Seeing Elastin: A Near-Infrared Zwitterionic Fluorescent Probe for In Vivo Elastin Imaging

Article abstract of DOI:10.1016/j.chempr.2018.02.016

Elastic fibers are present in a variety of tissues and are responsible for their resilience. Until now, no optical contrast agent in the near-infrared (NIR) wavelength range of 700–900 nm has been reported for the imaging of elastic fibers. Here, we report the discovery of a NIR zwitterionic elastin probe ElaNIR (elastin NIR) through fluorescent-image-based screening. The probe was successfully applied for in vitro, ex vivo, and in vivo imaging by various imaging modalities. Age-related elastin differences shown by in vivo fluorescent and photoacoustic imaging indicated that ElaNIR can be a potentially convenient tool for uncovering changes of elastin in live models. Technological advancement of microscopic imaging has spurred the development of molecular probes for the detection of biologically and environmentally important analytes. Among fluorescent imaging tools, near-infrared (NIR) fluorescent molecules have been popular for in vivo imaging because of their ability to penetrate tissue deeper with reduced scattering and autofluorescence. Here, we report the discovery of the NIR zwitterionic elastin probe ElaNIR. We found that ElaNIR showed selective binding for elastin, such as those in the aorta, lungs, and skin. ElaNIR also allowed visualization and quantification of elastin levels in young and old mice, which reflect age-dependent changes of elastin in vivo. This makes ElaNIR a unique chemical tool for monitoring changes of elastin in tissue or animal models and provides new opportunities to study the effects of chemical compounds on elastin levels in biological processes. The near-infrared zwitterionic elastin probe ElaNIR for in vivo imaging of elastin structures was found through fluorescent-image-based screening. ElaNIR is well suited for in vitro, ex vivo, and in vivo imaging of elastin and also allows visualization and quantification of elastin levels in young and old mice, which reflect age-dependent changes of elastin in vivo.

Full text of DOI:10.1016/j.chempr.2018.02.016

Article
Seeing Elastin: A Near-Infrared Zwitterionic
Fluorescent Probe for In Vivo Elastin Imaging
Dongdong Su, Chai Lean Teoh,
Sung-Jin Park, ..., Seong Soon
Kim, Myung Ae Bae, Young-Tae
Chang
ytchang@postech.ac.kr
HIGHLIGHTS
A zwitterionic NIR fluorophore,
ElaNIR, was found to selectively
bind elastin
ElaNIR is well suited for in vitro,
ex vivo, and in vivo imaging of
elastin
ElaNIR showed age-dependent
changes of skin elastin levels
The near-infrared zwitterionic elastin probe ElaNIR for in vivo imaging of elastin
structures was found through fluorescent-image-based screening. ElaNIR is well
suited for in vitro, ex vivo, and in vivo imaging of elastin and also allows
visualization and quantification of elastin levels in young and old mice, which
reflect age-dependent changes of elastin in vivo.
Su et al., Chem 4, 1–11
May 10, 2018 ª 2018 Elsevier Inc.
https://doi.org/10.1016/j.chempr.2018.02.016

Please cite this article in press as: Su et al., Seeing Elastin: A Near-Infrared Zwitterionic Fluorescent Probe for In Vivo Elastin Imaging, Chem
(2018), https://doi.org/10.1016/j.chempr.2018.02.016
Article
Seeing Elastin: A Near-Infrared
Zwitterionic Fluorescent Probe
for In Vivo Elastin Imaging
Dongdong Su,1,9 Chai Lean Teoh,1,9 Sung-Jin Park,1 Jong-Jin Kim,1 Animesh Samanta,1 Renzhe Bi,4
U.S. Dinish,4 Malini Olivo,4 Marie Piantino,5 Fiona Louis,6 Michiya Matsusaki,5,6,7 Seong Soon Kim,8
Myung Ae Bae,8 and Young-Tae Chang1,2,3,10,
*
SUMMARY
The Bigger Picture
Technological advancement of
microscopic imaging has spurred
the development of molecular
probes for the detection of
biologically and environmentally
important analytes. Among
fluorescent imaging tools, near-
infrared (NIR) fluorescent
Elastic fibers are present in a variety of tissues and are responsible for their
resilience. Until now, no optical contrast agent in the near-infrared (NIR) wave-
length range of 700–900 nm has been reported for the imaging of elastic fibers.
Here, we report the discovery of a NIR zwitterionic elastin probe ElaNIR (elastin
NIR) through fluorescent-image-based screening. The probe was successfully
applied for in vitro, ex vivo, and in vivo imaging by various imaging modalities.
Age-related elastin differences shown by in vivo fluorescent and photoacoustic
imaging indicated that ElaNIR can be a potentially convenient tool for uncover-
ing changes of elastin in live models.
molecules have been popular for
in vivo imaging because of their
ability to penetrate tissue deeper
with reduced scattering and
autofluorescence. Here, we report
the discovery of the NIR
INTRODUCTION
Elastin is a key structural protein found in the extracellular matrix (ECM). It is secreted
primarily by fibroblast cells, which assemble into fibers and cross-links to form a
mechanically flexible network of fibers and sheets.1 To yield a wide range of struc-
tures with tailored elastic properties, the arrangement of elastin in the ECM can
vary between tissues. Elastin in the form of lamina in the arterial wall is crucial for
the strength necessary for vessel expansion and regulation of blood flow, whereas
elastin fibers are enriched in the dermis of the skin to impart flexibility and
extensibility.
zwitterionic elastin probe ElaNIR.
We found that ElaNIR showed
selective binding for elastin, such
as those in the aorta, lungs, and
skin. ElaNIR also allowed
visualization and quantification of
elastin levels in young and old
mice, which reflect age-
Different from other connective tissue proteins such as collagens, which are mem-
bers of large and complex gene families, elastin is encoded only by a single tropoe-
lastin gene (ELN). As ELN expression is turned down substantially from a young age,
we rely mostly on the elastin that was produced before birth and in the first few years
of life.2,3 A low level of elastin production means that any damage cannot be
efficiently repaired as tissues gradually lose their elasticity. Visualization of elastin
architecture and its dynamic changes in live tissues will therefore provide better
understanding about its roles in biological processes.
dependent changes of elastin
in vivo. This makes ElaNIR a
unique chemical tool for
monitoring changes of elastin in
tissue or animal models and
provides new opportunities to
study the effects of chemical
compounds on elastin levels in
biological processes.
Despite its high importance, elastin is difficult to image. So far, methods for easy and
high-resolution imaging of elastin in vivo or in situ are limited. Reported methods of
studying their morphology have largely been restricted to either exploiting the
intrinsic fluorescence properties of elastin or using histologic dyes, which are incom-
patible with intravital studies. Recently, staining of elastin by fluorescent labeling,
such as sulforhodamine B, Col-F, and Alexa Fluor 633, has been reported in selected
tissues.4–6 Further in vivo applications, however, are impeded by the probes’ short
Chem 4, 1–11, May 10, 2018 ª 2018 Elsevier Inc.
1

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(2018), https://doi.org/10.1016/j.chempr.2018.02.016
emission wavelengths because visible light can penetrate only a few hundred micro-
meters below the tissue surface. Near-infrared (NIR) light, on the other hand, pene-
trates millimeters up to centimeters into living tissue and hence holds greater
promise over other small molecules.7–9
Inspired by the outstanding features of ZW800-1 in earlier reports,10–12 we synthe-
sized a zwitterionic NIR library, CyZW, by introducing structural diversity to the
key structure of ZW800-1 while retaining its superior chemical and photophysical
properties for bioimaging. The diversity-oriented fluorescence library (DOFL)
approach has proven its versatility in sensor development, especially during the
absence of mechanistic cues to rationally design probes.13–18 We present dye
CyZW-599 from DOFL screening, also dubbed ElaNIR (elastin NIR), as a bioimaging
probe for elastin.
RESULTS AND DISCUSSION
Library Design, Characterization, and Optical Properties
The recently developed zwitterionic NIR fluorophore, ZW800-1 has shown superior
in vivo properties, including low serum binding, ultra-low non-specific tissue back-
ground, and rapid clearance from the body via renal filtration, over other NIR
dyes.10 Despite that, limitations in its structural diversity have hindered ZW800-1
for potential downstream applications. To overcome this limitation and to expand
the application of zwitterionic NIR dyes, we designed a family of dyes based on
ZW800-1. Firstly, the key intermediate, ZW800-1, was successfully synthesized as
previously reported.10 A broad range of structural diversities were then constructed
quite efficiently by the reaction of a series of primary amines with tert-butyl 2-bro-
moacetate. Finally, ZW800-1 was coupled with the diversity amine building blocks
by a standard coupling protocol.19,20 The final step involved de-protection of the
1Laboratory of Bioimaging Probe Development,
Singapore Bioimaging Consortium, Agency for
acid linker to yield 64 members of the CyZW library (Scheme 1 and Figure S1).
Science, Technology, and Research (A*STAR), 11
Each CyZW compound contains a single carboxylic acid at the end of the structure,
which will be useful for further downstream modifications such as conjugation with
various reporter or affinity tags for bio-conjugation depending on the experimental
design requirements.
Biopolis Way, 02-02 Helios, Biopolis, Singapore
138667, Singapore
2Centre for Self-assembly and Complexity,
Institute for Basic Science, Pohang 37673,
Republic of Korea
3Department of Chemistry, Pohang University of
Science and Technology, 77 Cheongam-Ro,
Nam-Gu, Pohang 37673, Republic of Korea
Spectra properties of the newly synthesized library CyZW were measured in DMSO.
Because all the CyZW compounds share the same core structure, they display similar
optical properties. CyZW compounds show maximum absorption and emission
wavelengths at about 790 and 805 nm, respectively, in DMSO. The zwitterionic
CyZW dyes show a high extinction coefficient of more than 200,000 MÀ1 cmÀ1
and a much higher quantum yield (average of 0.39) than other NIR dyes
(Table S1). This is a great improvement because fluorescence intensity is crucial
for practical applications, in particular for achieving better-resolution images during
in vivo imaging.
4Bio-optical Imaging group, Singapore
Bioimaging Consortium, Agency for Science,
Technology, and Research (A*STAR), 11 Biopolis
Way, 02-02 Helios, Biopolis, Singapore 138667,
Singapore
5Department of Applied Chemistry, Graduate
School of Engineering, Osaka University, 2-1
Yamadaoka, Suita, Osaka 565-0871, Japan
6Joint Research Laboratory (TOPPAN) for
Advanced Cell Regulatory Chemistry, Graduate
School of Engineering, Osaka University, 2-1
Yamadaoka, Suita, Osaka 565-0871, Japan
ElaNIR, a NIR Fluorescent Elastin Probe
7JST, PRESTO, 4-1-8 Honcho, Kawaguchi,
Saitama 332-0012, Japan
Screening of the CyZW library revealed compounds with apparent specificity for
elastin. When frozen mouse aorta tissues were treated with CyZW dye solution,
we observed cases where a network of fibers were readily stained and became highly
visible under the fluorescence microscope (Figure 1A). These were identified to be
elastin fibers. The medial lamellar unit of the aorta wall is constructed with elastin
arranged in concentric lamellae and smooth muscle cells that lie in between.21,22
After evaluating the screened library for its fluorescence intensity and selectivity,
we selected ElaNIR (CyZW-599) as the best compound for further studies (Figures
S2 and S3) and selected CyZW-274, which ranked low in the image-based scoring,
8Bio&Drug Discovery Division, Korea Research
Institute of Chemical Technology, Yuseong-Gu,
Gajeongro 141, Daejeon 34114, Republic of
Korea
9These authors contributed equally
10Lead Contact
*Correspondence: ytchang@postech.ac.kr
https://doi.org/10.1016/j.chempr.2018.02.016
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O
O
NH2
H
a
R
N
Br
O
O
R
R
O
O
O
O
N
OH
b, c
O3S
SO3
O3S
SO3
O
O
N
N
N
N
N
N
N
N
CyZW library
ZW800-1
Scheme 1. Synthetic Scheme for the CyZW Library
Reagents and conditions: (a) 64 different aromatic and aliphatic primary amines, acetonitrile, N,N-diisopropylethylamine (DIEA), room temperature
(RT), 2 hr; (b) DMSO/DMF (1:4), HATU, DIEA, RT, 3 hr; (c) 20% trifluoroacetic acid in dichloromethane, RT, 3 hr. See also Figure S1.
as a negative control compound (Figure S4). On the other hand, when spectroscopy-
based screening was conducted, the in vitro screening results were unable to distin-
guish elastin specificity of different dyes within the library. There could be no change
in the structure and motion of the dye before and after analyte is added, resulting in
a small or no change in its fluorescence intensity or anisotropy. Thus, we found the
in vitro detection of elastin protein in solution by spectroscopy methods unsuitable;
the tissue-based screening approach was better suited for distinguishing an elastin-
selective probe. Optical properties of ElaNIR and CyZW-274 in different solvent
systems are detailed in Figure S5 and Table S2. Both dyes showed more fluores-
cence in the presence of warm serum than PBS alone. Optical parameters for both
dyes are similar in the same solvent system.
Elastin is a very non-polar protein, often with no negatively charged amino acids, and
most of the positively charged amino acids are lost when the lysines form cross-links
that convert the soluble monomer, tropoelastin, to insoluble, polymeric elastin in
tissues. Although it is still unclear, some elastin-staining dyes can be based on hydro-
phobic interactions that take place with many sites along the elastin molecule.23 We
evaluated the distribution coefficient (logD) of the CyZW library at pH 7.4, which is
an important parameter for evaluating the hydrophobicity of a compound. At pH
7.4, logD values varied from À5.12 (CyZW-414) to 2.94 (CyZW-656) (Table S1).
Comparison of the logD data with scoring from fluorescence image-based screening
(Figure S6) showed that logD alone is not a predictive measure of elastin specificity
fate. Nonetheless, some correlation existed near a logD value of 0, which implies
that the majority of compounds that fell on either side, with more negative or
positive logD values, might not have enough affinity for elastin or might have high
background signal toward neighboring tissues.
NMR and molecular dynamics data on elastin in both the monomeric and polymeric
states all support the conformational flexibility of elastin.24 There is no single struc-
ture for elastin; rather, the protein is made up of an ensemble of rapidly intercon-
verting conformations. Thus, it is not possible to designate a simple ‘‘structural
model,’’ which makes deducing a binding mechanism of ElaNIR for elastin espe-
cially challenging. On the basis of the structural-activity relationship observations
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Figure 1. ElaNIR, an NIR Fluorescent Elastin Probe
(A) Discovery of ElaNIR by image-based screening and its chemical structure.
(B) ElaNIR ex vivo staining of elastin lamellae. H&E staining of the aorta (left) and ElaNIR staining of
elastin (right) are shown. Scale bars, 50 mm. Lu, lumen.
See also Figures S2, S4, and S7.
from our screening results, we postulate that the biphenyl group in ElaNIR could
play a role in elastin specificity because the absence of the biphenyl structure
diminished the elastin selectivity of the other fluorophores within the CyZW library.
This is only speculative given that the precise molecular target for binding is not yet
known.
ElaNIR Enables Ex Vivo Detection of Elastin
As a preliminary bioimaging test for ElaNIR, the dye was administered intravenously
(i.v.) into mice, and CyZW-274-injected and non-injected animals were used as con-
trols. One hour after the compound was administered, the animals were sacrificed,
the organs were harvested, and cryosections were prepared for analysis. Figure 1B
shows H&E staining of the aorta cross-section. A strong NIR fluorescence signal was
observed in ElaNIR-injected mice, which showed clear staining of the elastic
lamellae (Figure 1B). In contrast, there was no detectable NIR signal in CyZW-274-
injected mice and non-injected control mice (Figure S7), reinforcing the notion
that ElaNIR exhibits affinity for elastin structures.
To confirm the observation separately by immunohistochemical staining with
elastin antibody, we prepared cryosections from organs with high-elastin content,
such as the lungs, skin, and bladder, in addition to the kidneys, aorta, and ears (Fig-
ures 2 and S8). An ElaNIR signal was captured, and consecutive sections were fixed
in paraformaldehyde for immunohistochemistry (IHC). Figure 2A shows abundant
ElaNIR-stained elastic fibers in the lungs, and fine, wispy elastic structures were
detected by ElaNIR in the skin dermis (Figure 2B). The urinary bladder also showed
the highly elastic urothelium brightly stained by ElaNIR (Figure 2C). In addition, the
kidney revealed an ElaNIR signal in the renal artery or vein, as well as calyx, without
much noticeable signal in other regions (Figure S8). Both aorta and ear tissue sec-
tions stained by ElaNIR gave clear strong signals consistent with antibody staining
(Figure S8). On the other hand, IHC was also performed on selected tissues with
4
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Figure 2. ElaNIR Enables Ex Vivo Detection of Elastin
Cryosections were prepared from organs including the lungs (A), skin (B), and urinary bladder (C). ElaNIR signal was detected in the NIR channel (left
panels). Consecutive sections were fixed with 4% paraformaldehyde, and IHC was performed with an anti-elastin antibody co-stained with Hoechst.
Merged images are shown in the right panels. White arrows show elastin fibers. Scale bars, 100 mm. See also Figures S8 and S9.
anti-collagen antibody, which is another major ECM component, and was found
to be inconsistent with signals displayed by ElaNIR (Figure S9). Cryosections pre-
pared from organs of a CyZW-274-injected mouse showed negligible signal
(Figure S10).
Elastin Detection in 3D Tissue Models
Elastin, a major component of the ECM, is a crucial but often overlooked element in
tissue regeneration. We applied ElaNIR to directly visualize the production of elastin
fibers in a 3D tissue model, where human umbilical artery smooth muscle cells
(UASMCs) were seeded in a suspension of collagen microfibers, leading to ball
tissues. Figure 3A shows a small amount of fibers externalized by UASMC ball tissues
after 3 weeks in culture. Despite the presence of another major extracellular protein
component, i.e., collagen, preferential staining of elastin fibers by ElaNIR further
supported its selectivity (Figure S11). As shown in Figure 3B, ElaNIR staining of
extracellular elastin fibers within the collagen mass is consistent with elastin antibody
staining. We envisage that when ElaNIR is combined with a 3D tissue model system,
it has the potential to serve as a powerful platform for assessing the potency of
compounds targeted at increasing elasticity.
In Vivo Elastin Targeting with ElaNIR
The observations so far give us strong confidence that ElaNIR could be further
explored for in vivo imaging. The pharmacokinetics of ElaNIR was studied by
quantification of the dye in blood plasma over several time points during a 24-hr
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Figure 3. Elastin Detection in 3D Tissue Models
(A) Bright-field signal (left) and ElaNIR signal in the NIR channel (right).
(B) ElaNIR signal detected in the NIR channel (left), signal from an anti-elastin antibody (middle),
and overlay imaging (right).
Scale bars, 100 mm. See also Figure S11.
period (Table S3 and Figure S12). More than 90% of the injected dose (ID) was elim-
inated from the plasma shortly after 4 hr (0.087 G 0.032 %ID/g), a decrease from
6.30 G 0.60 %ID/g at 0.083 hr after intravenous injection. At 24 hr, only 0.0040 G
0.0017 %ID/g dye remained in the plasma. At 4 hr after injection, organs were
also resected and dye distribution measured. The most intense uptake for ElaNIR
was measured primarily in the kidneys (5.01 G 2.14 %ID/g), consistent with active
renal excretion (Tables S4–S6). For in vivo visualization, a nude mouse received an
i.v. injection of ElaNIR, and the fluorescence signal was monitored by an IVIS Spec-
trum imaging system. Figures 4A and 4B show a robust fluorescence signal from the
whole body of the mouse injected with ElaNIR, and intense signal came from the
urinary bladder (Figure S13). On the other hand, a diminished fluorescence signal
was observed in a mouse injected with CyZW-274, and a non-injected control mouse
did not show any signal (Figure S14).
At the end of in vivo monitoring, the ElaNIR-injected animal was sacrificed and
observed ex vivo on the same imaging system. A fluorescence signal was no longer
observed at the head region after the skin was removed to reveal the skull. This
indicates that the fluorescence signal does not originate from the organ beneath
the skin. Instead, a strong signal was observed from the skin and the base of the
ear, both of which are rich in elastic structures. Also, by removing the organs
from the chest and abdominal cavity, we observed clear staining in the intact major
blood vessel joining the heart to the kidney (Figure 4C). Comparison of the fluores-
cence distribution in the various vital organs showed that the lungs and kidneys
gave strong signals (Figure 4D). This is unsurprising because the lungs are richer
in elastin than the other vital organs observed, whereas the kidneys are highly
vascularized. We also expect an ElaNIR signal from the urinary bladder because
it is an elastic organ, consistent with the immunohistochemical observations. In
addition, the zwitterionic NIR compound was excreted primarily through the renal
clearance pathway, which could also have contributed to the signals observed in
renal organs.
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Figure 4. In Vivo Elastin Targeting with ElaNIR
(A and B) Dorsal (A) and ventral (B) images of nude mice were captured in vivo on the IVIS Spectrum
imaging system 60 min after i.v. injection with ElaNIR. Non-injected mice were used as controls.
(C and D) Animals were sacrificed at the end of observation and imaged ex vivo. (C) ElaNIR staining
of elastic cartilage at the base of the ears (white arrow; top), in the aorta (yellow arrow), and in the
kidneys (bottom). (D) The distribution of ElaNIR in vital organs. Bl, bladder; Br, brain; H, heart; Ki,
kidney; Li, liver; Lu, lung; Pa, pancreas; Sp, spleen.
See also Figures S13 and S14.
ElaNIR Shows Differences between Young and Old Skin
Encouraged by the in vivo observations, we further explored the applicability of
ElaNIR by using a set of spectroscopic and optical imaging experiments performed
on young and old mice (1 and 10 months old, respectively).
Diffuse reflectance spectroscopy (DRS) provides a versatile platform for tissue char-
acterization because it is primarily dependent on tissue optical parameters. Because
of the strong light absorption of endogenous absorbers in the visible NIR region,
DRS offers great value for the classification and analysis of tissue samples.25
Figure S15A shows a difference in the DRS spectra between young and old mice
before ElaNIR injection. The relatively higher reflectance signal from the young
mouse can be explained by the presence of endogenous absorbers in the skin.
After ElaNIR injection, a dip observed in the spectra at ꢀ780 nm indicates the presence
of the probe in the skin (Figure S15B). Lower reflectance from the skin of the younger
mouse indicates higher absorption of light, which in turn implies greater content of
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Figure 5. ElaNIR Shows Differences between Young and Old Skin
(A) Ventral and dorsal images of 1-month-old and 10-month-old nude mice were captured in vivo on the In-Vivo Xtreme imaging system 1 min after i.v.
injection with ElaNIR. Non-injected mice were used as controls (not shown).
(B) Selected photoacoustic images of 1-month-old and 10-month-old nude mice were captured in vivo. The highlighted areas in the bottom panel show
the selected ROIs presented in (C). Ki, kidney; Li, liver; Sp, spleen.
(C) Reconstructed skin images for 1-month-old and 10-month-old mice from the selected ROIs of transverse slices in (B).
(D) ElaNIR signal for 1-month-old and 10-month-old mice by both imaging modalities. Data are represented as mean G SD; *p < 0.05.
See also Figures S15 and S16.
elastin in the skin. It was also noticed that the spectral profile is dominated by the signal
from ElaNIR after injection, which masks the contribution of endogenous markers in the
650–850 nm range. Because DRS is a point measurement, the difference in the reflec-
tance intensity value cannot be explicitly used for quantitative evaluation. However, it
can be used a fast approach to confirm the presence of the probe in the skin.
An ElaNIR fluorescence signal was first monitored by In-vivo Xtreme before injection
and immediately after the probe was injected (Figure 5A). We observed higher
8
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ElaNIR fluorescence signals from 1-month-old mice than from 10-month-old
animals. The mean of three sets of imaging experiments (dorsal and ventral com-
bined) gives a fold change of ꢀ1.68. In addition, photoacoustic imaging (PAI) was
also used for the relative quantification of ElaNIR targeting elastin in the skin of
young and old mice. PAI has recently emerged as an exciting imaging modality
because of its unique advantage in providing functional, anatomic, and molecular
information at microscopic resolution at a higher imaging depth than other existing
modalities.26–28 In PAI, samples are irradiated with short laser pulses, which are
absorbed by endogenous (e.g., hemoglobin and melanin) or exogenous contrast
agents, leading to a rapid and transient increase in temperature (in the order of
mK) and resulting in thermo-elastic expansion. These optically non-scattering acous-
tic waves can be detected by ultrasound transducers. The acoustic data can then be
used to reconstruct 2D or 3D optical absorption maps with a high spatial resolution
(<100 mm) and a penetration depth up to a few centimeters. Hence, PAI exploits the
advantage of an optical technique in terms of molecular specificity and resolution
along with that of high-frequency ultrasound for deep-tissue interrogation.28–30
In order to distinguish photoacoustic (PA) signal by ElaNIR in the skin area, we
selected a small region of interest (ROI) near the spine region (Figure 5B); it is
presented as the entire transverse slice in Figure 5C. Similarly, transverse slices
from CyZW-274-injected mice showed negligible signal (Figure S16). When the PA
intensity represented by the brighter region (pseudo color) is compared across
the skin, it is quite clear that localization of the probe signal is significantly higher
in the younger mouse, indicating the presence of a higher amount of elastin. We
found that the across all the slices, the average PA signal intensity from skin was
ꢀ1.8 times higher in the young mouse than in the older mouse, which is in good
correlation with the fluorescence imaging data (Figure 5D). An essential element
of the dermal connective tissue, elastin fibers, decreases with age as a result of
reduced synthesis and increased degradation, resulting in skin sagging and reduced
skin elasticity. This result indicates that ElaNIR is capable of showing differences
between young and old mice and the underlying change in elastin that comes
with aging.31
In summary, we have discovered ElaNIR as a NIR bioimaging probe suitable for
in vitro, ex vivo, and in vivo imaging of elastin structures. ElaNIR offers a simple
and convenient tool for fluorescence imaging of elastic structures, which will be
valuable for understanding the function of elastin.
EXPERIMENTAL PROCEDURES
All procedures for animal experiments were approved by the Institutional Animal
Care and Use Committee at the Agency for Science, Technology, and Research
(A*STAR) of Singapore. Full experimental procedures are provided in the Supple-
mental Information.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures, 20 fig-
ures, and 6 tables and can be found with this article online at https://doi.org/10.
1016/j.chempr.2018.02.016.
ACKNOWLEDGMENTS
We gratefully acknowledge intramural funding from the A*STAR (Agency for Sci-
ence, Technology, and Research of Singapore) Biomedical Research Council and
Chem 4, 1–11, May 10, 2018
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Please cite this article in press as: Su et al., Seeing Elastin: A Near-Infrared Zwitterionic Fluorescent Probe for In Vivo Elastin Imaging, Chem
(2018), https://doi.org/10.1016/j.chempr.2018.02.016
from National Medical Research Council grant NMRC/TCR/016-NNI/2016. We
would like to thank the SBIC-Bruker Preclinical Imaging Centre, Bruker Singapore
(http://www.pci-lab.com/), SBIC-iThera Medical Imaging Centre, and SBIC-Nikon
Imaging Centre for their state-of-the-art imaging equipment. We are also very grate-
ful for the help of Dr. Jun-Young Kim, Hui Shan Cheng, Anandh Kumar Raju, Lim
Hann Qian, and Tay Hui Chen.
AUTHOR CONTRIBUTIONS
D.S., C.L.T., and Y.-T.C. conceived the project. D.S. synthesized the CyZW library
and conducted photophysical characterization and analysis. C.L.T., S.-J.P., J.-J.K.,
and A.S. performed animal imaging experiments. R.B., U.S.D., and M.O. performed
the photoacoustic imaging. M.P., F.L., and M.M. performed the 3D ball-tissue imag-
ing. S.S.K. and M.A.B. performed the in vivo mouse pharmacokinetic study. All
authors participated in writing the manuscript.
DECLARATION OF INTERESTS
The authors declare no competing interests.
Received: May 8, 2017
Revised: September 28, 2017
Accepted: February 16, 2018
Published: March 29, 2018
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