Upconversion NIR-II fluorophores for mitochondria-targeted cancer imaging and photothermal therapy

Article abstract of DOI:10.1038/s41467-020-19945-w

NIR-II fluorophores have shown great promise for biomedical applications with superior in vivo optical properties. To date, few small-molecule NIR-II fluorophores have been discovered with donor-acceptor-donor (D-A-D) or symmetrical structures, and upconversion-mitochondria-targeted NIR-II dyes have not been reported. Herein, we report development of D-A type thiopyrylium-based NIR-II fluorophores with frequency upconversion luminescence (FUCL) at ~580 nm upon excitation at ~850 nm. H4-PEG-PT can not only quickly and effectively image mitochondria in live or fixed osteosarcoma cells with subcellular resolution at 1 nM, but also efficiently convert optical energy into heat, achieving mitochondria-targeted photothermal cancer therapy without ROS effects. H4-PEG-PT has been further evaluated in vivo and exhibited strong tumor uptake, specific NIR-II signals with high spatial and temporal resolution, and remarkable NIR-II image-guided photothermal therapy. This report presents the first D-A type thiopyrylium NIR-II theranostics for synchronous upconversion-mitochondria-targeted cell imaging, in vivo NIR-II osteosarcoma imaging and excellent photothermal efficiency.

Full text of DOI:10.1038/s41467-020-19945-w

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https://doi.org/10.1038/s41467-020-19945-w
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& Y u l in g X i a o
N I R -I I flu o ro p h o re s h a v e s h o w n g r e a t p r o m i s e f o r b i o m e d i c a l a p p l i c a t i o n s w i t h s u p er i o r
i n v i v o o p t i c a l p ro p e r t i e s . T o d a te , f e w s m a l l - m o l e c u l e N I R -I I flu or o p ho r e s h a v e b e e n d i s -
c o v e r e d w it h d o n o r- a c ce p to r- d o n o r ( D - A - D) o r s y m m et r i c a l s t r u c t ur e s , a n d u pc on v er s i o n -
m i t oc ho n d r ia - t a rg e t e d N IR -I I d y e s h a v e n o t b e e n r e p o r te d . H er e i n, w e r ep o rt d e v e l op m en t o f
D - A t y p e t h i o py r yl i um - b a s e d N IR -I I flu o r o p h o r e s w i th f re qu e nc y u pc on v e r s i on l u m i n e s c e n c e
( F U CL ) a t ~ 5 8 0 n m u p o n e x c i t a t i o n a t ~ 8 5 0 n m . H 4 - P E G - P T c a n n ot o nl y q ui c k l y a n d
e f fe c t i ve l y i ma g e m it o c h o n d r i a i n l i v e o r fix e d o s t e o s a r c o m a c e l l s w i t h s u b c e l l u l a r r e s ol ut i o n
a t 1 n M , b u t a ls o e f fic i e n tl y c o n v e r t o p ti ca l e n e r g y i n to h ea t , a c h i e vi ng m i to c h o nd r i a - t a r g e t e d
p h ot o t h e rm a l c a n c e r t h e r a p y w i th o u t R O S e f f e c ts . H 4 - P E G - P T h a s b ee n f ur t he r e v a lu a t ed
i n v i v o a nd e x h ib i t e d s tr o n g t u m o r u p ta ke , s p e c i fic N I R -I I s i g na l s w i t h h i g h s p a t i a l a n d
t e m p or a l r e so l u t i o n, a n d r e m a r ka b l e N IR -I I i m a g e - g u i d e d p ho t o th e rm a l t h e r a p y . T hi s r ep o rt
p r e s e nt s t h e fir st D - A t y p e t h i o p y r y l i u m N IR -I I t h e r a n o s t i c s f or s y nc hr o no u s u pc on v e r s i on -
m i t oc h on d ri a -t ar g e t e d c e l l i m a g i n g , i n v i v o N IR -I I o s t e o s a rc om a i m a g i n g a n d e xc e l l e n t
p h ot o t h e rm a l e ffic i e n cy .
1 State Key Laboratory of Virology, Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (MOE), Hubei Provincial Key Laboratory of
Developmentally Originated Disease, Hubei Province Engineering and Technology Research Center for Fluorinated Pharmaceuticals, Wuhan University School
of Pharmaceutical Sciences, Wuhan 430071, China. 2 College of Science, Innovation Center for Traditional Tibetan Medicine Modernization and Quality
Control, Tibet University, Lhasa 850000, China. 3 Shenzhen Institute of Wuhan University, Shenzhen 518057, China. 4 Key Laboratory for Organic Electronics
and Information Displays & Institute of Advanced Materials, Nanjing University of Posts and Telecommunications, Nanjing 210023, China. 5 Innovative
Institute of Chinese Medicine and Pharmacy, Chengdu University of Traditional Chinese Medicine, Wenjiang, Chengdu, Sichuan 611137, China. 6 Department
7
✉
of Chemistry, University of Aberdeen, Aberdeen, UK. These authors contributed equally: Hui Zhou, Xiaodong Zeng. email: xhy78@whu.edu.cn;
xiaoyl@whu.edu.cn
N A T U R E C OM M U N I C A T I ON S |
(2 0 2 0 ) 1 1 : 6 1 8 3 | h t t p s :/ / d o i . o r g / 1 0 . 1 0 3 8 / s 4 1 4 6 7 - 0 2 0 - 1 9 9 4 5 - w | w ww . n a t u r e . co m / n a t u r e co m m u n ic a t i o n s
1

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N A T U R E C O M MU N ICA T IO N S | h t t p s : / / d o i .o r g / 1 0 .1 0 3 8 / s4 1 4 6 7 - 0 2 0 - 1 9 94 5 - w
luorescence imaging in the second near-infrared window or high-energy laser excitation. Therefore, the exploitation of new
(NIR-II, 1000–1700 nm) has been widely used in biological mitochondria-targeted NIR-II fluorophores with a more bio-
and biomedical research, because the light in this wave- compatible upconversion strategy is greatly desired41.
F
length region is capable of deep penetration, and cellular auto-
As useful and versatile heterocyclic luminescent compounds
fluorescence is minimal1–17. Our initial progress toward the with a positive charge, pyrylium salts are widely utilized as photo-
first small-molecule organic NIR-II dye entailed the formation of sensitizers, Q-switchers, and NIR dyes for proteins and oligo-
the fluorophore CH1055 from benzo-bis(1,2,5-thiadiazole) saccharide labeling42,43. Thiopyrylium is analogous to the pyr-
(BBTD), which absorbed light at ~750 nm and fluoresced at ylium cation with the oxygen atom replaced by a sulfur atom.
~1055 nm18–20. The LUMO of CH1055 showed a strong con- Notably, thiopyrylium salts display physical and chemical prop-
tribution at the electron-accepting moiety BBTD with a typical erties typical of aromatic compounds and are much less reactive
optical bandgap (Egap) smaller than 1.5 eV, which was beneficial than analogous pyrylium salts due to the lower electronegativity
for bathochromic shift and large Stokes shift. Despite a panel of of the sulfur atom44,45. As is clearly seen in Supplementary
NIR-II emissive contrasts have been explored, the majority of Table 1, the Egap of thiopyrylium is much lower than that of
them had a BBTD core or were symmetrical fluorophores with a pyrylium, suggesting that thiopyrylium salts may become novel
D–A–D structure (Fig. 1a)21–29. It is imperative to search for new NIR acceptors. In the current work, we utilized thiopyrylium
types of small-molecule NIR-II probes with desirable chemical heterocycle 2 as a building block to construct a series of novel
and physical properties, minimal cellular toxicity, and clinical red-shifted thiopyrylium dyes 3 with donor–acceptor (D–A)
translation ability (Fig. 1).
structures (Fig. 1b). The electronic structure calculations and
Among all the fluorescent dyes, frequency upconversion experimental evaluation of the optical properties of 3a–3k and H4
luminescence (FUCL) materials can convert low-energy excita- were carried out to elucidate the structure–property relationship.
tion to high-energy emission with large anti-Stokes shift, limited Furthermore, the synthesized NIR-II fluorophores 3k-PEG, H4-
auto-fluorescence from biological samples, high signal-to-noise PEG, and H4-PEG-PT were highly resistant to photobleaching
ratio, and tunable excitation and emission30–32. Mitochondria are and well retained within the mitochondria even after osteo-
vital organelles responsible for the energy production and pro- sarcoma cell fixation and permeabilization at 1 nM based on
gramed cell death such as apoptosis33–35. Despite the develop- FUCL strategy with a short-wavelength luminescence at 580 nm
ment of mitochondria-targeted therapeutics, imaging of upon excitation at ~850 nm, which is among the most favorable
mitochondria is also crucial for providing important information characteristics of fluorescent dyes. Moreover, H4-PEG-PT
about disease presence, progression, and pathways for early dis- exhibited potent photothermal therapy effect toward osteo-
ease diagnosis and treatment36–39. To date, limited examples of sarcoma both in vitro and in vivo. To the best of our knowledge,
mitochondria-targeted FUCL organic dyes have been reported this report presents the first D–A type thiopyrylium NIR-II
and used for deep-seated tumor treatment40. Furthermore, no theranostics for synchronous FUCL mitochondria-targeted cell
research based on the FUCL and mitochondria-targeted NIR-II imaging with subcellular resolution, deeper tissue NIR-II osteo-
imaging technology has been developed for tumor imaging and sarcoma imaging in vivo and excellent photothermal efficiency.
antitumor therapy. Many small-molecule mitochondria-targeted
agents have been developed, including alkyltriphenylpho-
Results
sphonium cations, rhodamine, and both natural and synthetic
Synthesis and rational design. We first designed and synthesized
mitochondria-targeted peptides38. However, most currently
3a–3f (R2 = H) to confirm our hypothesis that these dyes fol-
available mitochondria-targeted fluorescent dyes emit only one
color in the visible or NIR-I window. Their applications are
somewhat limited due to poor photostability, small Stokes shifts,
lowed the D–A rather than D–A–D structure46,47. The chemical
structures of 3 are shown in Fig. 1b. The synthetic route for 3 is
shown in Fig. 2a. The key intermediate thiopyrylium
a
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A R T I C L E
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6a: R2 = H; 6b: R2 = OCH2CCH
5a: R2 = H; 5b: R2 = OCH2CCH
Phenyl ring A
Phenyl ring C
Phenyl ring B
7a: R2 = H; 7b: R2 = OCH2CCH
3a-3k, H4
3a: n = 0, R2 = H, R3 = H, 50%;
3c: n = 0, R2 = H, R3 = OME, 60%;
3b: n = 0, R2 = H, R3 = OCOMe, 54%
3d: n = 0, R2 = H, R3 = SMe, 58%
3e: n = 0, R2 = H, R3 = NMe2, 54%;
3g: n = 0, R2 = OCH2CCH, R3 = H, 48%;
3i: n = 0, R2 = OCH2CCH, R3 = OMe, 58%;
3k: n = 0, R2 = OCH2CCH, R3 = NMe2, 51%;
3f: n = 1, R2 = H, R3 = NMe2, 51%
3h: n = 0, R2 = OCH2CCH, R3 = OCOMe, 50%
3j: n = 0, R2 = OCH2CCH, R3 = SMe, 52%
H4: n = 1, R2 = OCH2CCH, R3 = NMe2, 47%
b
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37°
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3f-Geometry (S0)
48°
48°
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tetrafluoroborate 7a was obtained according to the literature46. 3a–3f was mainly localized on the phenyl ring C with a minor
Finally, subsequent condensation with various aldehydes under contribution from positively charged sulfur ions, while the
microwave irradiation resulted in the formation of the target LUMO was primarily centered on the thiopyrylium unit,
molecule 3a–3f in moderately high yields of 50–60%. In order to contributed by the p orbital on the sulfur ion and the phenyl
introduce a modifiable group at the R2 position instead of H, ring A (Fig. 2b). The lower Egap of 3e and 3k was 2.07 and 2.06
3g–3k, and H4 with a methoxyethyne group were then synthe- eV, which can be attributed to the increased internal charge
sized according to the general strategy outlined in Fig. 2a.
transferred by the introduction of a stronger electron-donating
DFT calculations with the B3LYP exchange functional employ- moiety of N,N-dimethylaniline group. However, the Egap of 3e
ing 6–31G(d) basis sets were performed to identify the highest and 3k was much higher than that of CH1055 (1.5 eV) with a
occupied molecular orbital (HOMO) and the lowest unoccupied typical NIR-II optical Egap based on previous results. Molecular
molecular orbital (LUMO) of 3a–3k and H4 (Supplementary engineering principles tell us that, to realize a lower Egap, the
Table 1, Fig. 2)48. From the calculation results, we found that the acceptor requires a higher LUMO energy level. Meanwhile, a
Egap of compounds 3a–3f without a methoxyethyne unit were higher HOMO energy level is also needed to reduce the Egap
similar to that of 3g–3k, H4 with a methoxyethyne unit, which value. The D–A-type fluorophore 3 can be further modified to
indicated that the presence of the methoxyethyne group did not achieve maximum emission within the NIR-II region by
have much effect on Egap. The electron density of the HOMO of extending the conjugation with the electron-rich thiophene
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1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
3a
Dichloromethane
3a
3b
3d
3c
3e
3f
Dichloromethane
3b
3c
3d
3e
3f
600 700 800 900 1000 1100 1200
Wavelength (nm)
400
500
600
700
800
900
Wavelength (nm)
c
d
1.0
0.8
0.6
0.4
0.2
0.0
3g
3h
3i
Dichloromethane
Dichloromethane
3g
3h
3i
0.8
0.6
0.4
0.2
0.0
3j
3j
3k
H4
3k
H4
600 700 800 900 1000 1100 1200
Wavelength (nm)
400
500
600
700
800
900
Wavelength (nm)
Fig. 3 Spectroscopic properties of 3a–3k and H4. a
d d g–
A
H
b
4
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spacer in compounds 3f and H4, and using N,N-dimethylaniline dimethylaniline substituted 3e and 3k. Quantum yields of 3a–3k
as an electron donor to strengthen the intramolecular charge- were measured in dichloromethane (Supplementary Fig. 2 and
transfer (ICT) effect. Moreover, 3f and H4 presented higher-lying Supplementary Table 2).
calculated HOMO (−5.33 eV, −5.30 eV) and LUMO (−3.63 eV,
The UV–Vis-NIR absorption bands of 3f and H4 were at
−3.60 eV) levels, and showed lower band gaps (1.7 eV, 1.7 eV), as 580–1100 nm in CH2Cl2 due to the formation of a strong charge-
the electron-rich thiophene spacer significantly lowers the transfer structure between D and A units, while the fluorescence
oxidation potential, resulting in a further bathochromic shift.
emission spectra of 3f and H4 demonstrated peak emission
wavelength at ~1100 nm (Fig. 3). Furthermore, NIR-II signals of
H4 were systematically investigated under various solvents and
acid conditions such as CH3CN, DMSO, MeOH, EtOH/HCl,
inorganic acids, organic acids, and Lewis acids (Fig. 4). The
absorbance of the H4 solution remained unchanged under
various organic solvents and acidic conditions. The absorbance
and fluorescence increased linearly with the increased concentra-
tion of H4 (Fig. 4g, h), suggesting no aggregation by accumulation
over a wide range of conditions. However, H4 exhibited a broad
peak with significant quenching of emission in polar solvents, and
increasing solvent polarity generally resulted in the shift of the
emission spectra to longer wavelengths. H4 exhibited high
photostability compared to ICG, with negligible decay under
continuous excitation for 40 min (Fig. 4i) in DMSO. The
quantum yield of H4 was 2.01% under 785 nm excitation in
dichloromethane, measured against an IR-26 reference (0.5%
quantum yield, Supplementary Fig. 3).
Spectroscopic properties of 3a–3k and H4. We then tested the
spectroscopic properties of 3a–3f in dichloromethane as shown in
Fig. 3a, b, and it was found that their emission wavelengths were
almost the same as 3g–3k, H4 (Fig. 3c, d). These results fully
demonstrated that the methoxyethyne group had minimal impact
on the spectra of thiopyrylium dyes, and the methoxyethyne
group did not act as a donor. The UV–Vis–NIR spectra of 3g–3k
in THF, CH3CN, Acetone, MeOH, EtOH, and DMSO were
similar to the spectra in dichloromethane solution, indicating that
the influence of these solvents on absorbance properties was
almost negligible (Supplementary Fig. 1A)49,50. As shown in
Supplementary Fig. 1, the magnitudes of the Stokes shift were
consistent with the relative electron-donating abilities of the
substituents on R3. Increased electron density of donors was
consistently accompanied by a shift in λex and λem to longer
wavelengths. If we considered compound 3a or 3g with unsub-
stituted benzene at R3 as a reference, introduction of electron-
donating substituents at the R3 position such as OAc (3b, 3h), In vitro evaluation. Osteosarcoma is the third most common
OMe (3c, 3i), and SMe (3d, 3j) led to a steady red-shift of malignant tumor and accounts for more than 60% of all malig-
fluorescence toward NIR-I wavelengths by 15–130 nm up to 31% nant bone tumors in children and adolescents51. Surgical excision
fluorescence quantum yield. Interestingly, photoluminescence of all primary and metastatic tumors remains the essential
excitation mapping performed on 3e and 3k in dichloromethane strategy behind osteosarcoma treatment. However, current diag-
showed maximum absorption peaks at ~741 and ~743 nm, and nostic tools allow detection of only 8–15% of bone or lung
maximum emission peaks at ~917 and 918 nm, with a tail metastatic patients52,53. Thus, it is urgent to develop new ther-
extending into the NIR-II region. Dimethylamino-substituent at anostic strategies for early diagnosis, therapy, and monitoring
the R3 position induced an additional strong bathochromic shift responses to osteosarcoma therapies54,55. The oligopeptide
of maximum absorption and fluorescence by enhanced electron- PPSHTPT (PT) was first designed to mimic the properties of the
donating ability and resonance zwitterionic structure of N,N- natural protein osteocalcin in vivo and possessed a high affinity
4
N
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a
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5.00E–012
4.00E–012
3.00E–012
2.00E–012
1.00E–012
0.00E + 000
5.00E–012
4.50E–012
4.00E–012
3.50E–012
3.00E–012
2.50E–012
2.00E–012
1.50E–012
1.00E–012
5.00E–013
0.00E + 000
Citric acid
H2SO4
BF3•Et2O
1 M HCl
0.20
0.15
0.10
0.05
0.00
1 μM HCl
10 μM HCl
50 μM HCl
100 μM HCl
Blank
CF3COOH
CH3COOH
FeCl3
BF3•Et2O
CF3COOH
CH3COOH
FeCl3
HCl
H2SO4
HCl
Blank
Citric acid
Blank
900 1000 1100 1200 1300 1400 1500 1600
400 500 600 700 800 900 1000
900 1000 1100 1200 1300 1400 1500 1600
Wavelength (nm)
Wavelength (nm)
Wavelength (nm)
e
d
f
2.48E–012
1.86E–012
1.24E–012
6.20E–013
0.00E + 000
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
3.50E–011
3.00E–011
2.50E–011
2.00E–011
1.50E–011
1.00E–011
5.00E–012
0.00E + 000
Acetone
Acetonitrile
Chlorobenzene
DCM
Chlorobenzene
Acetonitrile
Acetone
DCM
DMSO
EtOH
0 min
30 min
60 min
120 min
240 min
360 min
DMSO
EtOH
MeOH
THF
MeOH
THF
900 1000 1100 1200 1300 1400 1500 1600
500
600
700
800
900
1000
900 1000 1100 1200 1300 1400 1500 1600
Wavelength (nm)
Wavelength (nm)
Wavelength (nm)
g
i
h
2.0
1.5
1.0
0.5
0.0
80 µM
1.80E–011
1.60E–011
1.40E–011
1.20E–011
1.00E–011
8.00E–012
6.00E–012
4.00E–012
2.00E–012
0.00E + 000
80 μM
60 μM
40 μM
30 μM
20 μM
10 μM
5 μM
H4
1.0
0.8
0.6
0.4
0.2
60 µM
40 µM
30 µM
20 µM
10 µM
5 µM
ICG
1 µM
1 μM
1100
400 500 600 700 800 900
1000
900 1000 1100 1200 1300 1400 1500 1600
0
10
20
30
40
Wavelength (nm)
Wavelength (nm)
Time (min)
Fig. 4 Spectroscopic properties of H4. a
F
r
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b
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and specificity for osteosarcoma cell lines (e.g., 143B cells)56. H4- experiment (left tube, Fig. 5d) while negligible fluorescence was
PEG-PT was further synthesized through direct amidation and detected in the blocking experiment (right tube, Fig. 5d). The
Cu-catalyzed azide-alkyne cycloaddition (Fig. 5a). Azide-PEG16- toxicity of H4-PEG-PT was determined in vitro with an MTT
COOH was first conjugated with an amine group of PT to give assay on 143B and L929 cells at different concentrations (2, 4, 8,
PEG-PT, then H4 was reacted with PEG-PT via Cu-catalyzed 16, and 32 µM) (Fig. 5f). These results indicate that an aqueous
click reaction to obtain H4-PEG-PT. Normalized UV–vis spectra soluble, photostable, and biocompatible NIR-II fluorescence
showed that H4-PEG-PT possessed a maximum absorption peak probe H4-PEG-PT, is suitable for osteosarcoma imaging.
at ~800 nm and a maximum emission peak at ~1050 nm (Fig. 5g
and Supplementary Fig. 3). Meanwhile, under ~850 nm excita-
tion, H4-PEG-PT also showed an intense anti-Stokes FUCL signal
with a maximum emission peak ~580 nm (Fig. 5h). H4-PEG-PT
readily formed supramolecular assemblies in water with the
average length of 180.0 13 nm and the average width of 48 15
nm as determined by transmission electron microscopy (TEM,
FUCL Cellular localization of 3k-PEG, H4-PEG, and H4-PEG-
PT. Colocalization experiments involving Mito-Tracker Red
(MTR) or Mito-Tracker Green (MTG) were conducted to con-
firm the intracellular distribution of the synthesized compounds.
Specifically, the subcellular localization of 3k-PEG, H4-PEG, and
H4-PEG-PT in 143B cells was determined by co-staining with
Fig. 5i). The NIR-II quantum yield of H4-PEG-PT was 0.1
MTG and then imaged under confocal microscope, while the
0.03% in water. H4-PEG-PT also exhibited high photostability in
subcellular distribution of 3j-PEG was observed by co-staining
PBS and DMEM medium for 60 min (Fig. 5e). Furthermore,
with MTR (Supplementary Movie 1 and Supplementary Movie 2).
fluorescent signals of hydroxyapatite (HA) precipitates were
As shown in Fig. 6a–e, all of the fluorescence images for 3j-PEG,
enhanced57 when HA (10 mg) was incubated with different
3k-PEG, H4-PEG, or H4-PEG-PT with different concentrations
concentrations of H4-PEG-PT (4, 8, 16, 32, and 64 µM) for 4 h.
were found to overlap well with that of commercial mitochondrial
H4-PEG-PT has demonstrated a high affinity for HA in vitro in
dyes (MTR or MTG), suggesting the mitochondria-targeted
ability of those synthesized dyes. Moreover, it was found that the
fluorescent intensity of Mitotracker Green at concentrations
lower than 10 nM was too weak to be detected while 1 nM of H4-
PEG-PT still presented clear and strong fluorescent signals under
the same experimental conditions. NIR-II fluorescent confocal
studies (Fig. 6 and Supplementary Fig. 6) demonstrated that
bone-binding assays while much lower-intensity fluorescent sig-
nals were detected in HA precipitates with various concentrations
of H4 without the targeting ligand PT (Fig. 5b, c). In addition, the
binding ability of H4-PEG-PT to osteosarcoma was further
confirmed by co-incubation of 143B cells with H4-PEG-PT.
Much higher fluorescence was observed in the non-blocking
N
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NHS,EDCl,DMF (dry)
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PEG-PT
CuSO4 .5H2O, sodium ascorbate
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0.2
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800
1000
1200
10
20
30
40
50
60
Wavelength (nm)
µ
Time (min)
i
h
1.0
0.8
0.6
0.4
0.2
0.0
400
500
600
700
Wavelength (nm)
Fig. 5 In vitro evaluation of H4-PEG-PT. a
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mitochondria across the inner membrane and achieved be the first D–A type FUCL thiopyrylium mitochondria-targeted
mitochondria-targeted via the delocalized lipophilic cation thio- fluorophores with highly desirable characteristics including
pyrylium salt instead of the alkyltriphenylphosphonium moiety. mitochondria-targeted and low working concentration.
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In vivo imaging. The 143B mice tumor model was established In vitro photothermal efficacy. Photothermal therapy in the NIR
by orthotopic injection of green fluorescent protein (GFP)- optical window has received growing attention on account of its
transfected 143B cells. Orthotopic tumor growth was monitored convenient and efficient use of NIR light as well as its consider-
by GFP luminescence and X-ray imaging until the proximal able advantages of deeper tissue penetration and higher max-
tibia of the joints were phagocytosed by 143B cells (Fig. 7a). H4- imum permissible exposure to laser58–61. H4-PEG-PT was
PEG-PT was then intravenously injected (200 µg, 200 µL) into exposed to an 808 nm laser to investigate photothermal perfor-
143B tumor-bearing mice. From NIR-II imaging, 143B tumors mance via a mitochondrial pathway at various concentrations.
were clearly discernible from the surrounding background tis- Rapid photothermal heating occurred upon irradiation at
sues from 6 to 48 h (Fig. 7b, 1000 LP, 250 ms). Compared with 1.6 W cm−2 even at the dose of 32 µM (Fig. 8a). When the con-
the normal group, lower fluorescent signals were observed at all centration of H4-PEG-PT was increased to 64 µM, the tempera-
time points in the blocking group. The NIR-II intensity ratios ture increased significantly under laser irradiation by varying the
(T/N) for H4-PEG-PT were shown in Supplementary Fig. 7, and power density (1.4, 1.6, 1.8, and 2.0 W cm−2) as shown in Sup-
its T/N ratio reached 4.57 0.15 at 12 h. The targeting specificity plementary Fig. 10A. Exposure to 808 nm laser at 2.0 W cm−2 for
of H4-PEG-PT for osteosarcoma was confirmed by the blocking 12 min, the temperature of H4-PEG-PT was increased to 57 °C
experiment. After co-injection of free PT peptide (2 mg) with while the temperature of the pure water was increased only
H4-PEG-PT for NIR-II imaging, tumor fluorescent signals were slightly under the same laser irradiation. To further investigate
significantly reduced at all time points. H4-PEG-PT distribution the photostability of H4-PEG-PT, it was subjected to repeated
in major organs at 48 h was evaluated by an ex vivo biodis- heating-cooling cycles for 80 min (Fig. 8b), indicating excellent
tribution study (Supplementary Fig. 8). The predominance of stability under 808 nm laser irradiation (2.0 W cm−2). Then,
liver accumulation suggested that the hepatobiliary system was 143B cells were irradiated with an 808 nm laser for 4 min (1.8 and
the primary site for the clearance of H4-PEG-PT. The half-life of 2 W cm−2) after incubation with different concentrations of H4-
H4-PEG-PT in the blood circulation was assessed by quantita- PEG-PT. Cells were obviously destroyed when the concentration
tive determination of fluorescent intensity in the blood at dif- of H4-PEG-PT reached concentrations of 32 µM or higher
ferent time points, as shown in Supplementary Fig. 9. The time- (Fig. 8c). However, the control groups of H4-PEG-PT without
dependent relative fluorescent intensity in blood was fitted with 808 nm irradiation showed almost 100% cell viability. The pho-
a first-order exponential decay to evaluate the blood half-life. tothermal conversion efficiency of H4-PEG-PT was 18% (η ≈
The equation of the pharmacokinetic curve was y = 0.061*exp 18%) (Supplementary Fig. 10), indicating that H4-PEG-PT could
(−X/0.397) + 0.00994 (R2 = 0.989) and the blood half-life (t1/2
was found to be ~0.4 h. In addition, tumor uptake of H4-PEG- rapidly.
)
convert 808 nm light energy into heat energy efficiently and
PT reached a maximum at 12 h. Histopathological studies were
To explore the underlying mechanisms of H4-PEG-PT under
also carried out by ex vivo NIR-II fluorescent imaging and NIR laser irradiation against 143B cells, its effects on mitochon-
hematoxylin-and-eosin (H&E) tissue staining (Supplementary drial function and apoptosis were investigated (Fig. 8d–f).
Fig. 9).
Mitochondrial functioning was first assessed by determining the
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Fig. 7 In vivo imaging of osteosarcoma. a
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levels of ATP in 143B cells (Fig. 8d). Pretreatment with H4-PEG- combination with 808 nm laser irradiation. Notably, highest
PT or 808 nm irradiation, the levels of ATP were similar to those fluorescence of Cyto c was observed in the group treated with H4-
in control cultures. H4-PEG-PT under NIR irradiation treatment PEG-PT and 808 nm laser irradiation (Fig. 8q, Supplementary
significantly decreased 143B cellular ATP generation to ~5.6% Fig. 11). These results further demonstrated the mitochondrial-
(Fig. 8d). JC-1 assay was further used to analyze mitochondrial targeting photothermal effect of the synthesized NIR-II probes. It
membrane potential (ΔΨm). The ratio of J-aggregates to is well-known that caspase-9 and caspase-3 are important
monomers of H4-PEG-PT (60 μM) or NIR irradiation treatments members of the cysteine-aspartic acid protease family, which
for up to 3 min had no significant decrease. Treatment with H4- regulates cellular apoptosis, and their sequential activation plays a
PEG-PT (60 μM) under NIR irradiation or carbonyl cyanide m- key role in the mitochondria-mediated cell apoptosis pathway63.
chlorophenylhydrazone (CCCP) reduced the ratio of J-aggregates Western blot analysis showed that H4-PEG-PT dramatically
to monomers of 143B tumor cells to 25% and 63%, respectively reduced caspase-3 and caspase-9 protein levels of 143B cells
(Fig. 8e). The change in the permeability of mitochondrial under 808 nm irradiation (1 W cm−2, Fig. 8k). Afterwards, the
membranes is a significant event in the apoptosis process, which expression of catalytically active caspase-3 and cleaved caspase-9
is controlled by mitochondrial permeability transition (MPT). In were significantly increased. Besides, the increase of caspase-3/8/9
our experiment, calcein-AM cobalt assay was used to identify activities was also observed in the H4-PEG-PT group under NIR
MPT. The experimental results showed that, after 143B cells were irradiation by the caspase multiplex activity assay kit (Fig. 8g–i).
treated with H4-PEG-PT under an 808 nm irradiation for 3 min, Our findings further supported that cellular apoptosis of H4-
the calcein fluorescence intensity of 143B cells was much lower PEG-PT was triggered by the intrinsic mitochondrial pathway.
than that of H2O2-induced group (Fig. 8f). These results revealed Upon death stimuli, different apoptogenic factors such as
that H4-PEG-PT under NIR irradiation would cause serious apoptosis-inducing factor (AIF), Endonuclease G (EndoG) and
mitochondrial physiology dysfunction against cancerous 143B second mitochondria-derived activator of caspase/direct IAP
cells, while H4-PEG-PT or NIR irradiation treatment alone could binding protein with low pI (Smac/Diablo) are released during
not damage cells.
the early stages of apoptosis. These three characteristic proteins of
During apoptosis, Cytochrome c (Cyto c) was released from mitochondrial apoptosis were investigated by western blot. From
mitochondria to the cytosol and further activated the caspase- Fig. 8j, the AIF and EndoG expression levels in the H4-PEG-PT
dependent apoptosis pathway, which committed the cell to the group with NIR irradiation were significantly increased compared
death process62. In order to further understand the mechanisms with that of the same group without NIR light irradiation, control
of cell toxicity and mitochondrial-targeted properties of 3k-PEG, group or NIR laser group. Western blotting results indicated that
H4-PEG, and H4-PEG-PT, tests of subcellular distribution of there was burst release of AIF, EndoG and Smac/DIABLO from
Cyto c from the immunofluorescence technique and expression mitochondria into the cytoplasm with H4-PEG-PT under
levels of caspase-3 and caspase-9 as well as their activated irradiation, suggesting the disruption of mitochondria and
fragments were carried out by western blotting analysis. 3k-PEG, cellular metabolism. NIR light-triggered ROS generation of H4-
H4-PEG, and H4-PEG-PT were well retained within mitochon- PEG-PT under 808 nm laser irradiation was then evaluated by
dria even after cell fixation and permeabilization at 1 nM, and red 1,3-diphenylisobenzofuran (DPBF), dichloro-dihydro-fluorescein
fluorescence from Cyto c was clearly observed in all cells in diacetate (DCFH-DA) and MitoSOX assay (Fig. 8l–p, r and
8
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32 µM
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Water
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Time of irradiation (min)
Control
Concentration (μM)
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Caspase 3
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Caspase 9
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21
H4-PEG-PT (20 μg) + DPBF
H4-PEG-PT (50 μg) + DPBF
H4-PEG-PT (100 μg) + DPBF
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Irradiation time (min)
H4-PEG-PT
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DPBF
H4-PEG-PT(100 μg) + DPBF
H4-PEG-PT(20 μg) + DPBF
H4-PEG-PT(50 μg) + DPBF
0 min
2 min
4 min
6 min
8 min
10 min
12 min
14 min
0 min
2 min
4 min
6 min
8 min
10 min
12 min
14 min
3.0
2.5
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2 min
4 min
6 min
8 min
10 min
12 min
14 min
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2.0
1.5
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0.5
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8 min
10 min
12 min
14 min
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300
400
500
600
300
400
500
300
400
500
600
300
400
500
Wavelength (nm)
Wavelength (nm)
Wavelength (nm)
Wavelength(nm)
q
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Mitotracker green
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H4-PEG-PT
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H4-PEG-PT
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H4-PEG-PT
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H4-PEG-PT
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Supplementary Fig. 12)64,65. No significant amount of ROS and late-stage apoptotic versus necrosis. After an 808 nm laser
in vitro was observed in any group of H4-PEG-PT under 808 nm irradiation for 3 min, not only FITC signals were obviously
irradiation. This result is a positive finding regarding photo- emerged from the cell membrane but also PI signals were visible
thermal application of H4-PEG-PT since the concomitant in the group of H4-PEG-PT with laser irradiation. Then Hochest/
production of ROS during PTT has been proven to trigger PT staining assay was undertaken to examine the engagement of
detrimental side-effects to cells66,67. FITC-annexin V and apoptosis VS necrosis or mixed paths. 143B cells treated with
propidium iodide (PI) assay is usually used to identify early H4-PEG-PT under irradiation clearly demonstrated staining
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indicative of apoptosis (bright-blue particles). Because annexin V cm−2) continuously for 8 min after 12 h i.v. post-injection. The
could stain early-stage apoptotic cells while PI stains dead cells tumor surface temperature rapidly increased from 33.0 to 67.8 °C
or late-stage apoptotic cells, and Hochest could stain the nuclei in 8 min (Fig. 9b), while the normal skin exhibited only a slight
of apoptotic cells with a bright-blue fluorescence, these two temperature increase (Fig. 9a). Tumor sizes and body weights
results showed that the cells treated with H4-PEG-PT under were measured every 2 d as shown in Fig. 9c–f. No significant
irradiation were engaged with both apoptosis and necrosis body weight loss was observed in any group during the photo-
(Fig. 8s, t).
thermal therapy process. After 14 d of treatment, mice were
sacrificed and major organs collected for histopathological studies
with H&E staining. No obvious injury or inflammatory lesion was
observed in any major organ, as shown in Supplementary Fig. 13.
Overall, both in vitro and in vivo photothermal studies showed
that H4-PEG-PT efficiently converts 808 nm laser light into heat
with negligible production of ROS, and exhibits effective photo-
thermal efficacy against 143B tumors without a significant
adverse effect.
In vivo photothermal therapy. Encouraged by the findings of
excellent photothermal properties in vitro, the effects of in vivo
photothermal therapy in 143B tumor-bearing mice under an 808
nm NIR laser were also studied. H4-PEG-PT (200 μL, 400 μg) or
PBS (200 μL) were intravenously injected into 143B tumor-
bearing mice respectively (n = 4 per group). The right tumor
regions of the mice were irradiated with an 808 nm laser (1.5 W
10
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Discussion
Zhongnan Hospital of Wuhan University (Hubei, China), purchased from
American Type Culture Collection (ATCC). 143B, L929, and HepG2 cells were
cultured with Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Grand Island,
NY, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Grand Island,
NY, USA) and 1% (v/v) penicillin-streptomycin at 37 °C in a humidified incubator
containing 5% CO2. The orthotopic 143B tumor models were established by intra-
tibial injection of 143B cells (∼5 × 106 in 80 μL of PBS) into the left leg of 6-week-
old female Balb/c nude mice (Beijing Vital River Laboratory Animal Technology
Co., Ltd.) under anesthesia using isoflurane. The tumor-bearing mice were imaged
when the tumor volume reached 400–800 mm3 (about 4–6 weeks after
inoculation).
The fluorophores in this work composed of a series of novel red-
shifted thiopyrylium cores from heterocycles 7a and 7b. After
theoretical calculation and optical structure–property relationship
study, D–A thiopyrylium dye H4 derived by the conjugation with
the electron-rich thiophene spacer and N,N-dimethylaniline
substituent was discovered, and exhibited dramatic bathochromic
shifts of π–π* transitions in the NIR-II wavelength region
with emission wavelengths at ~1100 nm. In addition, a water-
soluble and biocompatible upconversion NIR-II probe H4-PEG-
PT was successfully constructed via amidation and click chem-
istry based on the thiopyrylium dye H4 and an osteosarcoma-
targeting oligopeptide PPSHTPT (PT), demonstrated specifically
mitochondria-targeted imaging of osteosarcoma in vitro and
in vivo. Moreover, the synthesized fluorophore functioned as a
photothermal agent by actively targeting tumor tissues and
mitochondria to selectively kill cancer cells. It has been demon-
strated that D–A thiopyrylium dyes such as H4-PEG-PT could
serve as effective mitochondrial-targeting photothermal agents
without ROS effects and achieve markedly enhanced antitumor
efficacy. In addition, H4-PEG-PT showed no observable toxicity
without laser irradiation, indicating their safety. Besides, H4-
PEG-PT showed super mitochondria-targeted FUCL bioimaging
in live cancer cells. Notably, the majority of commercially avail-
able mitochondrial dyes are suited for live samples. We developed
a small-molecule imaging agent that can quickly and effectively
image mitochondria in live or fixed cell samples with subcellular
resolution at a very low working concentration (1 nM). Western
blot analysis showed that both H4-PEG and H4-PEG-PT dra-
matically reduced caspase-3 and caspase-9 protein levels, which
further suggested that their photothermal effect was triggered by a
mitochondria-induced apoptosis pathway. Thus, combining
favorable clearance with biocompatibility, excellent photothermal
therapy, as well as NIR-II tumor-imaging ability, H4-PEG, and
H4-PEG-PT hold great promise for clinical translation. Impor-
tantly, chemical versatility makes H4-PEG and H4-PEG-PT sui-
table for various modes of tumor detection/imaging/image-
guided surgery with high safety profile, subcellular resolution, and
deep tissue penetration, and therapy by conjugating with different
targeting ligands.
Cellular toxicity of H4-PEG-PT by MTT assay. In vitro cytotoxicity studies of
H4-PEG-PT on 143B cells and L929 cells were performed by using an MTT
cytotoxicity assay. Cells were seeded in 96-well plates at densities between 5000 and
10,000 cells per well. The cells were incubated with 100 μL of fresh cell media
containing H4-PEG-PT for 48 h. The final concentrations of H4-PEG-PT in the
culture medium were fixed at 0, 2, 4, 8, 16, and 32 μM in the experiment. Then
MTT (0.5 mg/mL) was added into each well (10 μL) in order to convert into
formazan (purple). And the microplate was incubated at 37 °C for 4 h. After that,
using DMSO to resolubilize the formazan. The absorbance was measured at 490
nm by Perkin Elmer VICTOR X4. The following formula was used to calculate the
viability of cell growth: Cell viability (%) = (mean of absorbance value of treatment
group/mean of absorbance value of control) × 100.
Extracellular and intracellular ROS detection. The generation of extracellular
ROS was measured with a DPBF probe. Briefly, different concentrations of H4-
PEG-PT (20, 50, 100 μg, containing 100 μl EtOH solution) were added into 3 mL of
ethanol solution containing DPBF (10 mM), and then the solution was kept in the
dark with magnetic stirring and irradiated by an 808 nm NIR laser for various time
periods (0, 2, 4, 6, 8, 10, 12, and 14 min). The spectrum was collected at 410 nm
absorption.
Confocal laser scanning microscope (CLSM). 143B (50,000 cells) were seeded in
round discs and incubated in 1 mL of DMEM medium (pH = 7.4) containing 10%
FBS for 24 h. After replacing the medium with 1 mL of fresh medium, 3k-PEG, H4-
PEG, and H4-PEG-PT were added, respectively, and the cells were allowed to
incubate for 6 h. After removing the medium and subsequently washing with PBS
buffer (pH = 7.4) thrice, mitotracker green was added to stain the mitochondria for
30 min. The cells were viewed under a FV1200 CLSM (Olympus) and Leica SPE.
Detection of mitochondrial ROS formation. 143B cells were seeded on 15 mm
confocal dishes and allowed to stabilize for 24 h. The cells were then incubated with
medium containing H4-PEG-PT (64 μM) for 6 h and washed three times with PBS.
After 30 min incubation with or without N-acetylcysteine (5 mM) in 1 mL culture
medium, the cells were irradiated with a 808 nm laser (1.0 W cm−2) for 5 min. The
amount of ROS generated was determined using the MitoSOX (1 µM) staining kit
according to the manufacturer’s instructions (Invitrogen). The results in 143B cells
were monitored using a confocal fluorescence microscope. Fluorescence was col-
lected using an excitation wavelength of 488 nm and recording the emission at
550–650 nm (red).
Taken together, a small-molecule organic fluorophore with
cancer cell mitochondrial targeting, NIR-II upconversion ima-
ging, and photothermal effects was developed. The design and
synthesis of H4-PEG-PT will enrich and broaden the field of
NIR-II and FUCL-based probes. It is hoped that this integrated
small-molecule-based theranostic platform may become a prac-
ticable strategy for simultaneous cancer diagnostics, organelle
targeting, therapeutics, and fluorescence-guided surgery, as well
as postoperative monitoring.
Subcellular distribution of Cytochrome c (Cyto c). 143B cells were seeded onto
an 8-well Chambered Coverglass system at a density of 2 × 104 cells per well and
cultured for 24 h. 3k-PEG, H4-PEG, and H4-PEG-PT (32 μM) were added. To
assess the subcellular distribution of Cyto c, the 143B cells treated with and without
808 nm laser irradiation were further incubated for additional 48 h and then 200
nM MitoTracker Green was added to stain the mitochondria. Afterward, the cells
were fixed with 4% paraformaldehyde and then blocked with 1% BSA, 22.52 mg/
mL glycine in PBST (PBS + 0.1% Tween 20) for 1 h. Subsequently, the cells were
examined via the immunofluorescence technique using primary anti-Cyto c
monoclonal antibody (Abcam, 1:1000) and secondary Alexa-647-conjugated goat
anti-rabbit antibody (Life Technologies, 1:300). After staining with hoechst 33342
(Thermofisher Scientific, 1:10,000), the subcellular distributions of Cyto c in 143B
cells under different treatments were observed by confocal microscopy (FLUO-
VIEW FV1000, Olympus).
Methods
Information includes descriptions of thiopyrylium tetrafluoroborate 7, 3a–3k. H4,
3j-PEG-H4-PEG, H4-PEG-PT design and synthesis, bioconjugation, and pur-
ification are described in detail in SI Appendix.
Quantum yield measurements of H4 and H4-PEG-PT. In order to measure the
relative quantum yield of H4 in chlorobenzene, IR-26 (0.5%) in 1,2-dichloroethane
(DCE) was chosen as the reference. The quantum yield was calculated in the
following formula:
Western blot analysis and antibodies. Protein extracts were prepared using
modified radioimmunoprecipitation assay lysis buffer (50 mM Tris-HCl pH 7.4,
150 mM NaCl, 1% NP-40 substitute, 0.25% sodium deoxycholate, 1 mM sodium
fluoride, 1 mM Na3VO4, 1 mM EDTA), supplemented with protease inhibitor
cocktail (Cell Signaling) and 1 mM phenylmethanesulfonyl fluoride or complete
Mini protease inhibitor tablets (Roche). Equal amounts of protein, as determined
with the bicinchoninic acid (BCA) protein assay kit (Pierce/Thermo Scientific)
according to the manufacturer’s instructions, were resolved on SDS-PAGE gels and
transferred to nitrocellulose or polyvinylidene difluoride membrane. The blots were
blocked with 5% nonfat dry milk or 3% BSA in TBST (50 mM Tris-HCl, pH 7.4
and 150 mM NaCl, and 0.1% Tween 20) and then incubated with appropriate
Slopesample n2sample
ð1Þ
QYsample ¼ QYIR26
´
´
SlopeIR26
n2IR26
Five different concentrations were chosen at or below OD 0.1 and the integrated
fluorescence was plotted against absorbance for both IR-26 and H4. Comparison of
the slopes led to the determination of the quantum yield of H4.
Cell line and animal model. L929 and HepG2 cells were purchased from the China
Center for Type Culture Collection (CCTCC). 143B cells were obtained from
N A T U R E C OM M U N I C A T I ON S |
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11

A
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N A T U R E C O M MU N ICA T IO N S | h t t p s : / / d o i .o r g / 1 0 .1 0 3 8 / s4 1 4 6 7 - 0 2 0 - 1 9 94 5 - w
primary antibodies. Signals were detected with horseradish peroxidase (HRP)-
conjugated secondary antibodies and an enhanced chemiluminescence (ECL)
cell viability after laser irradiation. 143B cells were seeded in a 96-well plate at a
density of 5000 or 6000 cells per well for 12 h in a standard cell culture atmosphere.
The cells were added with H4-PEG-PT at its concentration of 8, 16, 32, and 64 μM.
After incubation for 6 h, a part of cells in the wells was irradiated with an 808 nm
NIR laser (1.8 and 2.0 W cm−2) for 0 and 4 min. Afterward, the cell viability of
143B was measured using a standard MTT assay.
detection system (Pierce). For biochemical analyses, the following antibodies were
used: Recombinant Anti-Smac/Diablo antibody (Abcam, 1:1000, ab32023); anti-
EndoG antibody (Abcam, 1:1000, ab9647); Recombinant Anti-AIF antibody -
Mitochondrial Marker (Abcam; 1:1000, ab32516); Recombinant Anti-Cytochrome
C antibody, (Abcam, 1:1000, ab133504), Secondary Alexa-647-conjugated Goat
anti-Rabbit Antibody (Life Technologies, 1:300, A27040); HRP conjugated Goat
Anti-Rabbit IgG H&L (HRP) (Abcam, 1:2000, ab6721); anti-caspase-3 (Cell Sig-
naling Technology, 1:1000, #9662S), anti-cleaved caspase-3 (Cell Signaling Tech-
nology, 1:1000, #9664S), anti-cleaved caspase-9 (Cell Signaling Technology, 1:1000,
#9509S), anti-caspase-9 (Cell Signaling Technology, 1:1000, #9504S), and anti-beta
actin antibody (Abcam, 1:2000, ab179467).
In vivo photothermal therapy. For photothermal therapy, the nude mice bearing
subcutaneous 143B tumors were randomly divided into three groups (n = 4 per
group): (a) PBS injection group with 808 nm laser, (b) H4-PEG-PT injection group
(200 μL, 400 μg per mouse), (c) H4-PEG-PT injection group (200 μL, 400 μg per
mouse) with 808 nm laser. For groups (a) and (b), after 12 h post-injection, right
tumors were irradiated by the 808 nm laser at a power density of 1.5 W cm−2
(CAUTION: this power density may cause skin hurt!) for 8 min. During the
treatment, the tumor lengths and widths were measured by a digital caliper every
2 days for 2 weeks. The tumor volume (mm3) was calculated by the formula: tumor
volume = length × (width)2/2.
Detection of the tumor by bioluminescence imaging. Tumor-bearing mice that
implanted 143B cells transfected with green fluorescent protein (GFP) were sub-
jected to anesthesia with 10% chloral hydrate, followed by whole-body real-time
bioluminescence imaging with In Vivo Xtreme imaging system (XTEREME BI,
Bruker). BLI signal was collected using the following settings: 480 nm excitation
and 535 nm emission; exposure time: 1 s; Bin: 1 × 1 pixels; FOV: 7.2 cm; fStop: 1.1;
focal plane: 0 mm.
Ethics statement. All animal studies were performed in accordance with the
Guidelines for the Care and Use of Laboratory Animals of the Chinese Animal
Welfare Committee and approved by The Institutional Animal Care and Use
Committee (IACUC), Wuhan University Center for Animal Experiment,
Wuhan, China.
In vitro bone-targeted assay. In order to evaluate the binding ability of H4-PEG-
PT and H4, various amount of the probes (2, 4, 8, 16, 32, and 64 μM, 500 μL) were
incubated with HA (10 mg), respectively, for 4 h at room temperature under mild
stirring. Then, the mixture was centrifugated at 1200 rpm for 10 min and washed
with 8-fold excess of PBS three times. The NIR-II images were collected by a Series
II 900/1700 In Vivo Imaging System (Suzhou NIR-Optics Co., Ltd., China).
Data analysis. All statistical data were expressed as means standard deviation.
Statistical differences were analyzed with Student’s t test to compare the differences
between two groups. Quantum chemistry calculation was performed using Gaus-
sian 09 program (Revision D.09) software, Image J (1.51j8) was used to analyze the
NIR-II images. Origin 9 was used to analyze the spectrum images. All statistical
analyses were performed by Graphpad Prism 8.0 and SPSS 20.0.
In vivo NIR-II fluorescence imaging of tumors. Two-hundred microliter portions
of H4-PEG-PT (200 μg) was injected intravenously into tumor-bearing nude mice.
NIR-II fluorescence signals were acquired using a two-dimensional InGaAs array
(Suzhou Optics) and thus fluorescence images were collected. Excitation light was
provided by the 808 nm diode laser. The emitted light from animals was filtered
through a 1000-nm long-pass filter for NIR-II imaging coupled with an InGaAs
camera. The exposure time for all images was 250 ms.
Reporting summary. Further information on research design is available in the Nature
Research Reporting Summary linked to this article.
Data availability
The authors declare that all data needed to evaluate the conclusion of this work are
presented in the paper and the Supplementary Information. Additional data related to
this paper may be requested from the authors. Source data are provided with this paper.
Ex vivo biodistribution analysis. Ex vivo fluorescence imaging of organs and
tissues were used Suzhou Optics NIR-II fluorescence imaging system with an
InGaAs camera at a power density of ~80 mW/cm2 at 808 nm laser diode irra-
diation. Forty-eight hours after injection of H4-PEG-PT for NIR-II imaging, 143B
xenograft mice were sacrificed, the NIR-II images of major organs were collected.
Received: 29 November 2019; Accepted: 9 November 2020;
Histological analysis. Major organs and tumors were taken from the tumor-
bearing mice and fixed with EDTA/formalin solution. After embedment and sec-
tion, tissue samples were further stained with hematoxylin and eosin and subse-
quently imaged using a NIKON Eclipse ci microscope with ×40 and ×200
magnification.
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Acknowledgements
The work was supported by the National Key R&D Program of China
(2020YFA0908800), NSFC (81773674, 81573383), Shenzhen Science and Technology
Research Grant (JCYJ20190808152019182), Hubei Province Scientific and Technical
Innovation Key Project, National Natural Science Foundation of Hubei Province
(2017CFA024, 2017CFB711), the Applied Basic Research Program of Wuhan Municipal
Bureau of Science and Technology (2019020701011429), Tibet Autonomous Region
Science and Technology Plan Project Key Project (XZ201901-GB-11), the Local Devel-
opment Funds of Science and Technology Department of Tibet (XZ202001YD0028C),
Project First-Class Disciplines Development Supported by Chengdu University of Tra-
ditional Chinese Medicine (CZYJC1903), Health Commission of Hubei Province Sci-
entific Research Project (WJ2019M177, WJ2019M178), the China Scholarship Council,
and the Fundamental Research Funds for the Central Universities.
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Author contributions
X.H. and Y.X. designed the research. Y.X., H.Z., X.Z., A.L., L.T., and W.Z. performed
research. X.H., Y.X., H.Z., X.Z., A.L., L.T., W.Z., X.M., W.H., Q.F., H.D., L.D., Y.L., and
Z.D. contributed analytic tools and data analyses. X.H., H.Z., Y.X., and X.Z. wrote
the paper.
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for selective detection of Hg2+. Sens. Actuators, B 188, 847–856 (2013).
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thiopyran rings. J. Jpn. Soc. Color. Mater. 78, 12–16 (2005).
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Competing interests
The authors declare no competing interests.
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N A T U R E C O M MU N ICA T IO N S | h t t p s : / / d o i .o r g / 1 0 .1 0 3 8 / s4 1 4 6 7 - 0 2 0 - 1 9 94 5 - w
Additional information
Supplementary information is available for this paper at https://doi.org/10.1038/s41467-
Open Access This article is licensed under
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020-19945-w.
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Correspondence and requests for materials should be addressed to X.H. or Y.X.
Peer review information Nature Communications thanks Xiaoyuan Chen and the other
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