Azide-Dye Unexpected Bone Targeting for Near-Infrared Window II Osteoporosis Imaging

Article abstract of DOI:10.1021/acs.jmedchem.1c00839

Azide is an important chemical functional group and has been widely used in chemical biology. However, the impact of azide on the in vivo behaviors of compounds has been rarely studied. Herein, azide was introduced into a fluorescent dye for the near-infr

Full text of DOI:10.1021/acs.jmedchem.1c00839

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Azide-Dye Unexpected Bone Targeting for Near-Infrared Window II
Osteoporosis Imaging
Xiaoqing Zhang,⊥ Aiyan Ji,⊥ Zhiming Wang, Hongyue Lou, Jiafeng Li, Lingling Zheng, Yujing Zhou,
Chunrong Qu,* Xingdang Liu,* Hao Chen,* and Zhen Cheng*
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ABSTRACT: Azide is an important chemical functional group
and has been widely used in chemical biology. However, the
impact of azide on the in vivo behaviors of compounds has been
rarely studied. Herein, azide was introduced into a fluorescent dye
for the near-infrared window two (NIR-II) bone imaging.
Specifically, we designed and synthesized the small-molecule
NIR-II dyes, N3-FEP-4T capped with azide and FEP-4T without
azide capping. In vitro assays revealed that N3-FEP-4T showed 5-
and 5.6- times higher hydroxyapatite accumulation and macro-
phage uptake than those of FEP-4T, respectively. Moreover, N3-
FEP-4T displayed higher bone uptakes and much better bone
NIR-II imaging quality, demonstrating the specific bone-targeting
ability of the azide-containing probe. N3-FEP-4T was then further successfully used for osteoporosis NIR-II imaging. Overall, our
study provides insights into the impact of azide on the in vivo behavior of azide-containing compounds and opens a new window for
biological application of azide.
window one, NIR-I), imaging at 1000−1700 nm (near-infrared
window two, NIR-II) has many significant advantages such as
lower autofluorescence background, less light absorption and
scattering in organisms, deeper tissue penetration capability,
higher image contrast, and sensitivity, making it a powerful tool
for bioimaging and studying the in vivo behaviors of
compounds.19−27
Fluorescent bone-imaging small molecular probes highly rely
on phosphonate-related bone targeting ligands.28−33 Moreover,
a class of fluorescent probes with structure-inherent targeting
has been reported for bone-specific imaging. For example,
squaraine-based rotaxane and heterocyclic polymethine cya-
nines possess both bone imaging and targeting properties.34
However, all the above bone imaging fluorophores are used for
NIR-I imaging, which suffers disadvantages including limited
tissue penetration, low resolution, and high background.35−37
Recently, we have successfully developed several bone-targeting
nanoprobes such as RENPs@DSPE-mPEG and AuNCs for
NIR-II imaging, which are both inorganic nanoparticles with
unknown long-term toxicity.38,39 Consequently, it is of great
INTRODUCTION
■
Azide is an important chemical group and can be converted into
various functionalities for broad applications. It has been widely
used in chemical biology and fluorescent labeling. Moreover, as
an essential active intermediate, organic azide plays an important
role in the synthesis of natural and biologically active
compounds and drugs.1−3 For example, organic azides are
essential components in the azide−alkyne “click” reaction.4−7
However, the impact of azide to the in vivo behaviors of
compounds has been rarely studied. In medicinal chemistry, a
nucleoside analogue named 3′-azido-3′-deoxythymidine (AZT,
zidovudine, retrovir) that contains an azide group has been
demonstrated to be an effective treatment for human
immunodeficiency virus (HIV) and approved by the Food and
Drug Administration (FDA).8,9 Interestingly, zidovudine causes
bone marrow toxicity,10 and further in vivo biodistribution study
verifies that 14C-AZT accumulates in the bone.9 Whereas its
counterpart drug, stavudine that does not contain an azide group
(Scheme 1), has not shown bone-related toxicity.11 These
findings inspire us to hypothesize that azide groups may render
AZT bone accumulation capability.
Received: May 9, 2021
Optical imaging has been widely used in biomedical research
because of many attractive properties such as nonradiation,
noninvasiveness, high resolution, high signal-to-noise ratio
(SBR), real-time imaging, low cost, and various function
fluorescent probes available.12−18 Compared with fluorescence
imaging in the traditional 700−900 nm region (near-infrared
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Scheme 1. Chemical Structure of Natural Deoxythymidine and Two Anti-HIV Drugs (on the Left); the 3D Model of N3-FEP-4T
(on the Right)
significance to develop novel NIR-II bone-targeting small-
molecule fluorophores.
the photostability of N3-FEP-4T in the PBS was lower than that
of FEP-4T, which is likely attributed to the azide group’s relative
high reactivity in electrophilic reagent-rich solutions, which is
hydroxyl (OH−) and phosphate (HPO32−) in PBS buffer.41
Therefore, we conducted a further investigation about the
stability of N3-FEP-4T and ICG in vitro. The result in Figure 1d
shows that N3-FEP-4T had superior photostability in the mice
serum and was suitable for in vivo imaging. The quantum yields
(QYs) of N3-FEP-4T and FEP-4T were determined, respec-
tively, to be 1.1 and 3.6%, using IR-26 as a reference (with a
nominal QY = 0.5%, Figure 1e).42 NIR-II fluorophores’ high QY
enables many exciting NIR-II medical imaging possibilities in
short exposure time, high imaging speed, and deep penetration
depth.
Calcium-Binding Property and Cell-Uptake Imaging. A
high affinity for binding to bone tissue minerals such as
hydroxyapatite (HA) is an essential criterion for bone targeting
imaging.33,38 Therefore, in vitro binding affinity of N3-FEP-4T
and FEP-4T toward inorganic compounds, including HA,
calcium carbonate (CC), calcium oxalate (CO), calcium
pyrophosphate (CPP), and manganese dioxide (MnO2) was
evaluated. The binding affinity of both probes toward inorganic
compounds is in the following order: HA > CPP > CO > CC >
MnO2 (Figure 2a,b). These results indicated that N3-FEP-4T
and FEP-4T bind more strongly and selectively to calcium
phosphate-containing bone minerals than other inorganic
compounds such as CO, CC, and MnO2, which do not contain
calcium phosphates. Moreover, it was found that N3-FEP-4T
displayed higher binding capability with HA than that of FEP-
4T, and the fluorescence intensity of the two groups showed
significant difference (P < 0.001). Considering that FEP-4T has
3 times higher QY (3.6%) than N3-FEP-4T (1.1%), N3-FEP-4T
showed ∼5 times higher HA accumulation than FEP-4T.
Encouraged by the above in vitro calcium-binding results, a
further test was performed at the cellular level. Three cell lines
related to bone metabolism, including embryonic fibroblasts
(3T3), macrophages (RAW264.7), and osteosarcoma (143B)
were selected for cellular uptake imaging.43−45 As shown in
Figure 2c,d, fluorescence signals were detected in three types of
the cell after 15, 30, and 60 min of incubation. The fluorescence
intensity of the two probes with macrophages at 60 min showed
a significant difference (P < 0.001). Considering that FEP-4T
has 3 times higher QY (3.6%) than N3-FEP-4T (1.1%), N3-FEP-
4T displayed 5.6 times higher uptake in RAW264.7 cells than
that of FEP-4T at 60 min (Figure 2e). Among the three cell lines,
macrophage cells’ fluorescent intensity reached much higher
than the other two types (3T3 and 143B) after 30 min of
incubation, suggesting that macrophage cells could more easily
Herein, based on the hypothesis that azide groups may equip
compounds bone-targeting capacity, we designed and synthe-
sized two novel NIR-II fluorophores, named N3-FEP-4T
(capped with an azide group) and FEP-4T (without an azide
group) (Figure 1a). After a series of experiments, including
optical characterization, calcium binding, cell imaging, in vivo
imaging, and ex vivo imaging, we have discovered that N3-FEP-
4T shows high bone-targeting property and is suitable for bone
imaging. Under the 808 nm laser excitation, the skeletal system,
such as the sternum, tibia, femur, and spine can be clearly
observed in the N3-FEP-4T based NIR-II imaging. Furthermore,
N3-FEP-4T has been successfully used for osteoporosis imaging,
which represents the first in vivo NIR-II osteoporosis imaging
study to the best of our knowledge. The fluorophore N3-FEP-4T
equipped with an azide can be used for real-time, long-term
observation of the whole skeletal system and help us monitor
bone-related diseases’ progress in preclinical biomedical
research.
RESULTS AND DISCUSSION
■
Design, Synthesis, and Characterization of the NIR-II
Probes. We designed and synthesized two novel donor−
acceptor−donor fluorescent dyes, N3-FEP-4T with azide and
FEP-4T. The synthetic routes are shown in Figure 1a. The key
steps used to assemble the core structure of the target
compounds included the Suzuki cross-coupling reaction, zinc
reduction, and ring closure.40 The products were characterized
and confirmed by nuclear magnetic resonance (NMR),
including 1H NMR, 13C NMR, and matrix-assisted laser
desorption ionizing time-of-flight mass spectrometry
(MALDI-TOF-MS) (Supporting Information Figures S1−S3).
The purity of both final compounds was 95% or higher, and it
was determined by high-performance liquid chromatography
(Supporting Information Figure S14). Also, some key properties
of the two probes are shown in Figure 1b. The absorbance
spectrum and fluorescent emission spectrum of the two dyes in
deionized water were almost identical due to their highly similar
structure (Figure 1c). Besides, different organic solvents have
minimal effect on the absorbance spectrum and fluorescent
emission spectrum under the 808 nm excitation for both N3-
FEP-4T and FEP-4T (Supporting Information Figure S4).
Photostability of N3-FEP-4T, FEP-4T, and indocyanine green
(ICG) in 1× phosphate buffered saline (PBS) was evaluated
under continuous 808 nm exposure for 1 h at a power density of
0.102 W cm−2 (Figure 1d). Compared with ICG, N3-FEP-4T,
and FEP-4T exhibited much higher photostability. Interestingly,
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Figure 1. Synthesis and characterization of N3-FEP-4T and FEP-4T NIR-II fluorophores. (a) Synthetic route of N3-FEP-4T and FEP-4T. (b)
Physicochemical properties of N3-FEP-4T and FEP-4T. (c) Normalized absorption and fluorescent emission spectrum of N3-FEP-4T and FEP-4T in
deionized water. (d) Photostability of N3-FEP-4T, FEP-4T, ICG in 1× PBS, N3-FEP-4T in NMS (normal mouse serum), and ICG in NMS under
continuous 808 nm exposure for 1 h at a power density of 0.102 W cm−2. (e) Plot of the integrated fluorescence spectrum of N3-FEP-4T and FEP-4T at
five different concentrations. Linear fits were used to calculate QY by comparing the slopes to reference IR-26 (QY = 0.5%).
uptake the fluorophores. The fluorescence intensity of N3-FEP-
4T with macrophages showed a significant difference separately
compared with embryonic fibroblasts and osteosarcoma at 60
min (P < 0.001). Compared to the two fluorophores’ cell
imaging results, macrophages showed a higher intake of N3-FEP-
4T, which may lead to a higher bone marrow uptake. The result
was highly consistent with the previous study that some
fluorescent probes are known to be readily taken up by
undifferentiated monocytes.38
The chemical and biological activity of azide may cause the
HA and RAW 264.7 cell accumulations. There are reports that
azide compounds can easily react with the phosphine group,
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Figure 2. In vitro calcium salt binding specificity and cell-binding characteristics. (a) Calcium-binding fluorescence image of N3-FEP-4T and FEP-4T.
(b) Quantitative fluorescent signals of N3-FEP-4T and FEP-4T bound inorganic compounds (n = 3 per group). (c) Cell binding fluorescence image of
N3-FEP-4T and FEP-4T. (d) Quantitative fluorescent signals of N3-FEP-4T and FEP-4T bound cells (n = 3). (e) N3-FEP-4T/FEP-4T accumulation
ratio of three types of cells at different times. All the NIR fluorescence images have identical exposure and normalizations. Abbreviations: HA =
hydroxyapatite; CPP = calcium pyrophosphate; CO = calcium oxalate; CC = calcium carbonate; and MnO2 = manganese dioxide. The detailed
imaging parameters for each image are listed in Table S2 (Supporting Information).
which was extensive existence in the HA.46,47 Meanwhile, azide
has some unique biological activities that are widely used for cell
metabolism inhibition.48 Compounds like this are usually easily
detected and phagocytized by macrophages.
reduced photon scattering, tissue absorption, and biological
autofluorescence result in the enhanced SBR for in vivo imaging.
Therefore, a 1250 nm long-pass (LP) filter was used for in vivo
imaging experiments. The N3-FEP-4T and FEP-4T were
intravenously injected (200 μL in 1× PBS, 12 mg kg−1) into
the tail of BALB/c nude mice (n = 3 per group). From NIR-II
imaging data shown in Figure 3a−d, Supporting Information
Figures S5 and S6, the N3-FEP-4T provided a higher sensitivity
and SBR in the normal bones, including the spine, sternum,
ilium tibia, and femur, compared with FEP-4T. The SBR of the
spine was obtained by the mean fluorescent intensity, using the
surrounding soft tissue (the white dotted-circle area in Figure
3a,c) as the background (n = 3). The SBR of N3-FEP-4T was 1.1
right after injection and reached a peak of 3.3 at 24 h after
injection (Figure 3e). Also, the SBR of FEP-4T was 0.98 right
after injection and reached a height of 1.1 at 24 h after injection.
The SBR of FEP-4T was much smaller than N3-FEP-4T by
almost all-time points (Figure 3e). NIR-II imaging also revealed
that the spine was easier to be recognized in the fluorescence
images of N3-FEP-4T than FEP-4T. Further quantitative
analysis results were found to be correlated with the above
fluorescence imaging results. Besides, for the mouse injected
with N3-FEP-4T, a vertical analysis spanning different thoracic
Macrophages exist in two major polarization states, classically
activated macrophages (M1) and alternatively activated macro-
phages (M2). Two groups, Gong et al. and Zhang Y et al., have
demonstrated that M2-polarized macrophages can stimulate
mesenchymal stem cells, precursor osteoblast cells, into mature
osteoblasts and increase bone mineralization in vitro.49,50 When
osteoporosis occurs, there is an imbalance between bone
formation and resorption, closely associated with osteoclasts
and osteoblasts.51 The ratio and number of M2/M1 macro-
phages may be changed under the circumstances, while further
in vivo and clinical studies are needed to elucidate the process of
change. Some studies indicate that M2-polarized macrophages
have higher endocytosis levels and may take up small particles
rapidly, while M1-polarized macrophages have a decrease in
particle uptake.52 Therefore, the accumulation efficiency of N3-
FEP-4T may be different in different types of macrophages,
which needs further study to verify.
In/Ex Vivo NIR-II Bone Imaging for Normal Mice. The
increase of the NIR-II fluorescence emission wavelength shows
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Figure 3. In/ex vivo bone-specific imaging using N3-FEP-4T and FEP-4T NIR-II fluorophores in mice. (a) Real-time NIR-II fluorescence imaging of
BALB/c mice (n = 3) by N3-FEP-4T in the prone position. White dotted circle: soft tissue. Scale bar, 1 cm. (b) Partially enlarged view of the hip in (a).
Scale bar, 1 mm. (c) Real-time NIR-II fluorescence imaging of BALB/c mice (n = 3) by FEP-4T in the prone position. White dotted circle: soft tissue.
Scale bar, 1 cm. (d) Partially enlarged view of the hip in (c). Scale bar, 1 mm. (e) Spine-soft tissue fluorescence ratio of N3-FEP-4T and FEP-4T.
Vertical analysis [along the red line in (a)] (f) and horizontal analysis [along the yellow line in (a)] (g) for the spine and the Gaussian fit to the vertebra
shown in cyan lines. The detailed imaging parameters for each image are listed in Table S2 (Supporting Information). (h,i) NIR-II fluorescence
imaging of the whole skeletal system using N3-FEP-4T. Scale bar, 1 cm. (j−q) Stereomicroscope fluorescence images of dorsal rib and thoracic
vertebra, lumbar vertebra and spinous/transverse process, ilium and sacral vertebra, caudal vertebra, skull and maxillary, and sternum and sternal rib,
tibia, and femur as well as the patella, metatarsal and digital bone, respectively, using N3-FEP-4T. Scale bar, 1 mm. The detailed imaging parameters for
each image are listed in Table S2 (Supporting Information).
vertebrae showed three fluorescence peaks. The full width at
half-maximums (FWHMs) were similar, ranging from 1.27 to
1.53 mm, high enough to discriminate different vertebrae
(Figure 3f). Horizontal analysis crossing the spine also
demonstrated an excellent differentiation between the vertebrae
and others, with a FWHM of 2.29 mm (Figure 3g).
To further confirm the in vivo results above, ex vivo imaging of
the isolated bone tissues of the BALB/c nude mouse after N3-
FEP-4T injection was performed following in vivo imaging. As
shown in Figure 3h,i, after removing the skin and internal organs,
the whole skeletal system was imaged in high clarity of the
supine position. The bone’s detailed imaging at various sites was
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Figure 4. In/ex vivo bone-specific imaging using N3-FEP-4T NIR-II fluorophores in osteoporotic mice. (a) Real-time NIR-II fluorescence imaging of
BALB/c mice (n = 3) and osteoporotic mice (n = 3) with images in the supine position acquired at 0.5 and 6 h postinjection. White dotted circle: soft
tissue. Scale bar, 5 mm. (b) Leg-soft tissue fluorescence ratio of the two groups. Stereomicroscope fluorescence images of thoracic vertebras in normal
(c) and osteoporotic mice (d). Scale bar, 1 mm. Stereomicroscope fluorescence images of the tibia and femur in normal (e) and osteoporotic mice (f).
Scale bar, 1 mm. (g) Cross-sectional normalized fluorescence intensity profiles along the cyan line in (c). (h) Cross-sectional normalized fluorescence
intensity profiles along the red line in (d). (i) Cross-sectional normalized fluorescence intensity profiles along the cyan line in (e). (j) Cross-sectional
normalized fluorescence intensity profiles along the red line in (f). (k) Bone density obtained by QCT of normal mice and osteoporotic mice. (l)
QCTimages of the tibia for normal and osteoporotic mice of the coronal and transverse sections. (m) Representative histological analysis of partial
femur and tibia from the normal mice and the osteoporotic mice. Scale bar: 500 μm. The detailed imaging parameters for each image are listed in Table
S2 (Supporting Information).
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then performed by a NIR-II stereomicroscope (Figure 3j−q).
Figure 3j−m shows the thoracic, lumbar, sacral, and caudal
vertebrae of the mouse clearly, and even the spinous and
transverse processes were visible in the images. Also, Figure 3j
displays the dorsal rib, and Figure 3l shows the ilium of the
mouse. The following pictures show the skull and maxillary
(Figure 3n), sternum and sternal rib (Figure 3o), femur and
tibia, as well as patella (Figure 3p) in turn. In the end, Figure 3q
displays not only the digital and metatarsal bone of the mice but
also the knuckle. These fluorescent images demonstrated that
the small-molecule N3-FEP-4T bound specifically to the whole
skeletal system. It can be used for real-time intraoperative
imaging, for example, placing pedicle screws in the accurate
position after detaching paravertebral muscles, real-time
positioning in scoliosis surgery, and open-reduction internal
fixation of phalanx fracture.
In/Ex Vivo NIR-II Imaging for Osteoporotic Mice.
Considering excellent bone imaging quality in normal mice of
N3-FEP-4T, osteoporotic model mice were chosen to study in
NIR-II imaging. NIR-II fluorescent images were taken at
different time points after tail vein injection (200 μL in 1×
PBS, 12 mg kg−1) of N3-FEP-4T into normal BALB/c mice (n =
3 per group) and osteoporosis BALB/c mice (n = 3 per group)
separately (Supporting Information, Figure S7). Figure 4l shows
the quantitative CT (QCT) images of the tibia for normal and
osteoporotic mice, and the bone density obtained by QCT
indicated significant difference between the two groups (P <
0.001, Figure 4k). Figure 4m shows the typical histological
analysis of partial femur and tibia separately from normal mice
and osteoporotic mice, and it was easy to see the cancellous bone
as the latter was sparser, agreeing with the QCT result.
Remarkable differences in the legs between the normal and
the osteoporotic mice were observed in N3-FEP-4T-based NIR-
II imaging 30 min after the injection (Figure 4a). It was found
that the SBR of the legs was significantly improved within 6 h of
the injection compared with the normal mice (Figure 4b), and
this may be related to osteoclasts. It is well known that the
imbalance between bone formation and resorption is thought to
underlie the pathogenesis of the reduced bone mass in
osteoporosis, which is closely related to osteoclast formation
and bone-resorbing activity.51,53 Before osteoclast formation,
osteoclast precursors exhibit a monocyte/macrophage pheno-
type; therefore, it is not surprising that the osteoclasts have the
same effect on macrophages of phagocytic function, which is
consistent with the cell-uptake experiments mentioned above.
Further research is still needed for clarifying the mechanisms
responsible for accumulation of N3-FEP-4T in osteoporosis.
The NIR-II stereomicroscope also performed the ex vivo NIR-
II fluorescent imaging after removing osteoporotic mice’s skin
and internal organs (Figure 4d,f and Supporting Information
Figure S8). Compared with the normal mice’s images (Figure
4c,e), the osteoporotic mice’s bone surface was pitted, especially
in the vertebrae. The results of quantitative analysis in the ex vivo
images confirmed this finding. Compared the quantitative
analysis of thoracic vertebrae in normal mice (Figure 4g) and
osteoporosis model mice (Figure 4h), the latter’s curve was
irregular and had several peaks. Figure 4i,j shows the quantitative
analysis of the tibia in normal mice and osteoporosis model
mice, respectively, and the curve of the normal mice looks like a
parabola. In contrast, the curve of the osteoporosis model mice
had a hollow. These suggested the uneven surface of the bone in
osteoporotic mice.
Cytotoxicity and Histological and Biodistribution
Analysis of N3-FEP-4T. To explore the potential toxicity of
N3-FEP-4T, standard (3-[4,5-dimethylthiazol-2-yl]-2,5 diphen-
yl tetrazolium bromide) (MTT) cell viability assays were
performed on macrophages (RAW 264.7), embryonic fibroblast
(3T3), and osteosarcoma (143B). No apparent cytotoxicity of
N3-FEP-4T was observed in these cells, even at a concentration
up to 100 μM (Supporting Information Figure S9). Moreover, as
shown in Supporting Information Figure S10, the tested mouse’s
main organs showed no apparent inflammation and abnormality
after N3-FEP-4T injection, indicating that the fluorescent probe
had excellent biocompatibility in vivo, agreeing with the
cytotoxicity results. The major organs such as the liver, lung,
spleen, kidney, heart, and lumbar vertebra were excised and
imaged at 7 and 14 days postinjection of N3-FEP-4T (n = 3 per
group, BALB/c mice) (Supporting Information Figure S11). It
revealed that the fluorophore’s signals were relatively stable in
the body, suggesting that the fluorophore had a prolonged in vivo
retention time. In bone imaging, the slow excretion can be used
to steadily monitor bone disease progression over a long period
on a nontoxic basis.
CONCLUSIONS
■
By comparing the in vivo behavior of N3-FEP-4T and FEP-4T
using NIR-II imaging, the azide group has been found to render
a compound bone targeting capacity. N3-FEP-4T demonstrates
superior calcium-binding and bone targeting properties, and it
can be used for noninvasive and nonradiative visualizing bone
tissues and for further monitoring skeletal system-related
diseases such as osteoporosis in vivo. Excellent biocompatibility,
high resolution, and favorable in vivo performance make N3-
FEP-4T a promising candidate for further preclinical applica-
tions in bone imaging. The finding of this research provides
more insights of the impact of the azide group to the in vivo
behavior of azide-containing compounds, and it opens a new
window for biological application of the azide group, such as
bone and bone-related disease imaging, which may provide
another design strategy for achieving in vivo bone targeting other
than the classical phosphonate modifications.
EXPERIMENTAL SECTION
Materials and Synthesis. For all materials and synthesis details,
please see Supporting Information.
■
Animal Handing. All vertebrate animals were purchased from the
Shanghai Experimental Animal Center (Shanghai). All animal
procedures were performed under the guideline approved by the
Institutional Animal Care and Use Committee (IACUC) of the
Shanghai Institute of Materia Medica, Chinese Academy of Sciences.
Six week old female BALB/c mice and female nude BALB/c mice were
obtained for all imaging studies and housed at the Shanghai Institute of
Materia medica under our approved animal protocols. Before imaging,
all mice were anesthetized with 2 L min−1 O2 gas mixed with 3%
isoflurane in a rodent anesthesia machine. Tail vein injection of contrast
agents was carried with a catheter and synchronized with equipment
that simultaneously started continuous image acquisition. The injected
dose was a 200 μL bolus in a 1× PBS solution at specified
concentrations. The mouse was kept anesthetized by a nose cone
during the imaging time, delivering 2 L min−1 O2 gas mixed with 3%
isoflurane. Mice were randomly selected from cages for all experiments.
No blinding was performed. All groups within the study contained n ≥ 3
mice.
Characterization of the N3-FEF-4T and FEP-4T. Spectral
Characterization of the N3-FEP-4T and FEP-4T. Absorbance spectra
of N3-FEP-4T and FEP-4T were taken on an ultraviolet−visible-2600
spectrometer (SHIMADZU), background-corrected for each solvent
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such as deionized water, MeOH, and dimethylformamide solution. The
NIR-II fluorescence emission spectra were captured on a spectrometer
(IHR320, HORIBA SCIENTIFIC) excited by an 808 nm laser.
Photostability of the N3-FEP-4T and FEP-4T. N3-FEP-4T, FEP-4T,
and ICG were dissolved in 1× pH 7.4 PBS, and N3-FEP-4T was also
dispersed in NMS. ICG was utilized as a reference fluorophore. They
were exposed to four solutions to continuous 808 nm excitation at the
same power density of 102 mW cm−2 and taking images every 5 min for
∼60 min. Stability was determined by measuring the regions of interest
(ROIs) and comparing the fluorescence intensity against the starting
fluorescence signal.
Measuring NIR-II QY. The fluorescence QY of N3-FEP-4T and FEP-
4T was measured similarly to the previous publications. Briefly, IR-26
was utilized as a reference fluorophore. The QY of IR-26 has previously
been determined as 0.5% in DCE.42 A serial dilution of five aqueous N3-
FEP-4T and FEP-4T solutions with an OD < 0.1 at 808 nm was carried
out to confirm absorbance values at 808 nm. The fluorescent emission
spectra were collected on a spectrometer (IHR320, HORIBA
Scientific). The fluorescent emission spectra were integrated and
plotted against the OD values at 808 nm, and a linear fit was applied to
verify the linearity between fluorescent intensities and concentrations.
For the brightest samples, interfilter effects were seen ∼OD 0.1 at 808
nm. Thus, lower concentration ranges were utilized. By comparing the
slope of the linear fit between IR-26 and N3-FEP-4T as well as FEP-4T,
the QY was determined based on the following supporting eq 1
Shanghai, China) at a dose of 10 mg kg−1, 3 times a week for 2 months
in the experimental group.54,55 The control group also received the
same volume (1× PBS) as each time’s experimental group.
In Vivo Bone NIR-II Imaging in Mice. Normal nude BALB/c mice
were injected, respectively, with N3-FEP-4T and FEP-4T (200 μL in 1×
PBS, 12 mg kg−1) via the lateral tail vein. Whole-body images were
collected on the MARS in vivo imaging system (Artemis Intelligent
Imaging, Shanghai, China) at 0, 2, 4, 6, 8, 12, 24, 48, 72, 96, 120, and
144 h postinjection. Imaging the mouse in a prone position (or a supine
position) with the chest and leg region well secured on the imaging
platform with a medical tape prevented any motion artifacts from
heartbeat or breathing motion. A fiber-coupled 808 nm laser diode
generated the excitation laser. Emission was typically collected with
1000, 1100, 1150, 1250, 1300, and 1350 nm LP filter (Thorlabs). The
detailed imaging parameters for each image are listed in Table S3 of
Supporting Information.
Ex Vivo Bone Imaging in Mice. For ex vivo bone imaging, skin and
all soft tissues except for muscle were removed from mice. The exposed
bone tissues were then imaged to verify bone tissue imaging of the N3-
FEP-4T using the MARS-Pathfinder in vivo imaging system (Artemis
Intelligent Imaging, Shanghai, China) for a large field of view. A fiber-
coupled 808 nm laser diode provided the excitation laser, and the
detailed imaging parameters for each image are listed in Table S2
(Supporting Information).
In Vitro Bone Density Measurement. The bone density was
calculated by Inveon CT (Siemens). For image-quality purposes, set
the voltage based on the sample’s density. Therefore, choose 80 kVp for
the tibia (hard bone). Calculation of bone density requires HU
calibrated CT data. A cylindrical model (Supporting Information
Figure S12) was used to get a formula between HU and bone density.
Each color corresponds to a region in the cylindrical model with a
different bone density (Supporting Information Table S1). Place the
mouse pallet model, and use 80 kVp for scanning to acquire the HU
data by drawing ROIs for each color in IRW General Analysis. Record
the mean value displayed for each of the ROIs, and match them to
corresponding bone mineral density (BMD) values yields a formula
(Supporting Information Table S1, Figure S13). Acquire the animal
data (tibial diaphysis) with the same protocol used to acquire the
model, and draw ROIs to obtain the HU data in IRW General Analysis.
Enter the mean HU value of the ROIs in the formula to get the tibia
bone density.
Cytotoxicity of the N3-FEP-4T toward Macrophage, Embry-
onic Fibroblast, and Osteosarcoma. In vitro cytotoxicity of the N3-
FEP-4T toward macrophages, embryonic fibroblast, and osteosarcoma
was evaluated using MTT. RAW 264.7 cells, 3T3 cells, and 143B cells
were all cultured in Dulbecco’s modified Eagle medium (DMEM)
supplemented with 10% fetal bovine serum (FBS) and 1% PS at 37 °C
and 5% CO2. Subcultured RAW 264.7 cells (1 × 104 cells well−1), 3T3
cells (1 × 104 cells well−1), and 143B (1 × 104 cells well−1) were
separately seeded in 96-well culture plates with a primary culture
medium and allowed to adhere for 1 day. The cells were then treated
with various concentrations of the N3-FEP-4T (10, 25, 50, 75, and 100
μM). After 24 h incubation, the old medium was taken out, and the
MTT assay solution was added to the cells according to the
manufacturer’s protocol. The medium was removed after further
incubation at 37 °C and 5% CO2 for 4 h, and then, 150 μL of dimethyl
sulfoxide (DMSO) was added to dissolve the formazan crystals
precipitates in each well. After shaking the cell plate for 15 min, the
optical density was measured at 490 nm using the enzyme-linked
immunosorbent assay (SynergyH1, BioTek, USA). Only active
reductase enzymes in living cells can reduce MTT; therefore, high
cell viability levels are indicated by high absorbance at 490 nm. The
relative cell viability (%) was calculated by the following supporting eq
3
2
slopesample insample
y
j
z
j
z
QY
= QY0 ×
× j
z
sample
j
j
z
z
slope0
n0
(1)
k
{
where QYsample is the QY of N3-FEP-4T and FEP-4T; QY0 is the QY of
IR-26; n0 is the refractive index of IR-26 solution, which is DCE (1.44);
nsample are the refractive indices of N3-FEP-4T and FEP-4T solutions,
which are both water (1.33).
In Vitro Calcium-Binding Experiments. CC, CO, HA, CPP, and
manganese dioxide (34.5 μmol) were incubated separately with 4.75
mM N3-FEP-4T and FEP-4T in 1× PBS. The calcium salts were
vortexed continuously with the fluorophores at room temperature for 2
h. The mixtures were then washed 3 times with PBS, followed by
centrifugation at 5500 rpm for 10 min to remove the unreacted
fluoroprobes. To compare the binding affinities, the collected
precipitate after centrifugation was dispersed in 200 mL of 1× PBS
(pH 7.4), and the fluorescence imaging system determined the
fluorescence intensities of the dispersed samples. All NIR fluorescence
images were collected at identical exposure times and are displayed with
equal normalization.
Intracellular NIR-II Imaging of N3-FEP-4T and FEP-4T.
Macrophages, embryonic fibroblast, and osteosarcoma were used to
examine the intracellular binding of N3-FEP-4T and FEP-4T. RAW
264.7 cells, 3T3 cells, and 143B cells were cultured on culture dishes.
Three kinds of cells were treated separately with both probes (25 μM)
and incubated respectively for 15, 30, and 60 min. After the time, the
cells were washed 3 times with 1× PBS. The cells were then digested
with trypsin and were transferred into EP tubes with 500 μL 1× PBS,
and the PBS was removed after centrifugation. The NIR fluorescence
images were collected on a MARS in vivo imaging system (Artemis
Intelligent Imaging, Shanghai, China) at identical exposure times,
which displayed equal normalization.
The N3-FEP-4T/FEP-4T accumulation ratio was determined based
on the following supporting eq 2:
AI1
QY2
AI2
AR =
×
QY
(2)
1
where AR is the N3-FEP-4T/FEP-4T accumulation ratio; AI1 is the
average intensity of N3-FEP-4T; AI2 is the average intensity of FEP-4T;
QY1 is the QY of N3-FEP-4T; QY2 is the QY of FEP-4T.
Osteoporosis Model of BALB/c Mice. Twelve 6 week old female
BALB/c mice were randomly divided into the experimental and control
groups, with six mice in each group. The osteoporosis induction was
done through the intraperitoneal injection of prednisolone (Macklin,
Asample − Ablank
Acontrol − Ablank
CV % =
× 100%
(3)
where Asample is the absorbance of wells with N3-FEP-4T and medium,
Ablank is the absorbance of wells with primary medium, Acontrol is the
H
https://doi.org/10.1021/acs.jmedchem.1c00839
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
absorbance of wells with PBS and medium. All samples were analyzed in
triplicate, and the experiment was replicated 3 times.
Hongyue Lou − Molecular Imaging Center, Shanghai Institute
of Materia Medica, Chinese Academy of Sciences, Shanghai
201203, China
Jiafeng Li − Molecular Imaging Center, Shanghai Institute of
Materia Medica, Chinese Academy of Sciences, Shanghai
201203, China
Lingling Zheng − Department of Nuclear Medicine, Huashan
Hospital, Fudan University, Shanghai 200040, China;
Molecular Imaging Center, Shanghai Institute of Materia
Medica, Chinese Academy of Sciences, Shanghai 201203,
China; Department of Nuclear Medicine, Pudong Hospital,
Fudan University, Shanghai 200120, China
Statistical Analysis. Data are expressed as mean SD (standard
deviation). Differences between groups were assessed using the paired,
two-sided Student’s t-test (IBM SPSS Statistics 20). *P < 0.05 was
considered significant. The fluorescence images were analyzed using
ImageJ 1.8.0 (NIH, Bethesda, MD). ROI analysis was performed by
drawing a shape around the specific site and using the software to
calculate the mean pixel intensity. The ROI values were plotted using
GraphPad Prism (GraphPad Software Inc., San Diego, CA). All NIR
fluorescence images for a particular fluorophore were normalized
identically for all conditions of an experiment. At least three animals
were analyzed at each time point.
Yujing Zhou − Department of Nuclear Medicine, Huashan
Hospital, Fudan University, Shanghai 200040, China;
Molecular Imaging Center, Shanghai Institute of Materia
Medica, Chinese Academy of Sciences, Shanghai 201203,
China; Department of Nuclear Medicine, Pudong Hospital,
Fudan University, Shanghai 200120, China
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00839.
Molecular formula strings (CSV)
Complete contact information is available at:
Synthetic procedures, compound characterization, addi-
tional in vivo bone images, cell toxicity, and histopathol-
ogy (PDF)
https://pubs.acs.org/10.1021/acs.jmedchem.1c00839
Author Contributions
⊥X.Z. and A.J. contributed equally.
Author Contributions
All authors have given approval to the final version of the
manuscript.
AUTHOR INFORMATION
■
Corresponding Authors
Chunrong Qu − Molecular Imaging Center, Shanghai Institute
of Materia Medica, Chinese Academy of Sciences, Shanghai
201203, China; Email: crqu@simm.ac.cn
Notes
The authors declare no competing financial interest.
Xingdang Liu − Department of Nuclear Medicine, Huashan
Hospital, Fudan University, Shanghai 200040, China;
Department of Nuclear Medicine, Pudong Hospital, Fudan
University, Shanghai 200120, China; Email: xingdliu@
fudan.edu.cn
Hao Chen − Molecular Imaging Center, Shanghai Institute of
Materia Medica, Chinese Academy of Sciences, Shanghai
201203, China; orcid.org/0000-0002-8470-5467;
Email: haoc@simm.ac.cn
Zhen Cheng − Department of Nuclear Medicine, Huashan
Hospital, Fudan University, Shanghai 200040, China;
Molecular Imaging Center, Shanghai Institute of Materia
Medica, Chinese Academy of Sciences, Shanghai 201203,
China; Molecular Imaging Program at Stanford (MIPS), Bio-
X Program, and Department of Radiology, Canary Center at
Stanford for Cancer Early Detection, Stanford University,
Stanford, California 94305-5344, United States;
orcid.org/0000-0001-8177-9463; Email: zcheng@
simm.ac.cn
ACKNOWLEDGMENTS
■
This work was partially supported by the Department of
Radiology Stanford University (Z.C.), the National Natural
Science Foundation of China under grant nos. 82071976 (H.C.)
and 81901799 (C.Q.), the Shanghai Pujiang Program
(19PJ1411100) (H.C.), the Shanghai Municipal Science and
Technology Major Project, and the National Key Research and
Development Plan Digital Diagnosis and Treatment Equipment
Research and Development Project (2017YFC0113300) (X.L.).
ABBREVIATIONS
■
AZT, zidovudine; BMD, bone mineral density; CC, calcium
carbonate; CO, calcium oxalate; CPP, calcium pyrophosphate;
d4T, stavudine; DMSO, dimethyl sulfoxide; FDA, Food and
Drug Administration; FWHMs, full width at half-maximums;
HA, hydroxyapatite; HIV, human immunodeficiency virus; ICG,
indocyanine green; LP, long pass; MALDI-TOF-MS, matrix-
assisted laser desorption ionizing time-of-flight mass spectrom-
etry; MnO2, manganese dioxide; NIR-I, near-infrared window
one; NIR-II, near-infrared window two; NMR, nuclear magnetic
resonance; NMS, normal mouse serum; PBS, phosphate-
buffered saline; QCT, quantitative computed tomography;
QY, quantum yield; ROI, region of interest; SBR, signal-to-
background ratio; SD, standard deviation
Authors
Xiaoqing Zhang − Department of Nuclear Medicine, Huashan
Hospital, Fudan University, Shanghai 200040, China;
Molecular Imaging Center, Shanghai Institute of Materia
Medica, Chinese Academy of Sciences, Shanghai 201203,
China; Department of Nuclear Medicine, Pudong Hospital,
Fudan University, Shanghai 200120, China; orcid.org/
0000-0002-0052-1629
Aiyan Ji − Molecular Imaging Center, Shanghai Institute of
Materia Medica, Chinese Academy of Sciences, Shanghai
201203, China
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