Equilibrium Loading of Cells with Macromolecules by
Ultrasound: Effects of Molecular Size and Acoustic Energy
HÉCTOR R GUZMÁN, DANIEL X. NGUYEN, ANDREW J. MCNAMARA, MARK R. PRAUSNITZ
School of Chemical Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332-0100
Received 25 October 2001; revised 12 February 2002; accepted 12 February 2002
ABSTRACT: Ultrasound has been shown to deliver small compounds, macromolecules,
and DNA into cells, which suggests potential applications in drug and gene delivery.
However, the effect of molecular size on intracellular uptake has not been quantified.
This study measured the effect of molecule size (calcein, 623 Da; bovine serum albumin,
66 kDa; and two dextrans, 42 and 464 kDa) on molecular uptake and cell viability in
DU145 prostate cancer cells exposed to 500 kHz ultrasound. Molecular uptake in viable
cells was shown to be very similar for small molecules and macromolecules and found to
correlate with acoustic energy exposure. Molecular uptake was seen to be heterogeneous
among viable cells exposed to the same ultrasound conditions; this heterogeneity also
correlated with acoustic energy exposure. In a fraction of these cells, molecular uptake
reached thermodynamic equilibrium with the extracellular solution for the small molecule and all three macromolecules. The results demonstrate that ultrasound provides
a means to load viable cells with large numbers of macromolecules, which may be of use
for laboratory and possible clinical drug delivery applications. ß 2002 Wiley-Liss, Inc. and
the American Pharmaceutical Association J Pharm Sci 91:1693–1701, 2002
Keywords:
ultrasound; macromolecule; drug delivery; cavitation; sonophoresis
INTRODUCTION
Ultrasound has been shown to enhance intracellular transport of small compounds,1–3 macromolecules,4,5 DNA,6,7 and therapeutics8,9 into
viable cells. However, absolute levels of intracellular delivery have been quantified only for the
transport of a small compound, calcein (623 Da),
which was observed to reach thermodynamic
equilibrium with the extracellular solution in
many cells.10 Absolute levels of intracellular
delivery of macromolecules by ultrasound have
not previously been measured. To determine
whether macromolecules can also be delivered
into cells with the high efficiency seen for calcein,
we measured levels of intracellular delivery of
Correspondence to: Mark R. Prausnitz (Telephone: 404-8945135; Fax: 404-894-2866;
E-mail: [email protected])
Journal of Pharmaceutical Sciences, Vol. 91, 1693–1701 (2002)
ß 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association
bovine serum albumin (BSA; 66 kDa) and two
high-molecular-weight dextrans (42 kDa and
464 kDa) into cells exposed to ultrasound.
Previous measurements of ultrasoundmediated delivery of calcein showed that intracellular uptake had great variability from cell to
cell.3,10 This variability, however, was reproducible and shown to correlate with acoustic energy
exposure. To determine whether the same predictive ability also applies to the delivery of
macromolecules, we quantified distributions in
uptake of BSA and dextrans, compared them with
distributions seen for calcein uptake, and looked
for correlation with acoustic energy exposure.
Although the ability of ultrasound to load
molecules into viable cells has been demonstrated
in numerous studies, the mechanism by which
it transiently disrupts cell membranes remains
poorly understood. It is generally believed that
ultrasound-mediated effects on cells are caused
by acoustic cavitation, in the absence of ultrasonic heating.11 Cavitation (i.e., bubbles created
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GUZMÁN ET AL.
by ultrasound) is typically generated through
the activation of small dissolved gas nuclei in
the presence of an acoustic pressure field.12 The
activated nuclei grow through rectified diffusion
and may oscillate and implode violently, thereby
releasing bursts of energy that may be sufficient
to disrupt cell membranes. These disruptions
may be reversible and result in viable cells with
uptake, or nonreversible and result in cell death.13
Although some applications may benefit from
killing cells, most drug and gene delivery scenarios seek to maximize intracellular uptake while
maintaining high cell viability.
MATERIALS AND METHODS
Experimental Protocol
DU145 human prostate cancer cells (DU145, lot
no. 1145858; American Type Culture Collection
(Manassas, VA) were cultured as monolayers in
RPMI-1640 media supplemented with 10% fetal
bovine serum (Cellgro; Mediatech, Herndon,
VA).1 The cells were harvested by trypsin/ethylenediaminetetraacetic acid (Cellgro) digestion at
80–90% mono-layer confluence, centrifuged at
1000g (model GS-15R; Beckman Coulter, Fullerton, CA) for 6 min, and then resuspended in fresh
culture media to a concentration of 1 106 cells/mL,
as determined by a Coulter counter (Coulter Multisizer II; Beckman Coulter). Assuming a spherical
cell geometry, cell volumes were calculated from
cell radii measured using the Coulter counter.
Four fluorescent model molecules that normally do not cross intact cell membranes were
used to monitor transport. Calcein (623 Da;
radius, r ¼ 0.6 nm; Molecular Probes, Eugene,
OR), fluorescein-labeled (FITC) BSA (66 kDa;
r ¼ 3.6 nm; Molecular Probes), 42 kDa FITClabeled dextran (dextran42; r ¼ 4.8 nm; Sigma,
St. Louis, MO), or 464 kDa FITC-labeled dextran
(dextran464; r ¼ 18.5 nm; Sigma) was added to the
cell suspensions to a final concentration of 10 mM.
Dynamic light scattering (DynaPro-LSR; Protein
Solutions, Charlottesville, VA) was used to measure sizes of dextran molecules. The radius of
calcein was obtained from a previous study,14 and
the radius of BSA was estimated by the Stokes
Einstein relation15 using a diffusivity of 6 107
cm2/s.16 To promote cavitation, albumin-stabilized gas bubbles (Optison; Mallinckrodt Inc., St.
Louis, MO) were added to the cell suspensions
(17 0.05 mL/mL; approximately 1.1 107 bubbles/mL; bubble diameter ¼ 2.0–4.5 mm).
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 7, JULY 2002
Before each experiment, cell samples were
slowly aliquoted via a 3-mL syringe (Becton
Dickinson, Franklin Lakes, NJ) with a 22-gauge
needle (Perkin Elmer, Foster City, CA) into
1.2-mL polyethylene transfer pipets (8.8-mm
inside diameter, 0.3-mm wall thickness, 3-cm
height; Samco, San Fernando, CA; catalog no.
241) and positioned using a three-way micropositioner (1-mm resolution; Velmex, Bloomfield, NY)
into the focal beam point of the ultrasound
transducer.
Cell samples were then exposed to ultrasound
using a focused 500-kHz ultrasound apparatus
described previously.1 Pressure (spatial-peaktemporal-peak negative pressure, P) was measured using a 0.2-mm aperture polyvinylidene
fluoride membrane hydrophone (model no.
MHA200A; NTR Systems, Seattle, WA). Acoustic
energy exposure (spatial peak, E) was approximated by the product of the acoustic intensity
(spatial-peak-pulse-average, I) of a plane traveling wave and the total ultrasound exposure
time, t.
P2
I ¼ rms
ð1Þ
rc
E¼It
ð2Þ
where Prms is rms pressure, r the density of water,
and c the speed of sound in water.1,17
Ultrasound was delivered using 60-ms pulses
at a 6% duty cycle with 120-, 540-, and 1000-ms
exposure times. Duty cycle refers to the percentage of time that ultrasound is applied (i.e., 60 ms
of ultrasound is followed by 940 ms of no ultrasound at a 6% duty cycle). Because a 6% duty cycle
was used, the actual duration of each experiment
was 2, 9, and 34 s. The pressures used were 0.6,
1.6, 2.4, and 3.0 MPa. ‘‘Sham’’ control exposures
were conducted using the same protocol, but with
no ultrasound. Post-exposure cell samples were
immediately transferred to 1.5-mL microcentrifuge tubes and left to incubate for 5 min at room
temperature to permit the cells to ‘‘recover.’’
Sonicated samples were then placed on ice until
all samples were exposed (10–30 min).
Cell samples were washed and centrifuged
(800g, 4 min; Eppendorf 5415C; Brinkman, Westbury, NY) three times to remove calcein, BSA, or
dextran molecules present in the extracellular
fluid (i.e., supernatant). Washed cell pellets were
resuspended in Dulbecco’s phosphate buffered
saline (Cellgro) to a final volume of 0.5 mL. Redfluorescent propidium iodide solution (Molecular
EQUILIBRIUM LOADING OF CELLS BY ULTRASOUND
Probes) was added to the cell suspension and
incubated for at least 10 min to stain nonviable
cells. Fluorescent calibration beads (catalog no.
L-14821; Molecular Probes) were added at a concentration of 2.4 104 beads/mL to serve as a
volumetric standard for determining cell viability,
as described below.
Analysis of Results
Viability and molecular uptake were measured
using flow cytometry, as described previously.1
Cell viability was determined by comparing the
ratio of viable cells (intact cells that excluded
propidium iodide) and fluorescent beads in each
sample to the ratio measured in control samples.18 Molecular uptake was quantified by measuring the green fluorescence emitted by calcein,
FITC-labeled BSA, or FITC-labeled dextran molecules. Quantitative fluorescein calibration beads
(catalog no. 825; Flow Cytometry Standards Corp.,
Fishers, IN) were used to convert the measured
fluorescence into an equivalent number of fluorescein molecules.18
The fluorescence emission of a calcein, BSA, or
dextran molecule differed in intensity from that of
a fluorescein molecule. To relate the fluorescence
of these molecules to that of fluorescein-based
calibration beads, the fluorescence intensity of
each molecule was measured at equal concentrations using a fluorometer (QuantaMaster; Photon
Technology International, Brunswick, NJ); a
bandpass filter identical to that used by the flow
cytometer was used to collect fluorescence emissions. The fluorescence of each uptake molecule
was then converted into equivalent fluorescein
fluorescence units by multiplying by the fluorescence intensity ratio of the uptake molecule to
fluorescein. In this study, the average standard
deviation intensity ratio for calcein-fluorescein
was 0.96 0.02, BSA-fluorescein was 0.99 0.03,
dextran42-fluorescein was 1.06 0.02, and dextran464-fluorescein was 1.25 0.03.
Confocal microscopy (Zeiss LSM510; Carl Zeiss,
Thornwood, NY) was used to image the uptake of
fluorescent molecules inside cells (optical section
made at a penetration depth of 8 mm, which is
near the center of each cell) excited using ultraviolet (351 and 364 nm) and 488-nm lasers lines.
Green fluorescence (calcein, BSA, or dextran)
indicated molecular uptake, red fluorescence
(propidium iodide) stained the nuclei of nonviable
cells, and blue fluorescence (Hoechst 33342;
Molecular Probes) nonspecifically identified the
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nuclei of all cells. Confocal images were used to
determine whether uptake occurred in the cytoplasm and/or nuclei of live cells, and to determine
whether BSA and dextran molecules aggregated on the cell membranes of both exposed and
control-sample cells. Anti-fluorescein monoclonal antibodies (catalog no. A-11090; Molecular
Probes), which do not cross intact cell membranes,
have blue fluorescence, and specifically bind to
fluorescein molecules, were used to identify possible aggregation of BSA and dextran molecules
on the surface of the cell membrane. Extracellular
aggregation can be identified by the superimposed
fluorescence of both BSA or dextran and antifluorescein molecules.
Flow cytometry showed distributions of intracellular fluorescence within samples of cells
exposed to ultrasound (Fig. 1). Control cells unexposed to ultrasound (Fig. 1A) exhibited a single
Figure 1. Fluorescence histograms of viable DU145
cell samples showing uptake of fluorescein-labeled BSA
(20,000 cells/sample). (A) Fluorescence of a control
sample shows Gaussian-distributed background fluorescence in the first decade of the histogram. Fluorescence signal, which is a measure of intracellular BSA
uptake, is heterogeneously increased for samples
exposed to ultrasound at (B) 8 J/cm2 (C) 90 J/cm2, and
(D) 817 J/cm2. As shown in (C), heterogeneous distributions can be divided into Gaussian-distributed regions
termed nominal (NUP), low (LUP), and high (HUP)
uptake subpopulations.
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cell population with a weak fluorescence distribution most likely due to autofluorescence, electrical
noise, and low-level surface binding of fluorescent
molecules. Cells exposed to ultrasound (Fig. 1, B–
D), however, exhibited heterogeneous cell fluorescence generally characterized by two peaks
and a valley in between. To describe the observed
phenomenon, the data were divided into three
fluorescent cell subpopulations, as described previously by Guzmán et al.,10 and shown graphically
in Figure 1C. Each cell sample was analyzed
and divided into subpopulations using WinMDI
(TSRI Flow Cytometry, San Diego, CA) and
MIX (Ichthus Data Systems, Hamilton, Ontario,
Canada) software as described previously.10
Molecular uptake within each subpopulation
was calculated by subtracting the mean fluorescence of control samples from the mean fluorescence of the subpopulations.
Statistical Analysis
At each condition tested, a minimum of three
replicate data points was collected. Replicates
were used to calculate experimental means and
standard errors. One-way analysis of variance
(a ¼ 0.05) was performed when comparing three
or more experimental conditions to a single factor.
RESULTS
This study compared the ability of ultrasound to
achieve intracellular delivery of macromolecules—BSA (66 kDa) and two large dextrans (42
and 464 kDa)—to previous results for intracellular delivery of a small molecule, calcein (623 Da).10
Specific questions addressed include (1) whether
sonicated cells take up macromolecules, (2)
whether macromolecular uptake shows cell-tocell heterogeneity, (3) whether any observed heterogeneity correlates with energy exposure, and
(4) whether intracellular molecular concentrations reach thermodynamic equilibrium with the
extracellular solution.
Sonicated Cells Take Up Macromolecules
To determine whether large macromolecules can
be delivered into sonicated cells, confocal microscopy was used to visualize molecular uptake into
the cell interior after exposure to ultrasound.
Figure 2 contains a set of confocal images which
show that extensive intracellular uptake occurred
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 7, JULY 2002
Figure 2. Confocal fluorescence micrographs showing intracellular uptake of (A) calcein, (B) BSA, (C)
dextran42, and (D) dextran464 into DU145 cells after
exposure to ultrasound. Calcein is distributed throughout the whole cell, whereas BSA and dextran molecules
are excluded from the nucleus. Hoechst nuclear stain
(not shown) was used to identify cell nuclei. Figures
A1–A3 show the simultaneous presence of three cells
having different levels of calcein uptake. (A1) The
brightly fluorescent cell is indicative of cells in the high
uptake subpopulation (HUP), (A2) the dimmer fluorescent cell is indicative of low uptake (LUP), and (A3)
the dimmest cell is indicative of nominal uptake (NUP).
All micrographs are shown at the same magnification.
for the three macromolecules tested, indicating
that ultrasound is able to load cells with molecules as large as 464 kDa. Uptake into the cell
nucleus, however, depended on molecule size.
Calcein was delivered throughout the cell (cytosol
and nucleus), whereas the larger BSA and dextran
molecules were delivered only to the cytosol.
Macromolecular Uptake Shows
Cell-to-Cell Heterogeneity
Previous work demonstrated that uptake of calcein showed significant cell-to-cell heterogeneity.10
This heterogeneity is also shown in Figure 2A,
where three neighboring cells exposed to the same
ultrasound conditions exhibit very different levels
of intracellular calcein uptake. In this study, we
similarly found heterogeneity in uptake of macromolecules. Figure 1 shows a representative set of
EQUILIBRIUM LOADING OF CELLS BY ULTRASOUND
histograms that contain heterogeneous distributions of intracellular BSA uptake over a range of
energy exposures (similar distributions were seen
for all molecules studied here). The histograms
show two peaks corresponding, respectively, to
low levels and high levels of uptake, with a broad
valley in between. As illustrated in Figure 1, the
locations of the peaks generally did not vary, but
their relative heights were strong functions of
ultrasound conditions.
As a means to concisely characterize and
display the observed heterogeneity, uptake histograms were divided into three uptake subpopulations as shown in Figure 1C.10 The region of
low intracellular fluorescence was termed the
nominal uptake subpopulation (NUP), the intermediate valley was termed the low uptake subpopulation (LUP), and the region of high
fluorescence was termed the high uptake subpopulation (HUP).
Macromolecular Uptake Heterogeneity
Correlates With Energy Exposure
Using our approach to characterize heterogeneous levels of uptake as three subpopulations,
Figure 3 shows the distribution of viable cells
among the NUP (white bars), LUP (gray bars),
and HUP (black bars) subpopulations for uptake
of (a) calcein, (b) BSA, (c) dextran42, and (d)
dextran464 as a function of energy exposure.
In a previous study, calcein uptake was
measured over a broad range of different ultrasound conditions and found to correlate with
energy exposure.10 As shown in Figure 3, uptake
of macromolecules also correlated with energy
exposure for all three macromolecules. For the
Figure 3. Distribution of molecular uptake subpopulations among cells remaining viable after exposure to
ultrasound as a function of acoustic energy exposure
and molecule size. The white bars indicate cells in NUP,
gray bars indicate LUP, and black bars indicate HUP.
The different molecules used were: a ¼ calcein, b ¼ BSA,
c ¼ dextran42, and d ¼ dextran464. With increasing
energy exposure, the fraction of cells in NUP decreased,
1697
macromolecules, NUP decreased, and LUP and
HUP increased with increasing energy exposure
( p < 0.01), indicating that intracellular delivery
occurred in a larger fraction of remaining viable
cells as energy exposure increased. Uptake of
macromolecules and calcein showed the same
dependence on energy exposure, except the LUP
subpopulation did not vary when using calcein as
the uptake molecule ( p > 0.05).
The distribution of uptake subpopulations was
also studied as a function of molecule size. Among
the three macromolecules considered, the fraction of cells within each uptake subpopulation
showed no significant dependence on molecular
size ( p > 0.05). However, if calcein is included
in the analysis, then the number of cells in the
LUP subpopulation decreased with molecular size
( p < 0.05) and the number of cells within the NUP
generally increased with molecular size at energies 49 J/cm2 ( p < 0.05). The fraction of cells
with the highest levels of uptake (HUP) remained
independent of molecular size ( p > 0.05).
These observations may give insight into the
mechanism of molecular delivery into cells.
Assuming that ultrasound creates physical disruptions in cell membranes,5 the finding that
all three macromolecules were delivered into
cells with equal efficiency suggests that membrane disruptions were larger than dextran464
(r ¼ 18.5 nm). The additional finding that calcein
was delivered into cells more easily than the
macromolecules suggests the possibility of two
different sizes of cell disruptions: (i) small disruptions that permit entry of calcein (r ¼ 0.6 nm) but
exclude BSA (r ¼ 3.6 nm) and the larger dextrans,
and (ii) large disruptions that permit entry of
molecules as large as dextran464 (r ¼ 18.5 nm).
whereas the fraction of cells in LUP and HUP increased
( p < 0.01). With increasing molecule size, the fraction of
cells in NUP increased for energy exposures < 49 J/cm2
( p < 0.05) and did not change for energy exposures
> 49 J/cm2 ( p > 0.05), LUP decreased ( p < 0.05), and
HUP generally did not vary ( p > 0.05). Error bars are
presented as SEM.
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GUZMÁN ET AL.
Intracellular Loading With Macromolecules
Approaches Thermodynamic Equilibrium
Figure 3 identifies the fraction of viable cells in
each uptake subpopulation, but does not quantify
the number of molecules delivered intracellularly
within each subpopulation. This information is
provided in Table 1, indicating uptake levels for all
molecules in NUP on the order of 104 molecules/
cell, in LUP of 105 –106 molecules/cell, and in
HUP of 106 –107 molecules/cell.
Among the HUP cells, intracellular macromolecule uptake appears to have reached thermodynamic equilibrium with the extracellular
solution for all molecules studied (Table 1). This
was determined by dividing the number of
molecules taken up by the intracellular volume
to yield an intracellular concentration. This concentration was then compared with the extracellular concentration and found to be similar. It
is important to note that this extremely high level
of uptake (i.e., the highest level possible without
binding or active transport) was achieved in cells
that remained viable.
Cell Viability Correlates With Energy Exposure
The data in Figure 3 and Table 1 quantify uptake
among the cells remaining viable after exposure
to ultrasound, because uptake by nonviable cells
is generally not relevant to applications. However, ultrasound can irreversibly damage cells,
especially at large energy exposures in which the
majority of cells can be killed. Figure 4 shows that
cell viability decreased with increasing energy
exposure ( p < 0.01), in agreement with previous
observations.1 Viability was independent of the
size of molecule being delivered ( p > 0.05), which
is consistent with our a priori assumption that the
molecules used in this study are biologically and
chemically inert to the cells. For practical applications, ultrasound conditions must be selected
that balance losses of viability against corresponding gains in intracellular delivery. The optimal
balance will depend on the constraints of each
application.
The propidium iodide viability test used in this
study assessed cell viability on the order of an
hour after exposure to ultrasound. It would be
of interest for most applications to additionally
determine whether there are any long-term
effects on cells. For this reason, we monitored cell
growth kinetics for up to 7 days after sonication
under sterile conditions. For cells exposed to
ultrasound at moderate energy (i.e., 49 J/cm2),
cell division times were the same for sonicated
and control cells (data not shown), indicating no
long-term effects on cell viability. However, for
cells exposed to ultrasound at high energy (i.e.,
408 J/cm2), many cells that were apparently
viable immediately after exposure to ultrasound
became nonviable within 36 h, as evidenced by
staining with trypan blue or their detachment
from the culture dish and floating in the media
(data not shown). As previously proposed by
others,19,20 this might indicate delayed death by
apoptosis. At later times, no differences between
cell division times were observed for sonicated
and control cells, consistent with previous observations.21,22 Together, this suggests that the
short-term viability data shown in Figure 4 are
representative of long-term viability at low to
moderate energy exposures, but underestimate
long-term viability at high energies.
Table 1. Average Number of Molecules Delivered Intracellularly
Calcein
BSA
Dextran42
Dextran464
Molecules/Cell NUPa
Molecules/Cell LUPa
Molecules/Cell HUPa
Percent HUP
Equilibriumb
4.8 5.9 104
1.2 0.3 104
2.2 0.3 104
5.4 0.9 104
3.0 1.9 106
6.4 0.4 105
6.0 0.7 105
1.0 0.03 106
1.4 0.5 107
7.8 0.3 106
6.7 0.3 106
1.0 0.04 107
103 37
105 3
98 4
154 6c
a
The average number of molecules delivered into cells in the nominal (NUP), low (LUP), or high (HUP) uptake subpopulations.
The intracellular molecule concentration in HUP cells expressed as a percent of extracellular molecular concentration. For this
calculation, the volume of distribution for calcein was the whole cell volume (2200 mm3) and for the macromolecules was only the
extranuclear cell volume (1600 mm3) (see Fig. 2).
c
Visual inspection by confocal microscopy using anti-fluorescein antibodies indicated significant membrane binding of
dextran464 (data not shown), which could explain the greater than 100% equilibrium value.
b
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EQUILIBRIUM LOADING OF CELLS BY ULTRASOUND
Figure 4. Cell viability as a function of acoustic
energy exposure. Viability decreased with increasing
energy exposure and did not vary with molecule size.
The molecules used were: * ¼ calcein, & ¼ BSA, ~ ¼
dextran42, and } ¼ dextran464. Error bars are presented as SEM.
DISCUSSION
The most remarkable finding in this study is that
ultrasound’s ability to deliver macromolecules
into cells was very similar to that for a small
molecule, calcein. This observation has important
implications for understanding mechanisms of
ultrasound’s effects and applications for drug and
gene delivery.
For all molecules tested, intracellular delivery
into many cells achieved approximate thermodynamic equilibrium with the extracellular solution. The size range covered by these molecules
(0.623–464 kDa; 0.6–18.5 nm radius) includes
the dimensions of most drugs and genes of therapeutic interest. A noteworthy observation was
the exclusion of macromolecules from the cell
nucleus, which is consistent with previous observations showing that globular proteins larger
than 60 kDa cannot enter into the cell nucleus
by diffusion.23 This suggests that ultrasound does
not damage the nuclear membrane of cells that
remain viable. Nevertheless, ultrasound has been
shown previously to increase DNA (i.e., > 60 kDa)
transfection of cells,6,7 suggesting that potential
gene therapy applications using ultrasound may
not be limited by this observation. Other studies
suggest that DNA incorporation and expression in
the cell nucleus occurs by a separate mechanism
that may be unrelated to ultrasound, and has
a pathway that may depend on the cell type,
plasmid type, plasmid promoters, and other biological factors.24
1699
The results from this study also showed that
molecule uptake was heterogeneous and correlated with acoustic energy exposure for all three
macromolecules, which is consistent with previously observed results for calcein.10 The additional observation that the fraction of NUP
and LUP cells depended on molecule size suggests the possibility of a bimodal distribution
of cell membrane disruptions: small disruptions
that only permit calcein-sized molecules to enter
cells and large disruptions that permit macromolecules to enter cells. Evidence for this comes
from Figure 3, which shows that at the same
energy exposure the fraction of cells that took up
macromolecules was less than the fraction that
took up calcein. This suggests that some cells
have disruptions large enough for calcein entry
but too small for entry of macromolecules. Other
cells permit entry of all the molecules tested,
suggesting disruptions at least as big as dextran464. Because there was no evidence for cells
that, for example, favored uptake of BSA over
dextran464, the data suggest that there were
no disruptions of intermediate size, i.e., between calcein and dextran464. For this reason,
we propose a bimodal distribution of membrane disruption sizes composed of small (i.e.,
calcein-sized) and large (i.e., dextran464-sized)
disruptions.
The results of this study have implications for
ultrasound-mediated drug delivery. First, they
show that molecules over a broad range of sizes
can be delivered into cells with similar efficiency.
This suggests the possibility of using ultrasound
to facilitate many different drug and gene therapies. In addition, the distribution of uptake of
all the molecules tested could be controlled in a
straightforward manner by adjusting the energy
exposure. The quantitative dependence on energy
exposure shown in Figure 3, for example, should
be useful for applications in which uptake needs
to be maximized in remaining viable cells regardless of viability loss. Such scenarios may occur for
in vitro laboratory settings or ex vivo protocols,
particularly those involving the transfection of
cells with genetic material. This information can
be combined with that in Figure 4, which shows
the viability dependence of cells on energy exposure if viability loss is a concern, as is the case
for most in vivo applications. Because these
results have only been observed on in vitro cell
suspensions, future studies need to determine
ultrasound’s effects on other cell types and tissues
in vitro and in vivo.
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CONCLUSIONS
In this study, intracellular uptake of macromolecules (i.e., BSA, dextran42, and dextran464) by
ultrasound was shown to occur at levels similar to
those seen with small molecules (i.e., calcein).
Moreover, macromolecule uptake was shown to be
heterogeneous among cells exposed to the same
acoustic conditions. These effects were weakly
dependent on molecular size. When molecular
uptake was quantified, intracellular concentrations were shown to reach approximate equilibrium with extracellular concentration levels in
a significant fraction of cells for all molecules
tested. In cells that had intracellular uptake
at subequilibrium concentrations, the smallest
molecule used, calcein, was generally able to
transport into a greater fraction of cells than
the larger BSA and dextran molecules. Together
these results suggest that ultrasound may provide a means to load cells with large numbers of
macromolecules for laboratory and possible clinical applications.
ACKNOWLEDGMENTS
We thank Dr. Paul J. Canatella, Steve I. Woodard,
Johnafel Crowe, and Dr. John Tsavalas for their
generous help and critical comments. This work
was supported in part by The Whitaker Foundation, National Science Foundation, and National
Institutes of Health.
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