Small-Molecule Absorber A1094 as a Stable and Fast-Clearing NIR-II Imaging Agent

Article abstract of DOI:10.1002/cmdc.202100125

Photoacoustic imaging (PAI) in the second near-infrared window (NIR-II) is conducive to deep-tissue imaging due to small scattering coefficients, but often requires exogenous imaging agents. At present, nanoparticle-based NIR-II imaging agents are mainly used in non-clinical studies, some basic components of which are resistant to metabolism in vivo. The aim of this study was to examine the ～600 Da croconaine absorber A1094, absorbing lights around 1094 nm, as a rare, small-molecule NIR-II imaging agent in vivo. The clinical translational potential of A1094 injection were systematically revealed, including sufficient solubility and freeness in blood, good anti-interference ability, and favourable pH/oxidation-reduction/metabolic stabilities. After intravenous administration of A1094 injection, PAI of murine ears exhibited comparable capillaries visibility to that of PAI with popular Au nanorods. The contrasts achieved with A1094 and Au nanorods were 1.78 and 1.29 times higher than before administration, in the healthy group, and 3.25 and 1.58 times higher in the inflammation group. Notably, A1094 demonstrated a desired faster liver clearance than Au nanorods. The PAI signal of A1094 was cleared by 74.2 % after 3 h, whereas Au nanorods were only cleared by 43.1 %. The main metabolic mechanisms of A1094 were identified as N-methylation and lipid hydrolysis by murine liver microsomes in vitro. Therefore, A1094 may have promising clinical potential as a stable and fast-clearing NIR-II imaging agent.

Full text of DOI:10.1002/cmdc.202100125

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Small-Molecule Absorber A1094 as a Stable and Fast-
Clearing NIR-II Imaging Agent
Yanfang Huang,[a] Huanhuan Liu,[a] Xueyuan Chen,[a] Yunming Zhang,[a] Xingyang Zhao,[a, b]
Wenchao Huang,[a] Zhaobiao Mou,[a] Chao Wang,[a] Kaiqiang Zhang,[a] and Zijing Li*[a]
Photoacoustic imaging (PAI) in the second near-infrared
window (NIR-II) is conducive to deep-tissue imaging due to
small scattering coefficients, but often requires exogenous
imaging agents. At present, nanoparticle-based NIR-II imaging
agents are mainly used in non-clinical studies, some basic
components of which are resistant to metabolism in vivo. The
aim of this study was to examine the ~600 Da croconaine
absorber A1094, absorbing lights around 1094 nm, as a rare,
small-molecule NIR-II imaging agent in vivo. The clinical transla-
tional potential of A1094 injection were systematically revealed,
including sufficient solubility and freeness in blood, good anti-
interference ability, and favourable pH/oxidation-reduction/
metabolic stabilities. After intravenous administration of A1094
injection, PAI of murine ears exhibited comparable capillaries
visibility to that of PAI with popular Au nanorods. The contrasts
achieved with A1094 and Au nanorods were 1.78 and 1.29 times
higher than before administration, in the healthy group, and
3.25 and 1.58 times higher in the inflammation group. Notably,
A1094 demonstrated a desired faster liver clearance than Au
nanorods. The PAI signal of A1094 was cleared by 74.2% after
3 h, whereas Au nanorods were only cleared by 43.1%. The
main metabolic mechanisms of A1094 were identified as N-
methylation and lipid hydrolysis by murine liver microsomes
in vitro. Therefore, A1094 may have promising clinical potential
as a stable and fast-clearing NIR-II imaging agent.
Introduction
organic absorbers are highly attractive, by virtue of their higher
biocompatibility, relatively small molecular mass, and rapid
clearance from the body.[3,7] However, due to disadvantages
such as low PA signal, poor photostability, and small optical
absorption cross sections,[14] the vast majority of the organic
absorbers used as contrast agents must be currently enclosed
in liposome polymer matrices to increase absorption efficiency,
stability and agent size beyond the renal clearance
threshold.[3,15,16] Liposomes are also prone to oxidisation and are
sensitive to temperature and pH, so this strategy is still
associated with some practical problems. Ideally, optical probes
for clinical use should be easily metabolised and should possess
Photoacoustic (PA) imaging (PAI) is a hybrid biomedical
imaging modality that integrates optical excitation and ultra-
sound detection.[1,2] The shifting of excitation wavelengths from
the first near-infrared window (NIR-I; 700–1000 nm) to the
second near-infrared window (NIR-II; 1000–1700 nm) can result
in reduced tissue scattering, deeper tissue penetration, mini-
mised background autofluorescence, and improved image
fidelity.[3–5] Due to the insufficiency of the NIR-II PA signal
generated by tissues, exogenous contrast agents are often
necessary to enhance the contrast and specificity of PAI in large
volumes of biological tissues.[6–8]
Researchers have endeavoured to explore efficient and
biocompatible contrast agents for NIR-II PAI. Most of the current
contrast agents are inorganic materials, including metal
sulphides, such as CuS, Ag2S, and ZnS,[9–11] carbon-based nano-
absorbers,[12] and semi-conductor nanocrystals.[13] However,
unfavourable biocompatibility and pharmacokinetics have im-
peded the clinical application of these materials. In contrast,
excellent
optical
properties,
light
stability,
and
biocompatibility.[17] Hence, further efforts are necessary to
develop small-molecule organic absorbers with sufficient
photostability, absorption, NIR-II PA signal generation and
favourable pharmacokinetics.[18,19]
A1094, a ring-open croconaine derivative first synthesised
by Tian et. al for use in optical communications, is perhaps the
smallest of the few small organic molecules with an absorption
wavelength greater than 1000 nm.[20,21] Its characteristic strong
NIR-II absorption peak at 1094 nm with a high molar extinction
coefficient (7.3×104 LmolÀ 1 cmÀ 1) is attributed to its dielectric
large π-conjugated structure, which greatly promotes electron
resonance and energy transfer.[22] A1094 is more stable than the
traditional long-straight-chain cyanine absorbers in vitro, prob-
ably due to its short-planar, rigid structure.[23–25] However, it is
not clear yet whether A1094 qualifies as a NIR-II PAI agent, i.e.
whether it possesses the stability, safety, and fast clearance
in vivo which are required of optical probes used for clinical
applications. Therefore, we aimed to provide a first comprehen-
[a] Y. Huang, H. Liu, X. Chen, Y. Zhang, X. Zhao, W. Huang, Z. Mou, C. Wang,
K. Zhang, Dr. Z. Li
Center for Molecular Imaging and Translational Medicine
State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics
School of Public Health
Xiamen University
Xiamen, Fujian 361102 (China)
E-mail: zijing.li@xmu.edu.cn
[b] X. Zhao
Department of Hepatobiliary Surgery
Zhujiang Hospital, Southern Medical University
Guangzhou, 510280 (China)
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cmdc.202100125
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sive preclinical evaluation of A1094 and its potential application
in basic blood pool imaging.
By observing the dissolution of solid A1094 and the colour
change before and after dissolution (Figure 1i), it was concluded
that A1094 was easily soluble in dichloromethane and
acetonitrile, soluble in methanol, DMSO and ethanol, and
slightly soluble in cyclohexane and n-hexane. It was also soluble
in aqueous solution containing 0.25% DMSO, with solubility up
to 0.5 mg/mL.
Results and Discussion
Characterization and physicochemical properties of A1094
The characterization of the deep-purple solid A1094 (yield:
1
8.3%) by MS, H and 13C NMR is shown in Figures S8, S9 and
Stability of A1094
S10. The UV-Vis-NIR absorption spectra of the same concen-
tration of A1094 in different organic solvents and aqueous
solution featured a broad, strong peak with 1094 nm as the
middle axis (Figure S1). A1094 possessed the strongest absorp-
tion capacity in acetonitrile (ɛ=7.3×104 L×molÀ 1 ×cmÀ 1), fol-
lowed by that in dichloromethane (ɛ=4.7×104 L×molÀ 1 ×cmÀ 1)
and DMSO (ɛ=3.6×104 L×molÀ 1 ×cmÀ 1) (Figure S4), probably
due to higher polarity. The absorption capacity in aqueous
solution was approximately the same as that in non-protonic
solvents such as dichloromethane (Figure S4). The absorption
intensity of A1094 was about three times higher that of the Au
nanorods at an equal mass concentration (0.05 mg/mL) (Fig-
ure 1j).
In terms of pH stability, the UV-Vis absorption spectra remained
almost unchanged in the range of pH 6–8 (Figures 1b, 1c), the
physiological pH interval. After the addition of various metal
ions, the UV-Vis absorption spectra were nearly consistent with
that of the control (Figure 1d). This observation indicated that
A1094 injection possessed a strong anti-interference ability in
the presence of biologically necessary metal ions. The absorp-
tion spectra of A1094 in solutions containing glutathione and
H2O2 indicated that the endogenous redox species had a
negligible effect on the chemical stability of A1094 (Figure 1d).
When A1094 was dissolved in FBS, the absorption intensity
decreased only slightly over a course of 120 min (Figure 1e).
These characteristics indicate that A1094 injection showed
Figure 1. Stabilities, photothermal and photoacoustic properties of small-molecular absorber A1094. (a) Absorption spectra of A1094 over 0–140 min with
10/20 min intervals in an aqueous solution (0.25% DMSO) and absorption spectra of endogenous tissue chromophores in the human body.[26] (b) Absorption
spectrum of A1094 at different pH values. (c) Absorption intensity of A1094 corresponding to (b). (d) Stability of A1094 in the presence of various metal ions
and re-dox species. (e) Retention of A1094 in serum over 120 min. (f) Serum protein loading capacity of A1094. (g,h) Photothermal conversion properties of
A1094, Blank=aqueous solution (0.25% DMSO). (i) Illustration drawing of the preparation of A1094 injection. (j) Comparison of absorption intensities between
A1094 and Au nanorods at the concentration of 0.05 mg/mL. (k) PA intensity of A1094 in a series of concentrations (1.0, 0.5, 0.25, 0.125, and 0.0625 mg/mL) in
aqueous solutions (0.25% DMSO). (l) PA intensity of Au nanorods in a series of concentrations (1.0, 0.5, 0.25, 0.125, and 0.0625 mg/mL).
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good stability, which is conducive to further study in vivo.
Furthermore, after the preparation of A1094 injection, it could
be stored for 24 h without change.
(Figure 2a–d). Local blood vessel dilation with little acute
exudate was clearly observed in inflamed ears. When Au
nanorods were injected in inflammation and control vessels
instead, 58% enhancement of PA signal was found in the
former case. This indicated the potentially at least comparable
sensitivity imparted by the use of A1094 in angiography
(Figure 2e–h).
Photothermal and PAI properties of A1094
Time-temperature curves and photothermal images showed
that the photothermal efficiency was linearly dependent on the
concentration of A1094 (Figures 1g, 1h). At 172 μM, there was
PAI of murine livers
2
°
an increase in temperature by 20 C after irradiation (1 W/cm ,
5 min), whereas the temperature of the blank (ultrapure water
The PA signal intensity was significantly enhanced during the
period 0–2 min after i.v. injection, indicating that A1094
possessed the ability to provide enough blood pool imaging
contrast. At 2–50 min period, the PA signal drops rapidly from
the peak. Moreover, the PA signal essentially disappeared at
50 min post-injection, which was cleared by 74.2% after 3 h,
indicating that A1094 was highly conducive to liver clearance
(Figure 3a). The rapid clearance may also be related to its low
plasma protein binding rate (15.1%, Figure 1f). Since liver is the
main metabolic organ of lipophilic small molecules, fast
clearance of absorber would not only reduce background
interference but also relieve liver burden. In contrast, after the
i.v. injection of Au nanorods, the PA signal increased slowly and
reached its peak value after 10 min. The PA signal also
decreased slowly, which was cleared by 43.1% after 3 h, and
was still perceptible on the seventh day, indicating poor hepatic
metabolism of Au nanorods (Figure 3b). Au nanorods were
easily ingested by the liver and not easily degraded after
ingestion. However, A1094 was easily degraded in liver, which
°
with 0.25% DMSO) increased by only 6 C. Besides excellent
photothermal conversion efficacy, the absorption intensity of
A1094 in 0.25% DMSO aqueous solution measured at 8
different time points over a course of 140 min was basically
unchanged, indicating the photothermal stability of A1094
(Figure 1a). Quantitative analysis of the PA signal showed that
there was a linear relationship between PA signal and the
concentration of A1094. In addition, the PA signal of A1094 was
superior to that of the Au nanorods at the same mass
concentration, an observation which highlights the potential of
A1094 for use in PAI (Figures 1k, 1l).
PAI of murine ears with and without acute inflammation
To evaluate the effect of A1094 injection as contrast agent
in vivo, it was intravenously injected to murine ear vessels. As
strong as 3.25 times PA signal was observed in acutely inflamed
murine ear vessels, compared with that in normal vessels
Figure 2. PAI of murine ears with A1094 and Au nanorods. (a) PAI of murine ears after i.v. injection of A1094 under 970 nm laser irradiation. (b) PAI of
murine ears with inflammation after i.v. injection of A1094 under 970 nm laser irradiation. (c) Increased PA signal in the inflammation ear after injection of
A1094 solution. (d) Corresponding PA intensity of (a) and (b). (e) PAI of murine ears after i.v. injection of Au nanorods under 970 nm laser irradiation. (f) PAI of
murine ears with inflammation after i.v. injection of Au nanorods under 970 nm laser irradiation. (g) Increased PA signal in the inflammation ear after injection
of Au nanorods. (h) Corresponding PA intensity of (e) and (f).
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Figure 3. Metabolism of A1094 in vivo and in vitro. (a) PAI of murine livers after i.v. injection of A1094 and corresponding PA signal value. (b) PAI of murine
livers after i.v. injection of Au nanorods and corresponding PA signal value. (c) Structure and mass spectrum of A1094 and its metabolites after metabolism by
murine liver microsomes in vitro.
lead to rapid elimination of PA signal in liver compared with Au
nanorods.
Biocompatibility and biosafety of A1094 injections.
Even at concentrations of up to 200 μM, the survival of N2a cells
remained high (Figure 4a), indicating that A1094 was signifi-
cantly safe and biocompatible. This observation laid the
foundation for further applications in vivo.
Evaluation of A1094 metabolites in mouse liver microsome
After incubation with mouse liver microsomes in vitro, the MS
molecular peak corresponding to A1094 completely disap-
peared from the MS spectra. Two new peaks appeared, with MS
(ESI) m/z values of 212.7 and 376.1 ([M+H]+). Based on the
structure of A1094, N-dealkylation and lipid hydrolysis were
considered to be the modes of A1094 metabolism (Figure 3c).
The metabolites were water-soluble and easily discharged from
the body, so they showed few toxic side effects. In addition, the
absorption intensity at 1094 nm was weakened after liver
hydrolysis, corresponding to the rapidly decreasing liver PA
signal value during 2–50 min.
When the main organs of the mice were collected for H&E
staining, tissue morphology revealed A1094 injection to be safe
for use (Figure 4b), as no obvious damage was observed. There
was no significant change in serum chemistry (Figure 4h–k) or
whole blood parameters (Figures 4d–g, S3). During long-term
toxicity testing, the skin colour and eyes of the mice were
normal, no abnormal phenomena were found, and no death
occurred, further indicating that the preparation had good
biocompatibility (Figure 4c).
Existing NIR-II PA contrast agents are inorganic nanomate- Conclusion
rials, which are usually not conducive to metabolism in vivo.
However, PAI in murine livers showed that A1094 was more
conducive to liver clearance than Au nanorods, suggesting that
A1094 might be a promising candidate for clinical use.
In summary, an injection of the small-molecular croconaine
absorber A1094 were prepared for NIR-II photoacoustic imag-
ing. In the NIR-II window, A1094 possessed an absorption
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Figure 4. Biocompatibility and toxicity of A1094. (a) Cell viability of N2a cells after treatment with different concentrations of A1094. (b) H&E staining of
major organs from mice administered A1094 for 3 days (15 mg/kg, n=3). (c) Amount of survival mice after 90 days. (d–g) Haematological analysis of mice
treated with PBS or A1094. MCH: mean corpuscular hemoglobin; MCHC: mean corpuscular hemoglobin concentration; RBC: red blood cell; WBC: white blood
cell. (h–k) Serum chemistry of mice treated with PBS or A1094. ALT: alanine transaminase; ALP: alkaline phosphatase; AST: aspartate transaminase; UREA:
blood urea nitrogen.
Experimental Section
efficiency comparable to that of the Au nanorods, and
produced effective NIR-II imaging. With deeper penetration into
the NIR-II window, the PAI of inflamed murine ears showed a
significantly enhanced PA signal and a rich contrast after
intravenous injection of A1094, and the images clearly showed
dilated vessels and blood exudate. Moreover, A1094 exhibited
good pH stability, metal ion stability, oxidation-reduction
stability, photothermal performance, and PA signal strength,
which are considerable advantages for use as a PAI contrast
agent.
N-dealkylation and lipid hydrolysis were identified as the
main metabolic pathways of A1094. Rapid hepatic clearance
was observed after the imaging window, indicating a high level
of biosafety and a reduced metabolic burden. Therefore, A1094
may be more easily approved for clinical use than other
imaging agents. Our tests of A1094 were repeatable, which is
difficult to achieve in nanomaterial investigations. The method
of purity identification was simple and easily reproducible.
Due to the limited water solubility of A1094, its structure
should be modified to improve its water solubility in follow-up
studies, making it more suitable for application in vivo. In
addition, some targeted modifications could be made to make
it specifically bind to biomarkers in vivo, an approach which is
valuable for the diagnosis of disease.
A1094 was obtained using a modified version of a previously
reported protocol,[27] and characterized by H, 13C nuclear magnetic
1
resonance (NMR) spectroscopies and mass spectrometry (MS).
Stabilities of A1094
Solubility of A1094 in different solvents. Equal amounts of A1094
(2.0 mg, 3.5 μmol) were added to each of several clean tubes. Then
0.5 mL each of methanol, DMSO, dichloromethane, acetonitrile,
cyclohexane, n-hexane, and ethanol were added to the tubes, and
the complete dissolution of A1094 after vortex vibration was
observed. Meanwhile, different amounts of A1094 were placed in a
clean tube, 50 μL DMSO was added, and the volume made up to
20 mL with ultrapure water; this produced a 99.75% aqueous
solution with 0.25% DMSO. The solubility of A1094 in aqueous
solution was observed to be up to 0.5 mg/mL (0.86 mM).
Stability of A1094 in different solvents. The absorption spectra of
A1094 at intervals of 30 or 40 min in each of the different solvents
(methanol, DMSO, dichloromethane, acetonitrile and ethanol) were
recorded.
pH stability. A1094 was dissolved in aqueous solutions (0.25%
DMSO, 99.75% aqueous solution: v/v, 25 μg/mL, 43.3 nmol/mL)
with different pH values (from 2 to 12).
Stability in the presence of different metals. A1094 was dissolved
in aqueous solutions (0.25% DMSO, 50 μg/mL, 86.6 nmol/mL) and
various metal ions (1.0 mM K+, Na+, Ca2+, and Mg2+; 25 μM Ba2+
,
Co2+, Fe3+, Mn2+, Ni2+, Pb2+, Al3+, Fe2+, Cu+, and Cu2+) were added
respectively to the solutions. The absorption spectra were meas-
ured after 2 h incubation.
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Stability in the presence of H2O2 and glutathione. A1094 was
dissolved in aqueous solutions (0.25% DMSO, 50 μg/mL, 86.6 nmol/
mL). H2O2 (100 μM) and glutathione (100 μM) were added respec-
tively to the solutions. The absorption spectra were measured after
2 h incubation.
0.125, and 0.0625 mg/mL were prepared. The PA signals were
measured at 1200 nm and 970 nm.
PAI of murine ears with and without acute inflammation
All of the animals used in the experiment were provided by the
Experimental Animal Center of Xiamen University. ICR mice were 4–
5 weeks old, weighed 18–22 g, and were male. The animal experi-
ments were conducted in accordance with the Guidelines for
Laboratory Animal Use and Care approved by the Xiamen
University Animal Care and Use Committee.
Stability in serum. A1094 was dissolved in aqueous solutions
(0.25% DMSO, 50 μg/mL, 86.6 nmol/mL) and 200 μL of the solution
was added to 2 mL of serum. The absorption spectra were recorded
at intervals of 30 min over a course of 120 min.
Determination of the plasma protein binding rate
Establishment of acute ear inflammation model in mice. 30 μL
xylene was daubed on the upper and lower sides of the right
auricle of ICR mice for 20 min to induce acute ear swelling as a
model of inflammation.
Specified amounts of A1094 (12.5, 25, and 50 μg/mL) were
dissolved in 50 μL of DMSO, 950 μL of foetal bovine serum (FBS)
was added, and the solutions were mixed using a vortex mixer. The
°
samples were incubated at a constant temperature of 37 C in a
water bath for 60 min. The incubated solutions were removed and
placed on ice, and the temperature was maintained at approx-
PAI of murine ears with and without acute inflammation. To
explore the in vivo PAI potential of A1094, PAI of murine ears with
and without acute inflammation was conducted. Firstly, the PAI of
normal murine ears were scanned, and PAI signals were collected
immediately after i.v. injection of A1094. Moreover, the PAI of the
inflamed murine ears were scanned, and PAI signals were collected
immediately after i.v. injection of Au nanorods. The PAI abilities of
A1094 and Au nanorods were compared in vivo by injecting each
agent into mice without acute inflammation.
°
imately 4 C. Next, 500 μL of each solution was transferred to an
ultrafiltration tube, placed in a low-temperature centrifuge, and
centrifuged at 4 C at 14000×g for 20 min to obtain ultrafiltration
solutions. The absorption spectra of the incubated and ultrafiltered
solutions were determined. The binding rate of plasma proteins
and the standard absorption-mass curve of A1094 was calculated
using formula (1).
°
Plasma protein binding rate ð%Þ ¼
½ðCðincubation solutionÞ-Cðultrafiltration solutionÞÞ=Cðincubation solutionÞ� � 100
Evaluation of metabolites of A1094
(1)
PAI of clearance of A1094 from murine livers in vivo. After an
intravenous injection of A1094, the dynamic processes of liver
metabolism were visualised, and the PA signal of the A1094 was
quantified, to measure the metabolic clearance rate.
C(incubation solution) represents the mass concentration (μg/mL) of A1094
in the incubation solution before ultrafiltration, and C(ultrafiltration solution)
represents the mass concentration (μg/mL) of A1094 in the ultra-
filtration solution.
Preparation of murine liver microsomes. After fasting for 16 h, the
mice were sacrificed using cervical vertebral dislocation, and a
laparotomy was performed. Then, normal saline cooled in an ice
bath was aspirated with a syringe, injected into the liver through
the thoracic artery or portal vein, and perfused until the liver turned
white, indicating that the blood in the liver had been removed. The
liver microsomes were prepared using differential centrifugation.
First, the liver tissue was weighed, cut into pieces, and washed
repeatedly with 0.1 mol/L potassium phosphate buffer (pH 7.4).
Then, the above buffer was added at a ratio of 1:4 (w/v), and the
tissue was placed in an ice bath with a glass homogeniser. The
Preparation and quality control of the A1094 injection
Preparation of the A1094 injection. Before i.v. injection, 1.0 mg
A1094 was dissolved in 20 μL DMSO. Then, 50 μL of Tween 80, a
commonly used solubilizer, and 930 μL of saline were added and
mixed using ultrasound to prepare a 1.0 mL solution, i.e., 1.0 mg/
mL A1094 injection.
Quality control of the A1094 injection. The A1094 injection – with
a pH between 5.0–8.0 – was a clear solution, and exhibited a main
peak at 1094 nm in the UV spectra. In order to ensure the sterility
of the injection, all A1094 injections were filtered through 0.22-μm
filter membranes before use. In addition, to ensure a uniform effect
of the injection dose, 100 μL was decided as the injection dose per
mouse throughout the whole experimental process.
°
supernatant was centrifuged at 9000×g for 20 min at 4 C and then
at 100 000×g for 60 min. The liver microsomes were resuspended
with buffer containing 20% glycerol and then placed in
a
°
refrigerator at À 80 C on standby.
Metabolite analysis. 100 μL diluent of the murine liver microsomes
was taken and 200 μL of an NADPH coenzyme regeneration system
(0.5 mmol/L NADP+, 5 mmol/L G-6-P, 2 unit/mL G-6-PDH, 6 mmol/L
magnesium chloride), which was freshly prepared and pre-aerated
for 1 min, was added and mixed well to yield the pre-incubation
Preparation of Au nanorod injection. Aqueous solutions of Au
nanorods with a 1125 nm absorption at the same mass concen-
tration gradient were prepared, and the PA signals were measured
at 1200 nm and 970 nm.
°
solution. The pre-incubation solution was placed in a 37 C water
bath and shaken for 3 min. Next, 100 μL of the 80 μmol/L A1094
solution was added to initiate the incubation reaction (the final
concentration of A1094 was 20 μmol/L). Lastly, 200 μL of cold
acetonitrile solution was added to the incubation tube, which was
then quickly immersed in an ice bath to stop the reaction.
Photothermal and PAI properties. To test the photothermal
effects, A1094 in aqueous solution (10.8, 21.5, 43, 86, 172 μM) was
exposed to a 980 nm laser (1 W/cm2 for 6 min). During laser
irradiation, the temperature was continuously monitored using a
charge-coupled device camera. To study photothermal stability, the
absorption spectrum of A1094 was evaluated before and after
treatment with laser irradiation. We recorded the absorption
spectrum of A1094 at intervals of 10 or 20 min over the course of
140 min. A1094 solutions with concentrations of 1.0, 0.5, 0.25,
Biocompatibility test
A1094 cytotoxicity assessment. Mouse neuroblastoma N2a cells
were seeded into 96-well plates at a concentration of 5×103 cells/
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[2] P. Beard, Interface Focus 2011, 1, 602–631.
well and cultured for 24 h. Then, N2a cells were treated with
different concentrations of A1094 (0, 12.5, 25, 50, 100 and 200 μM).
After 12 h, the viability of N2a cells was evaluated by standard CCK-
8 test.
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Haematological analysis. Samples of 500 μL whole blood were
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This study was financially supported by the National Natural
Science Foundation of China (81971674), Science Fund for
Distinguished Young Scholars of Fujian Province (2019 J06006),
Fundamental Research Funds for the Central Universities
(20720180050), and the Scientific Research Foundation of State
Key Laboratory of Molecular Vaccinology and Molecular Diagnos-
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Conflict of Interest
The authors declare no conflict of interest.
Keywords: croconaine absorber
·
small molecule
·
photoacoustic imaging · optical contrast agent · the second
near-infrared window
Manuscript received: February 22, 2021
Revised manuscript received: April 18, 2021
Accepted manuscript online: April 20, 2021
Version of record online: ■■■, ■■■■
[1] P. K. Upputuri, M. Pramanik, J. Biomed. Opt. 2019, 24, 1–20.
ChemMedChem 2021, 16, 1–8
www.chemmedchem.org
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FULL PAPERS
Because most current NIR-II imaging
agents are poorly metabolised
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