Design and Synthesis of a Ratiometric Photoacoustic Probe for in Situ Imaging of Zinc Ions in Deep Tissue in Vivo

Article abstract of DOI:10.1021/acs.analchem.9b05431

As a noninvasive deep-tissue imaging technique, photoacoustic (PA) imaging has great application potential in biomedicine and molecular diagnosis. The zinc ion (Zn²⁺), which is a necessary metal ion in the human body, plays a very important role in the regulation of gene transcription and metalloenzyme function. The imbalance of Zn²⁺ homeostasis is also associated with a variety of neurological diseases. Therefore, it is critically important to accurately image the steady-state changes of Zn²⁺ in vivo. However, no PA imaging method is currently available for Zn²⁺. To this end, we designed and synthesized the first PA probe of Zn²⁺, namely, CR-1 for in situ ratiometric imaging of Zn²⁺ in deep tissue in vivo. The CR-1molecule, combined with Zn²⁺, weakened the conjugation system of the π-electron in the CR-1 molecule, which resulted in the blue shift of its absorption peak from 710 nm to 532 nm. The PA signal intensity decreased at 710 nm and increased at 532 nm, and the ratiometric PA signal at these two wavelengths (PA₅₃₂/PA₇₁₀) showed a good linear relationship with the concentration of Zn²⁺ in the range of 0-50 μM, with a detection limit as low as 170 nM. Furthermore, this probe exhibits extremely fast responsiveness, is highly selective, and has excellent biocompatibility. We have used the developed PA probe for the ratiometric PA imaging of Zn²⁺ in the thigh tissue of mice, and we still can accurately image Zn²⁺ after covering chicken breast tissue on the surface of mice thigh. In light of these outstanding features, the developed PA probe has high potential for imaging Zn²⁺ in deep tissues; thus, it will open up new avenues for the study of the complex biochemical processes involving Zn²⁺ in vivo.

Full text of DOI:10.1021/acs.analchem.9b05431

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Article
Design and Synthesis of a Ratiometric Photoacoustic Probe
for in situ Imaging of Zinc Ions in Deep-Tissue in vivo
Chaobang Zhang, Rongkang Gao, Liangliang Zhang, Chengbo Liu, Zhengmin Yang, and Shulin Zhao
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b05431 • Publication Date (Web): 10 Mar 2020
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Design and Synthesis of a Ratiometric Photoacoustic Probe for
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in situ Imaging of Zinc Ions in Deep-Tissue in vivo
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Authors: Chaobang Zhang,†,# Rongkang Gao,‡,# Liangliang Zhang,† Chengbo
Liu,*,‡ Zhengmin Yang,† and Shulin Zhao*,†
Affiliation: † State Key Laboratory for the Chemistry and Molecular Engineering of
Medicinal Resources, Guangxi Normal University, Guilin, 541004,
China
‡
Research Laboratory for Biomedical Optics and Molecular Imaging,
CAS Key Laboratory of Health Informatics, Shenzhen Institutes of
Advanced Technology, Chinese Academy of Sciences, Shenzhen
518055, China.
Corresponding author: Professor Shulin Zhao and Chengbo Liu
E-mail: zhaoshulin001@163.com
cb.liu@siat.ac.cn
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ABSTRACT: As a non-invasive deep tissue imaging technique, photoacoustic (PA)
imaging has great application potential in biomedicine and molecular diagnosis. Zinc
ion (Zn2+), a necessary metal ion in human body, plays a very important role in the
regulation of gene transcription and metalloenzyme function. The imbalance of Zn2+
homeostasis is also associated with a variety of neurological diseases. Therefore, it is
critically important to accurately image the steady state changes of Zn2+ in vivo.
However, no PA imaging method is currently available for Zn2+. To this end, we
designed and synthesized the first PA probe of Zn2+ namely CR-1 for in situ
ratiometric imaging of Zn2+ in deep-tissue in vivo. The CR-1 combined with Zn2+
weakened the conjugation system of the π electron in the CR-1 molecule, which
resulted in the blue shift of its absorption peak from 710 nm to 532 nm. The PA signal
intensity decreased at 710 nm and increased at 532 nm, and the ratiometric PA signal
at these two wavelengths (PA532/PA710) showed a good linear relationship with the
concentration of Zn2+ in the range of 0 to 50 μΜ, with a detection limit as low as 170
nM. Furthermore, this probe exhibits extremely fast responsiveness, highly selective
and excellent biocompatibility. We have used the developed PA probe for the
ratiometric PA imaging of Zn2+ in thigh tissue of mice, and still can image accurately
Zn2+ after covering a chicken breast tissue on the surface of mice thigh. In light of
these outstanding features, the developed PA probe has high potential for imaging
Zn2+ in deep tissues, thus it will open up new avenues for the study of the complex
biochemical processes involving Zn2+ in vivo.
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Zinc ion (Zn2+), a necessary metal ion in human body, plays a very important
role in the regulation of the function of metalloenzymes, and gene transcription of
many proteins involved in a variety of cellular functions.1,2 The imbalance of Zn2+
homeostasis can leads to a variety of nervous system diseases.3,4 In addition, change
in Zn2+ concentration in the human body has been found to be closely associated with
the development of prostate cancer.5 Therefore, it is crucial to accurately image the
changes of Zn2+ in vivo.
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The traditional methods for the imaging of Zn2+ are mass spectrometry (MS)
imaging, magnetic resonance imaging (MRI) and photoluminescence (PL) imaging.6-8
Besides providing only general information on the total amount of Zn2+ and requiring
sample pretreatment, the MS technique cannot be used to image Zn2+ in the body in
real time. MRI can non-invasively image Zn2+ in real-time, but it is limited by lower
sensitivity and resolution. PL imaging has the advantages of high sensitivity and
resolution, but due to the strong scattering of light in biological tissues, its imaging
depth is only about 1 mm, and thus, its application in vivo is limited. The recently
developed photoacoustic (PA) imaging technique is a novel technology that combines
the high contrast of optical imaging with the high resolution and deep-tissue
penetration of ultrasonic imaging.9-11 Since the attenuation of sound waves in
biological tissues is three orders of magnitude lower than that of photons, PA imaging
can be used to perform real-time non-invasive high-resolution deep-tissue imaging.
Therefore, the PA imaging technique has great application potential in biomedicine
and molecular diagnosis.
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The PA imaging technology can achieve adequate resolution and image contrast
at a certain depth and provide morphological and functional information. Accordingly,
it has received increasing research interest in the field of biomolecule imaging. In the
past few years, PA imaging probes for various analytes have been developed and used
for imaging of biological small molecules,12,13 tumor tissue,14-16 tumor-associated
protease,17 calcium ion,18,19 copper ion20 and hypochlorous acid.21 These probes have
the potential to play an important role in biomedical research and early diagnosis of
target diseases. However, to the best of our knowledge, no PA imaging probe for Zn2+
imaging has been reported.
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Ratiometric PA imaging, through its built-in self-calibration system, can
eliminate the interference from the internal environment, such as uneven probe
accumulation and unstable excitation light intensity.22 Thus, the ratiometric PA
imaging has preferable application prospect for reliable imaging of biological tissue in
vivo. In this study, we designed and synthesized a ratiometric PA imaging probe of
Zn2+, namely CR-1, for highly selective ratiometric imaging of Zn2+ in vivo
deep-tissue. The complexation of CR-1 with Zn2+ resulted in the blue shift of its
absorption peak from 710 nm to 532 nm. The PA signal intensity decreased at 710 nm
and increased at 532 nm, and the ratiometric PA signal at these two wavelengths
(PA532/PA710) exhibited a good linear relationship with the concentration of Zn2+,
while other ions have no influence. We have demonstrated that the biological
application of the CR-1 by reversible ratiometric PA imaging of Zn2+ in the thigh
tissue of mice.
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EXPERIMENTAL SECTION
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Reagents and Materials. N,N-Dimethylformamide (DMF), phthalimide
potassium salt, 2,6-bis(chloromethyl)pyridine and di(2-picolyl)amine were purchased
from Accustandard Inc. (J&K Scientific Ltd., Beijing, China). Hydrazine
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monohydrate,
N,N,N′,N′-tetrakis
(2-pyridylmethyl)ethylenediamine
(TPEN),
nitrilotriacetic acid and indocyanine green (ICG) were obtained from Sigma-Aldrich
Co. (St. Louis, MO, USA) and used without any further purification. Milli-Q water
with a resistivity above 18 MΩ·cm was used in the experiments.
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Apparatus. Proton (1H) or C nuclear magnetic resonance (NMR) spectroscopy
was performed on a Bruker Avance III HD 400 MHz NMR spectrometer (Bruker
BioSpin GmbH, Ettlingen, Germany). MS analysis was performed on an Exactive
Liquid Chromatography-Mass Spectrometry (LC-MS) system (Thermo Fisher
Scientific Inc., Waltham, MA, USA). UV-vis absorption spectra were performed on a
Cary 60 UV-vis spectrophotometer (Agilent Technologies Inc., Santa Clara, CA,
USA). Cytotoxicity tests were performed using a Multiskan MK3 Enzyme Mark
spectrophotometer (Thermo Fisher Scientific Inc.). A homemade PA computed
tomography system was used in all PA imaging experiments.23
Synthesis of CR-1. The heptamethyl-carboline derivative (IR825, 0.38 mmol)
and tris (2-pyridylmethyl) amine (TMPA, 0.40 mmol) were added into a 100-mL
round bottom flask under nitrogen protection. Finally, 15 mL of DMF was added into
the round bottom flask, the mixed solution was heated to 70 °C with continuous
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stirring and refluxed for 1 h. Subsequently, the solvent was removed by vacuum
distillation. The crude product was then purified by column chromatography using
DCM/MeOH (v/v, 20:1) as eluent to produce a blue solid product, namely CR-1, with
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+
1
a yield of 31.3%. [M+H]+=894.520 (calcd. for C61H64N7 : 894.521). H NMR (400
MHz, CD2Cl2) δ=8.39 (d, J=4.9 Hz, 2H), 7.93 (d, J=8.5 Hz, 2H), 7.82–7.79 (m, 3H),
7.78 (d, J=2.3 Hz, 2H), 7.72 (d, J=7.7 Hz, 1H), 7.60 (td, J=7.5, 1.8 Hz, 3H), 7.54 (d,
J=7.8 Hz, 2H), 7.46–7.41 (m, 2H), 7.40 (d, J=8.7 Hz, 2H), 7.32–7.29 (m, 2H), 7.28 (d,
J=0.8 Hz, 1H), 7.22 (d, J=7.9 Hz, 1H), 7.18 (d, J=8.8 Hz, 2H), 7.08–7.06 (m, 1H),
7.05 (d, J=2.4 Hz, 1H), 5.68 (d, J=13.1 Hz, 2H), 4.02 (s, 2H), 3.94 (d, J=10.9 Hz, 6H),
2.51 (t, J=6.2 Hz, 4H), 2.17 (t, J=7.6 Hz, 2H), 1.92 (dd, J=10.5, 4.2 Hz, 2H),
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1.81–1.77 (m, 2H), 1.74 (s, 12H), 1.53–1.39 (m, 4H). C NMR (100 MHz, CD2Cl2)
δ=176.41, 169.14, 168.17, 157.97, 155.46, 149.21, 148.51, 148.33, 147.72, 147.66,
147.50, 145.86, 140.17, 139.42, 139.06, 138.78, 138.69, 138.24, 137.84, 137.51,
137.31, 131.60, 131.10, 130.23, 129.99, 128.60, 127.47, 127.30, 127.19, 124.84,
124.65, 124.05, 123.94, 123.66, 122.88, 122.73, 122.49, 121.99, 120.83, 120.53,
119.06, 118.99, 118.81, 113.92, 109.98, 94.35, 60.07, 59.71, 49.75, 34.94, 34.56,
32.04, 31.27, 30.09, 29.81, 29.48, 28.07, 22.81, 14.02, 11.82.
Determination of apparent dissociation constant of CR-1- Zn2+ complex. The
absorption spectra of CR-1-Zn2+ complex solutions were used to determine the
apparent dissociation constant (Kd),24 and the following equation is used to calculate
the Kd: R=(Rmax[Zn2+]+Kd Rmin)/(Kd+[Zn2+]). In the above formula, R is the absorption
ratio (Abs532/Abs710), Rmax is the maximum absorption ratio, Rmin is the absorption
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ratio without adding Zn2+, and [Zn2+] is the free concentration of Zn2+. 10 mM
nitrilotriacetic acid was used to regulate the concentration of free Zn2+.
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In Vitro Cytotoxicity Test and in Vivo Toxicity Assessment. The human
prostate carcinoma 22Rv1 cell line was selected as model cells, and the MTT
(methylthiazoly-ldiphenyl-tetrazolium bromide) assay was used to evaluate the
cytotoxicity of CR-1. The 22Rv1cells were incubated on 96-well cell culture plates
and incubated for 24 h at 37 °C with 5% CO2. Then, different concentrations of CR-1
(0, 5, 10, 25, 50, 100 μM) were added to the cells in the wells of the culture plate, and
incubated for another 24 h. Subsequently, 15 μL of MTT solution (5 mg/mL) was
added to each well, and the cells in plate was further incubated for an additional 4 h.
Afterwards, the cell culture medium was removed from the wells and 150 μL of
DMSO was added into each well. The absorbance of the solution in each well was
measured using a Multiskan MK3 Enzyme Mark spectrophotometer at a wavelength
of 570 nm. The in vivo toxicity of CR-1 was evaluated in female BALB/c mice (20-22
g) by monitoring the body weight of the mice every day for 14 days, after intravenous
injection into the tail vein of the mice of phosphate-buffered saline (PBS) for the
control group and CR-1 (3 mg/kg) for the experimental group.
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Animal. The 8-week old female BALB/c mice were supplied by Hunan SJA
Laboratory Animal Co., Ltd. (Changsha, China). Animal handling procedures were
approved by the Animal Ethics Committee of Guangxi Normal University (No.
20150325-XC). During the weight monitoring experiment, the weight of the mice and
any sign of clinical abnormality were recorded daily. Once a mouse was determined
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to be unable to eat, injured or dead, the mouse was excluded from the study.
In Vivo PA Imaging. Fifteen female BALB/c mice were divided into 5 groups
(3 mice in each group). All the solutions were injected subcutaneously into the thighs
of the mouse under the guidance of ultrasound imaging. The first group (control group)
received 50 μL HEPES buffer; the second group received 50 μL CR-1 solution (50
μM); the third group received a subcutaneous injection of a Zn2+ solution (0.25
mg/kg), followed by 50 μL of CR-1 solution (50 μM); the fourth group received a
subcutaneous injection of a Zn2+ solution (0.5 mg/kg), followed by 50 μL of CR-1
solution (50 μM); the fifth group received a subcutaneous injected of a mixture of
Zn2+ (0.5 mg/kg) and the same amount of TPEN, followed by 50 μL of CR-1 solution
(50 μM). The PA signals in the thigh of the mice were measured with the self-made
optical coherence tomography system (PACT) using a laser at the wavelengths of 532
nm and 710 nm. To further demonstrate that CR-1 still has the ability to image Zn2+ in
vivo deep tissue, subcutaneous injection of Zn2+ solution (0.5 mg/kg) into the thigh of
mice was followed by injection of CR-1 solution (50 μM，50 μL). PA signals at 532
nm and 710 nm were then measured after covering chicken breast in the injection
area.
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RESULTS AND DISCUSSION
Design and Synthesis of CR-1. The synthesis route of CR-1 is shown in Figure 1.
In developing a high sensitive PA imaging probe of Zn2+, firstly, an Zn2+ ligand
(TMPA) was synthesized according to the method reported by Yoon and
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co-workers.25,26 Then, a near-infrared dye molecule (IR825) was synthesized by the
condensation reaction (Supporting Information). Ultimately, the target product CR-1
was obtained by the nucleophilic substitution reaction between IR825 and TMPA.
Figures S1–S3 reveal the nuclear magnetic resonance (NMR) spectra and mass
spectrometry (MS) spectrum of IR825. Figures S4-S6 display the NMR spectra and
MS spectrum of CR-1. The synthesized CR-1 is very stable in aqueous solution
(Figure S7) and has a large molar absorption coefficient (Figure S8), thus it readily
yields a high photoacoustic signal.
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Principle for PA Imaging of Zn2+. The CR-1 consists of a heptamethyl-carboline
derivative (IR825) and the Zn2+ ligand (TMPA). The molecular structure of this probe
and the reaction mechanism with Zn2+ are illustrated in Scheme 1. When CR-1
interacts with Zn2+, the five nitrogen atoms on the TMPA group of the probe
coordinate with Zn2+, thus causing the nitrogen atom attached to the IR825 molecule
to remove one hydrogen atom. Subsequently, the double bonds on IR825 are
rearranged, which weakens the π electron conjugated system and shifts the absorption
peak of CR-1 from 710 nm to 532 nm (Scheme 1a). When the laser was used at the
wavelengths of 710 nm and 532 nm, the PA710 signal decreased, while the PA532
signal increased (Scheme 1b). In addition, there is a good linear relationship between
the ratio of the PA signal (PA532/PA710) and the concentration of Zn2+ in a certain
concentration range, so that the PA532/PA710 ratio-based PA imaging can be used to
monitor Zn2+in vivo.
Feasibility of CR-1 as PA Probe of Zn2+. To investigate the feasibility of using
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CR-1 as a PA probe for ratiometric PA imaging of Zn2+, we examined the changes in
the absorption spectrum after the reaction of CR-1 with Zn2+. The ultraviolet-visible
(UV-vis) absorption spectra of the CR-1 solutions with different concentrations of
Zn2+ are shown in Figure 2a. These spectra reveal that, with the increase of Zn2+
concentration in the range of 0-5 μM, the absorption intensity at 710 nm (Abs710) of
the CR-1 solutions decreases gradually, while the absorption intensity at 532 nm
(Abs532) increases gradually. The response process can be completed in seconds, and
the color of the solution changes from light blue to pink (inset in Figure 2a). The
reaction mechanism between CR-1 and Zn2+ at a 1:1 was confirmed by electrospray
ionization-mass spectrometry (ESI-MS) (Figures S9 and S10). The m/z peak of CR-1
is located at 894.52124 (calculated value was 894.5200), and the m/z peak of CR-1
combined with Zn2+ is located at 478.72183 (calculated value was 478.72). The
apparent dissociation constant (Kd) for CR-1 binding to Zn2+ was determined to be 4.3
nM by plotting the absorption ratio at 532 and 710 nm (Figure S11). Which is higher
than that of di(2-picol)amine (23 nM).27 In addition, we found a good linear
relationship between the ratio of absorption intensity (Abs532/Abs710) of CR-1 and the
concentration of Zn2+ in the range of 0-5 μM (Figure 2c), with a limit of detection as
low as 87 nM. Subsequently, the PA signals of CR-1 produced with different
concentrations of Zn2+ were measured in vitro, with the increase of Zn2+ concentration,
the PA signals of CR-1 solution at 710 nm was gradually weakened, while that at 532
nm was gradually enhanced (Figure 2b). These results reveal that the ratiometric PA
signal (PA532/PA710) of CR-1 has a good linear relationship with the concentration of
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Zn2+ in the range of 0-50 μM, with a detection limit as low as 170 nM (Figure 2d).
Since the gray matter region of the brain contains about 0.5 mM of Zn2+.28 Therefore,
we think that such a detection limit is much lower than the content of Zn2+ in the brain,
indicating that CR-1 has considerable potential to detect Zn2+ in the brain or other
biological tissues by ratiometric PA imaging.
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Photostability of CR-1 prob for Zn2+ detection. One of the biggest challenges of
using cyanine dyes for photoacoustic probe development is rapid photobleaching.
Therefore, we evaluated the photostability of using CR-1 as a photoacoustic probe of
Zn2+, the result is shown in Figure S12. Although the photostability of CR-1 is
slightly worse than that of ICG, the photoacoustic signal of the probe remains stable
after 50 cycles of photoacoustic imaging (Figure S13). More importantly, even though
the probe suffered severe photobleaching at the detection of Zn2+, the absorption ratio
of the probe (Abs532/Abs710) did not change significantly (Figure S14). Therefore,
CR-1 possesses sufficient photostability for photoacoustic imaging of Zn2+.
Reversibility of Binding Reaction Between CR-1 and Zn2+. The reversibility of
the binding reaction between CR-1 and Zn2+ was studied using N, N, N’, N’-tetrakis
(2-pyri-dylmethyl) ethylenediamine (TPEN) as a competitive reagent. The result
shown in Figure 3a reveals that after adding Zn2+ to CR-1 solution, the ratio of
absorption intensity (Abs532/Abs710) rapidly increased, and then, after adding an
excessive amount of TPEN, rapidly decreased to its original value. After adding
excessive Zn2+ again, the value of Abs532/Ab710 returned to the same level. The repeat
of such operation 5 times, all show the same phenomenon. When the ratiometric PA
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intensity of the solution (PA532/PA710) is measured, the reversible cycle process can
also be observed (Figure 3b), indicating that the binding reaction between CR-1 and
Zn2+ is reversible. In addition, we also found that at pH value between 6.2 and 8.2, the
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Abs532/Abs710 and PA532/PA
values of CR-1 solution remained basically
710
unchanged, while the Abs532/Abs710 and PA532/PA710 values of the complex increased
with the increase of the pH value of the solution (Figure S15). These findings show
that the CR-1 probe has good stability in the range of pH 6.2-8.2. The sensitivity of
PA imaging for Zn2+ increases with the increase of the environmental pH value.
Although the sensitivity increases with increasing pH value, the PA532/PA710 value is
about 0.36 in the presence of trace Zn2+ even at pH 6.2, which is still 3.2 times higher
than the probe itself (about 0.11). In general, the brain maintains its pH around 7.4 by
regulating acid-base balance.29 Accordingly, the CR-1 probe can monitor Zn2+ levels
by ratiometric PA imaging in physiological conditions (pH 7.4).
Specificity of the CR-1 for Zn2+. We investigated the specificity of the CR-1
probe for Zn2+, and the results are presented in Figure 4a and Figure S16a. The results
reveal that the maximum absorption of the CR-1 solution shifts from 710 nm to 532
nm only in the presence of Zn2+. At the same time, the PA signal of CR-1 solution at
710 nm was weakened, while that at 532 nm was enhanced. Apart from the slight
interference due to Cu2+, other metal ions including Ag+, Fe2+, Ca2+, Fe3+, Ni2+, Cd2+,
Na+, K+, Al3+, Pb2+, Cr3+, Mn2+, Hg2+, Co2+ and Mg2+ do not interfere with Zn2+
detection, and the ratio of PA intensity (PA532/PA710) and the ratio of absorption
intensity (Abs532/Abs710) remain basically unchanged (Figure 4b, Figure S16b).
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Moreover, other metal ions do not interfere with the combination of the CR-1 probe
and Zn2+, and the absorption intensity (Abs532/Abs710) and PA strength (PA532/PA710)
ratios of the CR-1 solution at the wavelengths of 710 nm and 532 nm in the presence
of Zn2+ with various metal ions remain essentially constant (Figure 4c and Figure
S17). These results show that the CR-1 probe is a highly-specific PA probe for the
detection of Zn2+.
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Cytotoxicity and Biocompatibility of the CR-1. We also evaluated the
cytotoxicity and biocompatibility of the CR-1. First, the cytotoxicity of CR-1 was
determined by the MTT assay. As shown in Figure 5a, the survival rate of 22Rv1 cells
incubated with CR-1 solution for 24 h was still more than 85%. Additionally, as
shown in Figure 5b, the assessment of the biocompatibility of CR-1 in mice by tail
vein injection of CR-1 found neither death nor significant weight loss in the mice after
14 days of administration of CR-1. These results indicate that the synthesized CR-1
probe has low cytotoxicity and good biocompatibility, suggesting that it can be used
for the imaging of Zn2+ in vivo.
Imaging of Zn2+ in Deep Tissue in Vivo. In order to study the feasibility of using
CR-1 to image Zn2+ in deep tissue in vivo, different substances were subcutaneously
injected into the mouse thigh tissue. Then, the PA signals in the mouse thigh tissue
treated with different substances were measured under laser light irradiation at 710
nm and 532 nm, and the results are shown in Figure 6. The mice treated with buffer
(control group) showed a negligible PA signal at 710 nm and a weak PA signal at 532
nm. This is due to the absorption of hemoglobin in the blood at the wavelength of 532
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nm. Therefore, when calculating the PA532/PA710 value, the background signal
intensity of the control group should be deducted first. The mice treated with CR-1
only had a strong PA signal at 710 nm and a weak PA signal at 532 nm, with a
PA532/PA710 ratio of about 0.15. The PA signal of mice treated with 0.50 mg/kg Zn2+
and CR-1 at 710 nm was significantly decreased, and the PA signal at 532 nm was
significantly increased, which increased the PA532/PA710 ratio value to 0.74.
Compared to mice treated with CR-1 alone, mice treated with 0.50 mg/kg Zn2+ and
CR-1 had a PA532/PA710 value 4.9 times higher. When Zn2+ was masked with TPEN,
the PA710/PA532 value was almost the same as that in mice treated with CR-1 alone,
indicating that the increase of the PA710/PA532 value was due to the presence of Zn2+.
To further evaluate the potential of CR-1 for imaging of Zn2+ in deep-tissue in
vivo, we simultaneously injected CR-1 and Zn2+ subcutaneously into mouse thigh
tissue and performed PA imaging after covering on the mouse thigh surface with
chicken breast tissue. As shown in Figure 7, despite the weakening of the PA signal
intensity, the distribution of Zn2+ in vivo can still be visualized in the deep tissue,
indicating that the synthesized CR-1 probe has the potential for imaging of Zn2+ in
deep-tissue in vivo.
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CONCLUSIONS
In summary, we have developed the first PA probe (CR-1) of Zn2+ for in vivo
ratiometric PA imaging. This probe reacts with Zn2+ resulting in the blue shift of its
absorption peak from 710 nm to 532 nm, which causes an increase in the PA signal at
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the excitation wavelength of 532 nm, while the PA signal decreases at the excitation
wavelength of 710 nm, and the ratiometric PA signal at these two wavelengths
(PA532/PA710) exhibited a good linear relationship with the concentration of Zn2+.
Therefore, the CR-1 can be used to monitor the steady state changes in Zn2+
concentration in the tissue of living animals by ratiometric PA imaging. Furthermore,
the designed probe showed a highly selective PA response to Zn2+ with a favourable
detection limit of 170 nM. In particular, this probe displayed an extremely fast
response, permitting the trapping of transient Zn2+ and real-time monitoring of
Zn2+-related biological processes. This study provides a new way for in situ
monitoring Zn2+ in vivo, contributing to the understanding of the relationship of Zn2+
to various neurological diseases. More importantly, as the imaging process is
reversible, it will open up new avenues for the study of the complex biochemical
processes involving Zn2+ in deep-tissue in vivo.
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications
website at DOI: 10.1021/acs.analchem.xxxxxxx.
Synthesis procedure of IR825; supplementary figures for 1H NMR spectrum of
1
IR825, 13C NMR spectrum of IR825, MS spectrum of IR825, H NMR
spectrum of CR-1, 13C NMR spectrum of CR-1, MS spectrum of CR-1,
absorption spectra of CR-1 at different time, absorption spectra of CR-1
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solutions at different concentrations, MS spectrum of CR-1-Zn2+ complex, the
detection of apparent dissociation constant (Kd) for CR-1 binding to Zn2+,
photostability of CR-1 and ICG solution, PA images of CR-1-Zn2+ solution at
different cycles, absorption spectra of CR-1-Zn2+ solutions at different time
(min) under continuous wave laser irradiation, the ratio of absorption intensity
(Abs532/Abs710) of CR-1 and CR-1-Zn2+ solutions under different pH, the ratio
of PA intensity (PA532/PA710) of CR-1 and CR-1-Zn2+ solutions under
different pH, absorption spectra of CR-1 solution in the presence of different
metal ions, the ratio of absorption intensity (Abs532/Abs710) of CR-1 solution in
the presence of different metal ions, the ratio of PA intensity (PA532/PA710) of
CR-1 solution in the presence of different metal ions.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: zhaoshulin001@163.com (S.Z.) and cb.liu@siat.ac.cn (C. L.).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was supported by the National Natural Science Foundations of China
(Grant No. 21874030) and BAGUI Scholar Program.
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Scheme 1. Schematic diagram of the CR-1 reaction with Zn2+ (a) and its application
for ratiometric PA imaging of Zn2+ in vivo (b).
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Figure 1. The syntheti c route of CR-1.
Figure 2. The absorption and PA characteristics of the CR-1 reaction with Zn2+. (a)
UV-vis absorption spectra of CR-1 solutions (10 mM HEPES containing
10% acetonitrile, pH=7.4) with different concentrations of Zn2+, inset
diagram shows the change of color of the solution before and after the
reaction between CR-1 and Zn2+; (b) the PA signals of CR-1 solution at 710
nm and at 532 nm with different concentrations of Zn2+; (c) linear
relationship between the ratio of absorption intensity (Abs532/Abs710) of
CR-1 solution and the concentration of Zn2+ in the range of 0-5 μM; (d) the
linear relationship between the ratio of PA intensity (PA532/PA710) of the
CR-1 solution (50 μM) and the concentration of Zn2+ in the range of 0-50
μM.
Figure 3. (a) The reversible change of the ratiometric absorption intensity after the
addition of Zn2+ or TPEN in the CR-1 HEPES solution; (b) the reversible
change of the ratiometric PA intensity after the addition of Zn2+ or TPEN in
the CR-1 HEPES solution.
Figure 4. Specificity of the CR-1 towards Zn2+. (a) PA imaging of the CR-1 solution
(50 μM CR-1, 10 mM HEPES containing 10% acetonitrile, pH=7.4) at 710
nm and 532 nm in the presence of different metal ions; (b) the ratio of PA
intensity in the CR-1 solution (50 μM) at 710 nm and 532 nm in the presence
of different metal ions. The metal ions 1→18: blank, Ag+ ，Fe2+ ，Ca2+ ，
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Fe3+，Ni2+，Cd2+，Cu2+，Na+，K+，Al3+，Pb2+，Cr3+，Mn2+，Hg2+，
Co2+，Mg2+, Zn2+ (Zn2+ is 50 μM, Na+，K+，Ca2+ and Mg2+ is 5 mM, and other
metal ions are 100 μM); (c) the ratio of absorption intensity (Abs532/Abs710)
of the CR-1 solution (5 μM) at 710 nm and 532 nm under the coexistence of
Zn2+ and different metal ions. The metal ions 1→18: blank, Zn2+，Zn2++Al3+，
Zn2++Ca2+，Zn2++Fe3+，Zn2++Cd2+，Zn2++Co2+，Zn2++Cr3+，Zn2++Hg2+，
Zn2++Fe2+，Zn2++K+，Zn2++Mg2+，Zn2++Cu2+，Zn2++Mn2+，Zn2++Na+，
Zn2++Ni2+，Zn2++Pb2+，Zn2++Ag+ (5 μM each).
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Figure 5. (a) Survival rate of 22Rv1 cells after 24 h incubation with different
concentrations of CR-1. (b) Body weight change of the mice during 14 days
following the intravenous injection of PBS (control group) and CR-1 (3
mg/kg) into the tail vein of the mice.
Figure 6. The imaging of Zn2+ in thigh tissue of living mouse. (a) In vivo ultrasound
(US) and PA imaging of mice treated with buffer (control), CR-1, 0.25 mg/kg
Zn2++CR-1, 0.50 mg/kg Zn2++CR-1，Zn2++TPEN+CR-1 at 710 and 532 nm;
(b) PA signal intensity of mice treated with different substances at 710 and
532 nm; (c) changes in the PA signal intensity ratio (PA532/PA710) of mice
treated with different substances at 710 and 532 nm, with an inline image of
the PA ratio. **p ＜ 0.01, the data were expressed as mean ± standard
deviation (n=3).
Figure 7. The PA imaging of Zn2+ in deep tissue of living animals. (a) PA imaging at
532 nm and 710 nm, and ultrasound (US) imaging of mice treated with Zn2+
and CR-1 after the mouse thigh surface was covered with chicken breast tissue;
(b) the PA signal strength corresponding to that in (a).
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