increased permeability of intestinal epithelial monolayers mediated by electroporation

www.elsevier.com/locate/jconrel

Journal of Controlled Releas

Increased permeability of intestinal epithelial monolayers

mediated by electroporation

Esi B. Ghartey-Tagoea, Jeremy S. Morganb, Andrew S. Neishc,*, Mark R. Prausnitza,b,*

aThe Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory University,

Georgia Institute of Technology, Atlanta, GA 30332, USAbSchool of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA 30332, USA

cDepartment of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA

Received 22 September 2004; accepted 17 November 2004

Available online 18 December 2004

Abstract

This study assessed whether electroporation enhances transport across intact intestinal epithelial monolayers that mimic the

intestinal epithelium. Confluent Caco-2 monolayers were exposed to electroporation pulses and then monitored over time for

transepithelial transport of calcein, a small fluorescent tracer, or fluorescein-labeled bovine serum albumin, a large protein.

Cumulative transport of both molecules across the monolayers increased significantly (up to 34-fold) after electroporation and

depended on electroporation voltage and pulse length and on molecular size. Increased transport was accompanied by a

decrease in the transepithelial electrical resistance of the monolayers. Further analysis of these results suggests that the increase

in transport observed after electroporation is due, at least in part, to the killing of a small fraction of cells, which increased

transport across bleakyQ dead cells that remained adherent and increased transport through small, temporary holes left by dead

cells that detached, but appeared to reseal within minutes by monolayer restitution. These findings could form the basis for the

development of electroporation as a clinical tool to increase intestinal permeability and, thereby, increase the absorption of

poorly absorbed drugs.

D 2004 Elsevier B.V. All rights reserved.

Keywords: Electroporation; Permeability; Intestinal epithelium; Caco-2; Transepithelial transport

0168-3659/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.jconrel.2004.11.021

* Corresponding authors. M.R. Prausnitz is to be contacted at

Georgia Institute of Technology, School of Chemical and Bio-

molecular Engineering, 311 Ferst Drive, NW Atlanta, GA 30332-

0100, USA. Tel.: +1 404 894 5135; fax: +1 404 894 2291. A.S.

Neish, Department of Pathology and Laboratory Medicine, Emory

University School of Medicine, 615 Michaels St., Atlanta, GA

30322, USA. Tel.: +1 404 727 8545; fax: +1 404 727 8538.

E-mail addresses: [email protected] (A.S. Neish)8

[email protected] (M.R. Prausnitz).

1. Introduction

Oral administration is the most common method of

drug delivery used today. Drugs given in this fashion

must be absorbed across the gastrointestinal barrier

before entering the systemic circulation and acting on

their target [1]. While many drugs are absorbed well,

e 103 (2005) 177–190

E.B. Ghartey-Tagoe et al. / Journal of Controlled Release 103 (2005) 177–190178

others may have poor oral bioavailability because of

first pass metabolism in the GI tract or liver, or poor

absorption across the intestinal epithelial barrier [2].

This is especially true for hydrophilic drugs and

macromolecules, such as proteins. To address this

problem, the physical characteristics of a drug can be

modified, e.g., charge, polarity, lipid solubility,

enzymatic stability, etc., or the drug can be encapsu-

lated within a polymeric or lipid-based vehicle to aid

absorption and prevent degradation [3].

As an alternative to modifying the drug or its

formulation to increase absorption, the barrier func-

tion of the intestinal epithelium itself can be altered by

chemical or physical methods. Several studies have

been conducted to evaluate chemical enhancers,

which aid absorption by increasing transcellular and/

or paracellular transport across the epithelial barrier

[4]. Iontophoresis has also been shown to increase

transport across intestinal epithelium, in part by

physically disrupting the tight junctions between cells

[5,6].

This study proposes to show that electroporation

can similarly enhance transport of molecules across

the epithelium. Electroporation involves the applica-

tion of short electric pulses to transiently permeabilize

cellular membranes [7] and has been used extensively

to promote gene uptake and expression in vitro [8], as

well as for clinical treatment of some types of cancer

in vivo [9]. Unlike iontophoresis, which is believed to

act primarily by providing an electrical driving force

to transport molecules across existing pathways

within a barrier, electroporation acts by transiently

disrupting the barrier and driving molecules through

the disruptions.

Previous experiments have shown that electro-

poration can be used to uniformly deliver membrane

impermeant molecules into in vitro epithelial mono-

layers that mimic the intestinal epithelium [10]. The

present study was conducted to determine whether, in

addition to transporting molecules into cells of the

monolayer, electroporation could also increase trans-

port across the cell layer. To answer this question,

intact intestinal epithelial monolayers were electro-

porated at various conditions with either a small

molecule, calcein, or a macromolecular protein,

bovine serum albumin (BSA), and monitored for

changes in transepithelial permeability. The Caco-2

intestinal epithelial cell line was used for this study

because it is the most widely used cell line for drug

absorption studies [11] and because transport across

these cells has been shown to correlate fairly well with

in vivo intestinal permeability [12–14].

2. Materials and methods

2.1. Cell and monolayer culture

Caco-2 cells (American Type Culture Collection,

Manassas, VA) were cultured as described previously

[10]. Briefly, cells were harvested and seeded onto

collagen-coated Transwell microporous cell culture

inserts with 0.4 Am pores (Corning Costar, Acton,

MA) and then cultured in Dulbecco’s Modified

Eagle’s medium (DMEM) supplemented with 10%

(v/v) heat-inactivated fetal bovine serum, 100 IU/ml

penicillin, 100 Ag/ml streptomycin, 2 mM l-gluta-

mine, 10 mM N-[2-Hydroxyethyl]piperazine-N’[2-

ethanesulfonic acid] (HEPES; Mediatech, Herndon,

VA), and 0.1 mM non-essential amino acids (unless

stated otherwise, media and supplements were

obtained from Invitrogen, Carlsbad, CA). Monolayers

were incubated in a 5% CO2, 37 8C environment and

allowed to remain in culture for 21–28 days. Growth

medium was replaced approximately every 48 h.

2.2. Transepithelial resistance measurements

To ensure monolayer integrity, the transepithelial

electrical resistances (TEER) of the monolayers were

measured before and during each experiment using a

Millicell ERS apparatus (Millipore, Bedford,MA). The

resistances of cell-free Transwell inserts were sub-

tracted from the total resistances and then multiplied by

the filter growth area (4.7 cm2) to obtain TEER values

for the monolayers alone. Measurements of approxi-

mately 500 V cm2 were considered normal.

2.3. Molecular transport experiments

Caco-2 monolayers were rinsed and equilibrated

for 30 min at 37 8C in Hanks’ Balanced Salt Solution

with calcium and magnesium (HBSS+) supplemented

with 25 mM HEPES buffer. Transport across the

monolayers was measured using calcein (623 Da, 0.6

nm radius) and fluorescein-labeled BSA (66,000 Da,

E.B. Ghartey-Tagoe et al. / Journal of Controlled Release 103 (2005) 177–190 179

3.5 nm radius) (Molecular Probes, Eugene, OR), both

of which are membrane-impermeant and fluoresce

green when excited at 488 nm. The markers were

dissolved in 1.5 ml HBSS+ for a final solution

concentration of either 10 AM or 100 AM, which

was then added to the apical side of the monolayers.

An appropriate number of six-well plates containing 2

ml of warmed HBSS+ in each well were kept ready

for each experiment.

2.3.1. Measuring permeability after electroporation

Caco-2 cells were electroporated as monolayers as

described previously [10]. Briefly, monolayers incu-

bated with the fluorescent marker solution on the

apical side were placed in an adherent-cell cuvette

with parallel, 4-mm gap, aluminum electrodes (InSitu

Electroporation System, Thermo Hybaid, Middlesex,

UK). The basal side of the monolayer was bathed in 3

ml of HEPES-buffered, serum-free DMEM. Expo-

nential-decay pulses were applied to the monolayers

at room temperature using a high voltage pulser (BTX

ElectroCell Manipulator 600, Harvard Apparatus,

Holliston, MA). An oscilloscope (HP54062B, Hewlett

Packard, Colorado Springs, CO) was used to measure

the applied voltages and pulse lengths.

Electroporation was initially carried out using a

single, 50 V–1 ms, bmildQ pulse or a single, 50 V–10

ms, bmoderateQ pulse. To determine whether perme-

ability could be increased even further, ten 50 V–1 ms

pulses were also delivered to the monolayers. Pre-

vious studies indicated that this condition has an effect

similar to a single 50 V–10 ms pulse (total exposure

time is 10 ms in both cases) [10], but allows more

time for diffusion of the markers across the cells

between pulses.

Immediately after electroporation, monolayer

inserts were transferred to a six-well plate containing

HBSS+. Marker transport in the apical to basal

direction was monitored by transferring each insert to

a fresh well of HBSS+ every 30 min over the course of

3 h to maintain sink conditions. With the exception of

the time required to transfer the inserts and perform

electroporation, monolayers were maintained at 37 8Con an orbital plate shaker (~90 rpm; RotoMix Model

M50825; Barnstead Thermolyne, Dubuqe, IA).

In the first set of experiments, unelectroporated

control monolayers were monitored for permeability

alongside the electroporated monolayers. However,

the monolayer-to-monolayer variability in baseline

permeability made it difficult to compare the two

sets of monolayers. To address this issue, each

monolayer served as its own control in subsequent

experiments, i.e., the baseline permeability for each

monolayer was monitored by transferring the insert

to a new well of HBSS+ at 30 min intervals for 3 h

prior to electroporation.

2.3.2. Sample collection and analysis

Samples of the apical donor solution (50 Al) werecollected at times t=0 and 3 h for the baseline

monitoring and at times t=30 s and 3 h for the post-

electroporation monitoring. Samples of the basal

receiver solutions (1 ml) in each well after each 30

min interval were also collected for analysis. The

transepithelial resistance of each monolayer was

measured every hour for the 3 h prior to and after

electroporation.

The apical and basal solutions were analyzed for

calcein or BSA concentration using a fluorescence

plate reader (FluoStar Galaxy, BMG Technologies,

Offenburg, Germany). Apical samples were diluted

1:1000 so that the fluorescence would not saturate the

reader. One hundred microliters of each sample were

dispensed into opaque, black, 96-well plates (Costar

#3944) along with known concentrations of the

markers to make a standard calibration curve. Plates

were analyzed using an excitation wavelength of 485

nm and an emission of 520 nm.

2.3.3. Calculating transepithelial permeability

The apparent permeabilities of Caco-2 monolayers

to calcein and BSA, Papp (cm/s), were calculated

according the following equation based on Fick’s Law

of Diffusion [15]:

Papp ¼dQ

dt� 1

AC0

ð1Þ

where dQ/dt is the rate at which the molecule of

interest appears in the receiver solution (mol/s), C0 is

the initial concentration of the donor solution (mol/

ml), and A is the growth area of the monolayer (4.7

cm2). The appearance rate was calculated by plotting

the cumulative number of moles of calcein or BSA in

the samples collected versus time and determining the

slope of the resulting line.


2.4. Evaluating paracellular permeability

A comparison was made between the permeabilities

of electroporated monolayers and those of monolayers

with tight junctions opened by calcium depletion.

Since calcium plays a significant role in maintaining

tight junctions [16,17], Caco-2 monolayers were

incubated in HBSS without Ca2+ (HBSS-). To main-

tain sink conditions, monolayers were transferred to

fresh wells of HBSS- at 5 min intervals for the period

of time required for the transepithelial resistance to

drop to approximately half of initial values (up to 40

min). The intervals and total incubation time were

much shorter in this case to minimize the risk of

irreparable damage to monolayer integrity caused by

incubating the monolayers without calcium for too

long. During these experiments, the transport of

calcein (10 AM apical donor concentration) across

the monolayers was monitored. Monolayers incubated

with 10 AM calcein in HBSS+ (i.e., with Ca2+) were

used as controls.

2.5. Delineating barriers to transport

The permeability values reported here are termed

apparent permeabilities (Papp) because they are not

just the permeabilities of the monolayer, but also

include the permeabilities of other barriers to trans-

port. The aqueous boundary layers (ABL), the

collagen matrix used to coat the filter, and the filter

support itself can all impede transport. Thus, it was

necessary to determine to what extent these additional

barriers to transport contributed to the permeabilities

obtained. To facilitate this analysis, the permeabilities

of empty filters and collagen-coated filters to calcein

and BSA were measured experimentally using the

same technique described above (Section 2.3).

If each of the transport barriers are considered to be

resistances (R) in series, then

Rtotal ¼ RABL þ Rmonolayer þ Rcollagen þ Rfilter: ð2Þ

Permeability, P, which is defined as the inverse of

resistance, can then be calculated as

1

Papp

¼ 1

PABL

þ 1

Pmonolayer

þ 1

Pcollagen

þ 1

Pfilter

ð3Þ

where Papp is the experimentally measured perme-

ability. To find the actual permeability of the mono-

layer alone, Pmonolayer, the values of each of the other

terms in the equation were determined using the

theoretical filter permeability [15] and experimentally

measured permeabilities of empty and collagen-coated

filters to yield Pfilter, PABL and Pcollagen, respectively.

When the resistance (1/P) of each barrier, i.e.,

ABL, filter, collagen, and monolayer, was divided by

the total resistance (1/Papp), the results showed that

ABL resistance was negligible and that the filter

support and collagen each contributed less than 1% of

the resistance to calcein and BSA transport across

unelectroporated monolayers (data not shown). For

electroporated monolayers, the filter and collagen

combined accounted for ~12% of the total resistance

to calcein transport and ~2% of the resistance to BSA

transport. Based on these results, it can be concluded

that the monolayer itself was the dominant barrier to

transport.

2.6. Statistical analysis

For all of the graphs presented in this study, each

data point represents the mean of at least three

replicates. Error bars are the standard deviations of

the means (SD). When a comparison between two or

more means was required, a Student’s t-test or a one-

way analysis of variance with a 95% level of

confidence (ANOVA, a=0.05) was used where appro-

priate. A p-valueb0.05 was considered to indicate

statistical significance.

3. Results

3.1. Increase in transepithelial transport due to

electroporation

In initial experiments, confluent Caco-2 mono-

layers incubated in 100 AM calcein were treated with

a bmildQ (50 V–1 ms–1 pulse) electroporation

condition or one of two bmoderateQ (50 V–10 ms–1

pulse and 50 V–1 ms–10 pulses) electroporation

conditions. The single pulse conditions were previ-

ously shown to cause reversible changes in monolayer

integrity that were restored within 6 h and 24 h,

respectively [10]. After electroporation, the rate of


transepithelial transport of calcein was monitored over

time and the cumulative amount delivered after 3 h

was determined.

Fig. 1 shows that electroporation increased calcein

transport across confluent Caco-2 monolayers as a

function of electroporation conditions ( p=0.0002).

When compared to unelectroporated controls, calcein

transport across electroporated monolayers increased

1.5-fold ( p=0.01), 2.5-fold ( p=0.07) and 19-fold

( p=0.01) for the three conditions tested. The corre-

sponding permeabilities of the electroporated mono-

laye r s showed a s imi la r dependence on

electroporation conditions ( pb0.05), resulting in a

1.4-, 1.8-, and 12-fold increase over the permeabilities

of the unelectroporated monolayers.

It is interesting to note that the two conditions

classified as bmoderateQ each had very different

effects on monolayer permeability. The first condition

applied a single pulse of 10 ms duration, whereas the

second condition applied ten pulses each of 1 ms

duration. These two conditions were both classified as

bmoderateQ because both have the same total exposure

time (10 ms) and previous studies showed that they

Fig. 1. Cumulative transport of calcein ( ) depended on the electroporatio

monolayers increased 1.5- and 2.5-fold over controls after treatment with

electroporation conditions, respectively. Calcein transport was significant

exposure time as the moderate pulse (50 V–1 ms–10 pulses, 15 s inter-p

permeabilities of the monolayers (o) showed a similar dependence on ele

calcein and monitored for 3 h after electroporation. Unelectroporated mon

both induced similar levels of intracellular delivery to

monolayer cells and resulted in similar levels of cell

viability [10]. In contrast, this study shows that the

multiple pulse protocol increased calcein transport

7.5-fold more than the single pulse condition

( p=0.002) (Fig. 1). It can be hypothesized that widely

spaced, multiple pulses are advantageous because

they provide time for diffusion across the transiently

permeabilized monolayer between pulses. As a result,

all subsequent experiments were carried out using the

multiple pulse protocol.

3.2. Dependence of transepithelial transport on

molecular size

To determine the dependence of transport on

molecule size, Caco-2 monolayers incubated with 10

AM apical solutions of either calcein (MW=623 Da,

radius=0.6 nm) or FITC-labeled BSA (MW=66,000

Da, radius=3.5 nm) were electroporated with ten, 50

V–1 ms pulses. Fig. 2 shows that cumulative transport

across electroporated monolayers was increased 10-

fold for calcein ( p=0.000) and 34-fold for BSA

n condition applied ( p=0.0002). Transport across confluent Caco-2

mild (50 V–1 ms–1 pulse) and moderate (50 V–10 ms–1 pulse)

ly higher after treatment with a condition that had the same total

ulse spacing), but resulted in a 19-fold increase over controls. The

ctroporation conditions. Each monolayer was treated with 100 AMolayers served as controls [n=3–5].

Fig. 2. Comparison of cumulative transport of calcein and BSA for unelectroporated (n) and electroporated ( ) Caco-2 monolayers. Calcein

transport increased 10-fold ( pb0.0001) after electroporation and BSA transport increased 34-fold ( p=0.13). Calcein transport was almost 9 and

3 times higher than BSA accumulation for unelectroporated and electroporated monolayers, respectively. Permeabilities of Caco-2 monolayers

(o) showed a similar dependence on molecule size. Monolayers served as their own unelectroporated controls. They were incubated with 10 AMof either calcein or BSA and monitored for baseline transport prior to being treated with a moderate electroporation condition (50 V–1 ms–10

pulses) [n=6–9].

Fig. 3. Transepithelial electrical resistance (TEER) of Caco-2

monolayers was monitored during transport studies. Resistance

was measured hourly before (n) and after ( ) electroporation a

moderate conditions (50 V–1 ms–10 pulses). Prior to electro-

poration, monolayers maintained TEER values close to 500 V cm2

Post-electroporation resistances were approximately 30% lower

than pre-electroporation resistance and remained relatively constan

( pN0.05). Previous studies have shown that TEER values return to

initial values within approximately 1 day after electroporation a

similar conditions [10] [n=18].


( p=0.13) relative to pre-electroporation controls.

Thus, the relative enhancement of transport was

greater for the macromolecule. On an absolute basis,

however, cumulative transport of calcein was three

times greater than BSA transport across electroporated

monolayers ( p=0.04). Again, the corresponding

monolayer permeabilities increased after electropora-

tion and showed a dependence on molecule size

( pb0.05).

3.3. Decrease in TEER of monolayers after

electroporation

As a companion to measurements of transepithelial

transport of molecules, the transepithelial electrical

resistances (TEER) of monolayers before and after

electroporation were also measured. As shown in Fig.

3, TEER was approximately constant at a level of

450–500 V cm2 ( p=0.83) before electroporation.

After electroporation, TEER dropped by ~30% and

remained approximately constant at 300–350 V cm2

( p=0.62). This post-electroporation drop in resistance

was found to be statistically significant ( pb0.0001).

Consistent with the molecular transport data (Figs. 1

and 2), these TEER data indicate that monolayer

t

.

t

t


barrier properties were reduced by electroporation and

remained that way for at least 3 h. Previous measure-

ments over longer time periods after electroporation of

monolayers with a single pulse of the same total

exposure time (and similar effects on cell viability)

showed that TEER returns to pre-electroporation

values within 24 h [10]. These data indicate that

electroporation creates a long-lived increase in trans-

epithelial permeability that could subsequently be

reversed.

Fig. 4. The raw data for calcein (A) and BSA (B) transport is shown

Electroporation took place at time t=0 h. Cumulative calcein and BSA trans

for most monolayers. However, in a few monolayers (solid black symbo

explained by transient detachment of cells from the monolayer, which wer

results in Fig. 2 were obtained from these data.

3.4. Kinetics of transepithelial transport due to

electroporation

Fig. 4 shows the cumulative transport of calcein

(A) and BSA (B) over time for the individual Caco-2

monolayers used to determine the total cumulative

transport plotted in Fig. 2. Before electroporation, the

rates of transport for calcein and BSA were very low.

After electroporation, the transport rates increased

significantly and, for most monolayers, the cumu-

over time before and after electroporation of Caco-2 monolayers.

port after electroporation increased approximately linearly over time

ls), there were sudden increases in transport (arrows) that could be

e rapidly repaired by restitution (see text). The cumulative transport


lative amount of each marker increased linearly over

time. For a few monolayers, however, there were

sudden increases in transport (indicated by heavy

black arrows) that lasted for just one sample time

interval (V30 min). These events do not appear to be

artifacts; possible reasons for this behavior are

discussed below. In Figs. 1 and 2, permeability values

were calculated based on rates of transport only

during linear increases in accumulation, whereas

cumulative transport was calculated based on all

transport during linear increases and sudden jumps.

3.5. Identification of pathways for increased trans-

epithelial transport

The results reported here show that electroporation

can increase the permeability of intestinal epithelial

monolayers, but the exact mechanism by which

transport increased is not clear. Based on known

mechanisms of electroporation in other cellular

systems, electroporation is believed to cause a

transient rearrangement of lipid bilayer structure in

the cell membrane, thereby increasing membrane

permeability, which can in turn have secondary effects

on cell function, including cell death [7].

Guided by this understanding, Fig. 5 illustrates

three potential pathways via which transport can occur

after electroporation. (1) Paracellular transport

through the tight junctions between cells is the

primary transport route for hydrophilic molecules

across intact epithelium [18] and could be enhanced

as a secondary effect of electroporation. (2) Although

transcellular transport across the cell interior is not a

common route of transport across intact epithelium for

hydrophilic molecules, electroporation’s primary

effect of increasing cell membrane permeability could

make this route available. (3) Finally, since electro-

poration can cause cell death, dead cells could lift off

Fig. 5. Illustration of three pathways that could contribute to the

increase in transport observed after electroporation. Paracellular

(between cells), transcellular (across cells), and transmonolayer

(through holes in monolayer) transport could each play a role.

the membrane and leave gaps through which transport

could occur by transmonolayer transport. Any, or all,

of these pathways could play a role in the increased

permeability observed after electroporation.

3.5.1. Paracellular transport

To determine whether increased paracellular trans-

port may contribute to the increase in permeability

observed after electroporation, permeability of elec-

troporated monolayers was compared to that of

monolayers incubated in calcium-depleted media to

open their tight junctions. Extracellular calcium plays

an important role in the formation and maintenance of

tight junctions and calcium depletion has been found

to decrease epithelial resistance and increase perme-

ability due to contraction of the cytoskeletal network

and subsequent opening of tight junctions [16,17].

As illustrated in Fig. 6, when Caco-2 monolayers

were incubated in calcium-free HBSS (HBSS-), the

paracellular permeability increased almost 6-fold

when compared to monolayers incubated in calcium-

supplemented HBSS+. This increase in permeability

was accompanied by a sharp (50–60%) decrease in

transepithelial resistance (data not shown). Control

monolayers incubated in HBSS+ maintained their

resistance for the duration of the experiments. When

the permeability of calcium-depleted monolayers is

compared to the permeability of electroporated mono-

layers, there is no significant difference ( p=0.60). The

same is true for the cumulative transport of calcein 40

min after calcium depletion and electroporation

( p=0.11). This suggests that the increased monolayer

permeability caused by electroporation could be

explained by paracellular transport through tight

junctions opened to an extent similar to that caused

by calcium depletion.

Although the magnitude of increased epithelial

permeability caused by electroporation and calcium-

depletion are similar, the kinetics are different. As

shown in other studies in which tight junctions were

disrupted by various means, including calcium deple-

tion, the permeability and TEER of Caco-2 mono-

layers returned to baseline within 1 h of being restored

to normal conditions [6,17,19]. Similar recovery

results were obtained in this study when calcium

was re-added to the monolayers (data not shown). In

contrast, electroporation’s effects on permeability

(Fig. 4) and TEER (Fig. 3) lasted for at least 3 h.

Fig. 6. Paracellular permeability of Caco-2 monolayers (o) with opened tight junctions was not significantly different from electroporated

monolayers ( p=0.60). Comparison of the cumulative transport of calcein ( ) 40 min after calcium depletion and electroporation also showed no

significant difference ( p=0.11). This suggests that electroporation could similarly disrupt the tight junctions between cells and that increased

paracellular transport could play a role in the increased permeability observed after electroporation. However, as discussed in the text, the

kinetics of tight junction resealing suggest otherwise. Caco-2 monolayers were incubated in calcium-free HBSS� to open tight junctions.

Monolayers were treated with moderate electroporation (50 V–1 ms–10 pulses) and 10 AM calcein. Total calcein accumulation 40 min after

electroporation was interpolated from the data [n=4–9].


This could suggest that electroporation opens tight

junctions by a mechanism that takes longer to recover.

Alternatively, the slower recovery kinetics could

indicate that electroporation does not increase epithe-

lial permeability via paracellular transport, and that

either the transcellular or transmonolayer mechanism

may be responsible.

3.5.2. Transcellular transport

In contrast to transport between cells, electro-

poration could increase transport across cells via a

pathway through nanometer-sized holes in the cell

membranes and traversing the cytosol. This transport

could occur through reversible electropores, which are

known to have a lifetime of up to 1 min in other

cellular systems [7]. Moreover, most transport, espe-

cially of macromolecules, is believed to occur by

electrically mediated transport during the electro-

poration pulse. Thus, transcellular transport through

reversible electropores is expected to occur predom-

inantly during and immediately after electroporation

of a monolayer.

Based on this expectation, the data are not

consistent with transcellular transport through rever-

sible electropores. As shown in Fig. 4, the rate of

transport during the first 30 min after electroporation

is generally no greater than rates of transport at later

times. To further assess the contribution of transport at

the time of pulsing, additional experiments were

performed to measure transepithelial transport within

30 s after electroporation (Fig. 7). Results showed that

the cumulative transport within 30 s after electro-

poration was significantly less than during the 30 min

that followed (34-fold less for calcein, p=0.002; 4-

fold less for BSA, p=0.0004) and did not provide a

statistically significant increase over the cumulative

transport immediately before electroporation ( p=0.41

for calcein; p=0.88 for BSA). Altogether, this

indicates that the increased apparent permeability

was not due to electrically driven transport through

membrane electropores.

Transcellular transport could alternatively occur

via non-reversible electropores in cells damaged, and

possibly killed, by electroporation, but not shed by the

monolayer. To estimate an upper limit on epithelial

permeability caused by non-reversible cell permeabi-

lization, Caco-2 monolayers were fixed with 95%

ethanol, which kills cells and permeabilizes their

membranes, as well as opens tight junctions [19].

Permeability of ethanol-permeabilized monolayers to

Fig. 7. The role of transcellular transport was assessed by

measuring the accumulation of calcein and BSA in basal samples

collected from unelectroporated (n) and electroporated ( )

monolayers 30 s and 30 min after treatment. Transcellular

transport of calcein and BSA within 30 s after electroporation

was negligible because there was no significant difference in

molecule accumulation for control and electroporated monolayers

( pN0.05). The relatively short lifetime of electropores (on the

order of 1 min [7]) suggests that the increased accumulation

observed 30 min after treatment was not due to transport through

long-lived electropores, but could be caused by transcellular

transport across leaky membranes of nonviable cells still attached

to the filter (see text) [n=6–9].

Fig. 8. Caco-2 monolayer imaged by fluorescence microscopy after

electroporation. Cells were stained with propidium iodide after

fixation with ethanol. Small holes, or gaps, (examples of which are

indicated by the white arrows) in the monolayers were observed

suggesting locations where cells had lifted off. However, non-

electroporated monolayers showed similar features (data not shown)

indicating that these holes may not be due to electroporation.


10 AM calcein and BSA was 33 and 68 times greater

than untreated controls ( pb0.0001 for both) and 4 and

10 times greater than electroporated monolayers

( pb0.0001 for both), respectively. The fact that the

permeabilities of ethanol-permeabilized monolayers

were up to an order of magnitude greater than

electroporated monolayers indicates that transcellular

transport through non-reversible electropores could

explain the data in Figs. 1–4. However, the degree of

permeabilization from electroporation must be con-

siderably less than from ethanol-fixation, possibly due

to a smaller fraction of cells affected or a lesser

density of membrane pores in each cell caused by

electroporation.

Consistent with the possibility that electroporation

makes non-reversible electropores, electroporated

monolayers exhibited patches of dead cells, as shown

by staining with the viability stain propidium iodide

(images not shown). Additional image analysis

showed that less than 10% of the cells in an

electroporated monolayer were dead, consistent with

the expectation that non-reversible electropores are

created in only a small fraction of cells.

3.5.3. Transmonolayer transport

Transmonolayer transport is hypothesized to occur

via transport through micron-scale holes left by cells

killed by electroporation and shed from the mono-

layer. As an upper limit in the case where all cells

were removed from the monolayer, the calcein

permeability of collagen-coated cell culture inserts

(i.e., with no cells) was measured and found to be 70

times greater than intact monolayers ( pb0.0001) and

9 times greater than electroporated monolayers

( pb0.0001). Assuming that permeability increases

linearly with the number of cells removed, these

measurements suggest that removing just over 10% of

cells from a monolayer can increase permeability to

the same level as electroporated monolayers.

Guided by this calculation, monolayers were

examined by fluorescence microscopy to look for

holes. To aid the identification of the cells, mono-

layers were washed, fixed with 95% ethanol, and then

stained with propidium iodide. When non-electro-

porated and electroporated monolayers were visually

,

,


examined, no evidence of holes created by electro-

poration was found. Given that holes are expected to

comprise only a tiny fraction of a monolayer, it is

perhaps not surprising that this was the case. Occa-

sionally, small dark areas that could represent

locations where cells had lifted off were observed

(Fig. 8, white arrows). However, these features were

found in both control and electroporated monolayers,

which suggests they are normal features of monolayer

histology.

More compelling evidence that cells may some-

times detach from the monolayer is illustrated in Fig.

4, which shows occasional jumps in transport at

isolated time points after electroporation. These jumps

can be best explained by the shedding of one or a

small number of cells that were killed by electro-

poration and subsequently detached from the support-

ing membrane. At the time points after the jump in

transport, the return to a linear rate of transport can be

explained by restitution of the monolayer, where cells

adjacent to a hole rapidly migrate to fill in the gap

[20].

4. Discussion

4.1. Efficacy of electroporation-mediated delivery

across intestinal epithelium

The results presented here demonstrate that electro-

poration can increase the permeability of polarized

intestinal epithelial monolayers, which increased

transepithelial transport by as much as 19-fold for a

small molecule, calcein, and up to 34-fold for a large

globular protein, bovine serum albumin (Fig. 2). Such

permeability increases could increase the oral bio-

availability of poorly absorbed drugs.

The significance of these permeability increases

can be evaluated by noting that the permeability of

Caco-2 monolayers has been shown to correlate with

oral absorption in humans [12]. Drugs with Caco-2

permeabilities less than 10�7 cm/s have absorption

levels below 1%. Those with permeabilities between

10�7 and 10�6 cm/s have absorption levels between

1% and 100% and, finally, those with permeabilities

greater than 10�6 cm/s demonstrate 100% absorption.

Based on these relationships, calcein, which had a

baseline (non-electroporation) permeability of

~3�10�7 cm/s in Caco-2 monolayers, would be

incompletely absorbed across the intestinal epithe-

lium. After electroporation, which increased perme-

ability well above 10�6 cm/s, calcein would be

completely absorbed. Likewise, BSA, with a baseline

permeability of ~3�10�8 cm/s would have essentially

no absorption without electroporation, and, with a

permeability of about 2�10�7 cm/s, would be

partially absorbed after treatment.

4.2. Analysis of pathways for increased transepithelial

transport

Overall, these studies and their analysis suggest

that transepithelial transport did not occur by a direct

effect of electroporation to transiently increase cell

membrane permeability. Note that reversible mem-

brane permeabilization almost certainly did occur, as

demonstrated previously when intracellular delivery

into viable monolayer cells was found to occur under

identical conditions [10], but reversible membrane

permeabilization did not appear to cause significant

transcellular transport.

Secondary effects of electroporation appear to be

responsible for the increase in transepithelial trans-

port. Although it is possible that increased para-

cellular transport through enlarged tight junctions

played a role, the differences in recovery kinetics

relative to calcium-mediated and other methods of

opening tight junctions suggests otherwise. A more

likely scenario is that electroporation increased trans-

epithelial transport by killing a small fraction of

monolayer cells. For this protocol and this particular

apparatus, most of these cells remained adherent,

which provided transcellular pathways through cell

membranes made leaky due to loss of viability [21].

Occasionally, some cells detached from the mem-

brane, temporarily creating a hole for transmonolayer

transport that rapidly resealed due to infilling by

neighboring cells [20]. Although this mechanistic

hypothesis is consistent with the data, more studies

are needed to fully confirm its accuracy.

4.3. Clinical applications for electroporation of

intestinal epithelium

To increase absorption in possible clinical appli-

cations, in vivo electroporation of intestinal epithe-


lium will probably require placing a pair of electrodes

within the intestinal lumen. For infrequent treatments

that justify physical intervention, a minimally invasive

approach involving an endoscopic probe could be

used. This approach, however, has limitations,

because it is time consuming, causes discomfort,

and is best suited for treating the lower GI tract.

A more useful approach could involve a bsmartQpill similar to the remotely activated Enterion capsule

used in human drug absorption studies [22] or the

video pill used to perform wireless endoscopy [23].

For electroporation of the intestinal wall, a capsule

could be designed to contain a drug reservoir and a

small electronic chip coupled to a battery, electrodes,

and, possibly, sensors. Given the exponentially grow-

ing capabilities of microelectronics and microfabrica-

tion, such a microdevice might be designed small

enough to be easily swallowed and mass produced at

low cost as a disposable device suitable for routine

use. This belectroporation pillQ could be ingested

orally to electroporate epithelial cells as it passes

through the intestinal tract and ultimately become

eliminated with the body’s waste. Additional research

would of course be needed to evaluate the feasibility

of such a device.

4.4. Safety of electroporation-mediated delivery

across intestinal epithelium

In other cellular systems, electroporation condi-

tions that cause extensive transport are often associ-

ated with at least some cell death [7]. Microscopy

observations in this study and previous flow cytom-

etry measurements [10] similarly show that a fraction

of epithelial cells are killed during monolayer electro-

poration too. In fact, this study hypothesized that

transcellular and transmonolayer pathways created by

these dead cells may provide the primary route for

increased transepithelial permeability.

For applications, especially clinical drug delivery

scenarios, it would be ideal to increase transport

without any cell loss for obvious safety reasons.

However, the type of cell loss caused by electro-

poration may be well tolerated by intestinal epithe-

lium. Electroporation appears to cause low levels of

cell death that is dispersed among many small patches

across the monolayer surface. This type of bdamageQto intestinal epithelium can be rapidly healed, as

observed in these and previous in vitro experiments

(Fig. 4 and [10]) and as established in other in vivo

studies as part of normal physiological processes [24].

Intestinal epithelium undergoes a continuous and

natural process of self-renewal in which epithelial

cells arise from stem cells located in crypts at the base

of each villus, migrate up the villi, and are eventually

shed at their tips. In situations where intestinal

epithelial cells are shed or killed due to physical,

chemical, or microbial injury, the epithelium regener-

ates quickly to replace lost cells and thereby maintain

function. During this process, which is called restitu-

tion, cells adjacent to a gap quickly dedifferentiate and

migrate to fill the gap [20]. In vitro experiments with

human colonic mucosa have shown that restitution

can begin within 30 min of a superficial injury

resulting in denudation of 95% of the mucosal

surface, and that after 3 h, approximately 80% of

the surface is repaired [25]. Smaller injuries, such as

those caused by electroporation, are expected to reseal

much faster.

There is evidence in these experiments that the

monolayers attempt to fill in gaps left by cells that

have shed due to electroporation. For example, in Fig.

4, the appearance of calcein and BSA in the receiver

compartments for some of the monolayers was

characterized by a slow and steady rate of transport,

then a sudden increase in permeability, followed again

30 min later by slow transport of the markers. The

slower rate of transport after the jump in permeability

could indicate cells attempting to fill holes left by

detached cells. Restitution does not require cell

proliferation and can take place within minutes to

hours [26,27]. As a result, it is possible that the 30

min intervals between sampling times in this study

were sufficient for restitution to occur.

5. Conclusion

In conclusion, this study showed that electro-

poration increases the permeability of intestinal

epithelial monolayers to calcein and bovine serum

albumin. This permeability increase resulted in an up

to 34-fold increase in molecular transport across the

monolayers that depended on the size of the mole-

cules and the strength of the electroporation condition

applied. The route by which transport occurred may


have involved paracellular transport through leaky

tight junctions, but was hypothesized to be due to

electroporation killing a small fraction of monolayer

cells and thereby increasing transcellular transport

through porous cell membranes of adherent dead cells

and transmonolayer transport through holes tempora-

rily left by detached dead cells. For applications, these

experiments suggest that electroporation could be

useful to increase the permeability of intestinal

epithelium and, thus, enhance the absorption of poorly

absorbed drugs, including macromolecules. Low

levels of cell death associated with increased perme-

ability could be rapidly repaired by restitution, a well-

known mechanism that heals intestinal injuries and

natural cell shedding as part of normal physiological

processes. Guided by future studies, intestinal electro-

poration might be achieved using a disposable bsmartQpill containing a drug reservoir and microelectronics-

controlled electrodes.

Acknowledgements

The authors would like to acknowledge Dr. Asma

Nusrat for helpful discussions. This work was

supported in part by the National Science Foundation.

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