A Dual-Modal Molecular Probe for Near-Infrared Fluorescence and Photoacoustic Imaging of Peroxynitrite

Article abstract of DOI:10.1021/acs.analchem.8b01879

Peroxynitrite (ONOO^-), a reactive and short-lived biological oxidant, is closely related with many pathological conditions such as cancer. However, real-time in vivo imaging of ONOO^- in tumors remains to be challenging. Herein, we de

Full text of DOI:10.1021/acs.analchem.8b01879

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Article
A Dual-modal Molecular Probe for Near-infrared
Fluorescence and Photoacoustic Imaging of Peroxynitrite
jianjian zhang, Xu Zhen, Jianfeng Zeng, and Kanyi Pu
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01879 • Publication Date (Web): 26 Jun 2018
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Analytical Chemistry
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A Dual-modal Molecular Probe for Near-infrared Fluorescence and
Photoacoustic Imaging of Peroxynitrite
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Jianjian Zhang,†,‡ Xu Zhen,‡ Jianfeng Zeng,*,§ and Kanyi Pu*,‡
†Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, College of Chemis-
try and Materials Science, Northwest University, Xi’an, Shaanxi 710127, People’s Republic of China
‡School of Chemical and Biomedical Engineering, Nanyang Technological University, 637457, Singapore.
§State key Laboratory of Radiation Medicine and Protection, Soochow University, Suzhou 215123, People’s Republic
of China
*Corresponding Author: Zeng J., jfzeng@suda.edu.cn; Pu K., kypu@ntu.edu.sg.
ABSTRACT: Peroxynitrite (ONOOˉ), a reactive and short-lived biological oxidant, is closely related with many pathological
conditions such as cancer. However, real-time in vivo imaging of ONOOˉ in tumor remains to be challenging. Herein, we de-
velop a near-infrared fluorescence (NIRF) and photoacoustic dual-modal molecular probe (CySO3CF3) composed of a water-
soluble hemicyanine dye caged with a trifluoromethyl ketone moiety for in vivo imaging of ONOOˉ. The trifluoromethyl ke-
tone moiety can undergo a series of ONOOˉ-induced cascade oxidation-elimination reactions, leading to sensitive and specif-
ic fluorescence and photoacoustic turn-on responses toward ONOOˉ; while a zwitterionic structure of the hemicyanine
component ensures good water-solubility. CySO3CF3 thus not only can specifically detect ONOOˉ in solution and cells with
the limit of detection down to 53 nM, but also allows for NIRF and photoacoustic dual-modal imaging of ONOOˉ in the tumor
of living mice.
Reaction oxygen and nitrogen species (RONS) play a vital
role in physiological and pathological processes of the liv-
ing system.1 Peroxynitrite (ONOOˉ) as a highly reactive
RONS can nitrate the biomacromolecules including pro-
teins, nucleic acids and lipids, and disorganize their biolog-
ical functions,2 leading to mitochondria malfunction and
cellular apoptosis. In tumor microenvironment, ONOOˉ is
closely related to tumor immunosuppression. Particularly,
ONOOˉ can nitrate p53 protein,3 a critical regulator of tu-
mor suppressor protein that is encoded by gene TP53 (tu-
mor protein p53) in human body, which causes the loss of
the control role of p53 protein to cell cycle followed by a
series of cytotoxic effects, ultimately accelerating the for-
mation of tumor.4 Thereby, real-time imaging of ONOOˉ in
tumor is essential for understanding the pathological func-
tions of ONOOˉ and developing innovative therapeutic ap-
proach to tumor treatment.
smart activatable probes with specific PA responses to-
ward ONOOˉ are less exploited.35,36 We recently developed
organic semiconducting nanoprobes for ratiometric PA
imaging of ONOOˉ.37 Although it can be applied for in vivo
imaging, this nanoprobe was doped with bulky borane in
addition to the chromophores to achieve selectivity. Be-
sides, the fluorescence is completely quenched, making it
incompatible for fluorescence imaging.
In this study, we report the design and synthesis of a wa-
ter-soluble small-molecular probe (CySO3CF3) for in vivo
near-infrared fluorescence (NIRF)/PA dual-modal imaging
of ONOOˉ. CySO3CF3 is composed of trifluoromethyl ketone
moiety (TFK),6-8,38 an ONOOˉ-responsive unit, caged NIR
hemicyanine dye with a zwitterionic structure39 (Scheme
1a). The probe is nonfluorescence because CySO3OH is in a
“caged” state with weak electron-donating ability of the
oxygen atom.37,40-44 In the presence of ONOOˉ, CySO3CF3 is
converted into the uncaged molecule (CySO3OH) through a
series of cascade oxidation-elimination reactions. Due to
the enhanced electron-donating ability from the oxygen
atom in CySO3OH, such an ONOOˉ-induced conversion re-
sults in not only fluorescent turn-on but also red-shifted
NIR absorption, enabling NIRF/PA dual-modal imaging.
Fluorescence imaging has been developed for detection
of ONOOˉ,5-11 but most of fluorescent probes are only re-
sponsive to visible light, which is not ideal for in vivo imag-
ing due to shallow tissue penetration. In contrast, photoa-
coustic (PA) imaging is a hybrid imaging modality that
combines laser excitation with acoustic detection, offering
higher tissue penetration depth (up to ≈ 5 cm) for in vivo
imaging.12-14 Although near-infrared (NIR) molecular
EXPERIMENTAL SECTION
dyes,15,16
polymer
nanoparticles,17-24
fluorescence
Chemicals. All chemicals and reagents were obtained
from Sigma-Aldrich unless otherwise stated. Sodium hypo-
chlorite (NaOCl, 5% aqueous solution). Hydrogen peroxide
(H2O2, 30% aqueous solution), sodium nitrite (NaNO2) and
proteins,25,26 porphysomes,27,28 upconversion nanoparti-
cles,29,30 quantum dots,31 gold nanoparticles,32 carbon
dots33 and 2D materials34 have been used for PA imaging,
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Analytical Chemistry
boron tribromide (BBr3) were purchased from TCI (Tokyo,
Page 2 of 8
2 H), 3.81 (s, 3 H), 3.81-3.56 (m, 2 H), 2.78-2.63 (m, 2 H),
2.18-1.96 (m, 2 H), 1.26 (td, J = 7.0, 0.9 Hz, 3 H), 0.24 (d, J =
0.9 Hz, 9 H). MS (ESI): m/z = 335.19 [M+H]+.
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Japan). Anhydrous dichloromethane (CH2Cl2) were treated
with CaH2 under reflux for 24 h and redistilled before use.
Milli-Q water was supplied by Milli-Q Plus System (Milli-
poreCorporation, Breford, U.S.A.). A stock solution (1
mmol/L) of CySO3CF3 was prepared by dissolving in dime-
thyl sulphoxide (DMSO).
Compound 6: To a solution of compound 5 (4.0 g, 12
mmol) in THF (2 mL) was added TBAF (1 M in THF, 13.0
mL) dropwise under nitrogen atmosphere. The mixture
was stirred at room temperature for 5 h and then was
treated with hydrochloric acid (4.0 M, 4.0 mL) for 3 h.
CH2Cl2 (30 mL) was added to the resulting solution, which
was washed with water and saturated sodium chloride
respectively. The organic phase was dried with anhydrous
Na2SO4, and then was concentrated under reduced pres-
sure. The residue was purified by silica gel column chro-
matography (ethyl acetate/hexane, 5%) to obtain trifluo-
ro-4-(4-methoxyphenyl)butan-2-one 6. (2.45 g, yield:
88%);1H NMR (300 MHz, CDCl3): δ = 7.11 (d, J = 8.7 Hz, 2
H), 6.84 (d, J = 8.7 Hz, 2 H), 3.78 (s, 3 H), 3.05-2.98 (m, 2 H),
2.96-2.89 (m, 2 H). MS (ESI): m/z = 233.09 [M+H]+.
Instrumentation. UV−vis spectra were recorded on a
Shimadzu UV-2450 spectrophotometer. Fluorescence
spectra were recorded on a Fluorolog 3-TCSPC spectroflu-
orometer (Horiba Jobin Yvon). NMR spectra were meas-
ured using a Bruker 300 MHz instruments (Germany). All
1H chemical shifts (δ) are relative to residual protic solvent
(CHCl3: δ 7.26 ppm). Mass spectra were measured using
Thermo LCQ Fleet LC-MS with ESI mode (America). The pH
values were measured using a digital pH-meter (Seven-
Compact S220, Zurich, Switzerland). High performance
liquid chromatography (HPLC) analyses were performed
on an Agilent 1260 system equipped with a G1311B pump,
UV detector and an Agilent Zorbax SB-C18 RP (9.4 × 250
mm) column, with CH3CN (0.1% of TFA) and water (0.1%
of TFA) as the eluent. Fluorescence images of cells were
acquired on Laser Scanning Microscope LSM800 (Zeiss) or
LX71-inverted fluorescence microscope (Olympus) with
Retiga-2000R CCD camera. Fluorescence images of tumor
were acquired with IVIS Spectrum imaging system (Perki-
nElmer, Inc.). In vivo PA images were performed with a
Multispectral Optoacoustic Tomography scanner (MSOT,
iThera medical, Germany) with excitation light of 680-850
nm.
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Compound 7: To a solution of 1,1,1-trifluoro-4-(4-
methoxyphenyl) butan-2-one 6 (2.4 g, 10.3 mmol) in
CH2Cl2 (20 mL) was added BBr3 (5.2 mL, 13.4 mmol) at –
78 °C under nitrogen atmosphere. The resulting mixture
was then warmed to room temperature and stirred 12 h.
Then ice-water was added slowly, and the mixture was
added ethyl acetate (50 mL). The resulting solution was
washed with H2O (3 × 20 mL), saturated sodium bicar-
bonate (30 mL) and saturated sodium chloride (30 mL),
respectively. The organic phase was dried with anhydrous
Na2SO4, and then concentrated under reduced pressure,
which was purified by silica gel column chromatography
(ethyl acetate/hexane, 25%) to obtain 1,1,1-trifluoro-4-(4-
hydroxyphenyl)butan-2-one 7 (1.84 g, yield 82%); 1H NMR
(300 MHz, CDCl3): δ = 7.06 (d, J = 8.6 Hz, 2 H), 6.77 (d, J =
8.6 Hz, 2 H), 3.05-2.97 (m, 2 H), 2.95-2.80 (m, 2 H). MS
(ESI): m/z = 219.16 [M+H]+.
CySO3Cl: A mixture of compound 1 (516 mg, 3.0 mmol),
compound 2 (1.76 g, 6.0 mmol) and sodium acetate (246
mg, 3.0 mmol) were dissolved in acetic anhydride (20.0
mL) under nitrogen atmosphere and heated to 90 °C for 1
h with a Dean-Stark condenser. After cooling to room tem-
perature, the resulting green solid was collected, washed
with Et2O (100 mL) and purified by silica gel column
chromatography using CH2Cl2/ 0-2% methanol as eluent to
afford desired product CySO3Cl as a blue solid. Yield: 1.59
g (73%). 1H NMR (300 MHz, CDCl3): δ = 8.43 (d, J = 14.2 Hz,
2H), 7.52 (d, J = 7.2 Hz, 2H), 7.48-7.32 (m, 4H), 7.28 (d, J =
7.0 Hz, 2H), 6.34 (d, J = 14.1, 2H), 4.23 (t, J = 7.2 Hz, 2H),
2.90 (t, J = 6.6 Hz, 2H), 2.75 (t, J = 4.2 Hz, 2H), 2.08-1.88 (s,
10H), 1.73 (s, 12H) ppm; ESI-MS, m/z: 727.48 [M]+.
CySO3OH: To a stirred solution of resorcinol (220 mg, 2.0
mmol) in acetonitrile (ACN, 15 mL) was added K2CO3 (276
mg, 2.0 mmol) at room temperature under nitrogen at-
mosphere and the resulting mixture was stirred for 20
min. Then, a solution of CySO3Cl (727 mg, 1.0 mmol) in
DMF (10 mL) was added to the above mixture via a sy-
ringe, and the reaction mixture was heated at 50 °C for 4 h.
Eventually the solvent was evaporated under reduced
pressure, and the crude product was purified by flash col-
umn chromatography (petroleum ether/CH2Cl2/MeOH =
25:25:1) on silica gel, affording the desired compound
CySO3OH as a blue-green solid (400 mg, yield 79%). 1H
NMR (400 MHz, CDCl3): δ = 8.75 (d, J = 14.8 Hz, 1H), 7.63
Synthesis
Compound 4: To a solution of 3-(4-methoxyphenyl)
propanoic acid (3, 2.7 g, 15.0 mmol) in ethanol (20 mL)
was added H2SO4 (0.7 mL, con.) at room temperature un-
der nitrogen atmosphere. The resulting mixture was re-
fluxed for 4 h. Then ethanol was evaporated under reduced
pressure, and the residue was added ethyl acetate (60 mL).
The resulting solution was washed with H2O (3 × 50 mL),
saturated sodium bicarbonate (30 mL) and saturated sodi-
um chloride (30 mL), respectively. The organic phase was
dried with anhydrous Na2SO4, and then concentrated un-
der reduced pressure, which obtained ethyl 3-(4-
methoxyphenyl)propanoate 4 (2.99 g, yield 96%) without
1
purification. H NMR (300 MHz, CDCl3): δ = 7.12 (d, J = 8.6
Hz, 2 H), 6.83 (d, J = 8.7 Hz, 2 H), 4.12 (q, J = 7.1 Hz, 2 H),
3.78 (s, 3 H), 2.89 (d, J = 7.8 Hz, 2 H), 2.58 (d, J = 7.8 Hz, 2
H), 1.23 (t, J = 7.2 Hz, 3 H). MS (ESI): m/z = 209.13 [M+H]+.
Compound 5: To
a
solution of ethyl 3-(4-
methoxyphenyl) propanoate 4 (2.91 g, 14.0 mmol) and
TMSCF3 (3.1 mL, 21.0 mmol) in tetrahydrofuran (THF, 10
mL) was added CsF (21.2 mg, 0.14 mmol). The mixture was
stirred at room temperature under nitrogen atmosphere
for 18 h and then CH2Cl2 (30 mL) was added to the result-
ing solution, the mixture was treated with water (3 × 10
mL) and saturated sodium chloride (10 mL) respectively.
The organic phase was dried with anhydrous Na2SO4, and
then was concentrated under reduced pressure. The resi-
due was purified by silica gel column chromatography
(ethyl acetate/hexane, 5%) to afford the desired product
(2-ethoxy-1,1,1-trifluoro-4-(4-methoxyphenyl) butan-2-
yl)trimethylsilane 5. (4.02 g, yield 86%). 1H NMR (300
MHz, CDCl3): δ = 7.14 (d, J = 8.2 Hz, 2 H), 6.86 (d, J = 8.0 Hz,
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Analytical Chemistry
(d, J = 14.8 Hz, 1H), 7.51-7.57 (m, 2H), 7.45-7.39 (m, 3H),
6.87-6.82 (m, 2H), 6.51 (d, J = 14.8 Hz, 1H), 4.37 (t, J = 7.2
Hz, 2H), 2.91 (t, J = 7.2 Hz, 2H), 2.78-2.72 (m, 4H), 2.11-
2.05 (m, 2H), 1.99-1.92 (m, 4H), 1.81 (s, 6H) ppm; ESI-MS,
m/z: 506.38 [M]+.
6-week-old female nude mice, 2 million 4T1 cells suspend-
ed in 1 mL DMEM (containing 10% fetal bovine serum, 1%
penstrep, 100 U/mL penicillin, and 100 μg/mL streptomy-
cin) were injected subcutaneously near the armpits of the
mouse. Tumors were grown until a single aspect was ~7
mm (approximately 10−15 days) before used for ꢀluores-
cence and PA imaging.
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CySO3CF3: To a stirred solution of BTC (17.8 mg, 0.06
mmol) dissolved in dichloromethane (1 ml) and cooled to
0 °C, was added drop-wise a solution of the compound 7
(17.4 mg, 0.08 mmol) in dichloromethane (2.0 ml) and
then triethylamine (8.0 mg, 0.08 mol) was added. During
addition, the temperature was kept at 0 °C. After the addi-
tion was completed, the mixture was allowed slowly to rise
to room temperature and stirring was continued for 2h.
Then a solution of CySO3OH (30 mg, 0.06 mmol) in di-
chloromethane (2 ml) was added to the above mixture and
stirred 30 min. The resulting mixture was concentrated
under vacuum, which was purified by silica gel column
chromatography (ethyl acetate/hexane, 10%) to obtain
CySO3CF3 (14.0 mg, yield 31%). 1H NMR (300 MHz,
CD3OD): δ = 8.64 (d, J = 19.6 Hz, 1H), 7.52-7.39 (m, 5H),
7.42-7.35 (m, 5H), 6.92 (d, J = 7.2 Hz, 1H), 6.89 (s, 1H), 6.43
(d, J = 19.6 Hz, 1H), 4.27 (t, J = 8.8 Hz, 2H), 3.25-3.05 (m,
4H), 2.90 (t, J = 9.2 Hz, 2H), 2.73 (t, J = 8.0 Hz, 2H), 2.68 (t, J
= 8.2 Hz, 2H), 2.11-1.85 (m, 6H), 1.77 (s, 6H). 19F NMR (300
M, CD3OD): = -79.28. MS (ESI): m/z = 750.49 [M]+.
In Vivo Fluorescence Imaging. Nude mice bearing 4T1
tumors were injected with CySO3CF3 (50 μM in 100 μL sa-
line) (n = 3) through the tail vein using a microsyringe.
Fluorescence imaging were performed on the IVIS spec-
trum imaging system at designated time points after
CySO3CF3 administration (ex: 675 ± 10 nm, em: 720 ± 10
nm). Images quantitation was performed using Living Im-
age software for IVIS imaging system.
In Vivo Photoacoustic Imaging. After the nude mice
were anesthetized using 2% isoflurane in oxygen,
CySO3CF3 (50 μM in 100 μL saline) (n = 3) was systemati-
cally injected through the tail vein using a microsyringe.
Multispectral Optoacoustic Tomography scanner was used
to acquire the PA images at 680 nm after systematical ad-
ministration of CySO3CF3. In vivo PA spectra with excitation
light from 680 to 850 nm were acquired from the tumor
regions after 3 h systemic administration of CySO3CF3.
Data Analysis. The fluorescence data were measured by
region of interest (ROI) analysis using Living Image 4.0
Software. In vivo and ex vivo PA intensities were quantified
with ROI analysis using postprocessing software
ViewMSOT. Results were expressed as the mean ± stand-
ard deviations (SD) unless otherwise stated.
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Cell Culture and Cytotoxicity Test. HeLa (Human Cer-
vical Adenocarcinoma Epithelial Cells), RAW 264.7 (Mouse
Leukaemic Monocyte Macrophage Cell Line) and NIH 3T3
(Mouse Embryonic Fibroblasts) cells were purchased from
the American Type Culture Collection (ATCC). These three
kinds of the cells were cultured in DMEM (Dulbecco’s Mod-
ified Eagle Medium) supplemented with heat-inactivated
fetal bovine serum (10%) in a humidified environment
containing 5% CO2 and 95% air at 37 °C, respectively. Cells
were seeded in 96 well plates (1×104 cells/well) for 24 h,
and then CySO3CF3 (final concentration 0, 5, 10, 20, 35, 50,
and 80 μmol/L) was added to the cell culture medium. The
corresponding cells were incubated with or without (con-
trol) CySO3CF3 for 24 h, followed by the addition of MTS
(100 μL, 0.1 mg/mL) for 4 h. The absorbance of MTS at 490
nm was measured by using a microplate reader. The cyto-
toxic effect (VR) of CySO3CF3 were assessed using the fol-
lowing equation: VR = A/A0 × 100%, where A and A0 are
the absorbance of the experimental group and control
group, respectively. The assays were performed in six sets
for each concentration.
Cell Imaging. After seeded RAW 264.7 cells reached
70−90% conꢀluency, the cells were washed three times
using prewarmed PBS buffer (10 mM, pH 7.4). Then the
cells were incubated with CySO3CF3 in an atmosphere of
5% CO2 and 95% humidified air at 37 °C for 30 min. After
that the cells were stained with 4',6-diamidino-2-
phenylindole (DAPI) (NucBlue Live ReadyProbes Reagent,
Thermo Fisher) for the nuclei as protocols. Fluorescence
images of the live cells were taken with the wide-field fluo-
rescence microscope Leica DMi8 (Leica, Germany)
equipped with scientific CMOS (sCOMS, Hamamatsu, Ja-
pan). Excitation/emission wavelengths were 405/490 ± 20
nm for DAPI, and 680/710 ± 20 nm for CySO3CF3.
RESULTS AND DISCUSSION
Scheme 1b describes the synthesis route of CySO3CF3.
(Z)-2-chloro-3-(hydroxymethylene)cyclohex-1-
enecarbaldehyde 1 was first reacted with 2,3,3-trimethyl-
1-(4-sulfobutyl)-3H-indol-1-ium 2 to afford compound
CySO3Cl, followed by reacted with resorcinol in the exist-
ence of K2CO3 to obtain CySO3OH directly for the following
reaction. The other component, the esterified ethyl 3-(4-
methoxyphenyl) propanoate was firstly attacked by (Tri-
fluoromethyl)trimethylsilane (TMSCF3) and then eliminat-
ed to form compound 6, which was further treated with
BBr3 to afford compound 7. Finally, the oxygen atom of
compound CySO3OH was caged with compound 7 using
phosgene to afford CySO3CF3. The chemical structure of
CySO3CF3 was characterized by proton nuclear magnetic
resonance (1H NMR) and electrospray ionization-mass
spectrometry (ESI-MS), as shown in the Experimental Sec-
tion and in the Supporting Information.
Tumor Mouse Model. All animal experiments were per-
formed under protocols approved by the Laboratory Ani-
mal Center of Soochow University. To establish tumors in
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Analytical Chemistry
fold higher than that at the initial state (0.051). In addition,
Page 4 of 8
a
CF3
O
O
1
2
3
4
5
6
7
8
the fluorescence at 712 nm dramatically enhanced upon
addition of ONOOˉ (Figure S5, Supporting Information),
which was ~59-fold higher relative to its initial state at the
saturation point. Such absorption and fluorescent respons-
es confirmed the ONOOˉ-induced conversion as proposed
in Scheme 1. Both absorption and fluorescent signals had
good linearity with the concentration of ONOOˉ (Figure 1b
and Figure S6, Supporting Information), showing the limits
of detection of 115 and 53 nM, respectively.
O
O
Caging moiety
NIR Dye
O
ONOO־
N
pH = 7.4
Dioxirane
Intermediate
Enhancement of
waterꢀsolubility
SO3H
CySO3CF3
NIRF/PA “off”
NIRF
680 nm
hν
712 nm
9
+
+
CO2
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Spiroꢀquinone
PA
hν
675 nm
680 nm
CySO3OH
NIRF/PA “on”
b
Scheme 1. Sensing mechanism and synthetic scheme. a)
Schematic illustration of peroxynitrite-induced oxidation
of a trifluoromethyl ketone moiety via a dioxirane inter-
mediate. b) Syntheses of CySO3CF3. Reagents and condi-
tions: (i) H2SO4 (con.), reflux, 4 h, EtOH, 96%; (ii) TMSCF3,
CsF, THF, 18 h, 86%; (iii) tetrabutylammonium fluoride
(TBAF), THF, 5 h, then 4 M HCl, 3 h, 88%; (iv) BBr3, -78 °C,
room temperature, 12 h, CH2Cl2, 82%; (v) sodium acetate,
acetic anhydride, 90 °C, 1 h, 73%; (vi) resorcinol, K2CO3,
DMF, 50 °C, 4 h, 79%; (vii) 7, triphosgene, Et3N, 0 °C, 2 h,
anhydrous CH2Cl2, 31%.
Figure 1. In vitro characterization. a) UV–vis absorption (dash
line) and fluorescence (solid line) spectra of CySO3CF3 (5 μM)
in the absence or presence of ONOOˉ (15 μM) for 3 min in PBS
buffer (10 mM, pH 7.4). Excitation: 675 nm. b) Fluorescence
intensities of CySO3CF3 (5 μM) as a function of the concentra-
tion of ONOOˉ in PBS buffer (10 mM, pH 7.4) (relative stand-
ard deviation δ = 1.16E5, n = 3; slope, 6.27E6). c) UV–vis ab-
sorption of CySO3CF3 (5 μM) after incubation with indicated
ROS for 3 min. Inset: white light images of CySO3CF3 (5 μM) in
the absence or presence of ROS for 3 min in PBS buffer (10
mM, pH 7.4). d) Ab686 of CySO3CF3 (5 μM) as a function of time
in the presence of ONOOˉ (15 μM) in PBS buffer (10 mM, pH
7.4). e) PA spectra of CySO3CF3 (5 μM) before and after addi-
tion of ONOOˉ in PBS buffer (10 mM, pH 7.4). f) PA686 of
CySO3CF3 (5 μM) as a function of the concentration of ONOOˉ
in PBS buffer (10 mM, pH 7.4) (relative standard deviation δ =
0.028, n = 3; slope, 0.58). The error bars represent standard
deviations of three separate measurements.
To validate ONOOˉ-induced oxidation of CySO3CF3, high
performance liquid chromatography (HPLC) analysis was
conducted (Figure S1, Supporting Information). The data
showed that CySO3CF3 (HPLC retention time, TR = 8.7 min)
was converted to CySO3OH (TR = 4.6 min). The ESI spec-
trometry analysis (Figure S2, Supporting Information) of
the probe treated with ONOOˉ further confirmed the for-
mation of the expected CySO3OH (m/z 506.38) and the
desired spiro-quinone (m/z 234.05). The reaction mecha-
nism was confirmed by the NMR spectra using trifluoro-(4-
methoxyphenyl)butan-2-one as a model compound (Figure
S3, Supporting Information).
To study the optical response along with ONOOˉ-induced
cleavage, the spectroscopic properties of CySO3CF3 under
physiological conditions (1×PBS: phosphate buffer, pH 7.4)
were measured before and after addition ONOOˉ. CySO3CF3
intrinsically had the absorption maximum at 602 nm (Fig-
ure 1a); upon addition of ONOOˉ, this peak gradually de-
creased, while a new peak at 686 nm appeared (Figure S4,
Supporting Information). At the saturation point, the ab-
sorbance at 686 nm for CySO3CF3 was 0.48, which was 9.4-
The specificity of CySO3CF3 towards ONOOˉ was tested
•
against other RONS (O2 ˉ, ClOˉ, 1O2, •OH, H2O2, NO, NO2ˉ)
2
and sulphur compounds (S2ˉ, GSH, Cys, Hcy, HSO3ˉ, SO3 ˉ).
The results showed that other RONSs and sulphur com-
pounds could not induce any absorption and fluorescent
changes for the probe (Figure 1c and Figure S7, Supporting
Information). Thus, CySO3CF3 could only be activated by
ONOOˉ, changing their solution colors from purple to cyan
(the inset of Figure 1c). To determine the activation speed
of the probe, the reaction kinetics of CySO3CF3 with ONOOˉ
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was studied by time-course absorption measurements
(Figure 1d and Figure S8, Supporting Information). The
corresponding pseudo-first-order rate constant (k) for the
present ONOOˉ reaction was determined to be 1.49 × 103
M-1s-1. Thus, CySO3CF3 should be able to specifically detect
ONOOˉ in real time.
contrast, the cells pretreated with N-acetylcysteine (NAC),
an effective RONS scavenger, only showed weak fluores-
cence signals. Moreover, the cytotoxicity of CySO3CF3 was
investigated by MTS assay. As shown in Figure 2c and Fig-
ure S11 (Supporting Information), the cells including HeLa,
RAW 264.7 and NIH 3T3 kept high survival rate upon ex-
posure to CySO3CF3 with concentrations below 50 μM.
These results illustrated that CySO3CF3 could detect en-
dogenous ONOOˉ in living cells and thus had a potential for
imaging application.
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The PA sensing ability of CySO3CF3 was also tested under
physiological conditions. After activation by ONOOˉ, the PA
band ranging from 680 to 750 nm appeared (Figure 1e),
consistent with the absorption response. At the saturation
point, the PA intensity at 680 nm (PA680) was 5.1-fold
higher than that at the initial state (1.73). In contrast, in
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•
the present of other ROS (O2 ˉ, ClOˉ, 1O2, •OH, and H2O2), no
obvious changes were observed in the PA spectra (Figure
S9, Supporting Information). Furthermore, PA680 was line-
arly correlated to the concentration of ONOOˉ with the
limit of detection of 145 nM, which was closed to that for
absorption response. As the concentration of ONOOˉ is in
the range of 50-100 μM per minute in living systems,45
CySO3CF3 should be ideal for sensing ONOOˉ in living cells
and mice.
The low pH (pH 6.8) of tumor tissues was caused by the
highly acidic metabolites, e.g. lactic acid produced by an-
aerobic glycolysis in the absence of oxygen during tumor
growth. To validate the feasibility of probe activation in the
acidic tumor environment, the absorption responses of
CySO3CF3 toward ONOOˉ was tested at pH ranging from 9.0
to 5.0. The probe presented weakened absorption signals
at 686 nm especially when pH decreased from 9.0 to 6.0
(Figure S10, Supporting Information). This could be as-
cribed to the protonation of the phenoxide product to its
conjugate acid at low pH as CySO3OH has pKa = 7.11. The
maximum absorption signals at 600 nm of the conjugate
acid were the same as the initially unactivated state. Note
that at pH = 6.8, in the present of ONOOˉ, the absorption
value at 686 nm was ≈ 0.290, 3.6-fold higher when com-
pared with its original state (0.081). Hence, despite its pH
response, CySO3CF3 could be applied to fluorescence and
PA imaging of ONOOˉ in acidic tumor microenvironments.
To validate the reactivity of CySO3CF3 toward ONOOˉ in
cells, Fluorescence imaging was conducted on murine
macrophage (RAW 264.7) cells (Figure 2a). After incuba-
tion with CySO3CF3 for 30 min, the cells were washed and
Figure 2. Imaging ONOOˉ in cells. a) Confocal microscope flu-
orescence images of live murine macrophages (RAW 264.7).
Cells successively treated with or without NAC (30 min) and
then CySO3CF3 (30 min), [CySO3CF3] = 10 μM; [NAC] = 1 mM.
b) Quantification of fluorescence intensities of RAW 264.7
cells incubated with CySO3CF3 and other reagents correspond-
ing to Figure 2a. The error bars represent the standard devia-
tion from three separate measurements. c) MTS assay for the
survival rate of RAW 264.7 cells treated with various concen-
trations (0, 5, 10, 25, 35, 50, 80 μM) of CySO3CF3 for 24 h. Er-
ror bars represent the standard deviations of 6 trials.
stained
with
nuclei
indicator,
4',6-diamidino-2-
phenylindole (DAPI), which was depicted in blue colors.
Images showed a remarkable red fluorescence in the cyto-
plasm, which implied that the probe underwent a cascade
oxidation-elimination reaction with intracellular ONOOˉ. In
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Figure 3. In vivo NIRF and PA imaging of ONOOˉ using CySO3CF3. a) Illustration of the mechanism for NIRF and PA imaging of
ONOOˉ in tumor using CySO3CF3. CySO3CF3 first accumulates into tumor and then is activated by ONOOˉ through cascade oxidation-
elimination reactions, eventually resulting in enhanced NIRF/PA signals. b) Real-time fluorescence imaging of tumor bearing
mouse after systemic administration of CySO3CF3 (50 μM in 100 μL saline) at 0, 3, and 4 h. c) Fluorescence intensity in tumor area
as a function of post-injection time for CySO3CF3 intravenous injected mice. d) Representative PA maximum imaging projection
(MIP) of tumor from a systemic administration of a living mouse at 0, 3, and 4 h post-injection of CySO3CF3 (50 μM in 100 μL sa-
line). e) In vivo real-time PA spectra extracted from the tumors in living mice after systemic administration of CySO3CF3 via intra-
venous injection for 3 h. f) PA intensity in tumor area as a function of post-injection time for CySO3CF3 intravenous injection. Error
bars represent standard deviations of three separate measurements (n = 3).
The feasibility of using CySO3CF3 as a NIRF/PA probe to
monitor ONOOˉ in the subcutaneous 4T1 xenograft tumor
of living mice was tested (Figure 3). After systemic admin-
istration of CySO3CF3 into tumor-bearing living mice
through tail vein, both NIRF and PA images were longitu-
dinally recorded and quantified. The NIRF and PA signals
gradually enhanced for CySO3CF3-treated mice and
reached its maxima 3 h post-injection (Figure 3b, 3d). This
showed that the probe was accumulated in the tumor,
which was also confirmed by ex vivo data (Figure S12,
Supporting Information). At t =3 h, the PA and fluorescence
intensities of CySO3CF3-treated tumor were respectively
2.1 and 5.3-fold higher than the background signals of tu-
mor (Figure 3c). The higher signal-to-noise ratio of fluo-
rescence imaging relative to PA imaging has been reported
before19,46-51, which should be mainly ascribed to the better
sensitivity of fluorescence imaging and higher tissue back-
ground for PA background at 680 nm from hemoglobin.
The real-time in vivo PA spectra extracted from the tumor
regions at t = 3 h showed a strong signal at 680 nm (Figure
3e); Moreover, the PA spectral profile resembled the solu-
tion spectrum of activated CySO3CF3. These results thus
not only confirmed the selective activation of CySO3CF3 by
ONOOˉ in tumor but also highlighted its ability for both
NIRF and PA imaging.
nanoparticles.32,43,44 CySO3CF3 also showed higher NIRF
(59-fold) and PA (5.1-fold) enhancement and fast activa-
tion kinetics in response to ONOOˉ. With its zwitterionic
structure, CySO3CF3 has good water-solubility and can be
systemically injected into living mice for NIRF and PA dual-
modal imaging of ONOOˉ. To the best of our knowledge,
this work represents the first smart molecular probe that
detects endogenous ONOOˉ in living system with NIRF and
PA dual-modal imaging.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS
Publications
website.
at
DOI:
10.1021/acs.analchem.xxxxxxx.
Additional information as noted in the text (Figures S1−S13);
Synthesis, characteristic (1H NMR and MS spectra), UV-Vis
absorption, fluorescence and photoacoustic spectra. (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail: kypu@ntu.edu.sg
*E-mail: jfzeng@suda.edu.cn
ORCID
CONCLUSIONS
Jianjian Zhang: 0000-0002-5220-1280
Kanyi Pu: 0000-0002-8064-6009
In summary, we synthesized a water-soluble small-
molecular probe (CySO3CF3) that can be specifically acti-
vated by endogenously overexpressed ONOOˉ to turn on
both NIRF and PA signals. CySO3CF3 is different from the
most reported RONS PA probes which are
Notes
The authors declare no competing financial interest.
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(25) Razansky, D.; Distel, M.; Vinegoni, C.; Ma, R.; Perrimon, N.;
ACKNOWLEDGMENT
Köster, R. W.; Ntziachristos, V. Nat. Photonics 2009, 3, 412-417.
(26) Filonov, G. S.; Piatkevich, K. D.; Ting, L.-M.; Zhang, J.; Kim,
K.; Verkhusha, V. V. Nat. Biotechnol. 2011, 29, 757-761.
(27) Lovell, J. F.; Jin, C. S.; Huynh, E.; Jin, H.; Kim, C.; Rubinstein,
J. L.; Chan, W. C.; Cao, W.; Wang, L. V.; Zheng, G. Nat. Mater. 2011,
10, 324-332.
1
2
3
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K.P. thanks Nanyang Technological University (Start-Up grant:
NTUSUG: M4081627.120) and Singapore Ministry of Educa-
tion (Academic Research Fund Tier 1: RG133/15 M4011559
and 2015-T1-002-091; and Tier 2 MOE2016-T2-1-098) for the
financial support.
5
(28) Muhanna, N.; Jin, C. S.; Huynh, E.; Chan, H.; Qiu, Y.; Jiang,
W.; Cui, L.; Burgess, L.; Akens, M. K.; Chen, J.; Irish, J.; Zheng, G.
Theranostics 2015, 5, 1428-1443.
(29) Liu, Y.; Kang, N.; Lv, J.; Zhou, Z.; Zhao, Q.; Ma, L.; Chen, Z.;
Ren, L.; Nie, L. Adv. Mater. 2016, 28, 6411-6419.
(30) Lv, R.; Wang, D.; Xiao, L.; Chen, G.; Xia, J.; Prasad, P. N. Sci.
Rep. 2017, 7, 15753.
(31) Bao, Y.-W.; Hua, X.-W.; Li, Y.-H.; Jia, H.-R.; Wu, F.-G. ACS
Appl. Mater. Inter. 2018, 10, 1544-1555.
(32) Qiao, Y.; Gumin, J.; MacLellan, C. J.; Gao, F.; Bouchard, R.;
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