A lysosome-targetable and two-photon fluorescent probe for monitoring endogenous and exogenous nitric oxide in living cells

Article abstract of DOI:10.1021/ja308967u

A lysosome-specific and two-photon fluorescent probe, Lyso-NINO, demonstrates high selectivity and sensitivity toward NO, lower cytotoxicity, and perfect lysosomal localization. With the aid of Lyso-NINO, the first capture of NO within lysosomes of macrophage cells has been achieved using both two-photon fluorescence microscopy and flow cytometry.

Full text of DOI:10.1021/ja308967u

Communication
pubs.acs.org/JACS
A Lysosome-Targetable and Two-Photon Fluorescent Probe for
Monitoring Endogenous and Exogenous Nitric Oxide in Living Cells
Haibo Yu,† Yi Xiao,*,† and Liji Jin*,‡
‡
†State Key Laboratory of Fine Chemicals and School of Life Science and Technology, Dalian University of Technology, Dalian
116024, China
S
* Supporting Information
shallow penetration depth, photodamage, and cellular auto-
fluorescence.10
ABSTRACT: A lysosome-specific and two-photon fluo-
rescent probe, Lyso-NINO, demonstrates high selectivity
and sensitivity toward NO, lower cytotoxicity, and perfect
lysosomal localization. With the aid of Lyso-NINO, the
first capture of NO within lysosomes of macrophage cells
has been achieved using both two-photon fluorescence
microscopy and flow cytometry.
Herein, based on the principle of photoinduced electron
transfer (PET), a lysosome-targeted and two-photon NO
fluorescent probe, Lyso-NINO, has been designed through
integration of NO-capturing o-phenylenediamine, lysosome-
targeting (aminoethyl)morpholine, and efficient two-photon
fluorophore naphthalimide, as shown in Scheme 1. o-Phenyl-
Scheme 1. Chemical Structure of Lyso-NINO and the
Reaction of Lyso-NINO with NO
ecently, it has been discovered that lysosomal functions
Rare subtly regulated by nitric oxide (NO), a ubiquitous
cellular messenger molecule in the cardiovascular, nervous, and
immune systems.1 NO exhibits complex effects on the catabolic
autophagy process,2 which involves the degradation of a cell’s
own components through the lysosomal machinery, to supply
energy and nutrient sources for cell growth,3 and is also closely
related to various disorders and diseases including lysosomal
storage disorders,4 Gaucher’s disease,5 and Danon disease.6
Endogenous NO, induced by some stimulants including
Lipopolysaccharide (LPS) and Interferon-r (IFN-r), has been
reported to react with several related proteins outside
lysosomes to drive the autolysosome process by releasing the
upstream signals.7 However, the effects of NO on lysosomes are
far from being completely understood. Owing to the dynamic
transformation of the morphology and components of the
highly heterogeneous lysosomes, it is difficult to find solid
evidence of direct interactions between NO and components
within lysosomes, but that does not mean they are non-existent.
So far, scientists have no tool to determine whether NO enters
into lysosomes, not to mention its quantification.
To our knowledge, fluorescent probespowerful tools in
cell biologycan hardly meet the requirements for monitoring
lysosomal NO. A variety of NO-sensitive fluorescent molecular
probes have already been developed to image intracellular NO
under one-photon microscopy (OPM),8 which has significantly
enriched our knowledge about NO homeostasis and NO’s
critical roles in many biological processes.9 Nevertheless, none
of these probes is well suited for lysosomal applications; they all
failed to specifically localize in lysosomes and monitor
lysosomal NO.
enediamine, employed not only as an electron donor (a
fluorescence quencher) for naphthalimide but also as a NO
trapper, was directly tethered to the imide position of
naphthalimide. Accordingly, the 4 position of naphthalimide
could be preserved and further modified with 4-(2-aminoethyl)-
morpholine. The alkylmorpholine group, possessing a lower
pKa value, targets lysosomes that contain a lot of acid
hydrolases in the pH range from 4.5 to 5.5.11,12
First, our study on Lyso-NINO and Lyso-NINO-T’s pH
responses confirms the probe’s suitability to lysosomal pH
range. As shown in Figure 1, the pKa of Lyso-NINO-T is 5.61
0.02, which is in good accord with the pKa value of the well-
known Lysosensor DND-189 (pKa = 5.80).12 This should be
attributed to the lower pH response of the morpholine group.
Compared with Lyso-NINO-T, Lyso-NINO has a lower pKa
value, 3.47 0.01, which is ascribed to the pH independence of
o-phenylenediamine. From these results, we could predict that,
in lysosomal pH ranges such as pH 4.5−5.5, there would be
very weak fluorescence for Lyso-NINO, yet Lyso-NINO-T
would exhibit a strong fluorescence at the same pH value.
Lyso-NINO exhibits high selectivity for NO over other
reactive oxygen species and high sensitivity, with a nanomolar-
scale limit of detection. Lyso-NINO in free form displays a
Besides the lack of lysosomal specificity, another disadvant-
age of the known NO probes is the lower two-photon
fluorescence activity, which gives them poor applicability in
two-photon microscopy (TPM). The TPM technique, exciting
the fluorophores with NIR laser pulses, has become a potent
alternative to OPM because it overcomes latter’s problems of
Received: September 10, 2012
© XXXX American Chemical Society
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dx.doi.org/10.1021/ja308967u | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
intensity (530 nm) and NO concentration is found in the range
of NO concentration from 1 to 1.3 μM (Figure 2b). The limit
of detection (S/N = 3) of Lyso-NINO is determined to be 5
nM (Supporting Information), indicating that Lyso-NINO
would be a potential tool to monitor endogenous NO in live
cells.
Lyso-NINO displays significant applicability under two-
photon excitation. The maximum two-photon action cross-
section (Φδ) value is 34 GM at 840 nm.13 Upon addition of
NO solution (5.0 equiv), the maximum two-photon action
cross section increases to 210 GM at 840 nm (Figure 2c). Such
a drastic enhancement in two-photon excitation fluorescence
indicates that Lyso-NINO is a potential two-photon probe for
monitoring and imaging NO in living specimens.
The cytotoxicity of Lyso-NINO is low in the first 12 h of
incubation. MTT assay (SI) has revealed that 80% of MCF-7
cells survive after incubation with Lyso-NINO (5.0 μM) for 12
h, and 24 h later the survival rate is over 60% (Figure 2d). As is
well known, most lysosomal probes bearing basic groups exhibit
an alkalinizing effect on the lysosomes, such that longer
incubation with these probes can induce an increase in
lysosomal pH and result in cell death. Although there is
significant cell death within 24 h incubation time, in the first 12
h the survival rate of these cells incubated with Lyso-NINO is
high: up to 80%. Compared with some commercial lysosomal
probes, the lower cytotoxic effect of Lyso-NINO on MCF-7
cells is a favorable characteristic of a practical NO probe for
application in living cells.
Figure 1. Change of intensity plots of (a) Lyso-NINO (11 μM) and
(b) Lyso-NINO-T (25 μM) at 530 nm vs different pH values. Inset:
change in fluorescence spectra of Lyso-NINO and Lyso-NINO-T in
aqueous solution containing 20% acetronitile as cosolvent vs different
pH values (λex = 440 nm; slit = 3 nm).
broad absorption band centered at 430 nm (ε = 14 500 M−1
cm−1) with almost no fluorescence (fluorescence quantum yield
Φ = 0.02) in aqueous buffer at pH 5.0. In the presence of 5.0
equiv of NO solution, the emission spectrum of Lyso-NINO
exhibited a remarkable enhancement (16 times) at 530 nm
(Figure S2), and the fluorescence quantum yield increased from
0.02 to 0.3. However, the presence of various reactive oxygen
−
−
and reactive nitrogen species, such as H2O2, NO2 , NO3 , 1O2,
•OH, and ONOO− (Figure 2a), does not cause an observable
Lyso-NINO exhibits a significant ability to target lysosomes.
Co-localization experiments are performed by co-staining
MCF-7 cells with Neutral Red (NR), a commercial
LysoTracker, and tetramethylrhodamine methyl (TMRM), a
mitochondria tracker. MCF-7 cells stained with Lyso-NINO for
10 min at 37 °C do not show green fluorescence in Channel 1
(Figure S5); after that, in the presence of 5.0 equiv of NO
solution, these cells display measurable levels of green TP
fluorescence in discrete subcellular locations (Figure 3a). The
TPM image merges well with the image of staining with NR
(Figure 3c) rather than TMRM (Figure S6), indicating that
Lyso-NINO can specifically localize in lysosomes rather than
mitochondria of living cells. The intensity profiles of the linear
regions of interest across MCF-7 cells stained with Lyso-NINO
and NR also vary in close synchrony (Figure 3d). The high
Pearson’s coefficient and overlap coefficient are 0.805 and
1.374, respectively, evaluated using the conventional dye-
overlay method (Figure S7).
Lyso-NINO and NR display a good coexistence in lysosomes
of MCF-7 cells, yet the differences in fluorescence intensity
between the two channels, which may provide more useful
information about the internal environment of organelles in
living cells, should be noted. Thus, an intensity correlation
analysis (ICA)14 is employed to assess the intensity distribution
of the two co-existing dyes. We plot the intensity of stain Lyso-
NINO against that of NR for each pixel. The dependent
staining in Figure 3c results in a highly correlated plot (Figure
3e), and the ICA plots for the two stains generate an
unsymmetrical hourglass-shaped scatterplot that is markedly
skewed toward positive values (Figure 3f,g). Moreover, the
intensity correlation quotient (ICQ) for the two dyes is 0.357,
very close to 0.5, suggesting that the staining intensities are
dependent on each other. From the product of the differences
from the mean (PDM) image with positive values in the pixels
(Figure 3h), we can easily identify those lysosomes with high
Figure 2. (a) Selectivity of Lyso-NINO (11 μM) fluorescence
response for NO over other reactive nitrogen and oxygen species (5.0
equiv) in 20:80 CH3CN−H2O solution at pH 5.0 (in 10 mM
phosphate buffer). (b) Intensity at 530 nm vs NO concentration under
the same conditions. Inset: changes in fluorescence spectra of Lyso-
NINO (11 μM) upon addition of different amounts NO solution (0−
2.0 equiv). (c) Two-photon action spectra of Lyso-NINO (in
phosphate buffer pH 5.0) before (black) and after (red) addition of
NO solution (5.0 equiv). (d) Cell viability of Lyso-NINO (5.0 μM) at
different times.
spectral change. The titrations of Lyso-NINO with NO are
examined in a mixture of acetonitrile and sodium phosphate
buffer. Upon addition of NO solution, a new emission peak at
530 nm increases gradually (Figure 2b); meanwhile, the
absorption spectra do not change during the titration of NO
(Figure S3), which indicates that nitric oxide reacts with o-
phenylenediamine moiety, a result of inhibition of the PET
process from the o-phenylenediamine moiety to the naph-
thalimide fluorophore. The in situ-formed NO product of Lyso-
NINO without any byproduct, as expected, is confirmed to be
Lyso-NINO-T, according to retention times and typical HPLC
absorption spectra (Figure S4). With increasing of NO, an
excellent linear correlation (R2 = 0.9992) between fluorescence
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Journal of the American Chemical Society
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Figure 4. (a) Time-dependent exogenous NO released from NOC13
(20 μM) in MCF-7 cells stained with Lyso-NINO (5.0 μM). (b) DIC
image and two-photon fluorescent image of RAW 264.7 macrophages
stained with Lyso-NINO (5.0 μM) for 4 h at 37 °C. (c) DIC image
and two-photon fluorescent image of RAW 264.7 macrophages co-
stained with Lyso-NINO (5.0 μM) and stimulants containing final
concentrations of 5000 μg/mL L-Arg, 150 units/mL INF-r, and 20 μg/
mL LPS for 12 h, λex = 840 nm, λem = 520−560 nm.
Figure 3. Lyso-NINO co-localizes to lysosomes in MCF-7 cells. MCF-
7 was stained with (a) 5.0 μM Lyso-NINO with NO solution (20 μM)
for 5 min at 37 °C (Channel 1: λex = 840 nm, λem = 520−560 nm) and
(b) 5.0 μM NR (Channel 2: λex = 559 nm, λem = 565−610 nm). (c)
Overlay of (a) and (b). (d) Intensity profile of regions of interest
(ROI) across MCF-7 cells. (e) Intensity correlation plot of stain Lyso-
NINO and NR. ICA plots of (f) stain Lyso-NINO. and (g) stain NR.
PDM images with (h) positive PDM values in the pixels and (i)
negative PDM values in the pixels.
endogenous NO in individual cells on a large scale, compared
with microscopy imaging which provides spatial information.
Histograms demonstrate intracellular accumulation of NO and
increase of intracellular fluorescence indicated by the shift of
the fluorescence signal measured in the FITC channel (Figure
5). Compared with control cells without staining (Figure
intensity distribution of the two dyes (white pixels in Figure
3h). There are also some lysosomes for which the intensity
distribution does not vary synchronously (Figure 3i). The
reason might be that nitrite ions, byproducts of NO, react with
NR in acidic conditions and deplete the fluorescence intensity
of NR in some lysosomes.15 On the other hand, the PDM
image with negative values in the pixels (Figure 3i) further
demonstrates that Lyso-NINO is a lysosome-specific probe for
NO and the byproduct of NO in lysosomes does not interfere
with its photoproperties.
Owing to its high selectivity and sensitivity, Lyso-NINO is
used as a fluorescent indicator to assess exogenous and
endogenous NO in living cells. In view of NO releaser widely
used in the treatment of cancer,16 herein, we employ Lyso-
NINO to monitor the releasing process of NO from NOC13 in
lysosomes of MCF-7 cells. NOC13 (5.0 equiv) is added into
MCF-7 cells incubated with Lyso-NINO, and the fluorescence
intensity gradually increases over time and reaches the
maximum after 15 min (Figure 4a). The gradually increasing
intensity in lysosomes demonstrates that NO releasing from
NOC13 is able to diffuse into lysosomes and then be captured
by lyosomes-targeting Lyso-NINO. The efficacy of Lyso-
NINO for endogenous NO is also investigated. Macrophages
RAW264.7 incubated with Lyso-NINO for 12 h at 37 °C
display a weak fluorescence (Figure 4b), which indicates that
Lyso-NINO could capture endogenous NO in lysosomes of
macrophages. Stimulation with L-arginine (L-Arg), interferon-r
(IFN-r), and Lipopolysaccharide (LPS) in the presence of
Lyso-NINO has led to a pronounced increase in fluorescence
intensity (Figure 4c), indicating that, despite the shorter
lifetime of NO, endogenous NO may easily diffuse into
lysosomes in living cells.
Figure 5. (a) Flow cytometric analysis of Macrophages loaded with
Lyso-NINO and different stimulants. (1) Control group: Macro-
phages loaded without Lyso-NINO. (2) Macrophages loaded with
Lyso-NINO (5.0 μM) for 12 h at 37 °C. (3) Macrophages loaded with
Lyso-NINO (5.0 μM), L-Arg(5000 μg/mL), and LPS (20 μg/mL) for
12 h 37 °C. (4) Macrophages co-incubated with Lyso-NINO (5.0
μm), L-Arg(5000 μg/mL), INF-r (150 units/mL), and LPS (20 μg/
mL) for 12 h 37 °C. (b) Mean fluorescence per macrophage cell of
(1), (2), (3), and (4) incubation conditions.
5a(1)), macrophages incubated only with Lyso-NINO for 12 h
show a significant fluorescence from the histogram (Figure
5a(2)). Incubation of cells with Lyso-NINO, L-Arg, and LPS
for 12 h shifts the histogram of green fluorescence in the
direction of higher intensity (Figure 5a(3)). However, in the
absence and presence of IFN-r, the histograms of fluorescence
intensity do not shift much higher (Figure 5a(4)). Quantifica-
tion by mean fluorescence of these macrophages (Figure 5b)
suggests that LPS would be a critical factor which effectively
induces an increase of endogenous NO in live macrophages.
In this paper, there are two significant aspects: (1) A
lysosomal-specific and two-photon fluorescent probe Lyso-
NINO has been first developed, and it demonstrates high
selectivity and sensitivity toward NO over other reactive oxygen
Lyso-NINO also shows good applicability in flow cytometry
(FCM). FCM can be used to quantitatively evaluate
C
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Journal of the American Chemical Society
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ASSOCIATED CONTENT
* Supporting Information
Synthesis, characterization, and experimental details. This
material is available free of charge via the Internet at http://
pubs.acs.org.
■
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AUTHOR INFORMATION
Corresponding Author
xiaoyi@dlut.edu.cn; jinliji@dlut.edu.cn
■
(14) Li, Q.; Lau, A.; Morris, T. J.; Guo, L.; Fordyce, C. B.; Stanley, E.
F. J. Neurosci. 2004, 24, 4070−4081.
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Notes
1000.
The authors declare no competing financial interest.
(16) (a) Safdar, S.; Taite, L. J. Int. J. Pharm. 2012, 422, 264−270.
(b) Lu, Y.; Sun, B.; Li, C. H.; Schoenfisch, M. H. Chem. Mater. 2011,
23, 4227−4233. (c) Chakrapani, H.; Wilde, T. C.; Citro, M. L.;
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ACKNOWLEDGMENTS
■
This work was supported by National Natural Science
Foundation of China (No. 21174022), National Basic Research
Program of China (No. 2013CB733702), and Specialized
Research Fund for the Doctoral Program of Higher Education
(No. 20110041110009).
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