, and the reported
mean pain scores were all comprised between 1 and 5.
When electric pulses are applied to tissues, distinct phenomena can
induce pain because of short- or long-term effects.
3.1. Short-term effects
The application of an electric field may activate any nerve fiber, and
can induce nerve stimulation, nociception and neuro-muscular stimu­
lation, as well as it can cause electroporation of cells in the treated zone.
3.1.1. Nerve stimulation and nociception
Applied electric field induces membrane depolarization and a
concomitant transmission of a nerve impulse to the spinal cord and
cortical areas via sensory nerve fibers [66,67]. These nociceptive fibers
have free specialized endings denuded of myelin and are called noci­
ceptors [68]. Nociceptors release glutamate and substance P, which
activate neurons directly connected to the brain, where the signal is
processed. The modulation of pain is carried out by the activation of
specialized receptors (such as opioid receptors) with their specific
A. de Caro et al. Bioelectrochemistry 156 (2024) 108629

Scheme 2. Representation of current and emerging protocols for reversible and irreversible electroporation A) When the cell is submitted to an electric field and the
electric field intensity E is higher than a threshold Ep, the cell membrane undergoes either transient or irreversible permeabilization. The «reversible» approach is
used to deliver chemotherapeutics or genes into cells, while irreversible electroporation is used to kill cancer cells or to ablate abnormal tissues. Specific pulse
parameters are dedicated to different procedures, with monopolar micro- and millisecond pulses being most commonly used. B) High pulse repetition rate (PRR) also
known as “high frequency” electroporation protocols can be used to deliver chemotherapeutics or to ablate tissues (this technique is known as “high frequency
irreversible electroporation” (H-FIRE).

4
A. de Caro et al. Bioelectrochemistry 156 (2024) 108629

Table 1
Pain sensation in patients treated by electroporation.
Reference Condition Type of Number of Mean pain score Observations
electroporation patients (between 0 and 10)

Narayanan et al. 2012 Hepatocellular Carcinoma Irreversible 21 1.96 In 12 % of cases, patients complain of pain
[53] greater than 5
El Kamary et al. 2011 Healthy Reversible 10 3 and less Shooting pain, burning or tingling for 2
[54] patients
Li et al. 2016 [55] Pancreatic Carcinoma Irreversible 48 4.95 and 4.85 –
Quaglino et al. 2015 Different types of tumors Reversible 121 2.3 and 1.3 13 % of patients with moderate pain and 13
[56] % with severe pain
Gasbarrini et al. 2015 Metastatic spinal melanoma Reversible 1 2 –
[57]
Campana et al. 2015 Metastatic breast cancer Reversible 55 ≥3 Post ECT pain not negligible
[58]
Claussen et al. 2021 Cutaneous tumors/metastases Reversible 716 0.6 to 2.5 Patients with ulceration lesions reported
[59] severe pain
Matthiessen et al. Cutaneous breast cancer Reversible 17 – Main side effect of treatment was pain
2012 [60]
Kjenken et al. 2004 Healthy Reversible 6 – Tested voltage range was painful but
[61] tolerable
Zupanic et al. 2007 Healthy Reversible 40 – 1 Hz pulse protocol provokes unpleasant
[8] pain
Sorokin et al. 2017 Renal tumors Irreversible 21 2.4 Significant pain in the perioperative period
[62]
Mir et al. 1998 [63] Cutaneous and subcutaneous Reversible 50 – Some muscle contractions and pain during
malignant tumors procedure
Mpendo et al. 2020 DNA vaccine in healthy patients Reversible 45 – 8.9 % of unacceptable pain reported during
[64] electrical stimulation
Lalanda et al. 2023 Subcutaneous and cutaneous Reversible 14 – Main complication was transitory local pain
[65] Kaposi sarcoma

ligands [69].
3.1.2. Neuro-muscular stimulation
During electrical stimulation, other types of mechanosensory neu­
rons, which are skeletal muscle proprioceptors and mechanoceptors,
may also be activated [66]. These have a lower activation threshold than
nociceptive sensory nerve fibers [70]. Skeletal muscle contraction, upon
microsecond electric pulse itself, can cause discomfort or soreness.
When electric pulses are applied in the vicinity of the heart muscle, atrial
and ventricular flutter, irregular heartbeats and, in particular, ventric­
ular fibrillation may occur [71,72]. Thus, it is necessary to use electro­
cardiograms so as to apply such treatments in synchronization with the
heartbeat [71] and general anesthesia is required, particularly for IRE
[73]. Indeed, anesthetic agents, which cause neuromuscular blockage,
are highly recommended to ensure that muscle excitation is minimized,
especially in the case of treatments for pancreatic, kidney and liver
cancers [2].
3.1.3. Electroporation
When transmembrane potential reaches a critical threshold, gener­
ally between 200 and 300 mV, electroporation occurs [74]. If this
threshold value is not reached, electric pulses of micro- and millisecond
duration will only cause electrostimulation.
When nanosecond pulse bursts are applied at high repetition rate,
with the intervals between pulses typically equal or shorter than 1 µs,
stimulation can occur at paradoxically low electric field intensities,
because of the charge build-up, as described in detail elsewhere [75,76].
This approach, known as MHz compression, has been shown to be
equivalent to the stimulation with a single long pulse with a field
strength that equals the average amplitude of the pulse train, in the case
of nerve fibers [77].
However, it is important to note that under specific conditions (e.g.
sufficiently intense electric fields), when nanosecond pulses are applied,
electroporation can also occur before electrostimulation [78]. This is
because, in order to achieve (electro)stimulation, the membrane must
remain depolarized for at least 11 µs, which corresponds to the mini­
mum activation time of sodium channels [78]. As nanosecond pulses (or
nanosecond pulse bursts) last less than that duration, they can cause
membrane disruption (electroporation) before inducing electro­
stimulation. Once the cell is electroporated, the resting membrane po­
tential is lost and electrostimulation follows as a downstream effect
[79–81].
The ratio between the electroporation and stimulation thresholds is
known as the “safety factor”. The safety factor drops when the pulse
duration is decreased (e.g. nanosecond pulses) and when the membrane
time constant is low [82].
3.2. Long-term effects
Electric pulses can damage tissues through several mechanisms,
depending on the characteristics of the pulses and tissue properties.
Some of the primary mechanisms by which electric pulses can cause
tissue damage include: 1) Electroporation-related modification of cell
homeostasis (cell death, leakage of cellular components into extracel­
lular space), 2) Thermal effect (causing tissue burns, denaturation of
proteins, and cellular damage), 3) Electrochemical reactions (leading to
the production of toxic by-products, harming the cells).
3.2.1. Modification of cell homeostasis
Application of an electric field can induce different cell death
mechanisms such as apoptosis, necroptosis, necrosis or pyroptosis as
reviewed by Brock et al. [83]. As an example, millisecond pulses can
induce massive myofiber necrosis with 5 ms pulses of 400 V/cm,
delivered at 200 ms interval, followed by potent muscle regeneration,
characterized by substantial myoblast engraftment [84].
In addition, when the tissue is damaged, many chemical substances
are released and participate to the process of inflammation [85]. Among
these substances, the most notable are histamine, which is secreted by
leucocytes such as mastocytes and basophilic granulocyte present in the
skin, prostaglandins secreted by epidermis cells, bradykinin from cap­
illaries, substance P from peripheral sensory nerve endings. These
chemicals can induce nerve depolarization and transmission of nerve
impulses [86].

5
A. de Caro et al.
3.2.2. Thermal effect
Tissue heating resulting from electrical power dissipation can remain
insignificant when pulses of 10 µs are applied at frequencies below 100
Hz (the increase of temperature at the surface of the electrode in a re­
ported setting was only 0.36 ◦ C) [87]. As delivering pulses at a high
repetition rate provides less time for heat to dissipate, and utilizing bi­
polar pulses requires a higher energy input to achieve equivalent out­
comes, this leads to increased heat generation, which can result in tissue
damage. This was observed during application of irreversible electro­
poration, when increasing pulse repetition rate resulted in tissue burns
and cellular damage [88,89] (see also section 5.3).
3.2.3. Electrochemical reactions
The electrochemical damage directly correlates with the amount of
charge per surface unit of the electrode used to deliver the electrical
stimulus [90]. However, the buildup of electrochemical reactions
byproducts can be reversed by an opposite polarity waveform [91].
3.3. Pain management in electroporation protocols
Due to notable nerve stimulation, pain and associated short- and
long-term effects, strategies are currently being developed to alleviate
electroporation-related discomfort (Table 1). Pharmacological strategies
include the administration of general or local anesthetics, paralytic
agents [2–4] and analgesics [55]. Yet pharmacological approaches may
be detrimental (e.g. general anesthesia can be dangerous for elderly
patients; local anesthesia can be inefficient, can cause allergies or
paresthesia, to mention but a few potential complications). Technical
strategies therefore emerged, and involved the development of different
pulse parameters, namely, the application of electric pulses at high pulse
repetition rate, ranging from kHz to MHz.
When electroporation pulses are applied at a repetition frequency of
1 Hz, the patient perceives a contraction (and the concomitant
discomfort) for every pulse delivered. In order to decrease the number of
unpleasant sensations, Miklavcic’s group [5,7,92] ingeniously suggested
to increase the electric pulses frequency above 100 Hz, which corre­
sponds to the frequency of tetanic contraction. In this way, for pulses
administered at frequencies at or above 100 Hz, only one muscular
contraction is felt, despite the fact that multiple pulses had been
delivered.
By increasing the pulse repetition rate, as shown by Arena et al. [6],
who applied bipolar bursts of pulses to the rat brain, delivered at a
repetition rate of 250 kHz and above, no overt muscle contraction could
be detected in muscles at the cervicothoracic junction.
In addition, pain management was studied thanks to numerical
stimulations as a complement of in vivo studies. Numerical simulations
indicate that short (1 or 2 µs) bipolar pulses can ablate tissues in a large
region without triggering action potentials in nearby nerves, because
stimulation thresholds in bipolar pulses protocols are higher than the
ones in classical IRE protocols, where monopolar pulses last about 100
µs [93]. Experimental studies confirmed this assumption and showed
that short (ns-range) bipolar pulses applied at high repetition rate not
only reduced stimulation thresholds, but also increased ablation
thresholds [94].
4. Technical features of high frequency electroporation
protocols
While a commercially available electroporator (Cliniporator (IGEA,
Carpi, Italy)) was used in one of the pioneering studies applying pulses at
the repetition rate of 5 kHz [8], the development of fast-switching power
electronics has promoted the conception of more powerful generators
that have enabled biological experiments at much higher repetition rate.
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Bioelectrochemistry 156 (2024) 108629
4.1. Generators
The first prototypes of generators used in electroporation protocols
were able to deliver exponential decay pulses, used mainly for bacterial
transformation [95]. Square wave generators were subsequently
developed. Because the charge capacity was not powerful enough,
however, these two types of generators did not allow the application of
high frequency electroporation protocols. Over time, increasingly better
generators have become available for electroporation [96]: analog
generators (producing a continuously varying electrical signal, such as a
sine wave or a triangular wave, where the output waveform is a smooth,
continuous signal with no sharp transitions), square wave generators
(generating a signal that alternates between two voltage levels, typically
a high voltage and a low voltage, with a fixed frequency and duty cycle,
where the output waveform is a series of square-shaped pulses with
equal rise and fall times) and capacitor discharge generators (which use
a charged capacitor to discharge a high-energy pulse of electrical cur­
rent). These generators are usually used to generate pulses longer than 1
µs [96]. Square wave pulse generators, which are now most often used in
electroporation protocols, use fast power metal oxide silicon field effect
transistor (MOSFET) or insulated gate bipolar transistor (IGBT) as
switches. Different strategies can be used to obtain generators appro­
priate for high frequency electroporation, such as the adaptation of high
voltage MOSFET switches [6,97,98] or the use of pulse forming net­
works or resonant charging generators, such as the Blumlein generator,
which can generate high frequency output pulses [96]. While important
progress has been made in generator development in the last decades,
technological efforts mainly leaded to custom made, in-house pro­
totypes [6,98,99]. These generators have a better performance in terms
of voltage amplitude, waveform, repetition rate and pulse width
[98–101]. Generators are designed to deliver pulses at high repetition
rate (up to a few MHz), and to apply pulse durations from nanoseconds
to milliseconds. A generator with four independent MOSFET based Marx
modulators was also developed and allows applying symmetric and
asymmetric pulses delivered at rates up to 5 MHz [97]. Novickij and
colleagues developed a high-power generator (3 kV, 60 A) allowing
pulses of variable duration (100 ns to 1 ms) with predefined repetition
frequency (1 Hz to 3.5 MHz) [98]. Generators where the polarity of
pulses can be reversed, were reported by Zeng and colleagues, where
pulses between 500 ns and 5 µs could be generated with a maximum
frequency of 500 kHz [100]. A different high repetition rate and high
voltage electroporator with bipolar pulses was also developed and tested
[99]. This electroporator is able to generate 4 kV pulses, with theoretical
131 A maximal current and 200 ns minimal pulse duration, the maximal
pulse repetition rate is 2 MHz and the burst maximal repetition rate is 1
MHz [99]. In order to avoid the uncontrolled discharge of the capacitor,
Folprecht et al. designed a high-frequency irreversible electroporation
(H-FIRE) generator, which is able to deliver bursts with variable length
from 50 to 150 µs where the gap between bursts can be set from 0.5 to
1.5 s. Pulse frequency can vary from 65 to 470 kHz and the output
voltage is controlled in two ranges from 0 to 1.3 kV or from 0 to 2.5 kV
[101].
In addition, frozen wave generation technology [102] can be used to
create high voltage generators applicable to electroporation protocols
[103]. These generators can apply adjustable delay of 5 to 200 ns be­
tween two nanosecond pulses [103,104].
4.2. Electrodes
Specific electrode designs have been suggested to minimize pain and
improve the precision of electrode placement. These could be used both
for high and low repetition rate electroporation protocols (Fig. 1), and
are highlighted in this review with the prospect to alleviate muscle
contraction and pain, and thus improve electroporation protocols.
The length, diameter, and distance between needle electrodes can be
reduced to minimize the volume of muscle submitted to the electric
A. de Caro et al. Bioelectrochemistry 156 (2024) 108629

Fig. 1. Examples of novel electrode designs applicable to standard and “high frequency” irreversible electroporation protocols (H-FIRE). These electrodes tackle two
principal issues: 1) They allow to limit the zone of application of high intensity electric pulses (A), and 2) They aim to avoid the insertion of multiple parallel needle
electrodes (B and C). A) Left: tips of conventional needle electrodes; right: insulated needle electrodes, painted with an insulating parylene coating on the outer
surface, with their respective electric field simulation results upon application of 5 μs pulses at 2000 V (shown in bottom panels of Fig. 3A), figure adapted from Yao
et al., 2017 [105]; B) Single needle dual-electrode device, which allows a single insertion, adapted from O’Brien et al., 2019 [106]. C) Top left: Tips of conventional
needle electrodes, which are generally used in IRE and are difficult to insert into organs in a parallel position, Top right: Array of single insertion electrode tines and a
distal grounding pad, which could potentially be used in H-FIRE protocols; Bottom left: Predicted ablation volume and corresponding conductivity map for two
electrodes, occurring at a specific IRE protocol; Bottom right: Predicted ablation volume and corresponding conductivity map for two electrodes, occurring at a
specific H-FIRE protocol; panel adapted from Sano et al., 2018 [107].

field, and consequently limit the number of muscle fibers that are trig­
gered by the pulse, and thus decrease pain.
Yao and colleagues described needle electrodes in which the tips are
partially coated with parylene, to confine the electric field distribution.
The zone treated is thus reduced between the two inner sides of the
electrodes. Consistently, less intense muscle contraction and a slightly
smaller ablation area was observed under their exposure conditions (see
Table 3 for protocol details), when a high pulse repetition rate IRE
protocol was used. [105]. This could, however, be applicable to IRE
protocols both at low and high repetition rate.
Alternatively, O’Brien and colleagues suggested the use of single
needle dual-electrode devices, which can enable a more accurate
placement and alignment and minimize the extent of ablated zones, to
avoid damage to underlying structures [106].
Sano et al. suggested the use of a single insertion device with an array
of expandable electrode tines and a distal grounding pad, which can, in
comparison to standard electrode geometry, produce a substantially
larger ablation volume at a given voltage [107]. The single multi-tined
electrode would be easier to place than multiple needle electrodes.
Indeed, the tines in the expandable tine electrode are not parallel, but
they have the advantage to be applied with one single insertion and the
induced ablation zone is bell-shaped and not oval, as is the case in
conventional IRE, which requires the placement of multiple needle
electrodes.
Moreover, whatever type of electrodes used, Vinzintin et al. showed
that application of irreversible electroporation with high frequency
protocols results in lower metal release from electrodes [108].
5. “High frequency” electroporation protocols
5.1. “High frequency” electroporation protocols applied with cytotoxic
drugs for chemotherapy enhancement
The main ambition of high frequency electroporation protocols is to
reduce muscle contractions and pain. Studies performed by different
groups showed that this goal could be fulfilled [5,7,8]. Yet, in order to be
translated to patients, the efficiency of electropermeabilization should
not be compromised and should be comparable to classical electropo­
ration protocols. In this section, we describe studies that evaluated high
electric pulse repetition rates electroporation with the perspective to
improve electrochemotherapy. The studies can be grouped to three
types of protocols used: microsecond monopolar pulses, nanosecond
monopolar pulses and bipolar pulses and are summarized in Table 2.
In order to attain the efficacy that is comparable to electric pulses
defined in ESOPE, high repetition rate (typically 5 – 8.3 kHz) protocols
with monopolar µs pulses were first investigated. In comparison to low
repetition rate (1 Hz) electroporation protocols, higher repetition rate
protocols (>10 kHz) employing 100 µs monopolar pulses showed similar
efficiencies. A greater number of pulses compensated for efficiency
when the pulse duration was reduced (e.g., 30 µs) or the intensity was
decreased (50 – 150 V/cm). This was first documented in 2002 by
Pucihar and colleagues. Studying high-frequency electroporation within
in vitro DC3F fibroblasts, they showed that 30 µs pulses applied at fre­
quencies up to 8.3 kHz were effective for cell permeabilization [92], but
a higher number of pulses was applied (N = 26). Shankayi and col­
leagues later also explored high frequency (in range 4–6 kHz) and low
intensity (50–150 V/cm) protocols with 100 µs monopolar pulses, where
the high number of pulses (N = 4000) resulted in increased uptake and
concomitant increased cytotoxicity of bleomycin in K562 lymphoblast
cells and MIA PaCa-2 human pancreatic cancer cell line. [109] (Table 2).
In another study, monopolar pulses of 100 µs with high repetition (5
kHz) and low intensity (50–150 V/cm) were also used in vivo and
showed tumor regression with increasing number of pulses (N = 4000).
A protocol of 4000 monopolar pulses of 100 µs at 5 kHz and field values
of 70, 100, 150 V/cm was tested on invasive ductal mice carcinoma
treated with bleomycin. The results obtained by this study indicated
maximal inhibition of tumor growth at 70 V/cm [110]. These parame­
ters were subsequently used in a clinical setting to treat a woman with
cervical lymph node metastasis of breast cancer, resulting in reducing
the tumor size and palliating the symptoms of the patient, while other
approaches such as chemotherapy, radiotherapy, and surgery had been
inefficient and hazardous [111]. Using the SA-1 mouse fibrosarcoma
model, a protocol of 100 µs monopolar pulses with a lower number of
pulses (N = 8, 1 Hz) and higher field intensity (1300 V/cm) was also
shown to result in tumor regression. Micklavcic and colleagues observed
after 26 days following the treatment, a decrease in tumor volume
compared to untreated mice, treated with bleomycin alone or electro­
poration alone [5]. The work of Sersa and colleagues revealed a similar
anti-tumor effect between 1 Hz and 5 kHz with optimal drug doses, but a
better efficiency of the 1 Hz protocol with use of sub-optimal drug doses.
Drug concentration during high frequency electrochemotherapy treat­
ment should be considered [112] (Table 2).

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Table 2
Summary of high frequency electrochemotherapy-like electroporation protocols.
Reference Study Type of pulses Pulse Field Cells/cancer type/tissue Main observation
repetition intensity
rate

Pucihar et al. 2002 In vitro 26 pulses of 30 µs 1 Hz to 8.3 400 to 2000 Chinese Hamster Fibroblasts Similar cells permeabilization for high and low
[92] (monopolar) kHz V/cm DC3F frequency pulses
Miklavcic et al. In vivo 8 pulses of 100 µs 1 Hz to 5 kHz 1300 V/cm Fibrosarcoma SA-1 tumor Inhibition of tumor growth with high frequency
2005 [5] (monopolar) model
Sersa et al. 2010 In vivo 8 pulses of 100 µs 1 Hz or 5 kHz 1300 V/cm Fibrosarcoma SA-1 tumor Treatment with 5 kHz led to identical antitumor
[112] (monopolar) model effectiveness as to 1 Hz pulses with optimal drug
doses
Shankayi et al. In vivo 4000 pulses of 100 µs 5 kHz 70 to 150 V/ Invasive ductal carcinoma Inhibition of tumor growth
2011 [110] (monopolar) cm
Shankayi et al. In vitro 4000 pulses of 100 µs 4 to 6 kHz 50 to 150 V/ Erythroleukemia K562 and Reversible effect of permeabilization observed
2014 [109] (monopolar) cm Pancreatic carcinoma Mia with low field intensity
Paca-2
Mofid et al. 2016 In vivo 4000 pulses of 100 µs 5 kHz 70 V/cm Cervical lymph Node Successful in reducing the size and palliating the
[111] (monopolar) metastasis of breast cancer symptoms
Novickij et al, In vitro 10 pulses of 200 ns 1 Hz to 1 MHz 10,000 to CHO Better electroporation in the MHz pulse repetition
2018 [113] (monopolar) 14000 V/cm frequency (PRF) region compared to the
conventional low frequency protocols
Baleviciute et al. In vivo 200 pulses of 200 or 1 kHz to 1 3500 V/cm LLC1 mouse lung carcinoma nsECT prolonged survival of the treated mice and
2022 [115] 700 ns MHz cells induced immune system changes
(monopolar)
Radzeviciute et al. In vitro 10 burst of 100 or 10 or 100 6000 to LLC1 mouse lung carcinoma Nanopulses result in comparable results as ESOPE
2022 [114] 500 ns kHz, 1 MHz 10000 V/cm cells in term of cell cytotoxicity
(monopolar)
Spugnini et al. In vitro 8 pulses of 50–10-50 8.3 kHz 1300 V/cm Lung cancer A549 Higher efficacy compared to previous ECT
2014 [118] and in µs treatment with high frequency pulses
vivo (bipolar)
Sweeney et al. In vitro 200 burst of 50 pulses 100 or 250 750 to 1250 Ovary cells Lower effective membrane permeabilization
2016 [116] of 1–1–1 or 1–4-1 µs kHz V/cm CHO-K1
(bipolar)
Scuderi et al. 2019 In vitro 8 burst of 50 pulses of 250 kHz 500 to 5000 Skin melanoma Need to apply high electric field to obtain similar
[117] 1–1–1 µs V/cm B16-F1 cytotoxicity as ECT
(bipolar)

Monopolar pulses were also studied in the nanosecond range, where
researchers applied varying numbers of pulses, from a dozen to a hun­
dred, delivered at pulse repetition rate up to 1 MHz and electric field
intensities of about one order of magnitude higher than the one specified
in the ESOPE protocols. These types of protocols for electro­
chemotherapy applications have proven to be efficient for in vitro cells
viability reduction and in vivo tumor regression. Increasing the pulse
repetition rate allows the charge on the membrane to accumulate and
result in electroporation when as little as 10 pulses are applied. Novickij
and colleagues showed that ten 200 ns pulses with high electric field
intensities (10–14 kV/cm) led to efficient permeabilization of CHO cells
in vitro [113]. Moreover, assays were also performed on lung carcinoma
cells, where it was shown that cytotoxicity with bleomycin and doxo­
rubicin provoked by ten nanosecond pulses (100 to 500 ns at 6–10 kV/
cm) was comparable to those induced by ESOPE protocol (8 pulses of
100 µs at 1.3 kV/cm) [114]. Nanosecond pulses compression in the MHz
range improved electrochemotherapy [113,114]. When the relaxation
of the transmembrane potential is longer than the delay between
consequent pulses, the charges accumulate at the membrane, which
results in electroporation [113]. An in vivo study using mice bearing
solid tumors induced with lung cancer cells showed that it is possible to
treat tumors efficiently with high-frequency electrochemotherapy using
pulses in the nanosecond range: 200 pulses of 200 ns at 1 kHz or 700 ns
at 1 MHz at 3.5 kV/cm [115]. The authors reported better survival of
treated mice and highlighted that the nanosecond pulses also induced
significant changes in the immune system such as upregulation of PD 1
expression on splenic CD4 + Tr1 cells or expansion increase of splenic
CD8 + T cells, CD4 + CD8 + T cells, encouraging future use of nano­
second electrochemotherapy combined with immunotherapy [115]
(Table 2).
Comparison between application of conventional 100 µs monopolar
pulses and 1 µs bipolar high frequency pulses revealed that 1 µs bipolar
pulses induced less cellular permeabilization compared to monopolar
pulses [116]. Nevertheless, if electric field intensities were increased (to
about twice the value of monopolar pulses protocols- circa 1250 V/cm),
the permeabilization was considerably improved [116]. Another study
performed on murine melanoma cells with cisplatin and protocols of 50
bipolar pulses of 1 µs with 1 µs pulse delay, revealed comparable cell
mortality rates between 1 Hz monopolar and 250 kHz bipolar pulses
only if the field value was increased in bipolar pulse protocols (between
0,5 and 5 kV/cm) [117]. Longer bipolar pulses (50 µs) were studied in
vitro in the A549 lung cancer cell model showing a significant increase in
apoptosis when tumor cells were treated with cisplatin after electropo­
ration. One in vivo assay showed that biphasic waveforms are much
better tolerated by animals and were effective in slowing tumor growth
with field intensity of 1300 V/cm, confirming the effectiveness of the
new parameters in increasing the uptake of drugs by cancer cells [118].
(Table 2).
To summarize, in vivo studies exploring the potential of high repe­
tition rate electroporation protocols combined with chemotherapeutics
have provided promising results, but much remains to be explored. In
the context of murine solid tumors treatment, but also in a case of cer­
vical lymph node metastasis of breast cancer in a human patient, these
protocols inhibited tumor growth and improved survival rates. These
findings suggest the potential for combining high repetition rate elec­
troporation with chemotherapy to treat tumors, opening avenues for
further research and clinical applications. These types of treatments are
promising alternatives to 1 Hz protocols, in particular regarding
reduction of muscle contractions and pain (Table 1).
5.2. “High frequency” electroporation protocols for gene transfer to
mammalian cells
To efficiently induce gene transfer with current electroporation

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Table 3
Studies evaluating high frequency irreversible electroporation.
Reference Type of pulses Pulse repetition rate Field intensity Cells/cancer type/ Main observations
tissue

Fesmire et al. In vitro 25 pulses of 2–1-2 µs ~ 166 kHz Not available Glioblastoma Tissue temperature can increase
2020 [89] and in (bipolar) (authors mentioned U118-MG ablation area
sillico voltages: 500 to
1250 V)
Sano et al. In vitro 100 bursts of 2–1-2 µs ~ 166 kHz 400 to 1500 V/cm Glioblastoma H-FIRE have the potential to
2018c [88] (bipolar) U-118 MG create larger ablations than IRE
with temperature increases
Rolong et al. In vitro A burst of 25 cycles of 1–2-1 µs ~ 71 to 166 kHz 250 to 350 V/cm Mouse ovarian surface H-FIRE induced death of tumor-
2017 [126] or 2–2–2 µs or 2–5-2 epithelial (MOSE) initiating cells
(bipolar) cancer model
Ivey et al. In vitro 50 bursts of 0.5–2-0.5 µs 200 kHz ~ 2500 V/cm Glioma stem cells GBM- GBM-10 were more sensible to H-
2019 [125] (bipolar) 10 FIRE than normal astrocytes:
selectivity
Wasson et al. In vitro 80 bursts of 1–1–1 to 10–1-10 µs 45 to 250 kHz 2000 V/cm Malignant glioma cells Enhancement of H-FIRE ablations
2020 [136] (bipolar) U251 using adjuvant calcium
Siddiqui et al. In vivo 100 to 300 pulses of 2–5-2 µs ~ 71 kHz 1500 V/cm Human liver Necrotic reproductible ablation
2016 [46] (bipolar) damage
Arena et al. In vivo 180 bursts of 50 or 100 pulses of 250 or 500 kHz 1000 to 4000 V/cm Rat brain Ablation lesions in brain tissue
2011 [6] 1 or 2 µs
(bipolar)
Dong et al. In vivo 2 to 100 bursts of 1–1 to 50–50 10 to 500 kHz 1000 to 3000 V/cm Rabbit liver Bursts with larger pulse width can
2018a µs ablate the liver tissues effectively
[128] (bipolar) with less extent of muscle
contractions
Heller et al. In vivo 60 or 20 bursts of 5–5 µs 100 or 150 kHz 1100 V/cm Rat beating heart Attenuation of muscle stimulation
2021 [127] (bipolar) and myocardial damage
comparable to monophasic 100 µs
pulses
Dong et al. In vivo 250 bursts of 20 pulses of 5–10- 33.3 kHz 1500 V/cm Human prostate cancer Validation of feasibility of tumor
2018b 5 µs ablation with H-FIRE in clinic
[129] (bipolar)
Wang et al. In vivo Not described Not described Not described Human prostate cancer Rate of prostate cancer cells
2022 [130] decreased with H-FIRE treatment
Latouche et al. In vivo 10 to 100 bursts of 0.5–0.5 to 250 kHz to 1 MHz 500 V/cm Intracranial H-FIRE can be used for brain
2018 [131] 2–2 µs meningiomas tumor
(bipolar)
Partridge In vivo 300 bursts of 2–5-2 µs ~ 71 kHz 1000 V/cm Hepatocellular H-FIRE results in predictable
et al. 2020 (bipolar) carcinoma ablation zone with recruitment of
[132] lymphocytes
Ringel-Scaia In vitro 200 bursts of 2–5-2 µs ~ 71 kHz 500 to 4000 V/cm Mammary tumor Tumor ablation with H-FIRE
et al. 2019 and in (bipolar) 4 T1 activates immune system
[133] vivo
Yao et al. In vivo 90 bursts of 50 or 20 or 10 or 4 5 kHz when 1 800 to 2000 V/cm Healthy rabbit liver H-FIRE causes less muscle
2017 [105] or 2 or 1 pulse(s) applied with 2 monopolar pulse, contraction and slightly smaller
µs delay (when > 1 pulse), or 9.6 to 125 kHz ablation area than IRE pulses used
bipolar (when > 1 pulse) when bursts of for comparison
bipolar pulses are
applied

protocols, tens of pulses ranging from 100 µs to a few millisecond, with
field values below 1 kV/cm trigger a process which unfolds through the
following stages [28]: 1) The electric pulses induce the electroporation
of the plasma membrane at the sides facing the electrodes, with the
negatively charged DNAs electrophoretically migrating towards the
plasma membrane facing the cathode side. 2) The accumulated plasmids
interact with the membrane. 3) Plasmids become effectively confined
with the plasma membrane in specific locations. 4) Subsequently, these
plasmids translocate to the cytoplasmic side of the plasma membrane
within a timeframe of a few minutes. Eventually, DNA, either aggre­
gated or in a released state, undergoes gradual diffusion towards the
nucleus [28].
Amidst the wealth of research experiments exploring gene delivery
with low frequency electroporation protocols, a recent study [119]
suggested the use of nanosecond monopolar pulses. These 200 ns long
pulses, applied at 10–––18 kV/cm at pulse repetition rates between 1 Hz
and 1 MHz [119] introduce a novel dimension to the field of gene
electrotransfer, with their distinctive attributes expanding conventional
insights and inviting for a closer examination of nanosecond pulses
potential. The authors of the study [119], Ruzgys et al., observed an
increase in gene expression rate of 17 % by increasing the frequency
from 100 kHz to 1 MHz, while only slightly decreasing cell viability.
Indeed, as nanosecond pulses are of short duration, electrophoretic drag
is less likely to be involved in their approach. Yet, as described in pre­
vious sections of this review, membrane electroporation does occur. In
addition to electroporation, the authors [119] hypothesized that the
increased average pore size and altered polarizability of the cells and/or
biomolecules during high frequency bursts might be accountable for the
positive gene electrotransfer outcome. Their findings therefore suggest
the potential existence of distinctions within the electrotransfection
mechanisms between low and high pulse repetition rates electropora­
tion protocols.
In a study, conducted by Potocnik and collaborators [120], 2 µs bi­
polar pulses applied with a delay of 2 µs and field value of about 1 kV/
cm were compared to standard ECT parameters (8 pulses, 100 µs, 1 Hz,
1600 V/cm) and gene electro transfer parameters (8 x 5 ms, 1 Hz, 500 V/
cm). Similar transfection rates were obtained for high frequency elec­
troporation protocols in comparison to monopolar pulses, but the
number of DNA copies was significantly lower in the high frequency
pulses protocol [120]. For the high repetition rate bipolar pulses

9
A. de Caro et al.
protocol, the lack of electrophoretic drag could be compensated with
higher pDNA concentrations, while this effect was not observed when
pDNA concentrations were increased during standard low repetition
rate protocols.
Despite the scarcity of studies in this specific research domain, the
scope for considerable progress remains noteworthy, suggesting the
potential presence of additional mechanisms involved. One plausible
rationale could be the influence of nanosecond pulses on the nuclear
membrane [121]. Experimental evidence demonstrates that nanosecond
pulses lead to reduced nuclear membrane distinctness [121], and it is
tempting to suppose that this might potentially increase its permeability
to DNA and consequently enhance gene expression. However, the val­
idity of this hypothesis requires further substantiation through empirical
validation and the relevance of the novel mechanism will have to be
tested in 3D cellular models as well as in vivo, where DNA will have to
migrate into deep layers to reach target cells, prior to its expression.
5.3. High frequency irreversible electroporation (H-FIRE) protocols for
tissue ablation
Tissue ablation by irreversible electroporation (IRE) relies on the
application of high intensity electric field to cause cell death. While IRE
protocols are considered as “non-thermal”, temperature does rise during
the procedure and can sensitize cells for electroporation [122,123]. In
addition, the procedure causes considerable muscle contraction, which
is currently minimized by pharmacological approaches. To decrease
muscle contractions induced in IRE procedures, high frequency irre­
versible electroporation (H-FIRE) emerged and consists in the applica­
tion of about 100 bipolar pulses of short duration (from nanoseconds to
microseconds).
A study conducted on potato tissue showed that pulses applied in H-
FIRE (several burst of 1 to 50 µs) required more energy to create lesions
equivalent to that caused by IRE (80 pulses of 100 µs, 1 Hz), pointing to
the need to optimize H-FIRE protocols [124]. As more energy is required
for H-FIRE protocols compared to IRE procedures, temperature eleva­
tion is expected. Using protocols based on H-FIRE 2 µs bipolar pulses,
Sano et al. reported temperature increases of approximately 16 ◦ C,
immediately after treatment which was more marked when the repeti­
tion rate increased [88]. Subsequently, a study proved that the effec­
tiveness of H-FIRE treatments on 3D in vitro tumor models of human
glioblastoma and pancreatic cancer cells was directly correlated with
the temperature of the culture medium. Authors emphasized that the
increase in initial medium temperature from 2 ◦ C to 37 ◦ C followed by
H-FIRE (25 bipolar pulses of 2 µs) resulted in a decrease in lethal electric
field threshold from approximately 1000 to 500 V/cm and observed an
139 % increase in ablated zone’s size [89] (Table 3).
H-FIRE protocols also appear to selectively ablate malignant cells,
while sparing some non-malignant cells. Three dimensionally arranged
glioma cells were more susceptible to H-FIRE treatment, while astro­
cytes were affected to a lesser degree. This indicates an opportunity to
preferentially target malignant cells [125]. H-FIRE treatment with bi­
polar pulses of 1 to 2 µs provoked the death of tumor-initiating cells
(TICs) embedded in collagen matrix, suggesting the treatment may
selectively target TICs and malignant late-stage cells, while sparing the
non-malignant mouse ovarian surface epithelial cells [126] (Table 3).
H-FIRE treatments have been studied in vivo across various animal
models (in rats, pigs, dogs) and in clinical trials in humans. The research
suggests the effectiveness of H-FIRE in ablating tumors while mini­
mizing unwanted muscle contractions and cardiac interference, show­
casing the potential of H-FIRE to selectively ablate tumors with short
bipolar pulses, whilst also leading to immune system stimulation. In vivo
studies began in 2011 with works on the quantification of pain felt
during the application of H-FIRE protocols on rat brains [6]. Muscle
contractions were quantified by accelerometers placed at the cervico­
thoracic junction. Ablation efficiency was also evaluated by histology.
The main conclusion of this study was that no apparent signs of muscle
10
Bioelectrochemistry 156 (2024) 108629
contraction were observed at the cervicothoracic junction during H-
FIRE treatment at 250 kHz or 500 kHz for H-FIRE protocols that pro­
duced ablative lesions in the brain [6]. Later, studies performed on
porcine livers with protocols of 50 pulses of 2 µs each with a delay of 5 µs
at a voltage of 2250 V (inter electrode diameter of 3 cm) [46] recorded
minor muscle twitch in one case and tissues necrosis in all cases. Post-
treatment autopsies revealed that the ablation areas were reproducible
and the resulting cell death was primarily apoptotic when less than 200
pulses were delivered. The authors also concluded that H-FIRE treat­
ments can be used for tissue ablation without the use of paralytic agents
and without cardiac gating [46]. On in vivo beating hearts, attenuation
of muscle stimulation was observed with protocols with 100 or 150 kHz
with efficient myocardial damage [127]. By testing increasing pulse
numbers (from 2 to 100) and durations between 1 and 50 µs, research on
rabbit liver concluded on the need to apply high field values (2000 V/
cm) for effective ablation with 5 µs bipolar pulses [128]. This protocol
led to similar results as IRE (90 pulses of 100 µs at 1000 V/cm, 1 Hz)
[128] (Table 3).
Moreover, clinical trials on humans and animals were conducted
with H-FIRE bipolar pulse protocols where efficiency of tumor ablation
and low muscle contractions were the main observations. The first
human trial was described by Dong et al in 2018. The study was con­
ducted on 40 men aged 51 to 80 with prostate cancer. The patients were
treated with protocols of 20 pulses of 5 µs at 1500 V/cm, which did not
cause any apparent muscle contraction when the patients concomitantly
received a mild dose of a muscle relaxant. In addition, they observed an
effective ablation while preserving the function of the urethra. This first
trial validated the use of short bipolar pulses for prostate tumor ablation
[129] and was later confirmed by Wang et al. [130]. Studies conducted
on dogs suffering from intracranial meningioma reported the efficacy of
10 to 100 burst of 0.5–0.5 to 2–2 µs bipolar pulses [131]. Among three
dogs treated, one dog had non-uniform ablation areas with some
remaining viable tumor cells. However, this study showed the effec­
tiveness of this method on brain tumors in treated animals [131].
Another example of H-FIRE protocols in dogs used 2–2 µs bipolar pulses
applied to 3 dogs with hepatocellular carcinomas without the addition
of paralytic agents and without cardiac synchronization [132]. This
study showed that a predictable ablation volume can be generated by H-
FIRE, as well as resulting in lymphocyte infiltration [132]. These two
studies on dogs concluded about the efficiency of bipolar pulses between
0.5 and 2 µs to ablate tumor with minimal muscle contractions and
negligible cardiac interference (Table 3).
As in IRE protocols, H-FIRE appear to preserve organ functions and
induce the recruitment of immune cells into the tumor and its micro­
environment. A study showed a correlation between H-FIRE-induced
cell death and increased expression of genes involved in inflammation,
cytotoxicity and leukocyte recruitment [133] (Table 3). These findings
are comparable with observations highlighted in standard IRE protocols,
which are known for their potential to elicit an immune response, as
recently reviewed [134]. In addition, beyond discomfort and pain, IRE
protocols might be characterized by incomplete ablation and suscepti­
bility to recurrence [135], whilst to the best of our knowledge, there is
currently no available data regarding recurrence assessment following
H-FIRE treatment..
Combination of H-FIRE treatment with calcium was studied to
enhance lethal effect. In fact, it has been shown that calcium electro­
poration led to a high tumor response with cell necrosis associated with
acute and severe ATP depletion [26]. Using an H-FIRE protocol of 10 µs
pulses coupled with 1 mM of calcium chloride, a cell mortality threshold
was comparable to that obtained with IRE (80 pulses of 100 µs, 1 Hz),
without adding calcium. This study suggests that calcium increases H-
FIRE-induced ablation with short pulse durations (1–10 µs) on glioma
cells [136] (Table 3).
A. de Caro et al.
5.4. High frequency irreversible electroporation (H-FIRE) protocols for
cardiac tissue ablation
Cardiac ablation is an established treatment modality for patients
with cardiac arrhythmias. Electroporation was studied for cardiac
ablation in particular to limit the use of thermal methods causing
damage to the heart. Irreversible electroporation protocols with fre­
quencies about 1 to 5 Hz have been shown to be effective against cardiac
arrhythmias by accurately targeting heart cells without generating
thermal effects [43,44] and were already tested in different clinical
trials, the PULSED AF Pivotal Trial being one example [137]. This
technique, also known as “pulsed field ablation” showed encouraging
results in a clinical trial involving 300 patients [137]. Tissue ablation
was shown to be effective at 1 year in 66.2 % of patients with parox­
ysmal atrial fibrillation and 55.1 % (95 % CI, 46.7 to 62.7) of patients
with persistent atrial fibrillation.
To go further, researchers have studied the effect of H-FIRE protocols
for cardiac tissue ablation. The efficacy of high frequency bipolar pulses
(167 kHz) using H-FIRE was described, indicating that deeper lesions
were observed with asymmetric pulses compared to symmetric ones
[138]. By applying protocols of 20 bipolar pulses in 100 µs bursts at 150
kHz, other authors observed specific lesions on myocardia tissue without
affecting the carotid artery and sciatic nerve [139].
6. Specific features of short bipolar pulses used in high pulse
repetition rate electroporation protocols: Permeabilization
cancellation
Permeabilization cancellation or bipolar cancellation is a phenome­
non that occurs with short bipolar pulses (in the nanosecond to a few
microseconds range) [140] in which the positive and negative phases of
a bipolar electrical pulse cancel each other, resulting in a weaker or
ineffective membrane permeabilization. This phenomenon was
observed both in individual cells [10] and in multicellular spheroids
[141] subjected to high intensity short bipolar electric pulses. This ef­
fect, not present if monopolar pulses are used, can be avoided by the use
of asymmetric bipolar pulses or by applying a sufficient time pulse delay
between the positive and negative phases of a bipolar electrical pulse.
Bipolar cancellation was reported first in low frequency regimes (1 to
5 Hz). Ibey et al. showed that bipolar pulses of short duration (of the
order of nanoseconds) were less effective in terms of cell per­
meabilization and in causing cell mortality compared to monopolar
pulses [10]. This was confirmed and explained with in vitro tests in
which intracellular calcium and cell viability were quantified using bi­
polar pulses of 60–60 ns and 300–300 ns. In both cases, they noticed
almost no effects, even after delivering twice as much energy as in
monopolar pulses [9]. This study was the first to postulate a cancellation
effect of the first phase of pulses by the application of a second opposite
polarity phase [9]. This observation was corroborated in later studies,
indicating that the second phase of bipolar pulses determines the effi­
ciency of electroporation with pulses of a few nanoseconds [142,143].
Bipolar cancellation was also present at intervals of more than 50 μs,
beyond the membrane discharge time [144].
The cancellation effect was subsequently also observed upon the
application of high-rate pulses spanning the frequencies from kHz to
THz. By modelling, Guo and colleagues revealed nanopores radius lower
with bipolar pulses compared to unipolar ones, with protocols between
10 and 100 MHz [145]. Using molecular dynamics simulation, Tang and
colleagues showed that the inversion of the electrical force of the bipolar
pulses in the range of THz delayed the movements of water molecules
and increased the time of formation of pores. According to their sug­
gestions, these phenomena would cause bipolar cancellation [140]. In
order to optimize electroporation using nanosecond pulses, researchers
sought to obtain electrical parameters that cause the strongest cancel­
lation effect [146]. They found that adding a second phase at 600 ns of
50 % lower intensity reduces the internalization of molecules by more
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Bioelectrochemistry 156 (2024) 108629
than 95 % at a high repetition (833 kHz) [146].
By increasing interpulse delay between bipolar pulses, cell per­
meabilization can be restored. This effect was studied also by Orlacchio
and collaborators, who created and tested high repetition rate and high
intensity nanosecond pulses (100 MHz, 11.5 MV/m) and demonstrated
that bipolar cancellation occurred for delays up to 30 ns and decreased
as a function of the interphase interval [104]. Vizintin et al. studied
different interpulse duration (0.5 to 10,000 µs) and concluded that
longer interphase and interpulse delay results in lower cell survival
[108].
In addition to the important role of the delay between bipolar
nanosecond pulses in modulating cancellation, with a 10-microsecond
delay between opposite polarity pulses being necessary to avoid bipo­
lar cancellation [147], Valdez et al. have shown that asymmetric bipolar
pulses could also avoid this phenomenon. A positive pulse of 300 ns
immediately followed by a negative pulse of 900 ns led to a total loss of
bipolar cancellation [148].
The occurrence of bipolar cancellation was also observed upon
application of high repetition rate bipolar microsecond pulses on CHO-
K1 in different electroporation buffers [149]. Polajzer et al. applied eight
bursts of 1–10 µs long pulses with inter-phase delays of 0.5 µs – 10 ms. In
permeability assays, cancellation effect was not observed in low-
conductivity buffer. Conversely, cancellation effect was observed in
viability experiments in all of the tested electroporation buffers. In their
setting, cancellation effect depended on inter-phase delay as well as on
pulse duration, i.e. longer pulses and longer interphase delay caused less
pronounced cancellation effect.
Importantly, Pakhomov and collaborators showed that it is possible
to cancel the cancellation effect of bipolar pulses, this phenomenon was
denominated the “CANCAN effect” [150,151]. The authors showed that
a unipolar pulse can be recovered by synchronizing and superimposing
bipolar pulses of a few nanoseconds.
The underlying reasons that would explain bipolar cancellation
remain uncertain, though the following mechanisms have been hy­
pothesized: assisted membrane discharge, reversed electrophoretic ion
transport, two-step oxidation of membrane phospholipids, localized
charging and discharging events across the membrane, reversed elon­
gation forces due to electrodeformation, and nanopore occlusion.
Indeed, from the cell’s perspective, bipolar cancellation is beneficial as it
protects the cell from damage caused by bipolar or biphasic pulses. The
phenomenon also helps reducing neuromuscular stimulation [94,152].
7. Discussion
Standard electroporation protocols allow effective cell per­
meabilization that promotes the diffusion of molecules through cells
(cytotoxics [24,25] or genes [27]) as well as cell death (in the context of
tissue ablation [37]), but they trigger adverse reactions. Electrical
stimulation causes muscle contraction and pain which triggers patient
discomfort (Table 1) and electrode displacement in some cases. The use
of muscle relaxants, anesthetics and analgesics [2–4] represents a partial
solution for pain management (Table 1). As an alternative, electrical
protocols with high pulse repetition rate have been considered in order
to decrease discomfort and pain [5,8].
While ECT and IRE protocol parameters (Schematic 2A) are well
defined, the current literature does not provide clear boundaries/spec­
ifications for high frequency electroporation protocols. Through studies
cited, electroporation protocols used in clinics to deliver chemothera­
peutics, the pulses are often delivered at repetition rates of 5 kHz
(Table 2), for which only one muscular contraction is felt. Therefore, in
this review, we set the boundaries as the ones using monopolar and
bipolar electric pulses delivered at repetition rate of 5 kHz and above.
The comparison between monopolar and bipolar pulses led us to
conclude that to minimize nerve stimulation, potential muscle damage
or electrochemical reactions, the utilization of bipolar pulses is partic­
ularly attractive (Schematic 4). Using microsecond monopolar pulses
A. de Caro et al. Bioelectrochemistry 156 (2024) 108629

administered at high frequency, the patient perceives one muscular Table 4

contraction but can still experience discomfort or pain [5,112]. The Comparison of IRE and H-FIRE protocols characteristics.
rationale behind employing bipolar pulses relies on their ability to IRE H-FIRE
reverse action potentials and thus suppress nerve excitation, and is
Voltage 1000 – 3000 V Voltage 3000 – 10000 V
achieved when an electric pulse is followed by an opposing polarity Monopolar Bipolar
pulse in a short time delay [91,153]. Decreasing pulse duration and Pulse duration 50 – 100 µs, Pulse duration < 10 µs, multiple bursts
conversely applying higher field intensity [115,125,133,136] or number generally a hundred pulses applied, generally a few thousand pulses
of pulses [109,110] decreases nerve stimulation. However, it is impor­ applied applied
Low repetition rate, typically 1 Hz Pulse bursts delivered at 1 Hz, typical pulse
tant to take into account that short bipolar pulses are less efficient for repetition rate within the burst 100 – 500 kHz
electroporation due to the cancellation phenomenon [125,142], which Considered as non-thermal method Higher heating than IRE, as higher energy
can occur but can be avoided by increasing interpulse delay required
[104,108,149] or by applying asymmetric bipolar pulses [147,148]. Intense muscle contractions Less muscle contractions
Low repetition rate allows heat dissipation between pulses, mini­
mizing thermal effects. To avoid thermal effect, decrease of repetition
lead to complete and long-lasting recovery of human patients. As we
rate and split pulse trains into short bursts with longer intervals to allow
summarized in previous sections, in order to perform tissue ablation
cooling should be considered. However, the electrical protocols also
without significant muscle contraction, bipolar pulses should be favored
depend on the desired applications. Monopolar pulses can be privileged
(Table 3), with pulse duration shorter than 10 µs, and pulses applied at
over bipolar pulses to avoid temperature increase [88,89] (Scheme 3).
repetition rates up to 1 MHz, but most often within hundreds of kHz. The
Regarding the therapeutic perspectives, in the context of high repe­
use of bipolar pulses allowed to elicit action potentials to a smaller de­
tition rate electrochemotherapy, monopolar pulses will need to be
gree, as the stimulation threshold in excitable cells is higher when bi­
favored, as has already been established and learned from standard ECT
polar pulses are used, while the ablation efficacy is maintained.
protocols (Table 2). However, a balance should be found in pulse pa­
rameters, and careful attention should be paid to the administration of
8. Conclusion
optimal drug doses in order to counteract possible decreases in mem­
brane permeability [112]. In gene electrotransfer approaches, high
Collectively, the results from the studies suggest that high repetition
repetition rate nanosecond pulses now appear to bring into play alter­
rate electroporation protocols may have future clinical applicability.
native options and mechanisms [119]. Nevertheless, it is yet to be
These protocols could be fine-tuned to yield outcomes comparable to
determined whether the effectiveness of such protocols will extend to
traditional low repetition rate protocols. Moreover, they hold the po­
more intricate tissue structures and deeper layers of closely neighboring
tential to minimize pain and discomfort, especially in the context of
cells. Standard protocols with 100 µs to few milliseconds pulse at low
tumor ablation procedures.
pulse repetition are somewhat unrivaled in case of nucleic acids de­
livery, which are negatively charged and require long pulses enabling
CRediT authorship contribution statement
the electrophoretic drag towards the cells. Finally, careful handling will
be required in H-FIRE protocols. High repetition rate pulse delivery
Alexia de Caro: . Franck Talmont: Investigation, Writing – original
gives little time for heat dissipation, and as bipolar pulses require more
draft. Marie-Pierre Rols: Conceptualization, Funding acquisition,
energy to produce the same effects, more tissue heating could be
Writing – review & editing. Muriel Golzio: Conceptualization, Formal
observed than in standard IRE protocols. Comparison between IRE and
analysis, Funding acquisition, Investigation, Supervision, Validation,
H-FIRE are summarized in Table 4.
Writing – original draft, Writing – review & editing. Jelena Kolosnjaj-
In addition, experiments yet have to show that H-FIRE protocols will
Tabi: Conceptualization, Formal analysis, Investigation, Supervision,

Scheme 3. Electroporation protocols in correlation with effects regarding pain, electrochemical reaction and heating. Protocols were resumed into five groups: low
frequency monopolar pulses (1 Hz), low frequency bipolar pulses (1 Hz), high frequency microsecond monopolar pulses (5–10 kHz), high frequency nanosecond
monopolar pulses (10 kHz to 1 MHz) and high frequency micro and nanosecond bipolar pulses (10 kHz to 1 MHz). These protocols are organized according to their
effects. Monopolar long (millisecond) pulses at low frequency results in to higher pain and electrochemical effects than high frequency, while thermal effect is low.
On the contrary, high frequency ns-µs bipolar pulses lead to reduced pain and electrochemical effect, while thermal effect becomes more prominent.

12
A. de Caro et al.
Validation, Writing – original draft, Writing – review & editing.
Declaration of competing interest
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.
Data availability
Data will be made available on request.
Acknowledgments
The authors would like to acknowledge the support from “Speed­
pores grant number 238252” and “Plan France Relance”, and support
from the grant Plan Cancer, Projet MECI, n◦21CM119-00. We are
grateful and kindly thank Mr Jean-Baptiste Leroy with whom we dis­
cussed technical aspects and Dr Etienne Joly and Mr Nicolas Mattei who
proof-read the manuscript. We sincerely thank the reviewers for their
relevant comments.
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