Ratiometric fluorescence imaging of cellular polarity: Decrease in mitochondrial polarity in cancer cells

Article abstract of DOI:10.1002/anie.201410645

Mitochondrial polarity strongly influences the intracellular transportation of proteins and interactions between biomacromolecules. The first fluorescent probe capable of the ratiometric imaging of mitochondrial polarity is reported. The probe, termed BOB, has two absorption maxima (λ_abs = 426 and 561 nm) and two emission maxima - a strong green emission (λ_em = 467 nm) and a weak red emission (642 nm in methanol) - when excited at 405 nm. However, only the green emission is markedly sensitive to polarity changes, thus providing a ratiometric fluorescence response with a good linear relationship in both extensive and narrow ranges of solution polarity. BOB possesses high specificity to mitochondria (R_r = 0.96) that is independent of the mitochondrial membrane potential. The mitochondrial polarity in cancer cells was found to be lower than that of normal cells by ratiometric fluorescence imaging with BOB. The difference in mitochondrial polarity might be used to distinguish cancer cells from normal cells.

Full text of DOI:10.1002/anie.201410645

Angewandte
Chemie
DOI: 10.1002/anie.201410645
Cellular Imaging
Hot Paper
Ratiometric Fluorescence Imaging of Cellular Polarity: Decrease in
Mitochondrial Polarity in Cancer Cells**
Na Jiang, Jiangli Fan,* Feng Xu, Xiaojun Peng, Huiying Mu, Jingyun Wang, and Xiaoqing Xiong
Abstract: Mitochondrial polarity strongly influences the intra-
cellular transportation of proteins and interactions between
biomacromolecules. The first fluorescent probe capable of the
ratiometric imaging of mitochondrial polarity is reported. The
probe, termed BOB, has two absorption maxima (labs = 426
and 561 nm) and two emission maxima—a strong green
emission (lem = 467 nm) and a weak red emission (642 nm in
methanol)—when excited at 405 nm. However, only the green
emission is markedly sensitive to polarity changes, thus
providing a ratiometric fluorescence response with a good
linear relationship in both extensive and narrow ranges of
solution polarity. BOB possesses high specificity to mitochon-
dria (Rr = 0.96) that is independent of the mitochondrial
membrane potential. The mitochondrial polarity in cancer
cells was found to be lower than that of normal cells by
ratiometric fluorescence imaging with BOB. The difference in
mitochondrial polarity might be used to distinguish cancer cells
from normal cells.
from one region to another. To better indicate local changes
in cells, the sensing of intracellular polarity (as well as other
environmental parameters, such as viscosity, and important
chemical species, such as Ca2+ and singlet oxygen) should to
the greatest possible extent be performed within distinct
organelles rather than in an unknown intracellular area.[5]
Mitochondria, the principal energy-producing compart-
ments in most cells, function in numerous vital cellular
processes, such as ATP production, central metabolism,
calcium modulation and redox signaling, and the apoptotic
process of cell death.[6,7] Mitochondrial polarity strongly
influences the intracellular transportation of proteins and
interactions between biomacromolecules. Furthermore,
polarity reflects the status and function of this kind of
organelle, whose location, morphology, and components are
always changing. Several features, such as mitochondrial
enzymes, proteins, and macromolecular substances, will not
be transmitted in cases of mitochondrial dysfunction.[8] For
example, the activity and stability of mitochondrial malate
dehydrogenases (mMDHs), important enzymes in numerous
mitochondrial proteins, are strongly affected by the amphi-
philic microenvironment.[9]
Fluorescent sensors have recently received considerable
attention owing to their high sensitivity, selectivity, and
nondestructive characteristics.[10] Thus, polarity-sensitive flu-
orescent probes are considered ideal candidates for sensing
polarity in cell biology. Several research teams have devel-
oped a number of polarity-sensitive probes based on intra-
molecular-charge-transfer (ICT) systems[11] to detect local
polarity around various proteins, sense the hydrophobic
cavities of numerous native proteins, and study the hydro-
phobic domains of biological macromolecules. Although
a few of them are cell-permeable,[12a,b] there are no probes
that show specific subcellular organelle distributions and that
the activity of a protein or enzyme might be influenced by the
local polarity. To date, the detection of mitochondrial polarity
in living cells remains a “blank space”, with no attempt
reported.
P
olarity is an important parameter in chemistry and
chemical technology.[1] Certain organic or inorganic processes
are markedly dependent on the surrounding polarities, which
greatly control the reaction processes.[2] In biological systems,
especially at the cellular level, polarity determines the
interaction activity of a large number of proteins and enzymes
or reflects the permeability of membrane compartments.
Furthermore, abnormal changes in polarity are closely linked
with disorders and diseases (e.g., diabetes, liver cirrhosis).[3]
However, polarity is a complex factor and encompasses
a range of noncovalent interactions, including dipolarity/
polarizability and hydrogen bonding.[4] Thus, its measurement
in live cells is difficult and necessitates the development of
new tools. Not surprisingly, local polarity differs considerably
[*] N. Jiang, J. Fan, F. Xu, Prof. X. Peng, H. Mu, X. Xiong
State Key Laboratory of Fine Chemicals
Dalian University of Technology
2 Linggong Road, 116024 Dalian (China)
E-mail: fanjl@dlut.edu.cn
Herein, we describe the development of the probe BOB
(Figure 1a), the first mitochondrial molecular probe of polar-
ity, which functions by the ICT mechanism.[13] On the basis of
the donor–p-bridge–acceptor (D–p–A) design philosophy, we
selected coumarin as the D group because of its high quantum
yields, high extinction coefficients, and the fact that this
moiety has been engineered to respond to environmental
polarity (solvatochromic probes).[14] Concerning the A group,
our choice fell on the benzothiazene group owing to its
electron-withdrawing aromatic system, which is also capable
of extending electron conjugation. Furthermore, the quater-
nized aromatic amino groups impart good water solubility to
Prof. J. Wang
School of Life Science and Biotechnology
Dalian University of Technology
2 Linggong Road, 116024 Dalian (China)
[**] This research was financially supported by the NSF of China
(21136002, 21376039, 21422601, and 21421005), the National Basic
Research Program of China (2013CB733702), the Ministry of
Education (NCET-12-0080), Liaoning NSF (2013020115), and
Fundamental Research Funds for the Central Universities
(DUT14ZD214).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201410645.
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a large polarity range, as expressed by the orientation
polarizability. The Lippert–Mataga polarity parameter Df is
related to solvent orientational polarization.[16] Only tiny
changes were discernible in the absorption maxima in all of
these solvents with different polarity (see Figure S3). In
contrast, solvent polarity had a dramatic effect on the
emission spectra, in which solvatochromic behavior usually
originates from large variations in the molecular dipole upon
photon absorption, thus leading to different stabilization
energies of the ground and excited states as a result of the
solvent shell around the molecule. When the solvent polarity
increases, this effect becomes more significant: The excited
state releases more energy to reach a more stable state, thus
resulting in low fluorescence intensity or/and a shift in the
absorption/fluorescence spectrum.[17] Furthermore, the larger
dipole moments of the excited state lead to a larger solvating
effect.[18] In our case, as the dipole moment of I in different
solvents is larger than that of BOB (see Table S3), the green
emission is more markedly sensitive to solution polarity
changes than the red emission upon the excitation of BOB at
405 nm. When the polarity (Df) of the solution was decreased
from 0.32 (water) to 0.013 (toluene), the fluorescence
intensity of BOB at 467 nm increased by a factor of 24, and
the red emission at 645 nm gave a slight fluorescence response
(see Figure S4 and Table S2), thus providing a ratiometric
response. Thus, a linear relationship exists between the
fluorescence intensity (I467/I645) and Df, which demonstrated
Figure 1. a) Molecular structures of BOB and I. b) Normalized absorp-
tion and emission spectra (excitation at 405 nm) of BOB in methanol.
the probe, and the benzyl group helps the probe to
accumulate in mitochondria.[15] BOB has a linear ratiometric
fluorescence response to solution polarity and is the first
fluorescent probe to image mitochondrial polarity. We found
for the first time that the mitochondrial polarity in cancer
cells tends to be lower than that of normal cells. The
difference in polarity could be discriminated to a remarkable
extent by ratiometric fluorescence imaging.
BOB has two absorption maxima (labs = 426 nm, e =
1.00 ꢀ 104 molÀ1 cmÀ1
L
and
labs = 561 nm,
e = 2.6 ꢀ
104 molÀ1 cmÀ1 L) and two emission maxima (lem = 467 and
642 nm, excitation at 405 nm) in methanol (Figure 1b; see
also Table S1 in the Supporting Information). To identify
which part of the molecule was responsible for the short-
wavelength emission of BOB, we studied the absorption and
emission properties of three parts of the molecule (see
Figure S1 in the Supporting Information) by DFT calculations
in methanol. The absorption and fluorescence emission of I
(Figure 1a) occur 424 and 488 nm, respectively (see Fig-
ure S2), which are very close to the wavelengths of BOB in
methanol (labs,test = 426 nm and lem,test = 467 nm). The calcu-
lated absorption and emission wavelengths of BOB (labs
=
560 nm and lem = 645 nm) are in complete accord with the test
results. Therefore, we deduced that the red emission is due to
the extended p-conjugation as well as strong ICT from the
diethylaminocoumarin moiety (represented by I) to the
conjugated hemicyanine moiety, whereas the emission band
at 467 nm can probably be ascribed to part I.
Figure 2. a) Fluorescence emission spectra in water/1,4-dioxane sol-
vent mixtures (the percentage in the box indicates the water content).
b) Linearity of I467/I645 versus the solvent parameter Df (with excitation
in all cases at 405 nm).
The absorption and fluorescence spectra of BOB were
then measured at 258C in a variety of solvents covering
2
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that
BOB
could
be
employed to ratiometrically
detect extensive solution
polarity.
In
a narrow polarity
range with different propor-
tions of water and 1,4-diox-
ane, the same result was
observed for the absorption
maxima as during extensive
solution polarity (see Fig-
ure S5). In contrast, when
the polarity (Df) of the solu-
tion decreased from 0.31
(80% water) to 0.29 (10%
water), the fluorescence
intensity of BOB at 467 nm
was increased by a factor of
3.5, whereas the red emission
at 645 nm showed only
a
slight response in the
form of a slight fluorescence
enhancement (from 31.4 to
45.8; Figure 2). This finding
also shows a good linear
correlation of the fluores-
cence intensity I467/I645 with
Df, which demonstrates that
BOB is highly sensitive to
solvent polarity and can
potentially reveal the polar-
ity of its immediate environ-
ment.
To assess the photophys-
ical behavior of the prepared
coumarin–hemicyanine
hybrids in a biological envi-
ronment, we examined com-
pound BOB in living cells.
Figure 3. a,b) Confocal fluorescence images of MCF-7 cells stained with Mito Tracker Green FM (2.0 mm) for
15 min (a) and BOB (2.0 mm) for 15 min (b). c) Merged image of (a) and (b). d–f) Enlarged representations
of cells in image (a), (b), and (c), respectively. g) Bright-field image. h) Intensity profile of regions of interest
(ROIs) across MCF-7 cells. i) Correlation plot of the intensities of Mito Tracker Green FM and BOB
(Rr =0.96).
Confocal fluorescence microscopy showed that MCF-7 cells
labeled with BOB emitted fluorescence with maximal wave-
lengths of 468 and 645 nm (see Figure S6). The wavelengths
were nearly identical to those observed for BOB in the
extracellular environment. Collected images showed that the
probe was emissive in subcellular compartments, which were
sometimes filamentous or slightly swollen (see Figures S6 and
S7). On morphological grounds, the compartments were
judged to be mitochondria.[19] Hydrophilicity–lipophilicity
was modeled by the logarithm of the water–octanol partition
coefficient (logP). Probes specifying mitochondrial accumu-
lation were numerically assigned on the basis of the following
criteria: electric charge Z > 0 and 0 < logP <+ 5.[20] For BOB,
the ZBOB and logPBOB values were 1 and 2.8, respectively,
which indicate that BOB can probably be trapped in
mitochondria. To further investigate the subcellular local-
ization of BOB, a commercially available, mitochondria-
localizing dye, MitoTracker Green FM, was used for a coloc-
alization study in two types of cell lines, MCF-7 cells
(Figure 3) and HepG2 cells (see Figure S8). Colocalization
was quantified by the use of Pearson sample correlation
factors (Rr). The intensity of the correlation plots revealed
a high Rr value of 0.96. We also performed the BOB
colocalization experiments with the commercial lysosome
fluorescent dye LysoTracker Green DND (see Figure S9) and
the endoplasmic reticulum fluorescent dye ER-Tracker
Green (see Figure S10). No colocalization was observed in
these two cases. All results confirmed that BOB was a true
mitochondrion-targeted probe.[21]
We examined BOB staining in MCF-7 cells treated with
the membrane-potential uncoupler 3-chlorophenylhydrazone
(CCCP), which can disrupt the mitochondrial membrane
potential.[22] Both the green and the red channel of confocal
fluorescence images were unchanged in the absence or
presence of CCCP (see Figure S11), thus suggesting that
BOB was independent of the mitochondrial membrane
potential. Given that cell toxicity is a key feature for live-
cell imaging, the cytotoxicity of BOB was then evaluated in an
MTT assay. The dye showed low cell cytotoxicity at low
(2.0 mm, cell viability ꢀ 96%) and high concentrations
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(10.0 mm, cell viability ꢀ 86%; see Figure S12). All results
showed that BOB was poorly cytotoxic to living cells under
our cell-imaging conditions. In a pH-titration study, the
fluorescence signal of BOB changed very little (see Fig-
ure S13), thus indicating that the fluorescence of BOB was
not influenced by the pH microenvironment. Photobleaching
is a common problem for most organic dyes and often
compromises the temporal monitoring of dynamic events
inside cells because of the fading of reporter dyes.[11h,23] Upon
irradiation for 10 h with a 500 W iodine–tungsten lamp, the
density of BOB remained at 89%, but that of MitoTracker
Deep Red FM (commercial) decreased to less than 65% (see
Figure S14). These results indicate the high photostability of
BOB under environmental conditions. Furthermore, the
fluorescence in both green and red channels did not decrease
significantly upon discontinuous laser irradiation for 30 min
(see Figure S15).
During the experiment, we found that mitochondrial
polarity varied as the cell status changed (Figure 4). In terms
of their morphology, the RAW 264.7 cells usually display an
irregular spindle shape or visible longer pseudopodia, so the
cells in area 1 were in the normal state. As the cells approach
death, they become circular, as in area 2. In Figure 4d, the
green region represents higher polarity (Df = 0.297), and the
color amaranth stands for lower polarity (Df = 0.284). Thus,
we deduce that mitochondrial polarity in dying cells is lower
than that in normal cells. To verify this phenomenon, we used
the drug etoposide to induce apoptosis. We found that during
apoptosis, the amaranth-colored areas increased in size, and
the size of the green region decreased (see Figure S16c–f),
which means that mitochondrial polarity was reduced.
The activity of mMDH in cirrhosis patients is higher than
that in a healthy person, and
Figure 4. a,b) Confocal fluorescence images of RAW 264.7 cells.
Images were acquired by using excitation and emission windows of
lex =405 nm, lem =435–535 nm (a) and lex =405 nm, lem =575–
675 nm (b). c) Bright-field images of cells. d) Fluorescence ratio
images.
In conclusion, we have developed the probe BOB, the first
fluorescent probe for mitochondrial polarity, on the basis of
a coumarin–hemicyanine dye. Significantly, BOB has a linear
ratiometric fluorescence response to both extensive and
narrow ranges of solution polarity. Furthermore, BOB local-
izes in mitochondria and is independent of mitochondrial
membrane potential, thus making it suitable as an indicator of
polarity changes in the mitochondrial microenvironment.
Mitochondrial polarity data for cos-7 cells, RAW 264.7 cells,
HeLa cells, HepG2 cells, and MCF-7 cells were obtained.
mMDH shows relatively high
activity in low-polarity micro-
environments.[24] Therefore, we
inferred that mitochondrial
polarity in cirrhosis patients is
lower than in healthy persons.
Thus, two types of normal cells
(cos-7 and RAW 264.7 cells)
and three kinds of cancer cells
(HeLa, HepG2, and MCF-7
cells) were selected to be incu-
bated with BOB. After the cells
were incubated with BOB
(2.0 mm) for 15 min, green- and
red-channel confocal images
were collected. The green area
in the two types of normal cells
was larger than that in the three
types of cancer cells, which
indicated that the mitochon-
drial polarity in both cos-7 and
RAW 264.7 (Df = 0.295) was
higher than in HeLa (Df =
Figure 5. Imaging of a–a3) cos-7 cells, b–b3) RAW 264.7 cells, c–c3) HeLa cells, d–d3) HepG2 cells, and
e–e3) MCF-7 cells. a–e and a1–e1) Confocal fluorescence images with lem =435–535 nm for (a–e) and
lem =575–675 nm for (a1–e1). a2–e2) Bright-field images of the cells. a3–e3) Fluorescence ratio images.
0.288), HepG2 (Df = 0.290),
and MCF-7 cells (Df = 0.287;
Figure 5).
4
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Differences in polarity could be discriminated to a remarkable
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lower than in normal cells. We hope that the detection of
mitochondrial polarity can be used as a new method in the
future to distinguish cancer cells from normal cells. Further
research is under way.
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Keywords: cancer sensing · dyes · mitochondria · polarity ·
ratiometric imaging
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Communications
Cellular Imaging
… and BOB’s your uncle: A fluorescent
probe of mitochondrial polarity, termed
BOB, showed a linear ratiometric fluo-
rescence response to solution polarity.
Various mitochondria of normal cells and
cancer cells were examined, and it was
found that mitochondrial polarity tends
to be lower in cancer cells than in normal
cells. The detection of mitochondrial
polarity could thus be used as a method
to distinguish cancer cells from normal
cells.
N. Jiang, J. Fan,* F. Xu, X. Peng, H. Mu,
J. Wang, X. Xiong
&&& — &&&
Ratiometric Fluorescence Imaging of
Cellular Polarity: Decrease in
Mitochondrial Polarity in Cancer Cells
6
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ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2015, 54, 1 – 6
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