Peroxynitrite Activatable NIR-II Fluorescent Molecular Probe for Drug-Induced Hepatotoxicity Monitoring

Article abstract of DOI:10.1021/acs.analchem.9b00317

Drug-induced hepatotoxicity represents an important challenge for safety in drug development. The production of peroxynitrite (ONOO^-) is proposed as an early sign in the progression of drug-induced hepatotoxicity. Currently, reported ONOO^- probes mainly emit in the visible range or the first NIR window, which have limited in vivo biosensing application due to the autofluorescence and photon scattering. Herein, we developed a peroxynitrite activatable second near-infrared window (NIR-II) molecular probe for drug-induced hepatotoxicity monitoring, based on the fusion of an NIR-II fluorescence turn-on benzothiopyrylium cyanines skeleton and the phenyl borate. In the presence of ONOO^-, the probe IRBTP-B can turn on its NIR-II fluorescence by yielding its fluorophore IRBTP-O and display good linear response to ONOO^-. Tissue phantom study confirmed reliable activated signals could be acquired at a penetration depth up to 5 mm. Using this probe, we disclose the upregulation of ONOO^- in a preclinical drug-induced liver injury model and the remediation with N-acetyl cysteine (NAC) in vivo. We expect that this strategy will serve as a general method for the development of an activatable NIR-II probe based on the hydroxyl functionalized reactive sites by analyte-specific triggering.

Full text of DOI:10.1021/acs.analchem.9b00317

Subscriber access provided by UNIV OF TEXAS DALLAS
Article
Peroxynitrite Activatable NIR-II Fluorescent Molecular
Probe for Drug-induced Hepatotoxicity Monitoring
Dandan Li, Shangfeng Wang, Zuhai Lei, Caixia Sun, Ahmed Mohamed
El-Toni, Mansour Saleh Alhoshan, Yong Fan, and Fan Zhang
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00317 • Publication Date (Web): 27 Feb 2019
Downloaded from http://pubs.acs.org on February 27, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 25
Analytical Chemistry
1
2
3
4
5
6
7
8
9
Peroxynitrite Activatable NIR-II Fluorescent
Molecular Probe for Drug-induced Hepatotoxicity
Monitoring
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Dandan Li1, Shangfeng Wang1, Zuhai Lei1, Caixia Sun1, Ahmed Mohamed El-Toni2, Mansour Saleh
Alhoshan3, Yong Fan1, Fan Zhang1*
1. Department of Chemistry, Shanghai Key Laboratory of Molecular Catalysis and Innovative
Materials, State Key Laboratory of Molecular Engineering of Polymers and iChem, Fudan
University, Shanghai 200433, P. R. China.
2. King Abdullah Institute for Nanotechnology, King Saud University, Riyadh 11451, Saudi Arabia.
3. Department of Chemical Engineering, King Saud University, Riyadh 11421, Saudi Arabia.
Tel: (+86)21-51630322; Fax: (+86)21-5163-0307
E-mail: zhang_fan@fudan.edu.cn.
ACS Paragon Plus Environment

Analytical Chemistry
Page 2 of 25
1
2
3
4
ABSTRACT
5
6
7
8
Drug-induced hepatotoxicity represents an important challenge for safety in drug development. The
production of peroxynitrite (ONOO-) is proposed as an early sign in the progression of drug-induced
hepatotoxicity. Currently, reported ONOO- probes mainly emit in visible range or the first NIR
window, which have limited in vivo biosensing application due to the autofluorescence and photon
scattering. Herein, we developed a peroxynitrite activatable second near-infrared window (NIR-II)
molecular probe for drug-induced hepatotoxicity monitoring, based on the fusion of an NIR-II
fluorescence turn-on benzothiopyrylium cyanines skeleton and the phenyl borate. In the presence of
ONOO-, the probe IRBTP-B can turn on its NIR-II fluorescence by yielding its fluorophore IRBTP-O
and display good linear response to ONOO-. Tissue phantom study confirmed reliable activated
signals could be acquired at a penetration depth up to 5 mm. Using this probe, we disclose the
upregulation of ONOO- in preclinical drug-induced liver injury model and the remediation with N-
acetyl cysteine (NAC) in vivo. We expect that this strategy will serve as a general method for the
development of activatable NIR-II probe based on the hydroxyl functionalized reactive sites by
analyte-specific triggering.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
KEYWORDS
NIR-II probe, fluorescence, activatable, peroxynitrite, hepatotoxicity
INTRODUCTION
Drug toxicity is not only an important safety evaluation target in preclinical research and
development of candidate drugs, but also a major cause for post-marketing drug withdrawals.1
Metabolic organs, especially liver, are the major site to convert drugs to various metabolites.2 In the
liver, drugs are enzymatically biotransformed into polar, water-soluble, and excretable metabolites
accompanied by the generation of reactive radicals or reactive electrophiles, such as reactive oxygen
species (ROS) and reactive nitrogen species (RNS), to affect normal liver physiology, which is the
ACS Paragon Plus Environment

Page 3 of 25
Analytical Chemistry
1
2
3
4
5
6
primary cause of drug-induced hepatotoxicity.3,4 The production of RNS, particularly peroxynitrite
(ONOO-), is proposed as an early sign in the progression of drug-induced hepatotoxicity.5
7
8
9
For the ONOO- detection, fluorescence imaging is a promising tool owing to the advantages of
being non-invasive, good sensitivity and selectivity.6-9 Up to now, quite a few fluorescence probes
for ONOO- detection in visible range and the first near-infrared window (NIR-I, 750-900 nm) were
reported.10-25 However, the fundamental barriers to bio-tissue fluorescence imaging in those windows
is the photon penetration depth which is limited by the wavelength-dependent behavior of scattering
and absorption.26 Recently, fluorescence imaging in the second near-infrared window (NIR-II, 900-
1700 nm) has come into the spotlight because it can benefit from the reduced autofluorescence,
absorption and scattering to reach deeper penetration depths, resulting in high image quality and
diffraction-limited resolution.27-30 Till now, the majority of NIR-II contrast agents for in vivo imaging
are comprised of nanomaterials such as carbon-nanotube31, quantum-dot32 and rare-earth doped
nanoparticles33,34. Owing to unknown long-term toxicity concerns, it is necessary to design organic
small-molecule NIR-II fluorophores to facilitate FDA approval and clinical translation. So far, there
are only a handful of organic molecules with fluorescence NIR-II emission.35-42 However, an intrinsic
issue of above NIR-II contrast agents is their always-on fluorescence behavior, which means that the
fluorescent signals have no response to specific target. This is one of the primary reasons for
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
hampering the in vivo biosensing applications43,44
.
Herein we developed ONOO- activatable NIR-II fluorescent probe (IRBTP-B) based on the
incorporation of phenyl borate group and benzothiopyrylium cyanines skeleton. In the presence of
ONOO-, the masking group phenyl borate can be removed to turn on the NIR-II fluorescence. Tissue
phantom study confirmed reliable activatable signal was acquired at a penetration depth up to 5 mm.
Using an acetaminophen (APAP)-induced liver injury model, we successfully realized the real-time
monitoring of preclinical drug-induced liver injury.
ACS Paragon Plus Environment

Analytical Chemistry
Page 4 of 25
1
2
3
4
EXPERIMENTAL SECTION
5
6
7
8
Synthesis of 7-bromo-2-(4-methoxyphenyl)-4H-thiochromen-4-one (1). A mixture of
polyphosphoric acid (PPA, 22 g), 3-bromothiophenol (2 g, 10.6 mmol) and ethyl 3-(4-
methoxyphenyl)-3-oxopropanoate (2.6 g, 11.66 mmol) was stirred at 95 oC for 2 h. After cooling to
room temperature, ice water was added to quench the reaction and extracted with DCM (100 mL ×
3). The combined organic extracts were dried with Na2SO4, filtered, and evaporated. The crude
product was further purified by a flash column chromatography [silica gel, PE/DCM = 1/1 to 0/1,
v/v] to give 1 (yield: 2.6 g , 70%). 1H NMR (400 MHz, CDCl3) δ 8.53 (d, J = 8.0 Hz, 1H), 7.86 (d, J
= 8.0 Hz, 1H ), 7.69 (d, J = 8.0 Hz, 2H), 7.43 (t, J = 8.0 Hz, 1H), 7.24 (d, J = 16.0 Hz, 1H), 7.02 (d,
J = 8.0 Hz, 2H), 3.88 (s, 3H).
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Synthesis of 7-(diethylamino)-2-(4-methoxyphenyl)-4H-thiochromen-4-one (2). A tube was
charged with compound 1 (694 mg, 2 mmol), Pd2(dba)3 (45.8 mg, 0.05 mmol), DavePhos (19.7 mg,
0.05 mmol), and Cs2CO3 (1.63 g, 5 mmol, 5 mmol). The tube was sealed and evacuated/backfilled
with nitrogen (3×). Anhydrous dioxane (10 mL) was added, following the addition of dibutylamine
(1.7 mL, 10 mmol). The reaction was stirred at 100 °C for 18 h. After cooling to room temperature,
the mixture was filtered, and the filtrate was evaporated. The crude product was further purified by
silica gel column chromatography (EA/DCM = 1/10, v/v), obtaining product 2 (685 mg, 86.6 %).1H
NMR (400 MHz, CDCl3): δ 8.32 (d, J=12 Hz, 1H),7.62 (d, J=12 Hz, 2H), 7.09 (s, 1H), 6.98 (d, J=12
Hz, 2H), 6.84-6.81 (dd, J1=4 Hz, J2=12 Hz, 1H), 6.64 (s, 1H), 3.87 (s, 3H), 3.36 (t, J=8 Hz,4H), 1.65-
1.58 (m, 4H), 1.44-1.35 (m, 4H), 0.99 (t, J=8 Hz, 6H). 13C NMR (100 MHz, CDCl3): δ 180.0, 161.4,
150.5, 149.9, 140.3, 129.8, 129.2, 128.1, 121.8, 119.4, 114.4, 112.9, 104.8, 55.4, 50.7, 29.2, 20.2,
13.9. Maldi-Tof/Tof-MS: calcd for C20H22NO2SH+ [M+H]+, 396.1992; Found, 396.3009. [M+H]+..
Synthesis of 7-(diethylamino)-2-(4-methoxyphenyl)-4-methylthiochromenylium perchlorate
(3). Compound 2 (395 mg, 1 mmol) was dissolved in anhydrous THF (10 mL) in a flame dried flask
ACS Paragon Plus Environment

Page 5 of 25
Analytical Chemistry
1
2
3
4
5
6
7
8
9
under nitrogen. 1.0 M CH3MgBr (3.6 mL) was added dropwise to this solution and allowed to stir at
room temperature for 2 h. The solution was poured into 10% aqueous HClO4 (10 mL) and extracted
with DCM (25 mL × 3). The combined organic extracts were dried with Na2SO4, filtered, and
evaporated to give 3 (yield: 480 mg, 97.3 %) without further purification. 1H NMR (400 MHz, CDCl3)
δ 8.27 (d, J = 8Hz, 1H), 7.91 (s, 3H), 7.38 (d, J = 8Hz, 1H), 7.32 (s, 1H), 6.99 (d, J = 8Hz, 2H), 3.84
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
13
(s, 3H), 3.55 (s, 4H), 2.90 (s, 3H), 1.67 (s, 4H), 1.46 (s, 4H), 1.00 (s, 6H); C NMR (100 MHz,
CDCl3) δ 164.1, 162.8, 159.9, 151.8, 145.5, 132.0, 129.7, 126.5, 124.4, 121.5, 119.6, 115.5, 105.8,
55.8, 51.7, 29.3, 23.0, 20.0, 13.8; Maldi-Tof/Tof-MS: calcd for C25H32NOS+ [M]+, 394.2199; Found,
394.2234. [M]+.
Synthesis of IRBTP-O. A mixture of 4-hydroxyisophthalaldehyde (50 mg, 0.33 mmol) and
compound 3 (329 mg, 0.66 mmol) was dissolved in Toluene/n-butanol (3 mL/3 mL). The reaction
o
mixture stirred for overnight at 100 C under an Ar atmosphere. After completion, the reaction
mixture was concentrated by evaporation under reduced pressure. The crude product was purified by
preparative RP-TLC (DCM/MeOH = 10:1, v/v) to give the desired product (yield: 105 mg, 35%). 1H
NMR (400 MHz, DMSO-d6) δ 8.97-8.76 (m, 2H), 8.66-8.17 (m, 5H), 8.05 (t, J = 8Hz, 2H), 7.95-
7.55 (m, 4H), 7.45-7.32 (m, 2H), 7.27-7.10 (m, 5H), 6.97-6.81 (m, 1H), 6.57 (d, J = 8 Hz, 1H), 6.50
(d, J = 8 Hz, 1H), 3.89 (s, 3H), 3.83 (q, J = 8 Hz, 2H), 3.74-3.67 (m, 2H), 3.62 (s, 2H), 3.53 (s, 2H),
13
3.19 (s, 3H), 1.60 (s, 4H), 1.41-1.19 (m, 12H), 0.93 (q, J = 8 Hz, 8H), 0.87 (t, J = 8 Hz, 4H). C
NMR (100 MHz, DMSO-d6) δ 164.0, 163.6, 159.9, 158.2, 158.0, 154.3, 154.2, 152.0, 147.3, 145.6,
145.3, 145.0, 134.1, 132.3, 132.2, 130.00, 127.7, 126.9, 119.4, 119.2, 119.1, 118.7, 118.5, 115.9,
115.8, 114.20, 107.2, 56.3, 55.5, 50.9, 50.0, 29.4, 20.1, 19.9, 14.2. HRMS (ESI) m/z calcd for
+
C58H65N2O3S2 [M]+, 901.4431; Found, 901.4459.
Synthesis of 2-(4-(iodomethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (4). (4-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methanol (2 g, 8.5 mmol) was dissolved in 60
ACS Paragon Plus Environment

Analytical Chemistry
Page 6 of 25
1
2
3
4
5
6
7
8
9
mL acetonitrile and cooled to 0 oC. NaI (3.84 g, 25.63 mmol) was added, and then TMSCl (3.25 mL,
25.63 mmol) was added dropwise. The reaction mixture stirred for 30 mins at room temperature, then
diluted with EtOAc, and washed with water, then brine, dried over Na2SO4, and evaporated under
reduced pressure. The crude product was purified by column chromatography on silica gel
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
(EtOAc/PE = 5:95, v/v) to give compound 4 as a white solid (yield:2.44 g, 83 %). H NMR (400
MHz, CDCl3) δ7.75 (d, J = 6Hz, 2H), 7.40 (d, J = 6Hz, 2H), 4.46 (s, 2H), 1.35 (s, 12H).
Synthesis of Compound 5. 4-hydroxyisophthalaldehyde (200 mg, 1.33 mmol) was dissolved in 4
mL dry DMF and cooled to 0 oC. K2CO3 (405 mg, 2.93 mmol) was added and the solution stirred at
0 oC for 10 min. Then compound 4 (916 mg, 2.66 mmol) was added. The reaction mixture stirred at
room temperature for 12h. After completion, the reaction mixture diluted with Et2O and was washed
with saturated solution of NH4Cl. The organic layer was separated, washed with brine, dried over
Na2SO4, and evaporated under reduced pressure. The crude product was purified by column
chromatography on silica gel (EA/PE = 3:7, v/v) to give compound 5 (yield: 380 mg, 78%). 1H NMR
(400 MHz, CDCl3) δ10.56 (s, 1H), 9.94 (s, 1H), 8.36 (d, J = 2Hz, 1H), 8.08 (dd, J1 = 2Hz, J2 = 8Hz,
1H), 7.87 (d, J = 8Hz, 2H), 7.45 (d, J = 8Hz, 2H), 7.18 (d, J = 8Hz, 1H), 5.32 (s, 1H), 1.35 (s, 12H).
13C NMR (100 MHz, CDCl3) δ 190.4, 188.7, 165.1, 138.1, 135.7, 135.6, 132.1, 130.0, 126.7, 125.4,
113.9, 84.2, 71.2, 25.1.
Synthesis of IRBTP-B. A mixture of compound 5 (100 mg, 0.27 mmol) and compound 3 (269
mg, 0.54 mmol) was dissolved in Toluene/n-butanol (3 mL/3 mL). The reaction mixture stirred for
overnight at 100 oC under an Ar atmosphere. After completion, the reaction mixture was concentrated
by evaporation under reduced pressure. The crude product was purified by preparative RP-TLC
(DCM/MeOH = 10:1, v/v) to give the desired product (yield: 98 mg, 32% ). 1H NMR (400 MHz,
DMSO-d6) δ9.08 (d, J = 8Hz, 1H), 8.88 (s, 1H), 8.80 (d, J = 8Hz, 1H), 8.46 (d, J = 16Hz, 1H), 8.28
(s, 3H), 8.20 (d, J = 16Hz, 1H), 8.06 (d, J = 8Hz, 3H), 8.02 (d, J = 8Hz, 1H), 7.90 (d, J = 8Hz, 2H),
ACS Paragon Plus Environment

Page 7 of 25
Analytical Chemistry
1
2
3
4
5
6
7
8
9
7.80 (d, J = 8Hz, 2H), 7.60 (d, J = 8Hz, 2H), 7.51 (s, 1H), 7.46 (s, 1H), 7.25-7.15 (m, 7H), 5.28 (s,
2H), 3.90 (d, J = 8Hz, 6H), 3.35 (s, 8H), 1.50 (s, 8H), 1.34 (s, 18H), 1.22 (s, 2H), 0.92 (s, 12H). 13C
NMR (100 MHz, DMSO-d6) δ 163.7, 163.5, 159.8, 158.2, 157.6, 153.9, 153.5, 151.7, 145.5, 145.3,
144.5, 140.0, 135.2, 133.0, 132.7, 129.9, 129.6, 127.4, 127.3, 125.0, 122.3, 119.4, 119.2, 119.1, 118.9,
118.0, 115.9, 115.8, 113.9, 107.0, 106.9, 84.3, 70.6, 56.3, 50.8, 29.5, 25.4, 25.1, 19.9, 14.2. HRMS
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
+
(ESI) m/z calcd for C35.5H41.5B0.5N1O2.5S1 [M/2]+, 559.2913; Found, 559.2922.
Preparation of dye-loaded phospholipid nanomicelles: The nanomicelles were prepared using
modified film hydration technique. In a typical procedure, 100 mg of DSPE-mPEG2000 was
dissolved in 0.8 mL DMF. Then 200 μL of dye solution (IRBTP-O, IRBTP-B or D632, 5 mM in
DMF) was mixed. 9 mL of deionized water was added under sonication to make the clear
nanomicelles solution with dye concentration of 100 μM. The nanomicelle solution was further
concentrated if necessary by using a 30 K Amicon Ultra filter (Millipore Corporation) under
centrifugation at 2,000 g for 5 min.
Determination of fluorescence quantum yields: Quantum yields (Φfl) were determined in
various solvents relative to IR26 (Φfl = 0.05% in DCE)36,45-49, from plots of integrated fluorescence
intensity vs. absorbance, according to the following relationship:
푛2푠 퐾푠
(1)
Φ푓푙,푠 = Φ푓푙,푟
×
( )
푛푟2
퐾푟
where subscripts r and s denote standard and test sample, respectively, Φ is the fluorescence
quantum yield, K is the slop of the integrated fluorescence intensity vs. absorbance plot, and n is the
refractive index of the solvent. Measurements were performed with the absorbance at 808 nm of all
dye solutions ≤ 0.08 in order to maximize illumination homogeneity and optical transparency. The
ACS Paragon Plus Environment

Analytical Chemistry
Page 8 of 25
1
2
3
4
5
6
808 nm laser was used as the excitation source and the emission spectrum in the 900-1500 nm region
was acquired in fluorescence spectrometer.
7
8
9
Photostability of dyes: Photostability of dyes were tested with ICG as the reference. The
absorbance at max of the dye solution at the corresponding maximum absorbing wavelength was
adjusted to 1 for both dyes and the reference. Both solutions were subjected to irradiation by a 2.8 W
cm-2 808 nm laser. The solution temperature was maintained steady by air-cooling. The absorption
spectrum of the solution was checked every 30 seconds.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Determination of pKa values: The calculation of pKa values was performed with Origin 2017
software (OriginLab, Northhampton, MA), using the Bolzmman fitting function:
퐴1 ― 퐴2
(2)
푦 =
+ 퐴2
(푥 ― 푥 )
0
푑
푥
1 + 푒
On the basis of the calculated equation of pKa, 0 corresponds to the pKa value.
푥
Cell viability: CaOV3 cell line was provided by American Type Culture Collection (ATCC,
Manassas, VA, USA). The cytotoxicity was measured by using Cell Counting Kit-8 (CCK-8) assay.
The cells (1×104) were incubated in each well of a 96-well plate for 24 h, then incubated with IRBTP-
B and IRBTP-O micelles (dye concentration: 0, 2.5, 5, 20, 40, 50 μM) with different concentrations
for 24 h, respectively. Enzyme dehydrogenase in living cells was oxidized by this kit to orange
carapace. The quality was assessed calorimetrically by using a multi-reader (TECAN, Infinite M200,
Germany). The measurements were based on the absorbance values at 450 nm. Following formula
was used to calculate the viability of cell growth: Viability (%) = (mean absorbance value of
treatment group/mean absorbance value of control group) × 100.
ACS Paragon Plus Environment

Page 9 of 25
Analytical Chemistry
1
2
3
4
5
6
7
8
9
Animal models of drug-induced hepatotoxicity: All animal experiments were in agreement with
the guidelines of Fudan University’s Administrative panel on Laboratory Animal Care. Five weeks
old female mice were purchased from Shanghai SLAC Laboratory animal CO. Ltd. And the animal
experiments were permitted by the Shanghai Science and Technology Committee. All groups within
study contained n=3 mice. Five weeks-old female nude mice (~15 g) were fasted for 12 h to avoid
the possible food fluorescence interference and randomly divided into three groups: For the control
group, the mice were administered an intraperitoneal injection of PBS buffer (200 μL) before being
given a tail-vein injection of probe (IRBTP-B or D632, 200 μL, 1 mM. The APAP group was given
an intraperitoneal injection of APAP (300 mg/kg), and then a tail-vein injection of probe (IRBTP-B
or D632, 200 μL, 1 mM) 2 h later. The mice in the last group were pretreated with an intraperitoneal
injection of NAC (300 mg/kg) 1 h before drug administration. Then all of the mice were anesthetized
and the livers of the mouse were imaged.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Histology staining assays: Five weeks-old female nude mice (~15 g) were treated with PBS,
APAP or NAC + APAP respectively following above procedure. The mice were dissected to isolate
the livers, which were then fixed with 10% PFA for 48 h. Then the samples were dehydrated with
ethanol and embedded in paraffin before 6 µm sectioning. H&E staining samples were prepared from
the mice stained by hematoxylin and eosin under standard protocols.
RESULTS AND DISCUSSION
Design and synthesis of activatable NIR-II probe for ONOO-. Polymethine dyes are one type
of the widely employed fluorophores for NIR fluorescent sensor design, due to their high extinction
coefficients, moderate quantum yields and facile synthesis and modification.50-54 By introducing
strong electron withdrawing groups (benzothiopyrylium) at both ends of the skeleton, a novel
fluorophore is expected to emit NIR-II fluorescence (Figure 1). In addition, the turn-on fluorescence
property of the fluorophore can be obtained by the fusion of a phenol moiety with methylene chain.
ACS Paragon Plus Environment

Analytical Chemistry
Page 10 of 25
1
2
3
4
5
6
7
8
9
The incorporation of phenyl borate can actualize the responsiveness of the probe towards ONOO-.
When the phenol is protected by phenyl borate, the conjugated π-electron system is divided in half,
which results in the masking of the NIR-II fluorescence emission. In the presence of ONOO-, the
dioxaborolane group is firstly oxidized to generate intermediate A, which converts to intermediate B
after electron rearrangement. Further electron rearrangement results in generation of the fluorophore
(IRBTP-O) with entire conjugation which is expected to emit NIR-II fluorescence. Moreover, the
incorporation of various analyte triggering groups to hydroxyl should allow design of different
functional NIR-II activatable probes.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Polymethine dyes are prepared through the introduction of an activated heterocycle to a
bis(aldehyde) equivalent (Figure 1 and S1). Compound 1 has been synthesized by condensation of
substituted 3-bromothiophenols and ethyl (4-methoxybenzoyl) acetate. The amine derivative 2 has
been obtained by the Buchwald–Hartwig amination of 1 with dibutylamine. Addition of MeMgBr to
the carbonyl group of 2 followed by elimination with HClO4 provides the important intermediate 3.
Condensation of compound 3 with 4-hydroxy-1,3-dialdehyde or 4 yields the final pentamethine
compounds IRBTP-B and IRBTP-O respectively.
Photophysical properties and penetration effect of IRBTP-O. Two absorption peaks at 575 and
905 nm are shown for IRBTP-O with its emission peak beyond 1000 nm (Figure 2a, 2b and Table
S1). In comparison, IRBTP-B does not exhibit any absorption or fluorescence emission in the NIR-
II region (Figure 2a and 2b). For in vivo applications, micelles containing IRBTP-O or IRBTP-B
were prepared based upon prior reports with mPEG-DSPE lipids.36 An average diameters of 8.3 and
6.3 nm, respectively, were obtained for IRBTP-O and IRBTP-B micelles (Figure S2 and S3). With
IR-26 as reference (QY= 0.05 %, Table S1), the quantum yield of IRBTP-O was determined to be
0.10 % in methanol (Figure S4). Then, the absorbance and fluorescence profiles of IRBTP-O at
different pH values were examined and a pKa of 6.75 was calculated from pH-fluorescence profile
ACS Paragon Plus Environment

Page 11 of 25
Analytical Chemistry
1
2
3
4
5
6
7
8
9
(Figure S5). As can be seen, IRBTP-O shows moderate fluorescence at physiological pH, suggesting
its suitability for biological applications. Compared with ICG, both IRBTP-O and IRBTP-B showed
excellent photostability (Figure 2c). Next, we investigated the penetration effect of IRBTP-O in
capillary tubes in different imaging windows through using long pass (LP) filters from 850 to 1300
nm on tissue phantom (1 % Intralipid in water to mimic biological soft tissue). Better resolution of
the capillary tubes and attenuation of image intensities were obtained when imaging in the window
with longer wavelength filters due to the reduced photon scattering (Figure 2d and S6). In addition,
we also evaluated the signal-to-noise ratio (SNR) of the capillary tubes in these imaging windows.
The optimal SNR was obtained at wavelength beyond 1100 nm when the covered phantom is more
than 5 mm due to the much weak signal in the imaging windows beyond 1200 and 1300 nm (Figure
2e). According to the Rose criterion, it can be concluded that emissions beyond 1100 nm is favorable
for in vivo applications with the relative higher SNR and higher penetration depth.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The fluorescence response of IRBTP-B towards ONOO-. Next, the time dependent fluorescence
response of IRBTP-B to ONOO- was studied (Figure S7), which showed the obvious enhancement
of fluorescence with the time and a plateau after 3 minutes. Then, the optical sensing toward ONOO-
at varied concentrations (0-11 µM) was studied in PBS buffer solutions (pH 7.4). Upon reaction
with ONOO-, the NIR absorption at 850 nm was generated, accompanied by the reduction of the peak
at 600 nm (Figure 3a). Meanwhile, the fluorescence intensity within NIR-II window was increased
gradually with increase of ONOO- concentration (Figure 3b), and a good linearity (R2= 0.9897) was
obtained with the detection limit determined to be 55.9 nM (Figure 3c). The selectivity experiment
showed that biothiols (cysteine and NaHS), ROS and other RNS had no apparent influences on the
fluorescence response of IRBTP-B, illustrating the good selectivity of IRBTP-B to ONOO- (Figure
3d and S8). The activatable fluorescence property of IRBTP-B was then assayed by comparison with
the widely used commercial ONOO- probe D63255 on a tissue phantom study (Figure 3e, S9 and
ACS Paragon Plus Environment

Analytical Chemistry
Page 12 of 25
1
2
3
4
5
6
7
8
9
S10). In the absence of ONOO-, both capillary tubes loading IRBTP-B and D632 showed no
detectable fluorescence signals before and after being covered by Intralipid. However, when ONOO-
was added, the fluorescence of IRBTP-B was turned on and the edges of the capillary tube could still
be resolved from the images obtained beyond 1100 nm even at 5 mm Intralipid (Figure 3e). On the
contrary, the fluorescence signal from D632 can only be detected when the thickness of Intralipid is
smaller than 2 mm. In both the capillary tubes with IRBTP-B and D632 in the presence of ONOO-,
activated ratio decreased with increasing the thickness of Intralipid, while the line width (FWHM,
full width at half maximum) showed an opposite dependence. Due to the longer emission wavelength
in the NIR-II region, imaging with IRBTP-B favors more clarity than that of D632 whose emission
lies in the visible region. Furthermore, the distance between the liver and skin of nude mouse is about
0.59 mm (Figure S11), which encouraged us to use IRBTP-B for in vivo monitoring of preclinical
drug-induced hepatotoxicity. All above results indicate that IRBTP-B can be used for ONOO-
detection under normal physiological conditions with deep penetration depth.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Imaging APAP-induced hepatotoxicity in vivo. Motivated by these promising results, we next
examined the ability of IRBTP-B probe to visualize production of ONOO- in a drug-induced
hepatotoxicity model in nude mice. A CCK8 assay with CaOV3 cells was first performed and
identified the low cytotoxicity of IRBTP-B and IRBTP-O (Figure S12). It is well-known that an
overdose of APAP, a commonly household antipyretic and analgesic agent, is the leading cause of
hepatotoxicity even acute liver failure due to the overproduction of RNS. Therefore, by guiding with
the imaging biomarker ONOO-, drug-induced liver damage can be visualized in nude mice with the
aid of IRBTP-B. Meanwhile, N-acetyl cysteine (NAC) was used as the antidote for APAP overdose,
which was reported to remediate cell injury via the depletion of the RNS levels by increasing the
GSH level and binding with the toxic metabolite of APAP. Generally, female nude mice were
randomly divided into three groups: PBS (control group), APAP, and NAC + APAP. 300 mg kg-1 of
ACS Paragon Plus Environment

Page 13 of 25
Analytical Chemistry
1
2
3
4
5
6
7
8
9
APAP and 300 mg kg-1 of NAC were Intraperitoneal injected as needed because those doses are
reported to efficiently induce and recover inflammation respectively.17 Weak fluorescence signal was
obtained in the mice liver of the control group, implying little production of ONOO- level under
normal conditions (Figure 4a). In contrast, after the administration of APAP, the fluorescence
intensities of the mice livers displayed gradual increments in a time-dependent manner and reached
a maximum after 15 minutes of injection of IRBTP-B (Figure 4a and 4b), indicating the increased
generation of ONOO- after drug treatment. Moreover, no obvious fluorescent intensity augmentation
was observed with time in the NAC pretreatment group, indicating effective NAC remediation.
Similar procedures were also performed to evaluate commercial ONOO- probe D632 (Figure 4c and
S13). In comparison, higher SNR and relative fluorescent intensity were obtained from IRBTP-B
due to the reduced autofluorescence and photon scattering in NIR-II window (Figure 4d and 4e),
indicating IRBTP-B is more sensitive than commercial D632 for in vivo imaging. Next, the
histological analysis on the liver tissues were carried out to identify the histological changes during
the drug administration (Figure 4f). The livers from the control group were found to have intact
centrilobular vein (arrow 1), healthy hepatocytes and liver cells were arranged in the radial shape
with the center of central vein. On the contrary, a non-uniform morphology of the hepatocytes,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
infla
atory cells infiltration (arrow 2), and hemorrhage (arrow 3) could be obviously observed
after the administration of APAP, implying APAP-induced liver injury. Significantly improved
symptoms could be observed with NAC pretreatment, indicating effective remediation with NAC.
These results were in good agreement with the observation of in vivo fluorescence imaging.
CONCLUSION
In summary, we have constructed an ONOO- activatable NIR-II probe for APAP-induced
hepatotoxicity monitoring. It was found that an apparent ONOO- level upregulation occurred during
the drug-induced liver damage. Therefore, we anticipate that IRBTP-B may be employed as a
ACS Paragon Plus Environment

Analytical Chemistry
Page 14 of 25
1
2
3
4
5
6
7
8
9
powerful tool to probe ONOO- for preclinical drug-induced organism injury. Preclinical interrogation
of drug hepatotoxicity will add benefits in improving patient safety and therapeutic outcomes. We
expect that this strategy may be extended for the development of a wide variety of activatable NIR-
II probes by masking the hydroxyl functionalized reactive site with an analyte-specific triggering
group.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ASSOCIATED CONTENT
Supporting Information paragraph.
Experimental section and additional information as noted in the text. This material is available free
of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
E-mail: zhang_fan@fudan.edu.cn.
Tel: (+86)21-51630322; Fax: (+86)21-5163-0307.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
The work was supported by the National Key R & D Program of China (2017YFA0207303), National
Science Fund for Distinguished Young Scholars (21725502), Key Basic Research Program of
Science and Technology Commission of Shanghai Municipality (17JC1400100), and China
Postdoctoral Science Foundation (Grant KLH1615151). The authors extend their appreciation to the
ACS Paragon Plus Environment

Page 15 of 25
Analytical Chemistry
1
2
3
4
International Scientific Partnership Program ISPP at King Saud University for funding this research
5
6
work through ISPP#0100.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
REFERENCES
(1) Liebler, D. C.; Guengerich, F. P., Elucidating Mechanisms of Drug-Induced Toxicity. Nat. Rev.
Drug Discov. 2005, 4, 410-420.
(2) Park, B. K.; Kitteringham, N. R.; Maggs, J. L.; Pirmohamed, M.; Williams, D. P., The Role of
Metabolic Activation in Drug-Induced Hepatotoxicity. Annu. Rev. Pharmacol. Toxicol. 2005, 45,
177-202.
(3) Lee, W. M., Drug-Induced Hepatotoxicity. New Engl. J. Med. 2003, 349, 474-485.
(4) Stefan, R.; Gerd, A. K.-U.; Ignazio, G., Current Concepts of Mechanisms in Drug-Induced
Hepatotoxicity. Curr. Med. Chem. 2009, 16, 3041-3053.
(5) Walsh, J. S.; Miwa, G. T., Bioactivation of Drugs: Risk and Drug Design. Annu. Rev. Pharmacol.
Toxicol. 2011, 51, 145-167.
(6) Schäferling, M., The Art of Fluorescence Imaging with Chemical Sensors. Angew. Chem. Int. Ed.
2012, 51, 3532-3554.
(7) Gorris, H. H.; Wolfbeis, O. S., Photon-Upconverting Nanoparticles for Optical Encoding and
Multiplexing of Cells, Biomolecules, and Microspheres. Angew. Chem. Int. Ed. 2013, 52, 3584-3600.
(8) Han, M.; Gao, X.; Su, J. Z.; Nie, S., Quantum-Dot-Tagged Microbeads for Multiplexed Optical
Coding of Biomolecules. Nat. Biotechnol. 2001, 19, 631-635.
(9) Liu, B.; Li, C.; Yang, P.; Hou, Z.; Lin, J., 808-nm-Light-Excited Lanthanide-Doped
Nanoparticles: Rational Design, Luminescence Control and Theranostic Applications. Adv. Mater.
2017, 29, 1605434.
(10) Li, Y.; Xie, X.; Yang, X. e.; Li, M.; Jiao, X.; Sun, Y.; Wang, X.; Tang, B., Two-Photon
Fluorescent Probe for Revealing Drug-Induced Hepatotoxicity via Mapping Fluctuation of
Peroxynitrite. Chem. Sci. 2017, 8, 4006-4011.
(11) Jia, X.; Chen, Q.; Yang, Y.; Tang, Y.; Wang, R.; Xu, Y.; Zhu, W.; Qian, X., FRET-Based Mito-
Specific Fluorescent Probe for Ratiometric Detection and Imaging of Endogenous Peroxynitrite:
Dyad of Cy3 and Cy5. J. Am. Chem. Soc. 2016, 138, 10778-10781.
(12) He, X.; Li, L.; Fang, Y.; Shi, W.; Li, X.; Ma, H., In Vivo Imaging of Leucine Aminopeptidase
Activity in Drug-Induced Liver Injury and Liver Cancer via a Near-Infrared Fluorescent Probe.
Chem. Sci. 2017, 8, 3479-3483.
(13) Cheng, D.; Xu, W.; Yuan, L.; Zhang, X., Investigation of Drug-Induced Hepatotoxicity and Its
Remediation Pathway with Reaction-Based Fluorescent Probes. Anal. Chem. 2017, 89, 7693-7700.
(14) Peng, J.; Samanta, A.; Zeng, X.; Han, S.; Wang, L.; Su, D.; Loong, D. T. B.; Kang, N. Y.; Park,
S. J.; All, A. H.; Jiang, W.; Yuan, L.; Liu, X.; Chang, Y. T., Real-Time In Vivo Hepatotoxicity
Monitoring through Chromophore-Conjugated Photon-Upconverting Nanoprobes. Angew. Chem.
Int. Ed. 2017, 56, 4165-4169.
(15) Zhang, Q.; Zhu, Z.; Zheng, Y.; Cheng, J.; Zhang, N.; Long, Y.-T.; Zheng, J.; Qian, X.; Yang,
Y., A Three-Channel Fluorescent Probe That Distinguishes Peroxynitrite from Hypochlorite. J. Am.
Chem. Soc. 2012, 134, 18479-18482.
ACS Paragon Plus Environment

Analytical Chemistry
Page 16 of 25
1
2
3
4
5
6
7
8
9
(16) Song, Z.; Mao, D.; Sung, S. H. P.; Kwok, R. T. K.; Lam, J. W. Y.; Kong, D.; Ding, D.; Tang,
B. Z., Activatable Fluorescent Nanoprobe with Aggregation-Induced Emission Characteristics for
Selective In Vivo Imaging of Elevated Peroxynitrite Generation. Adv. Mater. 2016, 28, 7249-7256.
(17) Shuhendler, A. J.; Pu, K.; Cui, L.; Uetrecht, J. P.; Rao, J., Real-Time Imaging of Oxidative and
Nitrosative Stress in the Liver of Live Animals for Drug-Toxicity Testing. Nat. Biotechnol. 2014,
32, 373.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(18) Oushiki, D.; Kojima, H.; Terai, T.; Arita, M.; Hanaoka, K.; Urano, Y.; Nagano, T., Development
and Application of a Near-Infrared Fluorescence Probe for Oxidative Stress Based on Differential
Reactivity of Linked Cyanine Dyes. J. Am. Chem. Soc. 2010, 132, 2795-2801.
(19) Kojima, R.; Takakura, H.; Kamiya, M.; Kobayashi, E.; Komatsu, T.; Ueno, T.; Terai, T.;
Hanaoka, K.; Nagano, T.; Urano, Y., Development of a Sensitive Bioluminogenic Probe for Imaging
Highly Reactive Oxygen Species in Living Rats. Angew. Chem. Int. Ed. 2015, 54, 14768-14771.
(20) Yang, D.; Wang, H.-L.; Sun, Z.-N.; Chung, N.-W.; Shen, J.-G., A Highly Selective Fluorescent
Probe for the Detection and Imaging of Peroxynitrite in Living Cells. J. Am. Chem. Soc. 2006, 128,
6004-6005.
(21) Liu, D.; Feng, S.; Feng, G., A Rapid Responsive Colorimetric and Near-Infrared Fluorescent
Turn-On Probe for Imaging Exogenous and Endogenous Peroxynitrite in Living Cells. Sens.
Actuators B 2018, 269, 15-21.
(22) Feng, S.; Liu, D.; Feng, G., A Dual-Channel Probe with Green and Near-Infrared Fluorescence
Changes for In Vitro and In Vivo Detection of Peroxynitrite. Anal. Chim. Acta 2019, 1054, 137-144.
(23) Feng, W.; Feng, G., A Lysosome-Targetable Fluorescent Probe for Imaging ONOO- in Living
Cells and Animals. Dyes Pigm. 2019, 164, 174-181.
(24) Cheng, D.; Pan, Y.; Wang, L.; Zeng, Z.; Yuan, L.; Zhang, X.; Chang, Y.-T., Selective
Visualization of the Endogenous Peroxynitrite in an Inflamed Mouse Model by a Mitochondria-
Targetable Two-Photon Ratiometric Fluorescent Probe. J. Am. Chem. Soc. 2017, 139, 285-292.
(25) Li, H.; Li, X.; Wu, X.; Shi, W.; Ma, H., Observation of the Generation of ONOO- in
Mitochondria under Various Stimuli with a Sensitive Fluorescence Probe. Anal. Chem. 2017, 89,
5519-5525.
(26) Ntziachristos, V.; Ripoll, J.; Wang, L. V.; Weissleder, R., Looking and Listening to Light: The
Evolution of Whole-Body Photonic Imaging. Nat. Biotechnol. 2005, 23, 313-320.
(27) Hong, G.; Antaris, A. L.; Dai, H., Near-Infrared Fluorophores for Biomedical Imaging. Nat.
Biomed. Eng. 2017, 1, 0010.
(28) Smith, A. M.; Mancini, M. C.; Nie, S., Second Window for In Vivo Imaging. Nat. Nanotechnol.
2009, 4, 710-711.
(29) He, S.; Song, J.; Qu, J.; Cheng, Z., Crucial Breakthrough of Second Near-Infrared Biological
Window Fluorophores: Design and Synthesis toward Multimodal Imaging and Theranostics. Chem.
Soc. Rev. 2018, 47, 4258-4278.
(30) Schnermann, M. J., Organic Dyes for Deep Bioimaging. Nature 2017, 551, 176.
(31) Yomogida, Y.; Tanaka, T.; Zhang, M.; Yudasaka, M.; Wei, X.; Kataura, H., Industrial-Scale
Separation of High-Purity Single-Chirality Single-Wall Carbon Nanotubes for Biological Imaging.
Nat. Commun. 2016, 7, 12056.
(32) Bruns, O. T.; Bischof, T. S.; Harris, D. K.; Franke, D.; Shi, Y.; Riedemann, L.; Bartelt, A.;
Jaworski, F. B.; Carr, J. A.; Rowlands, C. J.; Wilson, M. W. B.; Chen, O.; Wei, H.; Hwang, G. W.;
Montana, D. M.; Coropceanu, I.; Achorn, O. B.; Kloepper, J.; Heeren, J.; So, P. T. C.; Fukumura,
D.; Jensen, K. F.; Jain, R. K.; Bawendi, M. G., Next-Generation In Vivo Optical Imaging with Short-
Wave Infrared Quantum Dots. Nat. Biomed. Eng. 2017, 1, 0056.
ACS Paragon Plus Environment

Page 17 of 25
Analytical Chemistry
1
2
3
4
5
6
(33) Fan, Y.; Wang, P.; Lu, Y.; Wang, R.; Zhou, L.; Zheng, X.; Li, X.; Piper, J. A.; Zhang, F.,
Lifetime-Engineered NIR-II Nanoparticles Unlock Multiplexed In Vivo Imaging. Nat. Nanotechnol.
2018, 13, 941-946.
(34) Zhong, Y.; Ma, Z.; Zhu, S.; Yue, J.; Zhang, M.; Antaris, A. L.; Yuan, J.; Cui, R.; Wan, H.; Zhou,
Y.; Wang, W.; Huang, N. F.; Luo, J.; Hu, Z.; Dai, H., Boosting the Down-Shifting Luminescence of
Rare-Earth Nanocrystals for Biological Imaging beyond 1500ꢀn . Nat. Commun. 2017, 8, 737.
(35) Antaris, A. L.; Chen, H.; Cheng, K.; Sun, Y.; Hong, G.; Qu, C.; Diao, S.; Deng, Z.; Hu, X.;
Zhang, B.; Zhang, X.; Yaghi, O. K.; Alamparambil, Z. R.; Hong, X.; Cheng, Z.; Dai, H., A Small-
Molecule Dye for NIR-II Imaging. Nat. Mater. 2016, 15, 235-242.
(36) Cosco, E. D.; Caram, J. R.; Bruns, O. T.; Franke, D.; Day, R. A.; Farr, E. P.; Bawendi, M. G.;
Sletten, E. M., Flavylium Polymethine Fluorophores for Imaging in the Near- and Shortwave
Infrared. Angew. Chem. Int. Ed. 2017, 56, 13126-13129.
(37) Li, B.; Lu, L.; Zhao, M.; Lei, Z.; Zhang, F., An Efficient 1064 nm NIR-II Excitation Fluorescent
Molecular Dye for Deep-Tissue High-Resolution Dynamic Bioimaging. Angew. Chem. Int. Ed. 2018,
57, 7483-7487.
(38) Sheng, Z.; Guo, B.; Hu, D.; Xu, S.; Wu, W.; Liew, W. H.; Yao, K.; Jiang, J.; Liu, C.; Zheng,
H.; Liu, B., Bright Aggregation-Induced-Emission Dots for Targeted Synergetic NIR-II
Fluorescence and NIR-I Photoacoustic Imaging of Orthotopic Brain Tumors. Adv. Mater. 2018, 30,
1800766, https://doi.org/10.1002/adma.201800766.
(39) Wan, H.; Yue, J.; Zhu, S.; Uno, T.; Zhang, X.; Yang, Q.; Yu, K.; Hong, G.; Wang, J.; Li, L.;
Ma, Z.; Gao, H.; Zhong, Y.; Su, J.; Antaris, A. L.; Xia, Y.; Luo, J.; Liang, Y.; Dai, H., A Bright
Organic NIR-II Nanofluorophore for Three-Dimensional Imaging into Biological Tissues. Nat.
Commun. 2018, 9, 1171.
(40) Ding, F.; Zhan, Y.; Lu, X.; Sun, Y., Recent Advances in Near-Infrared II Fluorophores for
Multifunctional Biomedical Imaging. Chem. Sci. 2018, 9, 4370-4380.
(41) Tao, Z.; Hong, G.; Shinji, C.; Chen, C.; Diao, S.; Antaris, A. L.; Zhang, B.; Zou, Y.; Dai, H.,
Biological Imaging Using Nanoparticles of Small Organic Molecules with Fluorescence Emission at
Wavelengths Longer than 1000ꢁnm. Angew. Chem. Int. Ed. 2013, 125, 13240-13244.
(42) Shou, K.; Qu, C.; Sun, Y.; Chen, H.; Chen, S.; Zhang, L.; Xu, H.; Hong, X.; Yu, A.; Cheng, Z.,
Multifunctional Biomedical Imaging in Physiological and Pathological Conditions Using a NIR-II
Probe. Adv. Funct. Mater. 2017, 27, 1700995.
(43) Hori, Y.; Nakaki, K.; Sato, M.; Mizukami, S.; Kikuchi, K., Development of Protein-Labeling
Probes with a Redesigned Fluorogenic Switch Based on Intramolecular Association for No-Wash
Live-Cell Imaging. Angew. Chem. Int. Ed. 2012, 51, 5611-5614.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(44) Xu, G.; Yan, Q.; Lv, X.; Zhu, Y.; Xin, K.; Shi, B.; Wang, R.; Chen, J.; Guo, W.; Shi, P.; Fan,
C.; Zhao, C.; Tian, H., Imaging of Colorectal Cancers Using Activatable Nanoprobes with Second
Near-Infrared Window Emission. Angew. Chem. Int. Ed. 2018, 57, 3626-3630.
(45) Lin, Q. L.; Yun, H. J.; Liu, W. Y.; Song, H. J.; Makarov, N. S.; Isaienko, O.; Nakotte, T.; Chen,
G.; Luo, H. M.; Klimov, V. I.; Pietryga, J. M., Phase-Transfer Ligand Exchange of Lead
Chalcogenide Quantum Dots for Direct Deposition of Thick, Highly Conductive Films. J. Am. Chem.
Soc. 2017, 139, 6644-6653.
(46) Reineck, P.; Gibson, B. C., Near-Infrared Fluorescent Nanomaterials for Bioimaging and
Sensing. Adv. Optical Mater. 2017, 5, 1600446.
(47) Sun, Y.; Ding, F.; Zhou, Z.; Li, C.; Pu, M.; Xu, Y.; Zhan, Y.; Lu, X.; Li, H.; Yang, G.; Sun, Y.;
Stang, P. J., Rhomboidal Pt(II) metallacycle-based NIR-II theranostic nanoprobe for tumor diagnosis
and image-guided therapy. Proc. Natl. Acad. Sci. USA 2019, 116, 1968-1973.
ACS Paragon Plus Environment

Analytical Chemistry
Page 18 of 25
1
2
3
4
5
6
7
8
9
(48) Zhu, S.; Yung, B. C.; Chandra, S.; Niu, G.; Antaris, A. L.; Chen, X., Near-Infrared-II (NIR-II)
Bioimaging via Off-Peak NIR-I Fluorescence Emission. Theranostics 2018, 8, 4141-4151.
(49) Semonin, O. E.; Johnson, J. C.; Luther, J. M.; Midgett, A. G.; Nozik, A. J.; Beard, M. C.,
Absolute Photoluminescence Quantum Yields of IR-26 Dye, PbS, and PbSe Quantum Dots. J. Phys.
Chem. Lett. 2010, 1, 2445-2450.
(50) Hyun, H.; Wada, H.; Bao, K.; Gravier, J.; Yadav, Y.; Laramie, M.; Henary, M.; Frangioni, J.
V.; Choi, H. S., Phosphonated Near-Infrared Fluorophores for Biomedical Imaging of Bone. Angew.
Chem. Int. Ed. 2014, 53, 10668-10672.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(51) Carr, J. A.; Franke, D.; Caram, J. R.; Perkinson, C. F.; Saif, M.; Askoxylakis, V.; Datta, M.;
Fukumura, D.; Jain, R. K.; Bawendi, M. G.; Bruns, O. T., Shortwave Infrared Fluorescence Imaging
with the Clinically Approved Near-Infrared Dye Indocyanine Green. Proc. Natl. Acad. Sci. USA
2018, 115, 4465-4470.
(52) Yin, J.; Kwon, Y.; Kim, D.; Lee, D.; Kim, G.; Hu, Y.; Ryu, J.-H.; Yoon, J., Cyanine-Based
Fluorescent Probe for Highly Selective Detection of Glutathione in Cell Cultures and Live Mouse
Tissues. J. Am. Chem. Soc. 2014, 136, 5351-8.
(53) Karton-Lifshin, N.; Albertazzi, L.; Bendikov, M.; Baran, P. S.; Shabat, D., “Donor-Two-
Acceptor” Dye Design: A Distinct Gateway to NIR Fluorescence. J. Am. Chem. Soc. 2012, 134,
20412-20420.
(54) Li, H.; Li, X.; Shi, W.; Xu, Y.; Ma, H., Rationally Designed Fluorescence .OH Probe with High
.
Sensitivity and Selectivity for Monitoring the Generation of OH in Iron Autoxidation without
Addition of H2O2. Angew. Chem. Int. Ed. 2018, 57, 12830-12834.
(55) Szabó, C.; Virág, L.; Cuzzocrea, S.; Scott, G. S.; Hake, P.; O’Connor, M. P.; Zingarelli, B.;
Salzman, A.; Kun, E., Protection Against Peroxynitrite-Induced Fibroblast Injury and Arthritis
Development by Inhibition of Poly(ADP-ribose) Synthase. Proc. Natl. Acad. Sci. USA 1998, 95,
3867-3872.
ACS Paragon Plus Environment

Page 19 of 25
Analytical Chemistry
1
2
3
4
FIGURE CAPTION
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1. Design and synthesis of ONOO- activatable molecular probe with NIR-II fluorescence.
Conditions: a) Et2NH, Pd2(dba)3, DavePhos, Cs2CO3, dioxene, 95 oC, 18h; b) i CH3MgBr, ii HClO4;
c) toluene/n-butanol, 100 oC, 12h.
ACS Paragon Plus Environment

Analytical Chemistry
Page 20 of 25
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2. (a) Absorption and (b) fluorescence of IRBTP-O and IRBTP-B in CHCl3. (c) Photostability
comparison of ICG, IRBTP-B and IRBTP-O in PBS. (d) Fluorescence images of capillaries filled
with IRBTP-O in NIR-II (1100 nm LP) region immersed in 1% Intralipid with varying depth, Scale
bar: 2 mm. (e) Measured SNR of capillary images under different depth. SNR values were calculated
by dividing average signal intensity (ROI1) by the average back-ground signal intensity (ROI2).
Insert: SNR of capillary images under 5 mm.
ACS Paragon Plus Environment

Page 21 of 25
Analytical Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 3. (a) Absorption and (b) fluorescence changes of IRBTP-B (10 μM) upon addition with
ONOO- (0 - 11 μM) in PBS. (c) Plots of fluorescence intensity at 950 nm with varied concentrations
ACS Paragon Plus Environment

Analytical Chemistry
Page 22 of 25
1
2
3
4
5
6
7
8
9
of ONOO- in PBS. (d) The fluorescent response of IRBTP-B to bioreactive reagents. (e) Tissue
phantom study of the penetration depth of IRBTP-B (1100 nm LP) and D632 (540 nm band pass
(BP)) in the absence and presence of ONOO-. (f) Activated ratio comparison of IRBTP-B and D632
under different depth. Activated ratio values were calculated by dividing average signal intensity of
ROI1 by the average signal intensity of ROI2. (g) Feature width of capillary images filled with
IRBTP-B or D632 in the presence of ONOO- as a function of depth in 1% Intralipid.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

Page 23 of 25
Analytical Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 4. In vivo imaging of endogenous ONOO- in the livers of mice during an APAP-induced
hepatotoxicity by IRBTP-B and D632. (a) In vivo imaging of livers of mice from IRBTP-B treated
ACS Paragon Plus Environment

Analytical Chemistry
Page 24 of 25
1
2
3
4
5
6
7
8
9
with various substances: PBS, APAP, and NAC + APAP. (b) Relative fluorescence intensity of livers
of mice treated with various substances following by IRBTP-B over time. (c) In vivo imaging of
livers of mice from D632 treated with various substances: PBS, APAP, and NAC + APAP. (d) Signal
to noise ratios (SNR) obtained for IRBTP-B and D632 from different groups. (e) Relative intensity
of livers of mice after injection of IRBTP-B and D632 in different groups. (f) Representative
histology (hematoxylin and eosin staining (H&E)) of the livers of mice treated with PBS, APAP or
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
NAC + APAP (arrow 1: centrilobular vein, arrow 2: infla
hemorrhage, KC: Kupffer cell.) Scal bar: 50 µm.
atory cells infiltration, arrow 3:
ACS Paragon Plus Environment

Page 25 of 25
Analytical Chemistry
1
2
3
4
For TOC only
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment