[CANCER RESEARCH 51, 2710-2719, May 15, 1991]
Lysosomal Localization and Mechanism of Uptake of Nile Blue Photosensitizers in
Tumor Cells1
Chi-Wei Lin,2 Janine R. Shulok, Sandra D. Kirley, Louis Cincotta, and James W. Foley
Urology Research Laboratory, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114 [C-W. L., J. R. S., S. D. KJ, and Rowland
Institute for Science, Cambridge, Massachusetts 02142 ¡L,C., J. W. F.]
ABSTRACT
Nile blue derivatives have been shown to be potentially effective
photosensitizers for photodynamic therapy of malignant tumors. Results
of a previous study suggested that the high accumulation of these dyes
in cells may be the result of dye aggregation, partition in membrane
lipids, and/or sequestration in subcellular organelles. In this report,
results of studies are presented from an investigation of the subcellular
localization and mechanism of accumulation of these dyes in cells in
vitro. A video-enhanced fluorescence microscopy was used, and a punctate
pattern of fluorescence was seen, most of which was localized in the
perinuclear region with extracellular dye concentrations between 1 to 100
UM.These particles resembled characteristic particles identified by stand
ard lysosomal dyes. At higher dye concentrations (1 pM or above),
fluorescence in the perinuclear region was too intense to resolve into
discrete cellular structures, while fluorescence in other cellular structures
including mitochondria and cytomembranes was visible. At even higher
dye concentrations (10-100 MM).Nile blue derivatives were seen with a
light microscope as blue particles, the size and location of which resem
bled the punctate fluorescence described above. Results which further
suggest that the lysosome is the main site of dye localization include (a)
histochemical staining of dye-loaded cells with the lysosomal marker
enzyme acid phosphatase, which showed similar localization of the
enzyme-staining and dye-containing particles, (b) phototreatment of dyeloaded cells which obliterated the majority of the acid phosphatasestained particles, and (c) treatments with agents affecting the membrane
pH gradient reduced the uptake and enhanced the efflux of dyes, while
agents that alter cellular membrane potentials had no effect on dye
accumulation. The uptake of the dyes was partially inhibited by inhibitors
of oxidative phosphorylation indicating that at least part of the process
is energy dependent. These findings, together with previous results show
ing that the cellular uptake of these dyes is highly concentrative and
proportional to the extracellular dye concentration over a wide range, are
consistent with the hypothesis that the dyes are mainly localized in the
lysosomes via an ion-trapping mechanism. Results of the present study
also suggest that the lysosomes may be an intracellular target for pho
todynamic killing of tumor cells mediated by Nile blue photosensitizers
and that lysosomotropic photosensitization may be a strategy for effective
and selective destruction of tumor cells.
tosensitizers with high tumor selectivity will enable effective
treatment of multiple, infiltratili!*, and invisible tumors, thus
expanding the utility of PDT as a useful tool in cancer therapy
with intent to cure. Active research is under way to search for
more tumor-selective sensitizers (4-6) and to improve the sensitizer delivery system for better tumor targeting (7-10).
Several early studies with animal tumor models have shown
that benzophenoxazines, including several Nile blue analogues,
constitute a special class of dyes that are selectively localized in
tumors (11-15). Results of recent work have demonstrated that
Nile blue A can be converted to derivatives with substantially
increased photoactivity (16-18). Furthermore, structural mod
ifications of the parent dye can result in analogues having
substantially altered pKa values and hydrophobicities, proper
ties which may be significant in dye localization in tumors. In
a previous study (19, 20), we showed that Nile blue derivatives
having high "O^ yields are effective in mediating photocytotoxicity in vitro. Derivatives with "O2 quantum yields of 35-80%
can mediate a 90% in vitro photocytotoxicity with extracellular
dye concentrations as low as 5 x 10~8M. This is about 3 orders
lower than with hematoporphyrin derivative. The finding thus
suggests that these compounds are potentially effective photo
sensitizers for PDT. The cellular uptake of Nile blue derivatives
is rapid, highly concentrative, and directly proportional to the
extracellular dye concentration. The uptake can proceed at
temperatures below 2°C,thus excluding endocytosis or a car
rier-mediated mechanism for the uptake. The overall results
suggest that high cellular accumulation of these dyes may result
from dye aggregation, partition in membrane lipids, and/or
sequestration in certain intracellular organelles (20).
In the present study, the intracellular localization of Nile
blue derivatives and the mechanism of their accumulation in
human bladder carcinoma cells were examined. Findings from
this study suggest that the lysosome is the main site of localiza
tion and ion trapping is likely the process by which Nile blue
dyes are accumulated in cells.
INTRODUCTION
MATERIALS
PDT' is an investigational treatment procedure for malignant
tumors (1-3). The effectiveness of the treatment relies, to a
great extent, on the tumor selectivity of the photosensitizer
which, upon photoactivation, imparts a photodynamic action
for cytotoxicity and tumor destruction. The availability of pho-
Nile Blue Derivatives. Previous reports (16-18) indicated that struc
tural modifications of Nile blue A yielded derivatives with enhanced
photoactivity as well as different photochemical properties. The six
Nile blue derivatives used in this study and their designations are shown
in Table 1. The photochemical properties of these derivatives have been
described previously (16-20). All six derivatives were examined in
subcellular localization studies using fluorescence and light microscopy.
In studies involving uptake and sequestering mechanisms, derivatives
NBA and NBA-6I were used to represent derivatives with different pK.
values and hydrophobicities. In studies involving photodynamic treat
ment of the cell, derivatives with moderate photoactivity, NBA-6I and
NBS-6I, were used.
Tumor Cells. The cell line used for this study, MGH-U1, is a
subculture of I 24. a well established human bladder carcinoma cell
line (21). The cells were grown routinely in McCoy's 5A medium
Reccived 12/19/90; accepted 3/6/91.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by grants from the National Cancer Institute (CA
32259), the Beinecke Foundation, the Thomas Anthony Pappas Charitable Foun
dation, and the Rowland Institute for Science.
2 To whom requests for reprints should be addressed, at Urology Research
Laboratory, Massachusetts General Hospital, Boston, MA 02114.
3 The abbreviations used are: PDT, photodynamic therapy; PBS. phosphatebuffered saline at pH 7.4: DPBS. Dulbecco's phosphate-buffered saline at pH
7.4; 'Oi. singlet oxygen; SIT, silicon-intensified-target; TPP, tetraphenyl-phosphonium: FCCP.p-trifluoromethoxyphenyl
hydrazone; 2,4-DNP, 2,4-dinitrophenol. The designations of the Nile blue derivatives are listed in Table 1.
AND METHODS
supplemented with 5% fetal calf serum.
Microscopic Observations of Nile Blue Derivatives in Cells. Both
fluorescence and light microscopies were used to examine the subcel-
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LYSOSOMAL LOCALIZATION OF NILE BLUE PHOTOSENS1TIZERS
Table 1 Structures and designation of Nile blue derivatives used in this study
with their absorption maxima, pK,, and partition coefficients"
Designation
Structure
, (nm)
pK*
P/
NBA
(C2H5)2N
623
10.0
173
NBA-6I
(C2H5)2N
642
6.6
5625
NBS
645
10.0
356
NBS-6I
660
6.5
5027
Sat-NBS
628
11.0
109
examining dye localizations in cells (22), subconfluent cells grown on
coverslips were incubated for 30 min at 37°Cwith 10 ml of dye
solutions; dye concentrations ranging from 10 MMto 0.1 nM were used.
At the end of the incubation, the cells were removed from the dye
solution, washed with DPBS, mounted on Lab-Tek chambers, and
observed under the fluorescence microscope with the aid of a Hamamatsu C2400 SIT camera connected to a high resolution color monitor
and a Sony UP-5000 video printer.
At dye concentrations >10 MM,Nile blue dyes in ceils can be directly
seen under the light microscope. This was performed by incubating
subconfluent cells, grown on glass slides, in 100 mm culture dishes
with 10 ml of various dye solutions at 20 MM,at 37°Cfor 10-30 min
to permit dye uptake. Cells were rinsed to remove residual dye solution,
mounted with dye-free medium, and observed immediately under the
microscope. In experiments designed to examine the intracellular translocation of the dyes under conditions of short uptake times and rela
tively high dye concentration, cells grown on 22-mm coverslips were
incubated with 100 /il of 10-100 MMdye solutions at 37°Cfor 5 min.
Cells were rinsed twice to remove residual dye, placed in dye-free
medium, and returned to the incubator for specified times before
microscopic observation.
Acid Phosphatase Staining. To verify that the blue stained particles
seen under the light microscope were lysosomes, cells were stained with
a standard lysosomal marker enzyme, acid phosphatase, to identify the
organelle. In this experiment, cells were permitted to take up NBA-61
(20 MMat 37°Cfor 30 min), and locations of the dye-containing particles
were recorded by photomicrograph. The cells were immediately fixed
in cold 3% glutaraldehyde in 60% acetone for 3 min at -20°C,washed
Sat-NBS-6I
(C2HS)2N
637
9.5
1752
* Values of A„,„,
t, pK„and Pc are taken from Refs. 16-20.
* Solvent, methanol:acetic acid, 250:1.
' Partition coefficients between 2-octanol and phosphate-buffered saline at pH
7.4.
16
16
NBA-61
NBA
•12
12
—
8
500
600
700
500
600
700
Wavelength (nm)
Fig. 1. Excitation (
) and emission (
) fluorescence spectra of NBA
and NBA-61 in MGH-U1 cells. Monolayer cells were allowed to take up dyes by
incubating at 37"C with 2 ml of dye solution at 2.5 x 10"' M in serum-free
McCoy's 5A medium for 30 min. Cells were then removed from culture plates,
resuspended in DPBS, and placed in a cuvette equipped with a stirring device to
maintain cells in suspension. Excitation spectra were obtained with 725 nm
detection and emission spectra with 600 nm excitation. Autofluorescence was
corrected from the spectra by using cells without dye.
in deionized water, and air dried. Staining of acid phosphatase was
carried out with naphthol AS-BI phosphate (0.4 mg/ml) and Fast
Garnet GBC (0.3 mg/ml) in acetate buffer at pH 5.2 for 60 min at
37°C.When the reaction was completed, the cells were washed in
deionized water, air dried, and counterstained with méthylène
blue. The
stained slides were examined under the microscope to compare the sites
of dye localization with the pattern of the acid phosphatase-stained
lysosome particles.
Photodynamic Treatment of Cells. Cells to be treated were grown on
standard microscopic glass slides by placing one slide in a 100 mm cell
culture dish and adding 2 x IO6cells in 10 ml McCoy's 5A medium to
the dish. After the cells were allowed to attach overnight, the medium
was removed and the cells were washed once with DPBS. Ten ml of
DPBS containing photoactive Nile blue derivative, either 0.5 MMNBA61 or 0.2 MMNBS-6I, which was the extracellular dye concentration
determined from a previous study (20) to effect a 90% cell kill upon
photoirradiation, was added to the dish, and the cells were allowed to
take up the dye for 30 min at 37°C.Extracellular dye was removed and
the cells were rinsed once with PBS. The cells adhering to the slide
were placed in a new dish and covered with 8.5 ml of PBS, and light
treatment was performed immediately. The light source was a Polaroid
projector equipped with 590- to 700-nm band pass filters. The power
density of the light source was 8-10 mW/cm2, the light dose for the
treatment was 4.8 J/cm2, and the total treatment time was 8-10 min.
After photoirradiation, the cells were placed in medium for 10 min
before being fixed and stained for acid phosphatase to identify the
lysosomal particles.
Dye Uptake and Quantitation. The uptake of the Nile blue derivatives
by MGH-LJ1 cells was determined by plating 2 x IO6cells/60-mm dish
and allowing them to attach overnight. Dye solutions, 2 ml at 2.5 MM
in phenol-red free and serum-free McCoy's 5A medium, were added to
the cells at 37°Cand cellular dye concentrations were determined at
different time intervals as previously described (20). Briefly, cells were
removed from the cultured plates with 0.1% EDTA, dissolved in
lular localization of Nile blue derivatives. A Zeiss epifluorescence
microscope equipped with a xenon light source was used to observe the
concentrated HC1, and extracted with acidified chlorofornrmethanol
fluorescence of the Nile blue dyes in cells. Based on the excitation and
(1:1). Concentrations of the dye in the extracts were measured by
emission spectra of Nile blue dyes in cells (Fig. 1), a filter system
fluorescence spectroscopy.
consisting of a band pass excitation filter centered at 633 ±10 nm
Effect of Membrane Potential on Dye Uptake. Cellular membrane
(mean ±SD), a dichroic beam splitter at 650 nm, and a barrier filter
potential has been shown to affect the uptake and retention of cationic
dyes (23-25). We therefore examined the effects of this parameter on
at 675 nm, was used to permit specific observation of the fluorescence
emitted by these dyes. As described previously for the procedure of the uptake of the Nile blue analogues by subjecting the cells to three
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LYSOSOMAL LOCALIZATION OF NILE BLUE PHOTOSENSITIZERS
conditions known to reduce cell membrane potential: treatment with
valinomycin or ouabain or exposure to high K* medium. In the exper
iments in which the effects of valinomycin and ouabain were examined,
monolayers of cells at 2 x IO6 cells/60-mm dish were pretreated with
either valinomycin (1 fig/mi) or ouabain (1 HIM)for 30 min prior to the
addition of NBA or NBA-6I (2.5 ÕÕM
in phenol-red free and serum-free
McCoy's 5A medium), and the uptake was performed at 37°Cin the
presence of the agents. The cells were then removed from the plates
with 0.1% EDTA, and cellular dye was extracted and quantitated as
described above. TPP, a lipophilic cation, which has been used in a
variety of cellular and subcellular systems to measure membrane poten
tials (23), was used to verify that our experimental conditions produced
the expected changes in membrane potentials. Experiments in which
the effect of high K medium on dye uptake was examined were carried
out in DPBS with KC1 substituted for NaCI. The concentrations of K+
were 139 mM for the high and 3 mM for the low K+ DPBS. Cells were
pretreated with high or low K* DPBS for 30 min before addition of the
dyes (NBA or NBA-61 at 2.5 UM).
Effects of Membrane pH Gradient Modulators on Dye Uptake and
Efflux. Uptakes of many cationic compounds, particularly those that
are weak bases, are known to be affected by agents which reduce the
pH gradient or increase the intralysosomal pH (26, 27). Effects of these
agents on uptakes of NBA and NBA-61 were thus examined. Uptake
experiments were performed as described above for valinomycin with
the exception that nigericin (5 jig/ml), monensin (6 and 25 jiM), or
FCCP (1 and 10 JIM)was used.
The effect of nigericin on efflux of Nile blue dyes was also examined.
Monolayer cells plated at 2 x 106/60-mm dish were incubated with 2
ml of serum-free McCoy's 5A medium containing 2.5 fiM of NBA or
NBA-61 for 30 min at 37°Cto allow uptake of the dye. The medium
was removed, and the cell culture plates were washed twice in DPBS.
Dye-free medium with or without 5 ¿ig/mlof nigericin was added to
the plates and cellular dye concentrations were determined as above at
various times intervals for up to 80 min.
Effects of Oxidative Phosphorylation Inhibitors on Uptake. To deter
mine whether the uptake of Nile blue derivatives is an energy-dependent
process, effects of agents that inhibit oxidative phosphorylation on the
uptake of NBA and NBA-61 were examined in the absence of glucose
to reduce glycolysis as an alternative source of energy. Cells, 2 x IO6/
60-mm dish, were washed with DPBS twice, incubated with DPBS
without glucose for 30 min to deplete endogenous glucose, and then
pretreated with each of the inhibitors (2,4-DNP at 0.1 mM and sodium
azide at 10 mM) for 30 min in DPBS before the addition of dye (2.5
jiM). Uptake studies were performed at 37°Cin the presence of the
inhibitor.
RESULTS
Subcellular Localizations of Nile Blue Derivatives. The intracellular localization of Nile blue dyes was observed with both
fluorescence and light microscopy. A video-enhanced fluores
cence microscope equipped with a filter system for specific Nile
blue dye excitation and emission was used, and punctate fluo
rescence was seen in the cell with extracellular dye concentra
tions as low as 1 nM. Fig. 2, A-C, shows MGH-U1 cells stained
with 10 nM to 1 (¿Mof NBA, taken with the SIT camera,
demonstrating the dye distribution in these cells. At dye con
centrations of 1-10 nm, the fluorescence was seen as punctate
particles localized at the perinuclear area of the cell. These
particles resembled, both in the location and size, the particles
identified by standard lysosomal fluorescence dyes such as
Lucifer Yellow CH (28, 29) and acridine orange (30-32), as
well as particles identified by acid phosphatase staining shown
in Figs. 4 and 5. Aside from these particles, essentially no
fluorescence was observed in other parts of the cell in this
concentration range. As the dye concentration increased to 0.1
¿/M,fluorescence in the perinuclear region, where lysosomes
have been shown to localize, became dominant, and fluores
cence in other cytomembrane structures, including mitochon
dria, endoplasmic reticulum, and plasma membrane, became
visible (Fig. 2B). At even higher dye concentrations (>1 MM),
fluorescence in the perinuclear area was too intense to resolve
into discrete cellular structures, while fluorescence in mito
chondria! and cytomembrane structures was clearly visible (Fig.
2("). Examinations of these cells under the light microscope
revealed the appearance of blue particles in the perinuclear area
where intense fluorescence was seen.
Other Nile blue derivatives showed similar punctate patterns
of fluorescence at dye concentrations between 1 and 10 nivi; at
higher dye concentrations, cytomembrane structures, including
the plasma membrane and mitochondria, began to fluoresce
(Fig. 2, D-F).
With light microscopy, all six Nile blue derivatives appeared
mostly as blue particles localized at the perinuclear area (Fig.
3). The location and the size of these particles resembled the
punctate fluorescence particles seen under the fluorescence
microscope. With NBA, NBA-61, and NBS-6I, the paniculate
localization was highly exclusive and the sequestering occurred
almost immediately after the 5-min dye uptake period. With
NBS, sat-NBS, and sat-NBS-6I, however, diffuse cytoplasmic
and mitochondrial stains were also seen, and the sequestering
of the dye into particulate localizations usually required 15-40
min after the dyes entered the cells. Plasma membrane staining
can also be seen in cells treated with sat-NBS and sat-NBS-6I;
nucleolar staining can be observed in cells with NBS and satNBS.
Histochemical Identification of the Dye-containing Particles.
Further evidence for the lysosomal nature of Nile blue analoguecontaining particles was obtained from histochemical staining
of the dye-loading cells with a standard lysosomal marker
enzyme, acid phosphatase. The stained slides were examined to
determine whether the localization of blue particles matched
that of the acid phosphatase-staining particles. As shown in
Fig. 4, in most cells, particles containing both stains were
localized in the same areas. In some instances, individual
matching particles were identified with both stainings. This
result supports the overall conclusion that the dye-containing
particles observed under the light microscope were indeed
lysosomes.
Several technical difficulties prevented us from obtaining a
perfect match in localization between the dye-containing blue
particles and the acid phosphatase-staining
particles. First,
since cells were viable before fixation, some movement of the
lysosomes undoubtedly occurred during the time that lapsed
(2-5 min) between the photographic recordings of the blue
particles and fixation. Second, the fixation step tended to cause
shrinkage which may have altered the localization of subcellular
structures. Third, not all lysosomes took up adequate dye to be
seen under the light microscope. Thus, some lysosomes identi
fied by the acid phosphatase staining may not have been visu
alized as blue particles. Finally, the pattern of localization
recorded by photomicrograph is dependent on the depth of
focus under the microscope. Thus, different patterns of local
ization can exist for the same cell when a different layer of the
cell is in focus.
Photodynamic Evidence for Lysosomal Dye Localization. As
another test of the lysosomal localization of Nile blue dyes,
MGH-U1 cells were treated with NBA-61 or NBS-6I at concen
trations previously determined to elicit a 90% cell kill when
photoirradiated. The consequential elimination of the dye-eon-
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LYSOSOMAL LOCALIZATION OF NILE BLUE PHOTOSENSIT1ZERS
Fig. 2. Localizations of Nile blue derivative NBA in MGH-U1 cells seen under a video-enhanced fluorescence microscope equipped with a filter system for specific
excitation and emission of the dyes. Cells were incubated with various concentrations of dyes. At the end of the incubation, dye solution was removed and cells were
washed with DPBS and observed immediately. Photographs were taken with a Hamamatsu C2400 SIT camera connected to a color monitor and a Sony UP-5000
video printer. In A, with 10 nM of NBA, most of the fluorescence appeared as particles concentrated in the perinuclear region and an area around the nucleus. In B,
with 100 nM of NBA, the dye appeared mostly as punctate fluorescence, concentrated in the perinuclear region (arrows). Cytomembrane and mitochondria-like
structures also fluoresced. In C with 1 JJMof NBA. fluorescence at the perinuclear region was too intense to resolve into discrete cellular structures, while fluorescence
in mitochondria (arrows) and cytomembrane structures was clearly visible. In A with NBA-61 at 10 nM, punctate fluorescence, characteristic of lysosomes, appeared
as the main fluorescent structures. In E, with NBA-61 at I <JM.most of the fluorescence appeared in the perinuclear region and mitochondria-like structures were also
fluoresced. In F. with NBS at 10 nM, punctate fluorescence was seen as the main fluorescent structures.
taining lysosomes after photoirradiation of the cells may pro
vide another indication for the lysosomal localization of these
dyes acting as photosensitizers. Lysosomal particles, identified
by the acid phosphatase staining, in untreated cells were seen
distributed throughout the cells but, in most cases, were con
centrated in the perinuclear area (Fig. 5A). Photoirradiation of
dye-free cells (Fig. 5B) or dye treatment alone without photoir
radiation (photograph not shown) did not result in a noticeable
reduction in number or alteration in distribution of lysosomal
particles. However, most of the acid phosphatase particles were
obliterated with photoirradiation of dye-containing cells (Fig.
5, C and D). In these cells, only a small number of lysosomal
particles remained visible in the perinuclear area and virtually
all the peripheral lysosomes were no longer present. In about
10% of the cells, complete elimination of lysosomes was
observed.
Effects of Membrane Potential and pH Gradient on Dye
Accumulation. Previous studies have shown that cationic dyes,
such as rhodamine 123 and AyV'-bis(2-ethyl-l,3-dioxolane)kryptocyanine, which localize in the mitochondria are accu
mulated through an action of the mitochondrial membrane
potential. Alterations of the membrane potential changes the
uptake of these mitochondrial localizing dyes (23-25). Other
cationic dyes such as acridine orange, which localize in lyso-
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LYSOSOMAL LOCALIZATION OF NILE BLUE PHOTOSENS1T1ZERS
Fig. 3. Localizations of Nile blue derivatives
10-30 min. Cells were rinsed, mounted with
mitochondria-like structures without noticeable
61, 30-min dye incubation: E, sat-NBS. 30-min
observed under the light microscope. Cells grown on glass slides were incubated with 20 //\i dye solutions at 37"C for
dye-free medium, and observed immediately. Blue particles (arrowheads) signify sites of dye localization. Arrows,
stain. A, NBA, 30-min dye incubation: fi, NBA-6I, 30-min dye incubation; C, NBS, 20-min dye incubation; D, NBSdye incubation: F, sat-NBS-6I. I0-min dye incubation.
somes, are accumulated through the action of the pH gradient
across the lysosomal membrane. Agents perturbing the pH
gradient will affect the uptake of these compounds (26, 27). In
the present study, effects of agents and conditions which alter
either transmembrane potential or pH gradient on the uptake
of Nile blue analogues were studied in order to provide insight
into the mechanism of cellular dye uptake.
To verify that agents such as valinomycin, ouabain, and
nigericin caused the expected changes in membrane potentials
under our experimental conditions and cell system, a prelimi
nary experiment was conducted to test the effects of these agents
on MGH-U1 uptake of TPP, a well-studied lipophilic cation
whose uptake is highly dependent on membrane potential (23).
Under the conditions used, valinomycin (1 Mg/ml) and ouabain
(1 ITIM)caused, respectively, a 20 and 33% reduction of TPP
uptake, while nigericin (1 ^g/ml), which reduces the pH gra
dient and induces a compensatory increase in membrane poten
tial, caused a >2-fold increase in TPP uptake. Since high
concentrations of ionophores tend to produce nonspecific
permeability changes, studies with higher ionophore concentra
tions have not been performed.
Effects of cellular uptake of NBA and NBA-6I by valinomy
cin, ouabain, and high K+ medium were then examined. All
these are known to reduce the transmembrane potential but by
different mechanisms (23-25). Valinomycin is a membrane
ionophore which facilitates the efflux of K* ion. Ouabain is a
specific inhibitor for the membrane Na-K ATPase which pro
vides the energy for maintaining the membrane potential. High
k medium eliminates the transmembrane K" gradient and
hence reduces the potential. As shown in Fig. 6, none of these
treatments caused any significant reduction in dye uptake,
suggesting that the uptake of Nile blue dyes is not membrane
potential dependent. This result also implies that the mitochon
dria is not likely to be the main site of localization of these
dyes.
Effects of nigericin, FCCP, and monensin on the uptakes of
NBA and NBA-6I by MHG-U1 cells were then examined. All
of these agents are known to reduce the lysosomal transmem-
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LYSOSOMAL LOCALIZATION OF NILE BLUE PHOTOSENSITIZERS
B
Fig. 4. Acid phosphatase staining of NBA-61-loaded cells as histochemical
verification of the h sosomal nature of dye-containing particles. Cells were per
mitted to take up the dye (20 MMat 37°Cfor 30 min) and the locations of the
dye-containing particles were recorded by photomicrograph as shown in A. Cells
were immediately fixed and stained for acid phosphatase. The stained slides (B)
were examined under the microscope to compare the localizations of the dyeloaded particles and the acid phosphatase-containing lysosome particles. Imm \.
several matched particles.
of NBA and NBA-6I from dye-loaded cells (Fig. 7; P < 0.0001
for both NBA and NBA-6I at the end of 80 min). This result
suggests that the transmembrane pH gradient has a significant
effect on the retention of Nile blue dyes and further substanti
ates the hypothesis for the lysosomal localization of these dyes.
pH-dependent Dye Accumulation at Different Concentrations.
Since microscopic results described above indicate that the
intracellular distribution of the dyes may vary with different
dye concentrations and that nigericin appeared to be partially
effective in inhibiting the accumulation of dyes in the lysosomes, nigericin was used to examine whether lysosomal dye
accumulation varies as a function of dye concentration. Effects
of 5 ng/m\ of nigericin on 30-min uptakes of NBA and NBA61 were examined at different extracellular dye concentrations
ranging from 0.156 to 40 pM. Results (Fig. 8) show that, for
both NBA and NBA-6I, inhibitions of uptake by nigericin were
approximately 40-50% at dye concentrations <1 ^M, 60-80%
inhibitions between 1 and 10 ßM,and 60-70% inhibitions
between 10 and 40 //M. Although there appears to be higher
distributions of the dye in nigericin-sensitive sites at higher dye
concentrations, the overall result indicates that the pH-dependent accumulation constitutes a major portion of the total cell
ular uptake over the entire dye concentration range examined.
Effects of Oxidative Phosphorylation Inhibitors on Dye Up
take. To determine whether the uptake of Nile blue derivatives
is energy dependent, we examined the effects of two oxidative
phosphorylation inhibitors, 2,4-DNP and sodium azide, on the
uptakes of NBA and NBA-6I. Experiments were performed in
the absence of glucose to reduce glycolysis as an alternative
energy source. Both inhibitors caused significant reductions (P
< 0.0001 for all values) in the overall accumulation of dyes
(Fig. 9). At the end of the 20-min incubation, sodium azide and
2,4-DNP reduced NBA uptake by 32.9 and 49.5%, respectively,
and NBA-6I uptake by 28.2 and 50.0%, respectively. The cell
morphology at the end of the uptake period appeared to be
normal when observed by light microscopy, indicating that the
decreased uptakes with inhibitors of oxidative phosphorylation
were due to an energy-dependent process and not to cell death.
Although 2,4-DNP can also cause a reduction in mitochondria!
membrane potential, the observed decrease in uptake can imply
a mitochondria! localization of these dyes. Yet, the inhibition
did not occur in the presence of other energy source such as
glucose (data not shown), suggesting that the effect is related
to energy dependency rather than to membrane potential.
DISCUSSION
In a previous study (20), we suggested that the highly concentrative accumulation of Nile blue derivatives by tumor cells in
culture may be the result of dye self-aggregation, partition of
the highly lipophilic dyes into cellular lipids, and/or sequester
sosomal pH. All of these agents caused significant reductions
ing of the dyes into certain subcellular organdÃ-es. The first two
of the uptakes of the Nile blue dyes examined (Table 2). At the mechanisms are energy-independent processes, while the last is
end of a 30-min incubation, nigericin (5 ng/m\) reduced NBA likely energy dependent. Since the results of the present study
uptake by 72%, FCCP (10 ^M) by 75%, and monensin (25 MM) indicate that dye accumulation is energy dependent, it is un
by 54%. NBA-6I uptake was reduced 65% by nigericin, 67% by likely that molecular aggregation or lipophilicity is the primary
FCCP, and 40% by monensin (P < 0.0001 for all values, in mechanism for the cellular accumulation of these dyes, although
comparison to uptake without any of the agents). These levels lipophilicity is undoubtedly an important factor contributing to
of reductions were considerably greater than those reported for the uptake of the dye. The overall results of the present study
these agents with /V-dodecyl-(Ci2)-imidazole, a lysosomal local
indicate that the lysosome is the main site of intracellular
izing compound, in which reductions following 30-min uptakes
localization of Nile blue derivatives. At high dye concentrations
were 26 and 23% by nigericin (1.25 Mg/ml) and monensin (25 (1 fiM or higher), the cytomembranes, including plasma and
mitochondrial
membranes, are additional sites of dye
), respectively (28).
Nigericin also caused a rapid and significant increase of efflux localization.
brane pH gradient via different mechanisms (26, 27). Nigericin
is a membrane ionophore which facilitates the exchange of K+
and H+. FCCP is an ionophore which facilitates the efflux of
H+. Monensin acts by binding H+, thus increasing the intraly-
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LYSOSOMAL LOCALIZATION OF NILE BLUE PHOTOSENSITIZERS
B
,•.-*
l'
%
Fig. 5. Photodynamic evidence for lysosomal dye localization. Dye-treated MGH-U1 cells were photoirradiated (590-700 nm, 4.8 I/cm2), fixed 10 min later, and
stained for acid phosphatase. The elimination of acid phosphatase-staining particles (arrows) by this process is considered an indication for the lysosomal localization
of these dyes. In /. in untreated cells, the particles arc distributed throughout the cells but, in most cases, concentrated in the perinuclear area. B, cells treated with
photoirradiation alone without dye. C and D, cells treated with NBA-6I (0.5 JIM) or NBS-6I (0.2 /IM), respectively, and at 10 min postphotoirradiation showing
elimination of the majority of the particles.
The evidence for lysosomal localization for all of the dyes of
the present study is based on results which show (a) the simi
larity in locations, distributions, and morphologies of the Nile
blue-containing particles observed using fluorescence and light
microscopy to those identified by known lysosomotropic dyes,
including acridine orange and Lucifer Yellow CH (28-32), (b)
the similarity in localization between dye-containing particles
and acid phosphatase-stained lysosomal particles, (c) the results
of photoirradiation of dye-loaded cells which obliterated most
of the lysosomes identified by acid phosphatase staining, (d)
the actions of nigericin which inhibited rather than enhanced
the uptake and which promoted the efflux of the dyes, and (e)
the alterations in dye uptake by other agents which reduce
lysosomal pH gradients but not by those which change plasma
and/or mitochondria! membrane potentials. Other evidence
supporting this conclusion come from the results of our pre
vious study (20) which showed (a) highly concentrative cellular
accumulations of Nile blue derivatives and (b) a linear increase
of uptake proportional to the dye concentration in the medium
over a wide concentration range. Further supporting this sup
position that the majority of dyes are localized in the lysosomes
are: (a) under the same experimental conditions with videoenhanced fluorescence microscopy, the minimum dye concen
tration required for observing NBA fluorescence in lysosomes
was about two orders of magnitude less (IO'9 versus 10~7 M)
than that required for mitochondria or other cytomembranes
and (b) nigericin significantly inhibits uptakes of NBA and
NBA-6I at various dye concentrations ranging from 0.156 to
40 MM(Fig. 8).
Nigericin and FCCP affect both the lysosomal pH gradient
as well as the mitochondrial membrane potential. Nigericin
reduces lysosomal pH gradient and induces a compensatory
increase in mitochondrial membrane potential. Thus, it can
inhibit the uptake of lysosomal but enhance the uptake of
mitochondrial localizing compounds. Since the overall effect of
nigericin on dye localization to the two sites are opposite, the
inhibition on Nile blue uptake observed above can be clearly
attributed to the reduction of lysosomal pH gradient. On the
other hand, FCCP is a protonophore which increases the mem
brane permeability to protons and allows them to reach electro
chemical equilibration across the membrane. The overall effect
of FCCP is reductions of both lysosomal pH gradient and
mitochondrial membrane potential. Therefore, the inhibition
on the uptake of Nile blue dyes can be due to the reduction of
pH gradient and/or membrane potential. Since the former
interpretation agrees with other findings in this study, while
the latter does not, we attribute the FCCP action on Nile blue
uptake to its effect on the lysosomal pH gradient.
Although many cationic dyes are known to be localized in
the mitochondria (4, 25), there are a great number of other
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LYSOSOMAL LOCALIZATION OF NILE BLUE PHOTOSENSITIZERS
NBA-61
NBA
o
io
-» NBAonly
-*- VaJjnomycin
» Ouabajn
10
20
30
40
o.
0
10
20
30
i
40
wo Nigericin
w/ Nigericin
•w/ Nigericin
Uptake Time (min)
.1
1
10
100
1
10
Extracellular Dye Concentration
100
(uM)
Fig. 8. Effect of nigericin on uptakes of NBA (left) and NBA-61 (right) at
different dye concentrations. Cells were pretreated with 5 Mg/ml of nigericin for
30 min prior to the addition of the dye (NBA or NBA-61, from 0.156 to 40 MM).
The uptake was performed for 30 min at 37°Cin the presence of nigericin. For
comparison, uptakes of dyes at different concentrations but without nigericin
were also performed. Point, mean of 3 determinations; bar, SD when exceeding
symbol size.
10
20
30
40
O
10
20
30
40
Uptake Time (min)
1
Fig. 6. Effects of valinomycin (top) and ouabain (top) and high K* medium
(bottom) on uptakes of NBA and NBA-61. For the valinomycin and ouabain
studies, cells were pretreated with valinomycin (1 Mg/ml) or ouabain (1 mM) for
30 min prior to the addition of the dye. Uptake of NBA or NBA-61 (2.5 MM)was
performed for various time intervals at 37°Cin the presence of valinomycin or
ouabain. Uptake experiments in high K+ medium were carried out in DPBS with
KC1 substituted for NaCl. The K+ concentrations were 139 mM for the high and
3 mM for the low K+ DPBS. Cells were pretreated with high or low K+ DPBS for
30 min before addition of the dyes (NBA or NBA-61 at 2.5 UM)to the medium.
Point, mean of 3 determinations; bar, SD.
Table 2 Percentage inhibitions by nigericin, FCCP, and monensin on uptakes of
NBA and NBA-61 by MGH-U1 cells"
Nigericin (5 Mg/ml)
FCCP(1 MM)
FCCP (10 MM)
Monensin (6 MM)
Monensin (25 MM)NBA
" Uptake time: 30 min.
* Mean of 3 determinations ±SD.
uptake71.6±
2.0*
uptake64.5
36.4
75.4
42.3
54.3
30.8
66.9
33.3
40.1
±0.6
±2.1
±2.6
±2.3NBA-61
20
40
60
40
0
10
20
30
40
Uptake Time (min)
Fig. 9. Effects of oxidative phosphorylation inhibitors, sodium azide and 2,4DNP, on uptake of NBA and NBA-61. Cells, 2 X 10'/60-mm dish, were washed
with DPBS twice, incubated with DPBS without glucose for 30 min to deplete
the endogenous glucose, and pretreated with 2,4-DNP (0.1 mM) or sodium azide
(10 mM) for 30 min in DPBS before the addition of dye (2 ml, 2.5 MM).Uptakes
were performed at 37'C in the presence of the inhibitor. Point, mean of 3
±0.8
±2.0
±0.7
±0.5
±0.4
determinations; bar, SD when exceeding symbol size.
cationic compounds that are localized in the lysosomes (3337). Multiple factors may dictate the subcellular localization of
these compounds. Subtle changes in the chemical structure of
dyes can have profound influence on their localization. For
example, alkylation of acridine orange, a well-known lysosomotropic cationic fluorescent dye, can yield derivatives that are
localized specifically in the mitochondria. And the derivatives
can have different distributions between these two organelles,
depending on the degree of alkylation and apparently due to
the increase in hydrophobic interaction between the alkylated
derivatives and the mitochondrial membrane (32).
The localization of Nile blue derivatives in lysosomes may be
related to their capability of existing as uncharged weak bases
when deprotonated from the cationic species. This is common
to many weakly cationic "lysosomotropic agents" which become
o
0
30
800
20
40
Efflux Time (min)
Fig. 7. Effects of nigericin on efflux of NBA and NBA-61. Cells (2 X 10') were
incubated with 2 ml of serum-free medium containing 2.5 MMdye for 30 min at
37°C.The medium was removed, the cell culture plates were washed twice, and
dye-free medium with (bottom) and without (top) 5 Mg/ml of nigericin was
replaced. Cellular dye concentrations were determined at various time intervals.
Point, mean of 3 determinations; bar, SD.
highly concentrated within the lysosome because they can dif
fuse across cell membranes in their uncharged, nonprotonated
form but become trapped in their protonated, nondiffusible
form in the membrane-bound low pH compartment of the
lysosome (34). This ion-trapping mechanism is dependent on
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LYSOSOMAL LOCALIZATION
OF NILE BLUE PHOTOSENS1TIZERS
the pH gradient of the subcellular compartment and the ability
of the dye to undergo protonation-deprotonation
conversion.
From the findings of the present study and based on the iontrapping mechanism, we offer the following hypothetical
scheme for the uptake, distribution, and accumulation of Nile
blue derivatives in MGH-U1 cells. Dye molecules enter the cell
by diffusing across the plasma membrane in neutral nonprotonated forms. After they enter the cell, some of the dyes may
remain associated with the membrane and are distributed
throughout various cytomembrane systems. This part of the
dye distribution process probably relies on the lipophilicity of
the dye. Hence, it is not energy dependent and the steady-state
concentration depends on the extracellular dye concentration.
On the other hand, dye molecules which partition into the
cytoplasm are quickly sequestered into and accumulated in the
lysosomes. This process is mediated by the action of an iontrapping mechanism common to many weakly basic compounds
which concentrate in subcellular sites of lower pH. This process
relies on the pH gradient across the lysosomal membrane and
is maintained by an energy-dependent proton pump. Intrinsic
to this process is the ability of dyes to undergo protonationdeprotonation conversions at different pH values. In the acidic
environment of the lysosomes, the dyes are protonated and
cannot readily diffuse across the membrane. The difference in
transmembrane permeability coefficients between the proton
ated and deprotonated forms is the main reason for the high
accumulation of dyes in the lysosomes (34).
Although the lysosome appears to be the major site of local
ization for all of the Nile blue dyes examined, derivatives with
minor differences in structure also have subtle variations in
their distribution to other cell structures relative to the lyso
some. Sulfur substitution and D-ring saturation reduce, while
iodination increases, both the degree of specificity and speed of
sequestering into the lysosome. Of note, iodination also causes
reductions in pKa values and increased hydrophobicities of the
resulting derivatives. Both of these properties may influence
the ion-trapping process. However, the significance of pKa may
be limited only to the ability of the dye to be protonated. As
long as the pKa of the derivative is higher than the pH of the
lysosome, a sufficient amount of the dye will be in the proton
ated form and trapped in the lysosome. The increase in hydrophobicity, however, may increase the ratio of permeability
coefficients between the deprotonated and the protonated forms
moving across the lysosomal membrane and thus increase up
take and accumulation of the dye in the lysosome.
Since the subcellular localization of many photosensitizers is
currently a topic of active investigation (22, 38-41), it is im
portant to note that the present study demonstrated the de
pendence of apparent dye localization on both dye concentra
tion and method of detection. For example, under the light
microscope, Nile blue dye in cells can only be detected at high
extracellular concentrations (>10~5 M), which indicated mostly
intracellular sites for effective PDT of tumors (4, 24, 25, 38,
42). For example, the mitochondria has been considered an
important target for its possible high cationic sensitizer accu
mulations in tumors, because of its higher membrane potentials
in tumor cells (23, 25, 43) and its importance in supplying
energy for critical cell functions (38). Recently, the photosen
sitizers mono-L-aspartyl chlorin ce and sulfonated teraphenylporphyins have been found to be localized in the lysosomes,
although the uptake of these photosensitizers may occur
through endocytosis (39-40). These findings, taken with the
results of the current study, suggest the possibility of targeting
lysosomes as another strategy for PDT. There are a number of
advantages to this approach. One is the ability of the organelle
to accumulate very high concentrations of cationic photosensi
tizers. As shown in our previous study (20), with an initial
extracellular dye concentration of 2.5 ¿tM
the ratio of intracel
lular to extracellular Nile blue derivative concentrations after a
30-min incubation was on the order of 1000-6000. If most of
the dye is localized in the lysosomes, which constitute only a
small fraction of the cell volume, the intralysosomal dye con
centration can be many times higher. Our previous data (20)
also show that the capacity of the cell to take up these dyes can
be extended to 100 pM without reaching saturation, further
indicating the high capacity of lysosome to accumulate these
sensitizers. This permits the use of a lower sensitizer dose to
produce low systemic toxicity and effective cell killings, as
exemplified by the low (5 x 10~8 M) extracellular sensitizer
lysosomal localization. However, under fluorescence micros
copy, the fluorescence in the perinuclear region and area around
the nucleus was too intense to discern lysosomes at dye concen
trations between 10~6 and 10~7 M, while fluorescence in mito
The authors wish to thank Carl F. Schanbacher and Cindy Bachelder
for technical assistance and Dr. Irene Kochevar for critical reading of
the manuscript.
chondria and cytomembranes was clearly visible. Only when
lower dye concentrations were used did the punctate lysosomal
stain become evident, which was confirmed by histochemical,
photodynamic, and biochemical studies. The use of multiple
methods at a variety of dye concentrations to establish subcel
lular localizations of dyes is therefore emphasized.
A number of investigators have advocated targeting various
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Lysosomal Localization and Mechanism of Uptake of Nile Blue
Photosensitizers in Tumor Cells
Chi-Wei Lin, Janine R. Shulok, Sandra D. Kirley, et al.
Cancer Res 1991;51:2710-2719.
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