Intracellular uptake and trafficking of Pluronic micelles in drug-sensitive and MDR cells: Effect on the intracellular drug localization

Article abstract of DOI:10.1002/jps.10006

The intracellular uptake and localization of a fluorescently labeled Pluronic P-105 in HL-60 leukemia cells and in A2780 drug-sensitive and A2780/ADR MDR ovarian carcinoma cells were characterized by flow cytometry and fluorescence microscopy. Pluronic P-

Full text of DOI:10.1002/jps.10006

Intracellular Uptake and Traf®cking of Pluronic Micelles
in Drug-Sensitive and MDR Cells: Effect on the
Intracellular Drug Localization
NATALYA RAPOPORT,1 ALEXANDER MARIN,1 YI LUO,2 GLENN D. PRESTWICH,2 MD. MUNIRUZZAMAN1
1Department of Bioengineering, University of Utah, 20 S. 2030 E., Room 108, Salt Lake City, Utah
2Department of Medicinal Chemistry, University of Utah, Salt Lake City, Utah
Received 24 April 2001; revised 25 July 2001; accepted 1 August 2001
ABSTRACT: The intracellular uptake and localization of a ¯uorescently labeled
Pluronic P-105 in HL-60 leukemia cells and in A2780 drug-sensitive and A2780/ADR
MDR ovarian carcinoma cells were characterized by ¯ow cytometry and ¯uorescence
microscopy. Pluronic P-105 molecules were labeled with a pH-sensitive ¯uorescent
label, 5-(and 6-)carboxy-2070-dichloro¯uorescein. The ¯uorescence intensity of labeled
Pluronic was about twofold higher at pH 7.4 than at pH 5.5. At Pluronic concentrations
exceeding the critical micelle concentration (cmc), ¯ow cytometry histograms mani-
fested bimodal distribution of cell ¯uorescence for all types of cells. Cell population
characterized by higher ¯uorescence intensity presumably resulted from Pluronic
transfer from the acidic environment of cytoplasmic vesicles (endosomes or lysosomes)
into the neutral environment of the cytoplasm and cell nuclei, which suggested the
permeabilization of the membranes of acidic vesicle by Pluronic molecules. For the MDR
cells, the bimodal distribution of cell ¯uorescence was already observed at very low
Pluronic concentrations in the incubation medium (i.e., below the cmc). The data
suggest that the membranes of acidic vesicles of MDR cells are more susceptible to the
action of polymeric surfactants than those of drug-sensitive cells. Permeabilization of
acidic vesicles had a dramatic effect on the intracellular traf®cking of drugs: when
delivered in PBS, the anthracyclin drug ruboxyl (Rb) sequestered in cytoplasmic
vesicles and was excluded from cell nuclei; however, when delivered in Pluronic
micelles, drug accumulated in cell nuclei. Drug uptake from/with Pluronic micelles was
substantially enhanced by ultrasound. These ®ndings suggest that the nuclear
accumulation of drugs internalized via ¯uid-phase endocytosis can be enhanced by
the application of Pluronic micelles and can be further augmented by ultrasonic
irradiation. ß 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci
91:157±170, 2002
Keywords: endosomal release; intracellular traf®cking; Pluronic micelles; MDR cells;
drug-sensitive cells; HL-60 cells; A2780 cells; A2780/ADR cells
INTRODUCTION
properties that include (1) an appropriate size to
escape renal excretion, allowing however for the
extravasation at the tumor site; (2) an enhanced
penetration and retention (EPR) effect that
provides for a selective accumulation of micellar-
encapsulated drugs in solid tumors;1±3 (3) a
shielding effect that diminishes drug interaction
with healthy tissues;4±12 (4) the opportunity to
release drug from micelles in a spatially and
Polymeric micelles have been suggested as drug
carriers because of a number of advantageous
Correspondence to: Natalya Rapoport (Telephone: 801-581-
8990; Fax: 801-585-5151;
E-mail: natasha.rapoport@m.cc.utah.edu)
Journal of Pharmaceutical Sciences, Vol. 91, 157±170 (2002)
ß 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 91, NO. 1, JANUARY 2002
157

158
RAPOPORT ET AL.
temporally controlled manner by ultrasonic irra-
diation;4,5,7±13 (5) enhanced intracellular uptake
of a micellar-encapsulated drug under the action
of ultrasound;11±13 and (6) the uptake of micellar-
encapsulated drugs via a ¯uid-phase endocytosis
rather than a passive diffusion. The latter factor
is important for overcoming drug resistance,
which is a signi®cant problem in cancer che-
motherapy. Drug resistance and multidrug resis-
tance (MDR) of the cells are attributed to the work
of the ATP-dependent ef¯ux pumps localized in
the cytoplasmic membranes. Drugs internalized
by endocytosis bypass ef¯ux pumps;14,15 however,
endocytosed drugs are sequestered in the cyto-
plasmic acidic vesicles, which create a second
barrier on the way of the drug toward its target.
For anticancer drugs, the targets are commonly
located outside of the cytoplasmic acidic vesicles;
in particular, for anthracyclin drugs, the DNA
target is localized in cell nuclei.
Positively charged drugs usually end up in
acidic vesicles; this phenomenon is especially pro-
nounced in MDR cells because of the high pH
gradient between these vesicles and the cyto-
sol.16,17 It was reported that ionoforic compounds
that reduced the pH gradient between acidic vesi-
cles and cytosol reversed the multidrug resistance
of MDR cells.16 Enhancing drug release from the
acidic vesicles is an important task pertinent not
only to drug delivery but also to ef®cient gene
delivery.18±20
It was recently reported by Kabanov and
Alakhov's group that doxorubicin (DOX) formu-
lated with unimeric concentrations of Pluronic
L61 copolymer not only effectively penetrated
plasma membranes of drug-resistant cells but
also avoided sequestration in acidic vesicles.14,21
By using acridine orange as a ¯uorescent probe,
these authors showed that Pluronic L61 de-
creased the number of the cytoplasmic vesicles
characterized by a high pH gradient with cyto-
sol,21 which resulted in the hypersensitization of
drug-resistant cells.22±25 Pluronic (or Poloxamer)
copolymers are triblock copolymers of poly(ethy-
lene oxide) (PEO) and poly(propylene oxide)
(PPO), often denoted PEO-PPO-PEO. The funda-
mental relationship between the composition of
Pluronic block copolymers and their hypersensi-
tization effect in MDR cancer cells was recently
established by Kabanov et al.25 Importantly,
Pluronic copolymers have a relatively low in vivo
toxicity (see, e.g., ref. 21).
the appropriate molecular weight and PPO/PEO
block-length ratio, and by adjusting the concen-
tration.25±27 There is extensive information in
the literature on the structure and properties of
Pluronic micelles, including a comprehensive
review by Alexandridis et al.27
In our work, we use Pluronic copolymers at
concentrations that are above the critical micelle
concentration (cmc),4±13 which results in self-
assembly of Pluronic molecules into dense micel-
les; the core-forming block in aqueous solutions is
PPO, whereas PEO blocks form micelle coronas.
The hydrodynamic radii of Pluronic micelles at
physiological temperatures range between 10 and
20 nm.
The application of Pluronic micelles rather
than unimers (i.e., individual molecules) has a
number of advantages, the most important of
which are (i) the possibility of drug targeting to
tumors while avoiding damage to healthy tissues,
and (ii) the chances of overcoming drug resistance
by switching drug transport from passive diffu-
sion to endocytosis. We use ultrasound to enhance
the intracellular uptake of micellar-encapsulated
drugs by tumor cells.
Important information on the Pluronic uptake
and distribution in drug-sensitive and MDR cells
is absent in the literature. In the present paper we
report on the effect of the extracellular Pluronic
concentration, which controls the aggregation
state of Pluronics, on the intracellular uptake
and distribution of Pluronic P-105 in drug-
sensitive and MDR cells. For this study, Pluronic
P-105 was ¯uorescently labeled with a pH-
sensitive ¯uorescent label, and its uptake and
distribution were studied by ¯ow cytometry.
MATERIALS AND METHODS
Materials
Pluronic P-105, with an average molecular weight
of 6500 and the number of monomeric units in
PEO and PPO blocks being 37 and 56, respec-
tively, was kindly supplied by the BASF Corpora-
tion (Mount Olive, NJ. Pluronic solution in
phosphate-buffered saline (PBS) was used for
the experimennts. Dulbecco's PBS, pH 7.4, was
purchased from Sigma and used as received.
Anthracycline Drug
The aggregation state of Pluronic copolymers
can be controlled by choosing the copolymer with
Two anticancer anthracycline drugs, doxorubicin
(DOX) and ruboxyl (Rb), were used in this study.
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INTRACELLULAR TRAFFICKING OF PLURONIC MICELLES
159
Rb, a paramagnetic analog of DOX, was synthe-
sized by conjugating a paramagnetic Tempo-
type nitroxyl radical (1-oxo-2,2,6,6-piperidone-4-
hydrazone) to DOX (US Patent 4,332,934, 1982;
see Scheme 1). This drug is presently in phase II
of clinical trials in Russia. Rb was found to be
effective against breast and colon carcinoma and
bone sarcoma. Rb is an intercalating drug that
stacks between paired bases in DNA. In this
study, Rb was chosen because it is more lipophilic
than DOX; within the cells, Rb partitions between
DNA and phospholipid membranes.7,11. We were
interested in the effect of Pluronic micelles on the
intracellular distribution of the drug that not only
intercalates DNA but is also partially localized in
cell membranes.
50 mg/mL gentamicin at 37 8C in humidi®ed air
containing 5% CO2.
Drug-sensitive A2780 and multidrug resistant
(MDR) ovarian carcinoma A2780/ADR cells grow-
ing in adherent monolayers were kindly provided
by Dr. T.C. Hamilton (Fox Chase Cancer Center,
PA). Cells were cultured in a complete RPMI 1640
medium, supplemented with 10% fetal bovine
serum, 100 U/mL penicillin, 100 mg/mL strepto-
mycin, and 10 mg/mL insulin, which in the case of
A2780/ADR cells included 100 ng/mL DOX for
maintaining resistance.
Synthesis of a Fluorescently-Labeled
Pluronic P105
Pluronic-105 (MW ˆ 6500; 28.7 mg, which corre-
sponds to 8.8 mmol of free OH group) was dissolved
in 10 mL of dimethylformamide (DMF), and
3.94 mg (8.8 mmol) of 5-(and 6-)carboxy-2070-
dichloro¯uorescein mixed isomers (Molecular
Probes, Inc., Eugene, OR) in 5 mL of DMF was
added. Next, 18.3 mg of dicycohexylcarbodimide
(8.8 mmol) and 2.2 mg of dimethylaminopyridine
(1.7 mmol) were added in solid form. The reaction
was stirred at room temperature for 3 days, then
diluted with 100 mL of ice-cold water. The
mixture was dialyzed against water for 7 days
(3 changes/day), and the purity of ¯uorescently
labeled Pluronic 105 was monitored by gel per-
meation chromatography (GPC). The product was
lyophilized to obtain the dry powder of a ¯uores-
cently labeled Pluronic 105. The reaction is
schematically shown in Scheme 2.
Cell Incubation With Fluorescently
Labeled Pluronic
Cell were harvested, washed with PBS, and re-
suspended in a solution of a ¯uorescently labeled
Pluronic; overall Pluronic concentration in a
solution ranged from 0.0002 to 5 wt %.
Two sets of experiments were made. In the ®rst
set, HL-60 cells were incubated with progres-
sively increasing concentrations of ¯uorescently
labeled Pluronic. In the second set, the concentra-
tion of a ¯uorescently labeled Pluronic in the
incubation medium remained constant at 10, 20
or 50 mg/mL, and nonlabeled Pluronic was added
to the incubation medium at concentrations below
and above the cmc of Pluronic P-105 (which is
0.004 wt % at 378C). Following incubation with a
Pluronic solution, cells were analyzed by ¯ow
cytometry and ¯uorescence microscopy. Pluronic
uptake by the cells was quantitatively measured
by ¯uorescence depletion from the incubation
medium.
Cell Lines
HL-60 promyelocytic cells were kindly provided
by Dr. B.K. Murrey (Department of Microbiology,
Brigham Young University, Provo, UH). They
were cultured in RPMI-1640 medium supplemen-
Sonication
ted with 10% fetal bovine serum,
L-glutamine, 0.2% sodium bicarbonate, and
2
mM
Polystyrene test tubes (diameter, 10 mm) contain-
ing cell suspensions were positioned along the
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160
RAPOPORT ET AL.
circumference of a circular holder; they were
placed in water bath thermostated at 378C.
Ultrasound at 20 kHz was generated by a microp-
robe transducer (tip diameter, 13 mm; Sonics and
Materials, Newton, CT) positioned in the center of
the sample holder at a distance of 3 cm from the
samples. Power density was controlled by varying
the amplitude settings of the instrument and was
measured with a hydrophone (Bruel & Kjaer, type
8103). Sonication of adherent monolayers of
A2780 and A2780/ADR cells at 67 kHz was per-
formed in Sonicor SC 100 sonication bath (Sonicor
Instruments, Copaique, NY) in 6-well plates. We
could not measure power density received by the
monolayer, but indirect evidence indicates that
cells experienced mild insonation because no
detachment of cells from monolayers was ob-
served in our experiments.
Figure 1. Fluorescence spectra of aqueous solutions
of ¯uorescently labeled Pluronic P-105 at pH 5.5
and 7.4.
RESULTS AND DISCUSSION
Fluorescence Spectra
Flow Cytometry
The ¯uorescence spectra of ¯uorescently labeled
Pluronic at neutral (pH 7.4) and acidic (pH 5.5)
pHs are presented in Figure 1. The ¯uorescence
intensity of labeled Pluronic at neutral pH more
than doubles compared with the acidic environ-
ment. This feature was used to study the intracel-
lular distribution of ¯uorescently labeled Pluronic.
Fluorescence histograms were recorded with a
Facscan (Beckton Dickinson) ¯ow cytometer and
analyzed using CellQuest software supplied by
the manufacturer. Minimums of 10,000 events
were analyzed to generate each histogram. To
prevent Pluronic ef¯ux from the cells at washing,
the cells were injected into the ¯ow cytometer in
the initial incubation medium.
Intracellular Pluronic Distribution in HL-60 Cells
Fluorescence histograms of drug-sensitive pro-
myelocytic HL-60 cells incubated with various
concentrations of ¯uorescently labeled Pluronic
P-105 are presented in Figure 2. A clearly bimodal
character of ¯uorescence histograms is observed
at Pluronic concentrations ꢀ 0.2%; these concen-
trations are above the cmc of Pluronic P-105 and
correspond to micellar solutions.
Fluorescence Microscopy
The cells were ®rst ®xed with 3% formalin, then
washed with PBS containing 3% formalin, sealed
on glass slides, and visualized at 100Â magni®ca-
tion by ¯uorescence microscopy with 527±552 nm
excitation and 577±632 nm emission wavelengths
(Eclipse E800, Nikon).
Figure 2. Fluorescence histograms of HL-60 cells incubated with ¯uorescently
labeled Pluronic P-105; initial concentration of a labeled Pluronic P-105 in the
4
3
incubation medium (left to right) 2 Â 10
%wt, 2 Â 10
%wt, 0.02%, 0.2%, and
0.2 %wt; incubation time: 60 min.
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INTRACELLULAR TRAFFICKING OF PLURONIC MICELLES
161
We hypothesize that the population of the cells
with a higher ¯uorescence intensity resulted from
the disruption of the pH gradient on acidic vesicle
membranes followed by the release of Pluronic
molecules from the acidic environment of the
cytoplasmic vesicles into the neutral environment
of the cytosol. This result may, in turn, be caused
by the ¯uidization of phospholipid membranes
under the action of the internalization Pluronic
micelles. The electron paramagnetic resonance
(EPR) spectra of a membrane-localized spin probe
16-doxyl stearic acid methyl ester (16-DSME) in
HL-60 cells indicated that Pluronic intracellular
uptake from micellar solutions resulted in a de-
crease of a solid±¯uid transition temperature and
intensi®cation of probe motion in phospholipid
membranes.10
The hypothesis of the self-release of Pluronic
from acidic vesicles was con®rmed by the results
of the experiment presented in Figure 3 and by a
direct observation of the cells by ¯uorescence
microscopy (Figure 4). In the experiment pre-
sented in Figure 3, the cells were incubated with a
mixture of ¯uorescently labeled and unlabeled
Pluronic P-105. The initial concentration of
labeled Pluronic in the incubation medium was
Figure 4. Effect of Pluronic P-105 concentration in
the incubation medium on its intracellular distribution.
The concentration of ¯uorescently labeled Pluronic P-
105 is maintained constant at 0.005%. The overall
Pluronic concentration is either (A) 0.1% or (B); 10%.
The incubation time is 10 min. The cells of the same
sample are characterized by a different intracellular
distribution of a ¯uorescently labeled Pluronic P-105.
With increasing Pluronic concentration, release of
Pluronic from sequestering compartments is observed.
the same for both A and B samples, but the
concentration of unlabeled Pluronic was an order
of magnitude higher in sample B than in sample A
(both concentrations being above the cmc). As
seen from Figure 3, the population of the cells
with a stronger ¯uorescence was much higher in
sample B than in sample A, presumably because
of the Pluronic-induced permeabilization of the
endosomal (and/or lysosomal) membranes, which
resulted in Pluronic transfer to the environment
with a higher pH.
Figure 3. Fluorescence histograms of HL-60 cells
incubated with a mixture of ¯uorescently labeled and
unlabeled Pluronic P-105. The concentration of labeled
Pluronic in the incubation medium is maintained
constant at 0.002%; the concentration of unlabeled
Pluronic is 0.02% (a) and 0.2% (b); the incubation time
is 30 min.
For the samples characterized by bimodal ¯uo-
rescence histograms, ¯uorescence micrographs
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162
RAPOPORT ET AL.
revealed distinctly different distribution of ¯uor-
escence for the cells belonging to the same sample
(Figure 4). Figure 4B shows that ¯uorescence of
some cells is relatively low and concentrated
around the nucleus, suggesting that Pluronic is
sequestered in cytoplasmic vesicles. In contrast,
¯uorescence of other cells is higher and more
evenly distributed, suggesting that Pluronic is
released from cytoplasmic vesicles. The observed
differences may be due to the differences in the
intracellular Pluronic concentration within the
cell ensemble; alternatively, for the cells that are
at different stages of the cell cycle, the mem-
branes of acidic vesicles may have different
susceptibility to the action of a polymeric surfac-
tant. The fraction of the cells with stronger and
more evenly distributed ¯uorescence grew with
Pluronic concentration, as indicated by ¯ow
cytometry (Figure 2) and con®rmed by ¯uores-
cence microscopy.
for drug-sensitive cells (Table 1). For both types of
cells, the uptake of a ¯uorescently labeled Pluro-
nic dropped with increasing concentration of the
unlabeled Pluronic (Table 1).
Fluorescence histograms are presented in
Figure 5; intensities and fractions of the respec-
tive ¯uorescence peaks are presented in Table 2.
Fluorescence histograms revealed differences in
Pluronic distribution in drug-sensitive and MDR
cells. There is an obvious discrepancy between the
data of Tables 1 and 2: whereas the uptake of
¯uorescently labeled Pluronic dropped with in-
creasing concentration of the unlabeled Pluronic
(Table 1), the mean intracellular ¯uorescence of
the cells went through a ¯at maximum in drug-
sensitive cells and even increased in drug-resis-
tant cells (Table 2). In other words, the uptake of
the unlabeled Pluronic resulted in a higher
¯uorescence of the cells that comprised a lower
intracellular concentration of a ¯uorescently
labeled Pluronic. This result presumably occurred
because of the increase of pH of the probe environ-
ment, which, as shown in Figure 1, resulted in an
increase of probe ¯uorescence.
The duration of cell incubation with Pluronic
substantially affected the intracellular distribu-
tion of Pluronic. For all Pluronic concentrations,
Pluronic release from acidic vesicles increased
with the incubation time.
The data just presented con®rm the permeabi-
lization of the membranes of acidic vesicles under
the action of Pluronic copolymer, which may allow
the release of Pluronic molecules from acidic
vesicles into cytosol.
Ultrasound was another important factor that
modulated the intracellular uptake and distribu-
tion of Pluronic molecules.12 Figure 12 of ref. 12
suggests that ultrasound increased Pluronic
transfer from acidic vesicles, presumably due to
the perturbation of their membranes resulting in
their enhanced permeabilization; the latter was
observed even for a short-term (10 min) cell
sonication with relatively low Pluronic concentra-
tions (0.005%). The application of ultrasound
allowed decreasing Pluronic concentration and
shortening of the incubation time needed for the
permeabilization of the cytoplasmic vesicles.
Some indications of the transfer of Pluronic
from the cytoplasmic acidic vesicles into the
cytosol were obtained in ref. 28; the author con-
jugated peroxidase to Pluronic P-85 and intro-
duced the conjugate encapsulated in Pluronic
P-85 micelles into Jurkat cells.28 The conjugate
was taken up by the cells via endocytosis and was
localized in the acidic vesicles. However, the
insoluble product of the peroxidase reaction with
its substrate was observed not only in the vesicles
but also in the cytosol, indicating that the con-
jugate was partly released from acidic vesicles.
This result is consistent with our results already
presented.
For the MDR cells, a bimodal distribution of
cell ¯uorescence was already observed at a very
low Pluronic concentration in the incubation
medium that corresponded to Pluronic unimers
(Figure 5). In contrast, for the sensitive cells, the
distinctly bimodal distribution of cell ¯uorescence
was observed only at Pluronic concentrations
corresponding to micellar solutions. These data
suggest that MDR cells are more susceptible to
the action of a Pluronic copolymer than drug-
sensitive cells, which may point to the differences
Pluronic Uptake and Distribution in Ovarian
Carcinoma Cells
These experiments were performed with drug-
sensitive A2780 and MDR A2780/ADR ovarian
carcinoma cells. Cells were incubated with a
mixture of a ¯uorescently labeled and unlabeled
Pluronic P-105; the concentration of the labeled
Pluronic in the incubation medium was main-
tained constant at 0.001% whereas the concentra-
tion of the unlabeled Pluronic progressively
increased, as indicated in Figure 5.
For drug-resistant cells, the overall Pluronic
uptake measured by ¯uorescence depletion from
the incubation medium was slightly lower than
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INTRACELLULAR TRAFFICKING OF PLURONIC MICELLES
163
Figure 5. Fluorescence histograms of drug-sensitive A2780 cells (left column) and
MDR A2780/ADR cells (right column) incubated with a mixture of a ¯uorescently
labeled and unlabeled Pluronic P-105. The concentration of labeled Pluronic is
maintained constant at 0.001%, and the concentration of unlabeled Pluronic P-105 is
indicated in the Figure.
Table 1. Pluronic P-105 Uptake by Drug-Sensitive and MDR A2780 Cells Measured
by Depletion From the Incubation Mediuma
Uptake of a
Total Pluronic P-105
Concentration, % wt/v
Fluorescently Labeled
Pluronic, mg/106 cells
Cell Type
Drug-sensitive A2780 cells
0.001
0.011
0.1
0.53 Æ 0.02
0.19 Æ 0.02
0.11 Æ 0.01
0.08 Æ 0.01
0.44 Æ 0.02
0.18 Æ 0.02
0.08 Æ 0.01
0.04 Æ 0.01
2.0
Drug-resistant A2780/ADR cells
0.001
0.011
0.1
2.0
aConcentration of ¯uorescently labeled Pluronic P-105 is 0.001% for all samples; incubation
time 30 min.
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164
RAPOPORT ET AL.
Table 2. Flow Cytometry Data for A2780 and A2780/ADR Cells Incubated With a Mixture of a Fluorescently
Labeled and Unlabeled Pluronic P-105a
Drug-Sensitive A2780 Cells
Drug-Resistant A2780/ADR Cells
Pluronic
Concent-
ration, %
Mean-
Fluores-
cence
Mean-
Fluores-
cence
Fraction
M1,%
Fraction
M25,%
Fraction
M1,%
Fraction
M25,%
b
M1
M2
M1
M2
0.001
0.01
0.1
1.0
2.0
Ð
Ð
Ð
24
26
Ð
Ð
Ð
57
56
Ð
Ð
Ð
79
80
Ð
Ð
Ð
43
44
35
46
57
40
43
13
20
20
23
24
47
51
49
43
47
55
82
87
96
96
53
49
51
57
53
28
40
43
53
50
aConcentration of ¯uorescently labeled Pluronic P-105Ð0.001% for all samples; incubation time 30 min.
bM1 and M2 are mean ¯uorescence intensities of the ®rst and the second pick, respectively.
in the structure of the acidic vesicle membranes in
drug-sensitive and drug-resistant cells. This
suggestion is in agreement with Kabanov and
Alakhov's results.21 These authors observed a
reduction of high pH gradients in MDR cells
under the action of unimeric concentrations of
Pluronic L61; the reduction of pH gradients
indicated that Pluronic had ionoforic properties,
which resulted in the increased release of posi-
tively charged compounds (e.g., DOX) from acidic
vesicles.21 Our data suggest that not only ions but
also neutral polymeric molecules (i.e., Pluronic P-
105 molecules; MW 6500 Da) can experience
Pluronic-induced enhanced release from acidic
vesicles. The data just presented support Kaba-
nov and Alakhov's hypothesis that the permeabi-
lization of the cytoplasmic vesicles of MDR cells by
Pluronic molecules may play an important role in
the hypersensitization of drug-resistant cells by
Pluronic unimers.21
Rb are the DNA-intercalating drugs; DOX has
6,10
signi®cantly higher af®nity to DNA than Rb
;
in HL-60 cells, DOX preferentially accumulates in
cell nuclei while Rb partitions between nuclei and
cell membranes.10
Drug intracellular drug distribution in ovarian
carcinoma cells depended on cell type (sensitive,
resistant), drug type (Rb, DOX), and delivery
system (PBS, Pluronic micelles). In drug-sensitive
A2780 cells, both DOX and Rb preferentially
accumulated in cell nuclei independently on the
delivery system; location of the nuclei was veri®ed
by the DAPI uptake. For Rb, some fraction of the
drug also partitioned into the membranes and
perinuclear compartments. These data suggest
that in drug-sensitive cells, drug accumulation in
acidic vesicles was not signi®cant, presumably
due to the low pH gradient between acidic vesicles
and cytosol.17 This gradient provided for drug
traf®cking into cell nuclei.
For drug-resistant A2780/ADR cells, drug dis-
tribution was distinctly different for DOX and Rb.
Whereas DOX accumulated in cell nuclei (Fig. 6A),
Rb was completely excluded from the nuclei
(Fig. 6B). Confocal microscopy revealed Rb parti-
tioning into plasma membranes, intracellular
membranes, and intracellular vesicles (data not
Effect of Pluronic on Drug Traf®cking in
Drug-Sensitive and MDR Cells
Anthracyclin drugs DOX and Rb were used in
these experiments; conventional and confocal
¯uorescence microscopy was exploited. DOX and
Figure 6. Phase contrast and ¯uorescence micrographs of A2780/ADR cells incu-
bated for 30 min with (A) DOX (10 mg/mL) dissolved in PBS or Rb (10 mg/mL) dissolved
in (B) PBS, (C) 1.0% Pluronic P-105, or (D); 0.1% Pluronic P-105. When delivered in
PBS, DOX accumulates in cells nuclei whereas Rb is excluded from cell nuclei [compare
(A) and (B)]; when delivered in 1.0% Pluronic micelles, Rb penetrates into cell nuclei
[compare (B) and (C)]; at 0.1% Pluronic solution, Rb does not penetrate into nuclei
but accumulates in plasma membranes, cytoplasmic vesicles, and perinuclear
compartments (D).
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INTRACELLULAR TRAFFICKING OF PLURONIC MICELLES
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RAPOPORT ET AL.
shown). The Rb molecule is bulkier and more
hydrophobic than that of DOX and much less
DNA-tropic.6 Our previous experiments using
HL-60 cells showed that ꢁ40% of the intra-
cellular Rb delivered in PBS was retained in cell
membranes.10
micelles on Rb release from acidic vesicles and
cell membranes.
Effect of Pluronic on the Intracellular
Drug Uptake by Drug-Sensitive and MDR Cells
Introduction of Rb in Pluronic micelles drama-
tically changed the intracellular traf®cking of Rb
in MDR cells. In this case, Rb preferentially
accumulated in the cell nuclei (Fig. 6C). No drug
was observed in cell membranes, vesicles, or
cytosol, suggesting that Pluronic induced Rb
release from acidic vesicles and extraction from
membranes. Interestingly, substantial nuclear
accumulation of Rb in MDR cells required much
higher Pluronic concentrations than those that
caused disruption of the pH gradient of acidic
vesicles of MDR cells. At Pluronic concentration of
0.1%, Rb accumulated in the perinuclear com-
partments of the MDR cells and did not penetrate
into the nuclei (Fig. 6D); nuclear accumulation
was observed at Pluronic concentrations of 1%
and higher (Fig. 6C). It seems that for lipophilic
drugs like Rb, substantial nuclear accumulation
involves not only drug release from acidic vesicles
but also drug extraction from cell membranes,
which in turn requires rather high concentrations
of polymeric surfactants.
It should be noted that Rb delivery in a
complete RPMI-1640 medium rather than in
PBS increased Rb accumulation in cell nuclei.
Moreover, if the cells were ®rst drug-loaded
in PBS and then incubated in a complete RPMI-
1640 medium, Rb gradually accumulated in the
nuclei. This result indicates that some component
(or components) of the complete RPMI-1640
medium exerted the same effect as Pluronic
Drug-sensitive and MDR cells differed dramati-
cally in their response to drug introduction in
Pluronic micelles. When DOX was delivered in
Pluronic micelles, its uptake by drug-sensitive
cells dropped, whereas the uptake by MDR cells
increased. Typical ¯ow cytometry results are
presented in Tables 3 and 4 (the data of Tables 3
and 4 were acquired with different cell batches;
only minor quantitative differences were observed
for non-sonicated samples, the general pattern
being the same). It should be noted that for DOX,
¯ow cytometry results agree with the direct
measurements of DOX uptake based on drug de-
pletion from the incubation medium. This agree-
ment allows using ¯ow cytometry for comparing
DOX uptake by various samples.
The micellar-induced decrease of DOX uptake
by drug-sensitive cells was previously reported by
us,5±13 and the mechanism of this effect is
discussed in details in a recent paper.12 The
increase of DOX uptake by MDR cells in the
presence of Pluronic micelles is observed for the
®rst time; the effect was presumably caused by a
Pluronic-induced deactivation of drug ef¯ux
pumps in MDR cells, as was previously reported
for Pluronic unimers.14,21±26 Note that the
increased DOX uptake was more pronounced at
Pluronic concentration of 0.1% than at 10%
(Tables 3 and 4). At a concentration of 0.1%,
Pluronic P-105 does not exert a shielding effect on
drug uptake and therefore the main effect of
Table 3. Effect of Pluronic Micelles and Ultrasound on DOX Uptake by Adherent
A2780 and A2780/ADR Cellsa
Pluronic P-105
Mean Fluorescence, arb.u.
Concentration in
RPMI-1640, wt %
Cell Line
Non-sonicated
Sonicated
Drug-Sensitive
A2780
0
233.1 Æ 17.7
233.6 Æ 18.7
111.6 Æ 11.2
31.8 Æ 0.1
230.8 Æ 21.8
238.0 Æ 26.0
132.2 Æ 6.1
35.5 Æ 0.1
0.1
10
0
0.1
10
Drug-resistant
A2780/ADR
125.5 Æ 1.5
137.9 Æ 12.4
90.0 Æ 0.8
66.5 Æ 4.0
aInitial DOX concentration, 50 mg/mL; continuous wave sonication for 10 min; ultrasound
frequency, 69 kHz; ultrasound power density in the incubation medium, 3.2 W/cm2. The
experiments were made in duplicate; average values Æ standard deviations are presented.
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INTRACELLULAR TRAFFICKING OF PLURONIC MICELLES
167
Table 4. Effect of Pluronic Micelles and Ultrasound on DOX Uptake by Adherent
A2780 and A2780/ADR Cellsa
Pluronic Concentra-
tion in RPMI-1640,
wt %
Mean ¯uorescence, arb.u.b
Cell Line
A2780
Non-sonicated
Sonicated
0
229.2 Æ 15.4
237.4 Æ 16.0
124.4 Æ 11.2
52.3 Æ 5.0
294.1 Æ 21.8
274.7 Æ 24.0
160.0 Æ 6.1
58.2 Æ 5.3
0.1
10
0
0.1
10
A2780/ADR
143.6 Æ 15.4
191.8 Æ 15.6
104.7 Æ 5.2
72.8 Æ 4.1
aInitial DOX concentration, 50 mg/mL; continuous wave sonication for 10 min; ultrasound
frequency, 69 kHz; ultrasound power density in the incubation medium, 3.2 W/cm2. The
experiments were made in triplicate; average values Æ standard deviations are presented.
bStatistically signi®cant differences (p < 0.05) were observed between drug-sensitive and drug-
resistant cells in all cases. Across various Pluronic concentrations, for the drug-sensitive cells,
statistically signi®cant differences were observed between a 10% Pluronic solution and a RPMI-
1640 or 0.1% Pluronic solution. For the MDR cells, across various Pluronic concentrations,
statistically signi®cant differences were observed in all cases. The sonication effect was statistically
signi®cant in all cases with the exception of the MDR cells incubated in RPMI-1640 medium; in the
latter case, substantial cell sonolysis was observed, indicating considerable damage of plasma
membranes that could cause drug leaking from the cells.
Pluronic is that of deactivating ef¯ux pumps. In
contrast, at a concentration of 10%, Pluronic
micelles exert a shielding effect on the drug
uptake; this effect is opposite in direction to the
deactivation of ef¯ux pumps. The ®nal result
depends on the competition of two processes; as
follows from the data of Tables 3 and 4, for DOX in
the MDR cells, the deactivation of ef¯ux pumps
prevailed over the shielding effect of Pluronic
micelles.
For Rb, the situation was more complicated. As
already mentioned, Rb partitions between various
cellular compartments (i.e., nuclear DNA, mem-
branes, cytoplasmic vesicles). Rb ¯uorescence de-
pends strongly on its localization. Compared with
an aqueous environment, ¯uorescence is enhanc-
ed in phospholipid membranes and quenched
with Rb intercalation into DNA.6 The resulting
¯uorescence of Rb-loaded cells depends on Rb
partitioning between various cellular compart-
ments. Therefore, for Rb, in contrast to DOX,
¯uorescence intensity is not a direct function of
the intracellular concentration. This difference
complicates quantitative measurements but pro-
vides additional information on Rb partitioning.
At a low Rb concentration in the incubation
medium (10 mg/mL), the effect of Pluronic micelles
on Rb uptake by drug-sensitive and MDR cells
was similar to that observed for DOX. Based on
the direct measurements of drug depletion from
the incubation medium, the uptake of Rb by the
drug-sensitive A2780 cells dropped whereas
that by the MDR cells increased when Rb
was delivered in Pluronic micelles (Table 5). In
the drug-sensitive cells, Rb predominantly accu-
mulated in cell nuclei independently of the
delivery system; in this case, a decrease in drug
uptake was in agreement with a decrease in cell
Table 5. Effect of Pluronic Micelles on Rb Uptake and Intracellular Fluorescence for
Drug-sensitive and MDR Cellsa
Pluronic Con-
centration in
RPMI-1640, %
Mean Fluores-
cence, arb.u.
(¯ow cytometry)
Fluorescence/
Uptake Ratio,
arb.u.
Rb Uptake,
mg/106 Cells
Cell Line
Drug-Sensi-
tive A2780
MDR A2780/
ADR
0
10
0
1.30 Æ 0.13
0.38 Æ 0.04
0.17 Æ 0.02
0.30 Æ 0.03
79.8
22.9
31.2
24.7
62
61
185
82
10
aRb concentration in the incubation medium, 10 mg/mL.
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168
RAPOPORT ET AL.
¯uorescence (Table 5). In contrast, for the MDR
cells, the increased drug uptake in the presence of
Pluronic micelles was accompanied by a slight
drop of cell ¯uorescence (Table 5), indicating the
enhanced nuclear localization. This result is in
agreement with the data provided by ¯uorescence
microscopy (see Figure 6).
sensitive and MDR cells was much more pro-
nounced in the presence of 10% Pluronic micelles
than in PBS or 0.1% Pluronic P-105 solution
suggesting that Pluronic micelles and ultrasound
acted synergistically. The mechanism of the
ultrasonic enhancement of drug uptake from
micellar solutions was previously discussed in
detail.12 Brie¯y, a new equilibrium between the
external and internalized drug is established
under ultrasound, being shifted towards the
internalized drug. Upon turning ultrasound off,
the initial external±internal drug equilibrium is
not restored if ultrasound-induced plasma mem-
brane defects are sealed, which results in the
``supersaturation'' of cells with drug. We have
reported that 10% Pluronic micelles exerted a
protective effect on the cells during the sonication
process, presumably because of the membrane
¯uidization that resulted in the accelerated
sealing of the ultrasound-induced membrane
defects.11,12 No cell protection was exerted by
PBS, RPMI-1640, or a 0.1% Pluronic solution;
therefore, the internalized drug could partly leak
out of the cells through the ultrasound-induced
membrane defects, thus decreasing the resulting
drug uptake. These data show that drug delivery
in polymeric micelles combined with the appli-
cation of ultrasound might be an effective way
of drug targeting to drug-sensitive and MDR
tumors.
An important difference between DOX and Rb
is that the uptake of DOX gradually levels off with
increasing drug concentration in the incubation
medium, whereas the uptake of Rb signi®cantly
increases in the same concentration range.12 For
DOX, the upper limit of drug uptake is controlled
by drug intercalation into DNA. For Rb, uptake
proceeds above this limit12 because of the drug
accumulation in the membranes and cytoplasmic
vesicles. Rb localization in mitochondrial mem-
branes was con®rmed by the intracellular bior-
eduction of the nitroxide moiety that proceeded in
mitochondrial membranes.10 For Rb, the differ-
ence between the ¯uorescence of drug-sensitive
and MDR cells signi®cantly dropped with increas-
ing drug concentration in the incubation medium
(Table 6), presumably because of Rb accumulation
in the membranes and vesicles of the MDR cells.
These data suggest that ef¯ux pumps do not affect
a fraction of drug localized in vesicles and
membranes.
Effect of Ultrasound on the Intracellular Uptake
of DOX and Rb by Drug-Sensitive and MDR Cells
As was previously observed for drug-sensitive
cells, ultrasound substantially enhanced the
intracellular DOX and Rb uptake by the MDR
cells in the presence of 10% Pluronic micelles.
Typical results for DOX are presented in Tables 3
and 4. It is noteworthy that the effect of
ultrasound on the drug uptake by both drug-
CONCLUSIONS
The intracellular uptake of Pluronic P-105 result-
ed in the permeabilization of acidic vesicles and
disruption of a pH gradient at their membranes.
In MDR cells, the permeabilization occurred at a
signi®cantly lower Pluronic concentrations than
Table 6. Effect of Rb Concentration in the Incubation Medium on the Intracellular Fluorescence of Rb in A2780
Drug-Sensitive and MDR Cells
Fluorescence Ratio,
Mean Fluorescence, arb.u.
Drug-Sensitive/MDR
Rb Concentration in the
Rb Concentration in the
Incubation Medium, mg/mL
Incubation Medium, mg/mL
Pluronic P-105
Cell Type
Concentration, wt%
10
50
10
50
Drug-Sensitive
0
10
0
79.8
22.9
31.2
24.7
367
95
284
60
2.6
0.9
Ð
1.3
1.5
Ð
MDR
10
Ð
Ð
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INTRACELLULAR TRAFFICKING OF PLURONIC MICELLES
169
in drug-sensitive cells, suggesting a higher sensi-
tivity of drug-resistant cells to the action of
polymeric surfactants. The permeabilization of
acidic vesicles caused dramatic changes of the
intracellular localization of the anthracyclin drug
Rb. When delivered in PBS, the drug was
excluded from the nuclei of MDR cells, whereas
delivery in Pluronic micelles resulted in drug
release from the cytoplasmic vesicles and cell
membranes and accumulation in cell nuclei. Drug
delivery in Pluronic micelles combined with the
application of ultrasound substantially increased
drug uptake by the MDR cells. These ®ndings
suggest that the nuclear accumulation of drugs
internalized via ¯uid-phase endocytosis can be
enhanced by the application of Pluronic micelles
and can be further augmented by ultrasonic
irradiation.
7. Munshi N, Rapoport N, Pitt WG. 1997. Ultrasonic
activated drug delivery from Pluronic P-105
micelles. Cancer Lett 118:13±19.
8. Rapoport NY, Herron JN, Pitt WG, Pitina L. 1999.
Micellar delivery of doxorubicin and its paramag-
netic analog, ruboxyl, to HL-60: Effect of micelle
structure and ultrasound on the intracellular drug
uptake. J Controlled Release 58:153±162.
9. Husseni GA, Myrup GD, Pitt WG, Christensen DA,
Rapoport NY. 2000. Factor affecting acoustically
triggered release of drugs from polymeric micelles.
J Controlled Release 69:43±52.
10. Rapoport N, Marin AP, Timoshin AA. 2000. Effect
of a polymeric surfactant on electron transport
in HL-60 cells. Arch Biochem Biophys 384:100±
108.
11. Marin A, Muniruzzaman M, Rapoport N. 2001.
Acoustic activation of drug delivery from polymeric
micelles: Effect of pulsed ultrasound. J Controlled
Release 71:239±249.
12. Marin A, Muniruzzaman M, Rapoport N. 2001.
Mechanism of the ultrasound activation of micellar
drug delivery. J Controlled Release 75:69±81.
13. Muniruzzaman Md, Marin A, Luo Y, Prestwich GP,
Pitt WG, Husseini G, Rapoport N. Intracellular
uptake of Pluronic unimers and micelles: Effect of
the aggregation state and ultrasound. Colloids
Surfaces B: Biointerfaces, in press.
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ACKNOWLEDGMENTS
This work was supported by the NIH grant R01
CA76562-01A1. Productive discussions with
Dr. A. Kabanov are greatly appreciated. The
authors acknowledge valuable editorial com-
ments of Dr. D.A. Christensen.
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