F?rster Resonance Energy Transfer Switchable Self-Assembled Micellar Nanoprobe: Ratiometric Fluorescent Trapping of Endogenous H2S Generation via Fluvastatin-Stimulated Upregulation

Article abstract of DOI:10.1021/jacs.5b03248

H₂S produced in small amounts by mammalian cells has been identified in mediating biological signaling functions. However, the in situ trapping of endogenous H₂S generation is still handicapped by a lack of straightforward methods wi

Full text of DOI:10.1021/jacs.5b03248

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Article
FRET-Switchable Self-Assembled Micellar Nanoprobe:
Ratiometric Fluorescent Trapping of Endogenous H2S
Generation via Fluvastatin-Stimulated Upregulation
Chunchang Zhao, Xiuli Zhang, Kaibin Li, Shaojia Zhu, Zhiqian Guo, Lili Zhang,
Feiyi Wang, Qiang Fei, Sihang Luo, Ping Shi, He Tian, and Wei-Hong Zhu
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.5b03248 • Publication Date (Web): 12 Jun 2015
Downloaded from http://pubs.acs.org on June 18, 2015
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FRET-Switchable Self-Assembled Micellar Nanoprobe:
Ratiometric Fluorescent Trapping of Endogenous H2S
Generation via Fluvastatin-Stimulated Upregulation
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Chunchang Zhao,*,† Xiuli Zhang,† Kaibin Li,† Shaojia Zhu,‡ Zhiqian Guo,† Lili Zhang,† Feiyi Wang,†
Qiang Fei,† Sihang Luo,† Ping Shi,*,‡ He Tian,† and WeiꢀHong Zhu*,†
†Key Laboratory for Advanced Materials and Institute of Fine Chemicals, Shanghai Key Laboratory of Functional Materials
Chemistry, Collaborative Innovation Center for Coal Based Energy (iꢀCCE), East China University of Science &
Technology, Shanghai 200237, P. R. China.
‡State Key Laboratory of Bioreactor Engineering, East China University of Science & Technology, Shanghai 200237, P. R.
China.
ABSTRACT: H2S produced in small amounts by mammalian cells has been identified in mediating biological signaling functions.
However, the in situ trapping of endogenous H2S generation is still handicapped by a lack of straightforward methods with high
selectivity and fast response. Here we encapsulate a semiꢀcyanineꢀBODIPY hybrid dye (BODInD-Cl) and its complementary
energy donor (BODIPY1) into the hydrophobic interior of an amphiphilic copolymer (mPEGꢀDSPE), especially for building up a
ratiometric fluorescent H2S nanoprobe with extraordinarily fast response. A remarkable redꢀshift in the absorption band with a gap
of 200 nm in the H2S response can efficiently switch off the Förster resonance energy transfer (FRET) from BODIPY1 to
BODInD-Cl, subsequently recovering the donor fluorescence. Impressively, both the interior hydrophobicity of supramolecular
micelles and electronꢀwithdrawing nature of indolium unit in BODInD-Cl can sharply increase aromatic nucleophilic substitution
with H2S. The ratiometric strategy based on the unique selfꢀassembled micellar aggregate NanoBODIPY achieves an extremely
fast response, enabling in situ imaging of endogenous H2S production and mapping its physiological and pathological consequences.
Moreover, the amphiphilic copolymer renders the micellar assembly biocompatible and soluble in aqueous solution. The
established FRETꢀswitchable macromolecular envelope around BODInD-Cl and BODIPY1 enables cellular uptake, and makes a
breakthrough in the trapping of endogenous H2S generation within raw264.7 macrophages upon stimulation with fluvastatin. This
study manifests that cystathione γꢀlyase (CSE) upregulation contributes to endogenous H2S generation in fluvastatinꢀstimulated
macrophages, along with a correlation between CSE/H2S and activating Akt signaling pathway.
sensitivity, selectivity and chemostability/photostability with
currently available probes.25 Another bottleneck encountered
with most reported probes is critically limited to the response
rate of H2S.46 Given the transient endogenous generation of
H2S in live cells, the slow response characteristic seriously
retards their practical application in realꢀtime imaging of
H2Sꢀrelated biological processes.
INTRODUCTION
Hydrogen sulfide (H2S) is produced in small amounts by
mammalian cells and has been identified in mediating many
biological signaling functions, such as modulating blood
pressure, exerting antioxidation and antiꢀinflammation effects,
and regulating the central nervous, respiratory and
gastrointestinal system.1ꢀ4 Endogenous H2S can be generated
enzymatically by cystathionine γꢀlyase (CSE), cystathionine
βꢀsynthase (CBS), and 3ꢀmercaptopyruvate sulfurtransferase
(3ꢀMST).5ꢀ7 These enzymes digest cysteine or cysteine
derivatives to produce H2S within different organs and
tissues.8ꢀ10 As is well known, abnormal H2S production is
tightly associated with a variety of human diseases, such as
Alzheimer’s disease and Down’s syndrome.11ꢀ13 Therefore, it is
of scientific interest to develop selective methods for real time
monitoring of H2S in living cells, mapping its physiological
and pathological consequences. Fluorescence trapping with
H2Sꢀresponsive fluorescent probe offers an attractive
molecular imaging technique for the in vivo detection,14ꢀ20
mostly based on its unique chemical reactivity, such as
exploiting selective H2Sꢀmediated reduction of azides,21ꢀ36
trapping H2S by proximate bisꢀelectrophile centers,37ꢀ43 and
employing copper sulfide precipitation strategies.44ꢀ45
However, trapping endogenous production of H2S in living
cells is still a challenge due to common problems in
With this in mind, we here develop a fluorescent boron
dipyrromethene (BODIPY)ꢀbased platform for construction of
selective and rapidly responsive H2S probes. The design
strategy relies on the thiol−halogen nucleophilic substitution of
a monochlorinated boron dipyrromethene (BODIPY) core,
fully employing the nucleophilic feature of H2S. Generally,
increasing the electron deficiency within the BODIPY
chromophore is an effective strategy to enable aromatic
nucleophilic substitution (SNAr) proceeding more readily.47ꢀ48
In this regard, we specifically incorporated an
electronꢀwithdrawing unit of indolium through a vinylene
bridge to the BODIPY core, thus creating
a
semiꢀcyanineꢀBODIPY hybrid dye BODInD-Cl (Scheme 1).
Indeed, BODInD-Cl realized a distinct increase in the reaction
rate of thiol−halogen nucleophilic substitution, exhibiting a
highly selective and fast response to H2S. Unfortunately, its
fluorescence turnꢀoff nature leads to low signal/noise (S/N)
ratios, which are detrimental to intracellular H2S detection.
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Noteworthy, a remarkable redꢀshift up to 200 nm in absorption generation within raw264.7 macrophages upon stimulation
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spectra was observed upon treatment of BODInD-Cl with H2S,
enabling BODInD-Cl as a component in a Förster resonance
energy transfer (FRET) based probes, which can address the
limitation in fluorescence turnꢀoff probes.
with fluvastatin, a cholesterolꢀtype drug used widely in
treating cardiovascular diseases. This study demonstrates that
fluvastatinꢀtriggered CSE upregulation is responsible for the
endogenous H2S generation in macrophages. Further, a
correlation between CSE/H2S and activating Akt signaling
pathway is also identified. To the best of our knowledge, the
selfꢀassembled micelle NanoBODIPY is the first
supramolecular nanoprobe with an H2Sꢀtriggered FRET switch,
enabling the endogenous modulation of H2S to be rapidly and
accurately tracked via a ratiometric fluorescent method.
Scheme 1. Chemical Structures of BODInD-Cl, BODIPY1 and
mPEGꢀDSPE, and Formation of Micellar Aggregate
NanoBODIPY for Modulation FRET from the Complementary
Energy Donor (BODIPY1) to the Responsive Energy Acceptor of
BODInD-Cl.
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RESULTS AND DISCUSSION
Incorporating an Electron-Withdrawing Semi-Cyanine
Unit for Fast Response to H2S. The in situ trapping of
endogenous H2S generation remains handicapped by a lack of
straightforward methods. Keeping in mind that H2S is
catabolized rapidly in biological systems, it is therefore highly
desirable to develop specific probes with extremely fast
reactivity towards H2S for real time monitoring of H2Sꢀrelated
biological processes. Here we exploited an approach which
utilizes thiol−halogen nucleophilic substitution of
a
monochlorinated BODIPY. It is known that the reaction
between monochlorinated BODIPY and nucleophilic reagent
can be modulated via changing the electronic nature of the
substituents.47ꢀ48 Accordingly, it is possible to design an
optimal probe for H2S via modifying the electron deficient
nature of BODIPY core. In our preliminary search for such a
probe, we first tested our previously reported monochlorinated
BODIPY (BODIPY2, Scheme 2). Indeed, no fluorescence
response of BODIPY2 to H2S could be observed under
physiological conditions, indicative of a lack of nucleophilic
substitution tendency between the chloro group and H2S. Since
enhancement of the electron deficiency at the 3ꢀposition can
increase reactivity towards nucleophiles, careful selection of
an electronꢀwithdrawing unit for appending to BODIPY2 core
would allow a judiciously designed probe for monitoring H2S.
To prove this concept, an electronꢀwithdrawing semiꢀcyanine
group was incorporated into the parent structure of BODIPY2
through a vinylene unit, and BODInD-Cl was thus designed.
As shown in Scheme 2, BODInD-Cl was obtained via
condensation of Nꢀethylꢀ2,3,3ꢀtrimethylindolenine with
aldehyde BODIPY3, which was prepared according to our
previously established procedure.53
Considering that a supramolecular micelle or cluster of
amphiphilic polymers can entrap multiple chromophores
within the same interior of the macromolecular envelope,49ꢀ52
we further encapsulated the semiꢀcyanineꢀBODIPY hybrid dye
(BODInD-Cl) and its complementary energy donor
(BODIPY1) into the hydrophobic interior of a micelle based
on
amphiphilic
copolymer
mPEGꢀDSPE
(1,2ꢀdimyristoylꢀsnꢀglyceroꢀ3ꢀphosphoꢀethanolamineꢀNꢀ(meth
oxy(polyethyleneglycol)ꢀ2000)), imposing the trapped
chromophores in close proximity. In this way, the unique
selfꢀassembled micellar aggregate NanoBODIPY is designed
to comprise BODIPY1 as an energy donor and BODInD-Cl
as dynamic energy acceptor due to the good spectral overlap
between the emission spectrum of the donor and absorption
spectrum of the acceptor, thus guaranteeing that the specific
FRET from BODIPY1 to BODInD-Cl occurs efficiently in
the absence of H2S (Scheme 1). In contrast, the presence of
H2S induces a remarkable shift up to 200 nm from 540 to 738
nm in the absorption of BODInD-Cl, resulting in the poor
overlap with the emission of BODIPY1, and subsequently loss
of FRET with recovering the donor fluorescence. The change
of FRET efficiency is reflected in an enhancement of
fluorescence intensity of the donor BODIPY1 and a decrement
of that of the acceptor BODInD-Cl. In particular, the
macromolecular envelope around BODInD-Cl and BODIPY1
enables cellular uptake of living cells, which was successfully
exploited for in situ ratiometric imaging of endogenous H2S
With BODInD-Cl in hand, we then evaluated the
spectroscopic responses of BODInD-Cl toward H2S under
model conditions (acetonitrile / PBS buffer, 1:1, v/v, 20 mM,
pH 7.4, 37 °C) by monitoring the changes in absorption and
emission spectra. Here acetonitrile was chosen as coꢀsolvent to
improve the aqueous solubility of BODInD-Cl. As expected,
BODInD-Cl responded to H2S very rapidly when using NaHS
as a H2S donor. Time dependent fluorescence quenching of
BODInD-Cl in the presence of H2S showed that the reaction
could be completed within minutes (Figure S1 in the
Supporting Information). Furthermore, a fascinating feature
was observed in the absorption, that is, addition of NaHS
triggered a rapid redꢀshift from 540 to 738 nm (Figure 1a),
resulting in the possible colorimetric and ratiometric detection
even with naked eyes. Both high performance liquid
chromatography (HPLC)
and
highꢀresolution
mass
spectrometry (HRMS) experiments were carried out to confirm
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the reaction mechanism of BODInD-Cl and NaHS (Figure S2
in the Supporting Information). Upon incubation with NaHS
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(100 µM) in reaction buffer, BODInD-Cl (5 µM, HPLC
retention time at 5.4 minutes) was converted efficiently and
rapidly into BODInD-SH (retention time at 7.3 minutes). Also
the HRMS spectrum of BODInD-Cl solution in the presence
of NaSH showed a m/z signal at 554.2772, corresponding to
[BODInD-SH – Br]+. All these data suggest the attachment of
H2S to BODInD-Cl by replacement of the chloro group
(Scheme 2). Furthermore, BODInD-Cl displayed good
selectivity to NaHS with minimum interference from other
biologically relevant analytes in the PBS buffer (Figure 1b and
Figure S3 in the Supporting Information). Here the specific
reaction with H2S was retained even in the presence of 1 mM
GSH, suggesting the high selectivity to H2S over GSH.
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Scheme 2. Synthesis of BODInD-Cl and Proposed Reaction
Mechanism with NaHS
.
Figure 1. (a) Absorption changes of BODInD-Cl (5 µM) upon
addition with 100 µM NaHS, and (b) changes in absorbance of
BODInD-Cl (5 µM) in the presence of 100 ꢁM NaHS and other
biologically relevant reactive sulfur and anions (500 µM) in PBS
(pH 7.4, 50% CH3CN, v/v). Data were recorded at 30 min after
addition of analytes.
Supramolecular Strategy to Construct Nanoprobes for
Fast and Selective Response to H2S. It is obvious that H2S
triggered fluorescence turnꢀoff nature (Figure S1 in the
Supporting Information) renders this probe less attractive in
detection of endogenous H2S within living cells due to the low
signal/noise (S/N) ratio. Fortunately, a remarkable redꢀshift in
absorption spectra was observed when BODInD-Cl was
treated with NaHS, showing a distinct gap of 200 nm (Figure
1a). This distinct gap in the absorption makes BODInD-Cl
promising for construction of FRETꢀbased probes, which
enables analytes to be evaluated via the ratiometric change in
two emission bands. It then can be envisioned that a
fluorophore (energy donor) and BODInD-Cl (energy acceptor)
can be integrated within a covalently linked skeleton to form a
FRET pair. However, this covalent system requires tedious
multistep synthesis procedure. In contrast, the supramolecular
assemble approach offers an opportunity to overcome this
limitation. Indeed, selfꢀassembling nanoparticles of
amphiphilic polymers can entrap multiple chromophores
within the same interior of the macromolecular envelope,
imposing the trapped chromophores in close proximity. 50ꢀ52 In
this way, we can establish a specific FRETꢀbased nanoprobe
for fast and selective response to H2S via encapsulating
BODInD-Cl and its complementary donor in the hydrophobic
part of the amphiphile.
To test this design hypothesis, we utilized an amphiphilic
copolymer mPEGꢀDSPE as the host and BODIPY1 as the
energy donor for BODInD-Cl because of the good spectral
overlap between the emission spectrum of the donor and
absorption spectrum of the acceptor. BODIPY1 and
BODInD-Cl are almost insoluble in water at ambient
temperature, limiting their biological applications. However,
when the amphiphilic polymer mPEGꢀDSPE was introduced to
the PBS buffer, they were readily dissolved due to the ability
of this amphiphilic polymer to form hydrophilic micelles,
which are capable of capturing BODIPY1 and BODInD-Cl
inside their hydrophobic interiors (Scheme 1). The PEG
function of the formed micelles also improved the
biocompatibility. The average hydrodynamic size of these
micelles (named as NanoBODIPY) is about 10 nm as
measured by dynamic light scattering and transmission
electron microscopy (Figure S4 in the Supporting
Information).
The absorption spectra of the micelles containing either
BODIPY1 or BODInD-Cl in PBS reveal the typical features
of BODIPY derivatives in organic solvents, with the
absorption bands at 500 and 548 nm, respectively (Figure 2).
Correspondingly, the emission spectra also showed the
characteristic bands at 511 and 589 nm. Notably, the emission
band of BODIPY1 is overlapped well with the absorption
band of BODInD-Cl, suggesting that the encapsulation of both
chromophores within the same micellar aggregates might
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result in an efficient energy transfer from BODIPY1 to BODInD-Cl was used in the entrapment protocol, the FRET
BODInD-Cl upon excitation of the donor BODIPY1.
1
efficiency among the encapsulated BODIPY1 and
BODInD-Cl was found to be BODIPY1 doseꢀdependent.
Here the optimized ratio of BODInD-Cl to BODIPY1 was
thus evaluated to be around 6 with the FRET efficiency up to
96% (Figure S5 in the Supporting Information). Compared
with the simple mixture of BODInD-Cl with BODIPY1 under
the same condition, the minimal fluorescence decrement at 511
nm or enhancement at 589 nm was further confirmed that the
successful encapsulation of the two chromophores in close
proximity contributes to efficient FRET. Given BODInD-Cl
as a responsive energy acceptor, the presence of H2S can lead
to a remarkable redꢀshift with a gap of 200 nm in the
absorption of BODInD-Cl, which would trigger poor overlap
between the emission spectrum of BODIPY1 and absorption
spectrum of the acceptor, thus quickly abolishing FRET with
the recovery of donor fluorescence. In this way, we can
transduce the trapping of H2S by BODInD-Cl from the
intensityꢀbased method in the unfavored fluorescence turnꢀoff
characteristic to the ratiometic fluorescence mode. Based on
the specific FRET “on–off” sensing system, the ratiometric
mode is expected to exactly allow for fast and accurate
measurements with elimination of the influence in
experimental conditions, such as light source, dye
concentration and background interference effects.54ꢀ58
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The photostability of the selfꢀassembled micellar nanoprobe
was firstly investigated before exploiting its potential
applications. Under continuous irradiation with a Hg/Xe lamp
for 3 h (Figure S6 in the Supporting Information), minimal
change was observed in the fluorescence intensity ratio of the
two emission bands at 511 and 589 nm (I511/I589), indicative of
excellent photostability of NanoBODIPY. This result suggests
the superiority of the nanoprobe in bioimaging. Then, we
evaluated the fluorescence responses of NanoBODIPY toward
H2S in aqueous solution under physiological conditions.
Figures 3a and 3b shows the representative fluorescence
spectral changes of NanoBODIPY upon addition of NaHS. As
Figure 2. (a) Absorption and (b) emission spectra (λex = 490 nm)
of micelles entrapped with BODIPY1 (1.7 ꢁM), BODInD-Cl
(10.0 ꢁM), or both to form NanoBODIPY in PBS (pH 7.4) at 37
°C. Inset: enlargement of the fluorescence intensity between
0ꢀ100.
expected, the addition of NaHS elicited
a remarkable
ratiometric fluorescence change. The emission intensity
increased gradually at 511 nm with a concomitant loss of
emission at 589 nm. Interestingly this process became very fast,
reaching completion within 140 s (Figure 3a). Generally, the
response rate of small fluorescent BODInD-Cl toward H2S, a
typical nucleophilic substitution, is dependent upon the solvent
polarity, for instances, the response reaction was completed
from within 1000 to 4000 s when the water content increased
from 50% to 80% (Figure S7 in the Supporting Information).
Therefore, we can conclude that the interior hydrophobicity of
supramolecular micelles from the amphiphilic copolymer can
decrease the solvent interaction (polarization) with the
nucleophile of H2S, thus promoting the reaction rate between
BODInD-Cl and H2S. In this regard, both the interior
Also NanoBODIPY showed two typical absorption bands
at 500 and 548 nm, corresponding to that of BODIPY1 and
BODInD-Cl, respectively. Notably, the predominant
absorption of NanoBODIPY is similar as that of the simple
mixture of micelles containing BODIPY1 or BODInD-Cl
exclusively. Upon excitation at 500 or 490 nm, NanoBODIPY
exhibited two wellꢀresolved emission bands at 511 and 589 nm
(Figure 2b). The occurrence of FRET from BODIPY1 to
BODInD-Cl within the micellar aggregates can be identified
by comparing the spectra of NanoBODIPY with that of
micelles containing exclusively either BODIPY1 or
BODInD-Cl. As can be seen from Figure 2b, the fluorescence
intensity of NanoBODIPY at 511 nm was much weaker than
that of BODIPY1 in micelle, showing a decrease close to 90%.
Compared with the fluorescence intensity of BODInD-Cl in
micelle, significant increment of the fluorescence intensity at
589 nm was observed for NanoBODIPY. These data
highlighted the efficient occurrence of FRET. Within the
interior of supramolecular micelles, the entrapped organic dyes
with small Stokes shifts often show selfꢀquenching of
fluorescence behavior at relatively high local concentrations.
To minimize the selfꢀquenching among BODIPYs in
NanoBODIPY, the optimal ratio of these dyes to
mPEGꢀDSPE was evaluated as 0.0025–0.01 by weight (Figure
S5 in the Supporting Information). Specifically, when 10 µM
hydrophobicity
of
supramolecular
micelles
and
electronꢀwithdrawing nature of indolium unit in BODInD-Cl
can sharply increase aromatic nucleophilic substitution with
H2S, thus enabling H2S to be rapidly and accurately tracked
within 140 s. As
a matter of fact, the response of
NanoBODIPY is faster than most reported H2S probes, which
fulfill the response in the range of 30ꢀ60 min.20ꢀ21,39 Notably,
the fluorescence intensity ratio of the two emission bands at
511 and 589 nm (I511/I589) increases from 1.8 to 16.5,
corresponding to a 9ꢀfold enhancement factor, indicative of an
efficient ratiometric response of NanoBODIPY to H2S.
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Obviously, the H2Sꢀtriggered redꢀshift in absorption of in different cellular compartments, experiments were carried
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BODInD-Cl (Figure S8 in the Supporting Information)
switched off the FRET process, and recovered the donor
fluorescence (BODIPY1) at 511 nm. In addition, upon the
titration of NaHS with the ratiometric fluorescence responses
(Figure S9 in the Supporting Information), NanoBODIPY can
sensitively detect submicromolar concentrations of H2S with a
detection limit of 7 nM, which is sufficiently sensitive to trap
endogenous generation of H2S in living cells.25 Since the pH
value of living cells can vary from neutral to acidic condition
out to interrogate pH effects on the ratiometric response of
NanoBODIPY toward H2S. Fortunately, it was found that pH
exerted little influence on the response properties within a
physiological range from pH 8 to approximately 4.5 (Figure
S10 in the Supporting Information). The FRETꢀswitchable
selfꢀassembled micellar nanoprobe of NanoBODIPY endows
the low detection limit and fast response to H2S within several
seconds.
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Figure 3. (a) Timeꢀdependent ratiometric fluorescence changes of NanoBODIPY in the presence of NaHS (100 µM) in PBS (pH 7.4) at
37 °C, inset is changes of fluorescence spectra in the absence and presence of NaHS; (b) Fluorescence spectra of NanoBODIPY in the
presence of various concentrations of NaHS (0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0
ꢁM, respectively) in PBS buffer (pH 7.4) at 37 °C; (c) Ratiometric fluorescence changes of NanoBODIPY in the presence of 100 ꢁM
NaHS and other biologically relevant competing analytes in PBS (pH 7.4) at 37 °C. R0 represents the fluorescence intensity ratio (I511/I589
)
in the absence of an analyte, and Rt represents the ratio in the presence of an analyte. Bars represent fluorescence changes 140 s, 10 min
and 30 min after addition of analytes. Data shown are for 1 mM glutathione, 1 mM cysteine, and 100 ꢁM for other analytes. λex = 490 nm.
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Figure 4. Confocal microscopy images of H2S in live raw264.7 macrophages cells using NanoBODIPY. (A) cells incubated with
NanoBODIPY for 30 min; (B) cells incubated with NanoBODIPY for 30 min, then 100 µM NaHS was loaded for another 30 min; (C)
cells were pretreated with 2.0 ꢁM fluvastatin for 48 h, further incubated with NanoBODIPY for 30 min; (D) cells were pretreated with 1
mM PAG for 1 h, further incubated with 2.0 ꢁM fluvastatin for 48 h, cells were then incubated with NanoBODIPY for 30 min; (E) cells
were pretreated with 1 mM cysteine for 1 h, further incubated with NanoBODIPY for 30 min; (F) cells were pretreated with 1 mM
cysteine for 1 h, further incubated with 2.0 ꢁM fluvastatin for 48 h, cells were then incubated with NanoBODIPY for 30 min; (G) cells
were pretreated with 10 ꢁM perifosine for 1 h, further incubated with 2.0 ꢁM fluvastatin for 24 h, cells were then incubated with
NanoBODIPY for 30 min. Green channel at 500–550 nm; red channel at 560–650 nm; ratio images generated from green channel to red
channel. (H) Average intensity ratios from green to red channel in fluorescence images. Data represent mean standard error
.
In good agreement with the small molecule probe
BODInD-Cl, the micelle nanoprobe NanoBODIPY also
showed high selectivity to H2S over other competing analytes
(Figure 3c), including reactive sulfur (RSS), oxygen (ROS),
imaging applications.59 When murine raw264.7 macrophages
were incubated with NanoBODIPY for 30 min, a bright
fluorescence in the green or red channel was observed upon
excitation at 488 nm. The ratio of the two emissions from
green channel to red channel is about 0.8 (Figure 4A),
indicative of the low level of H2S in macrophages. Further
treatment of these cells with NaHS for 30 min resulted in
significant increment of the bright green fluorescence,
accompanied by the faint red fluorescence signal (Figure 4B).
A remarkable 6ꢀfold enhancement of the emission ratio was
observed. These imaging experiments indicate that
NanoBODIPY can be used to track H2S in dualꢀcolor imaging
modality.
and nitrogen species (RNS). Only NaHS triggered
a
remarkable enhancement in the fluorescence intensity ratio
(I511/I589) while other analytes gave minimal ratiometric
changes. In addition, the ratiometric enhancement elicited by
H2S was retained even in the presence of 1 mM GSH and 1
mM Cys.
Taken
together,
the
encapsulation
of
a
semiꢀcyanineꢀBODIPY hybrid dye (BODInD-Cl) and its
complementary energy donor (BODIPY1) into the
hydrophobic interior of an amphiphilic copolymer
(mPEGꢀDSPE) can realize ratiometric fluorescent sensing with
rapid kinetics, enabling the trapping of endogenous H2S
production. Furthermore, the amphiphilic copolymer endows
the micellar assembly with biocompatibility and aqueous
solubility.
Next, our selfꢀassembled nanoprobe was explored for
monitoring endogenous H2S generation with fluvastatin
stimulated murine raw264.7 macrophages as a model cell
system (Figure 4). Fluvastatin is a cholesterolꢀtype drug and
widely used in treating cardiovascular diseases. Recent studies
have demonstrated that it stimulates the H2S formation in
raw264.7 macrophages.59 However, the underlying molecular
action mechanism of fluvastatin remains poorly understood. In
this study, we employed NanoBODIPY to investigate the role
of fluvastatin on elevating H2S generation in raw264.7
macrophages. Macrophages cells were firstly incubated with
fluvastatin (2.0 µM) for 48 h, and subsequently treated with
NanoBODIPY for 30 min. H2S generation was determined by
monitoring the changes in ratiometric fluorescence signal.
Compared with the untreated cells, the fluorescence ratio from
Trapping Endogenous H2S Generation in Fluvastatin
Stimulated Macrophages. After investigating the promising
responsive behavior of NanoBODIPY toward H2S in vitro, we
then explored the ratiometric characteristic of NanoBODIPY
for fluorescent trapping of cellular H2S (Figure 4). Since the
H2Sꢀproducing enzyme CSE was identified as generating H2S
in macrophages, here raw264.7 macrophages were explored to
interrogate the capability of NanoBODIPY for fluorescence
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green to red channel in fluvastatin–stimulated cells was fluvastatinꢀstimulated CSE upregulation and subsequent H2S
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increased by 4ꢀfold from 0.8 to 3.2 (Figure 4C). The
fluorescence ratio was greatly decreased to 0.9 when
level elevation. To interrogate this process, we assayed the
effects of changes in H2S production via ratiometric
florescence image in the absence and presence of Akt
inhibitor, perifosine. Figure 4H displayed the distinctive
reversal effect of perifosine on the elevation of H2S elicited by
fluvastatin. The fluorescence ratio was found to be
significantly decreased to 1.0 when macrophages are
pretreated with perifosine before incubation with fluvastatin, a
3ꢀfold decrease relative to that in fluvastatin–stimulated cells
(3.2). These imaging data imply a correlation between
CSE/H2S elevation and activating Akt signaling pathway.
fluvastatinꢀinduced
cells
were
pretreated
with
DLꢀpropargylglycine (PAG, 1 mM, Figure 4D), a commercial
CSE irreversible inhibitor, before incubation with
NanoBODIPY. A marked increase of H2S generation was
noted upon introduction of cysteine (1 mM), the substrate for
CSE in producing H2S, to fluvastatinꢀstimulated cells (Figure
4F), with a nearly 4ꢀfold increase in fluorescence ratio relative
to that of normal cells in the presence of Cys (Figure 4E).
These results manifested that fluvastatin stimulated H2S
generation can be attributed to CSE upregulation activity in
raw264.7 macrophages. Western blot analysis further
demonstrated the positive effect of fluvastatin on CSE
upregulation in raw264.7 macrophages. Treatment with 2.0
µM fluvastatin for 48 h stimulated a distinct elevation of CSE
protein expression (Figure S11 in the Supporting Information).
As a consequence, NanoBODIPY has been proven as a
promising probe for fluorescent trapping of endogenous H2S
with the preferable ratiometric mode.
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As known, intracellular location of particleꢀbased
fluorophores is an important issue.60,61 Here, the localization of
NanoBODIPY in living cells was identified by coꢀstaining
experiments using commercial available LysoꢀTracker Green,
MitoꢀTracker Green and Hoechst blue, respectively (Figure 5).
Obviously, the red fluorescence from NanoBODIPY
colocalized well with green fluorescence from LysoꢀTracker
Green or MitoꢀTracker Green. A poor overlap between
NanoBODIPY and Hoechst was also noted. Therefore, it is
rational to infer that the nanoprobe is delivered to the
cytoplasm with nonꢀspecific intracellular localization.
Since fluvastatin can increase the phosphorylation of Akt, it
is hypothesized that the Akt signaling pathway is involved in
Figure 5. Confocal fluorescence images for intracellular localization of NanoBODIPY in cells. Cells were incubated with NanoBODIPY
for 1 h and then coꢀstained with (a) 1 µM LysoꢀTracker Green, (b) 200 nM MitoꢀTracker Green, or (c) 100 nM Hoechst for 30 min. In the
case of coꢀstaining with MitoꢀTracker, no commercial available tracker is suitable for colocalization with NanoBODIPY, thereby, we here
employed the strategy to encapsulate BODInD-Cl only within interior of supramolecular micelles, but keeping the hydrodynamic size of
formed micelles similar as that of NanoBODIPY.
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Municipality (15XD1501400), and Shanghai Scientific and
Technological Innovation Project (14520720700).
CONCLUSIONS
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In summary, we have developed a BODIPYꢀbased platform
for the construction of selective and fast responsive H2S
probes. The designed small molecular probe, BODInD-Cl,
showed fascinating feature with a rapid redꢀshift in the
absorption from 540 to 738 nm upon interaction with H2S.
Although the fluorescence turnꢀoff nature renders this probe
less sensitive in detection of endogenous H2S generation
within living cells, the remarkable redꢀshift in the absorption
makes this probe promising for construction of FRETꢀbased
probes. Selfꢀassembling micelles of amphiphilic polymers can
capture multiple chromophores within the same interior of the
macromolecular envelope, imposing the trapped chromophores
in close proximity. By encapsulating BODInD-Cl and its
complementary donor BODIPY1 in the hydrophobic part of
mPEGꢀDSPE, a FRETꢀswitchable nanoprobe for fast and
selective response to H2S was established with features of
aqueous solubility and biocompatibility. Both the interior
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hydrophobicity
of
supramolecular
micelles
and
electronꢀwithdrawing nature of indolium unit in BODInD-Cl
can sharply increase aromatic nucleophilic substitution with
H2S, thus enabling H2S to be rapidly and accurately tracked via
a ratiometric method. Moreover, this supramolecular envelope
holds promising potential in cell imaging due to several
advantages such as favorable cellular uptake, aqueous
solubility, biocompatibility and ratiometric measurement,
which has been successfully exploited for in situ trapping of
endogenous H2S generation within raw264.7 macrophages
upon stimulation with fluvastatin. Based on the unique
responsive energy acceptor of BODInD-Cl, we provide a clear
clue to increasing the H2Sꢀresponse rate, building up
preferable ratiometric fluorescent sensors based on the
unfavored fluorescence turnꢀoff chromophore, and making full
use of the amphiphilic assembled micelles to guarantee
biocompatibility and aqueous solubility. The novel
nanoprobeꢀbased platform for H2S could be further constructed
by simply employing versatile energy donors.
ASSOCIATED CONTENT
Supporting Information. Detailed characterization data,
experimental procedures including preparation of NanoBODIPY
and cell imaging, supplementary figures. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Eꢀmail: whzhu@ecust.edu.cn.
*Eꢀmail: zhaocchang@ecust.edu.cn.
*Eꢀmail: ship@ecust.edu.cn.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We gratefully acknowledge the financial support by National 973
Program (No. 2013CB733700), NSFC for Creative Research
Groups (21421004) and Distinguished Young Scholars
(21325625), NSFC/China, the Oriental Scholarship, the
Fundamental Research Funds for the Central Universities
(WJ1416005), Science and Technology Commission of Shanghai
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