An activatable probe for detecting alcoholic liver injuryviamultispectral optoacoustic tomography and fluorescence imaging

Article abstract of DOI:10.1039/d0cc04635c

A probe has been developed for imaging alcoholic liver injury through detecting the overexpressed cytochrome P450 reductase in hypoxia in the hepatic region. Upon response to the enzyme, the activated probe displays turn-on fluorescence and near-infrared absorption and generates prominent optoacoustic signals.

Full text of DOI:10.1039/d0cc04635c

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Activatable Probe for Detecting Alcoholic Liver Injury via
Multispectral Optoacoustic Tomography and Fluorescence Imaging
a
a
a
a
a
Junjie Chen, Yichang Fang, Lihe Sun, Fang Zeng* and Shuizhu Wu*
Received 00th January
0xx,
2
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
1
4-
A probe has been developed for imaging alcoholic liver injury α reaches 4 for tissues) reduces the imaging depth and resolution,
1
8
which would erode the performance of fluorescent imaging for
through detecting the overexpressed cytochrome P450 reductase
in hypoxia in hepatic region. Upon response to the enzyme, the
activated probe displays turn-on fluorescence and near-infrared
absorption and generates prominent optoacoustic signals.
liver, as liver is relatively deep seated in the body. Therefore, the
imaging method with deep penetration depth and high temporal and
spatial resolution for alcohol-induced liver injury would be ideal.
Optoacoustic imaging is a rapidly developing and non-invasive
imaging technique with great potential for preclinical/clinical
1
9,20
applications.
In-vivo optoacoustic imaging could combine the
Alcoholic liver diseases (ALD) as a result of excessive alcohol
consumption are the most prevalent cause for advanced liver
diseases and have become a major cause of liver-related mortality
advantages of optical imaging and ultrasound imaging, such as high-
contrast, reduced tissues’ light scattering and deep imaging depth. In
particular, as a spectral optoacoustic technique, multispectral
optoacoustic tomography (MSOT) operates by irradiating a sample
with multiple wavelengths and then detecting ultrasound waves
produced by photoabsorbers in the sample; with spectral unmixing
MSOT can thus separately visualize individual photoabsorber in the
1
worldwide, as liver is the main site for alcohol metabolism and the
major target organ of alcohol-induced liver injury. Alcohol-induced
liver injury is the early stage of ALD which include hepatic lesions,
fatty liver, steatohepatitis, progressive fibrosis, cirrhosis and even
2
liver failure. Therefore, it is of great importance to detect alcohol-
8
,20
target sample.
Moreover, three-dimensional (3D) images are
induced liver injury, so as to plan optimal rehabilitation program and
efficacious treatment. Recent researches have shown that, in the
case of alcoholic liver injury, hepatic cytochrome P450 (CYP450)
provided with a stack of cross-sectional images being rendered as the
maximum intensity projection (MIP) images. Hence, with a suitable
probe that is responsive to hepatic CYP450, MSOT imaging method
would be conducive to accurately detecting alcohol-induced liver
injury with 3D information in a non-invasive and spatiotemporal way.
In the light of the foregoing, herein we developed an activatable
and water-soluble probe (NIR-NO) for detecting alcohol-induced liver
injury using the optoacoustic/fluorescent dual-mode turn-on
imaging. As for the chemical structure of this probe in terms of the
design rationale, a benzothiazole-xanthene chromophore serves as
the reporter, while an N-oxide moiety acts as both the fluorescence
quencher and the responsive unit that can undergo facile bio-
reduction by CYP450 reductase with oxygen deficiency; as a result,
upon response to CYP450 reductase in hypoxia, the N-oxide-
containing probe NIR-NO turns into the tertiary-amine-containing
3
-5
reductase is overexpressed along with enhanced hypoxia extent.
Therefore, the overexpression of hepatic CYP450 along with hypoxia
could act as an in-situ biomarker for alcohol-induced liver injury, and
methods for sensitive detection of CYP450 reductase in hypoxia in
hepatic region could aid in the early diagnosis of alcohol-induced
liver injury, so that effective treatment measures could be taken to
prevent the liver injury from worsening into severe ALD.
Usually diagnostic methods for liver injury include blood
6
biochemistry, histopathological examination and liver biopsy.
However, these methods still have some limitations; for example, as
for blood chemistry, it usually measures the levels of the enzymes
that liver releases into blood, however, the enzymes also are present
in other organs or tissues besides the liver, which could compromise
the detection specificity and thereby could give false positive results;
2
chromophore (NIR-NEt ) which elicits an enhanced fluorescence at
7
-10
745 nm as well as a red shift in the absorption from 550 nm to 718
nm and correspondingly strong optoacoustic signals (as shown in
Scheme 1,). Owing to the change in intramolecular electronic push-
pull state with the electron-withdrawing N-oxide element turning
into electron-donating group diethylamino, both the fluorescent and
optoacoustic signals are turned on. The probe exhibits additional
beneficial features, e.g. the introduction of sulfonate group
while histopathological examination and liver biopsy are invasive
and require relatively time-consuming and complicated operation
1
1
process. Hence non-invasive fluorescent imaging method has
1
2,
recently been employed for detecting alcohol-induced liver injury.
1
3
-α
However, strong light scattering by tissues (which scales with λ ,
a. State Key Laboratory of Luminescent Materials and Devices, Guangdong Provincial
Key Laboratory of Luminescence from Molecular Aggregates, College of Materials enhances water solubility that facilitates biological applications
Science and Engineering, South China University of Technology, Guangzhou 510640,
China. E-mail: mcfzeng@scut.edu.cn; shzhwu@scut.edu.cn.
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
commonly in aqueous media, and it also is conducive to the probe’s
reaching and accumulating in liver, furthermore the dual-mode
(MSOT and fluorescent) imaging allows mutual corroboration. In vivo
imaging results indicate that, the probe can quickly respond to the
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overexpressed CYP450 reductase in the liver region and thereby
provide temporal and 3D spatial information of the disease foci for we therefore evaluated the probe’s responsDeOtI:o1w0a.1r0d39th/De0eCnCz0y4m6e35iCn
alcohol-induced liver injury. vitro by incubating NIR-NO with MLM with nitrogen bubbling. As
As mouse liver microsomes (MLM) are rich in CYP4V5ie0wrAertdiculecOtanlsinee,
The activatable probe was synthesized by coupling of a sulfonate- shown in Fig S13, for the probe, after treatment with MLM, the
containing benzothiazole derivative with xanthene via Knoevenagel changes in fluorescence and absorption spectra are similar to those
reaction, and then N-oxide was attached onto the xanthene side measured for the probe upon reaction with CYP450 reductase in PBS.
through oxygenation with m-chloroperbenzoic acid. The detailed
synthetic route of the NIR-NO is given in Scheme S1. The probe NIR-
1
NO and some intermediates were characterized by H NMR and high-
resolution
mass
spectroscopy
(Fig.
S1-S8).
Fig. 1 (A) Fluorescence spectra (excitation: 700 nm) and (B)
Absorption spectra of NIR-NO (10 μM) before and after incubation
with different concentrations of CYP450 reductase with nitrogen
bubbling. (C) Fluorescence intensity at 745 nm for NIR-NO (10 μM) as
a function of CYP450 reductase level (n=3). Inset: Representative
fluorescent images of the probe towards varied concentrations of
Scheme 1 Schematic display of the probe’s response to the
overexpressed CYP450 reductase in hepatic region (A) and
fluorescent and MSOT imaging of alcohol-induced liver injury in
mouse model (B).
0 0
CYP450 reductase. (D) Relative optoacoustic intensity [(I-I )/I ] for
The spectral properties of NIR-NO were investigated with
CYP450 reductase in PBS solution (pH=7.4, 10 mM, 37 C) with
oxygen deficiency. As shown in Fig. 1, in the absence of CYP450
reductase, NIR-NO is weakly emissive at 745 nm and exhibits a strong
absorption at 550 nm; while incubation with CYP450 reductase
gradually increases the fluorescence emission at 745 nm and the
absorption red-shifts, correspondingly optoacoustic signal at around
the probe upon incubation with varied concentrations of CYP450
reductase (n=3). Inset: Representative optoacoustic images of the
NIR-NO in phantom after being incubated with varied concentrations
of CYP450 reductase with nitrogen bubbling.
The research by Patterson et al. demonstrated that N-oxides
could undergo two-electron reduction by CYP450 reductase in
hypoxia with a heme-iron center as an active site and thus transform
7
18 nm enhances. These results demonstrate that the probe NIR-NO
could be employed for both fluorescence and optoacoustic imaging
via responding to CYP450. Additionally, the time-dependent spectral
measurements were performed after incubating the probe with
CYP450 reductase with nitrogen bubbling for varied time. As shown
in Fig. S9, with the prolonged time of reaction between the probe
NIR-NO and CYP450 reductase, the fluorescence intensity increases
at 745 nm and the absorbance increases at 718 nm and reaches the
plateau in about 10 min, indicating the fast response of the probe. In
addition, the probe shows good pH stability (as shown in Fig. S10).
To test the sensing selectivity, the probe NIR-NO was treated
with various potential and biologically-relevant interfering species,
such as hyaluronidase, γ-glutamyl transferase, β-D-galactosidase,
alkaline phosphatase, acetylcholinesterase, nitroreductase,
heparinase and sulfhydryl-containing compounds like GSH and then
fluorescence intensities and relative optoacoustic intensities
measurements were conducted. As shown in Fig. S11-S12, NIR-NO
shows high selectivity towards CYP450 reductase. Moreover, the
21
into tertiary amines. Based on this, the proposed mechanism for
the probe’s response (CYP450 mediated N-oxide reduction of the
probe) is presented in Fig S14. To confirm this mechanism, high-
performance liquid chromatography (HPLC) measurement was
conducted. As shown in Fig. S15, upon treatment with CYP450
reductase under hypoxia, the peak for NIR-NEt
appeared while that for the NIR-NO (at 2.41 min) decreased
accordingly. This demonstrates that NIR-NEt is generated after
2
(at 3.64 min)
2
incubation with CYP450 reductase under hypoxia condition.
Prior to the cell imaging, the cytotoxicities of the probe were
tested by MTT assay. As shown in Fig. S16, the probe NIR-NO displays
low cytotoxicity towards the HepG2 cells and L929 Cells. Notably, the
cell viability remains nearly 90% even after being incubated with 50
μM of the probe. The low-toxic probe is suitable for imaging in live
22
cells. HepG2 cells are rich in CYP450 reductase. Therefore, imaging
of CYP450 reductase under hypoxia in HepG2 cells by the probe was
conducted. From Fig. S17, we can see that the cells incubated with
the probe under hypoxia for 4 h exhibit evident fluorescence
extinction coefficient for NIR-NEt
was determined to be 2.2×10 M cm .
2
(the activated probe) at 718 nm
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(
excitation filter: 710 nm). While for the cells incubated in normoxia, different doses of alcohol and the rehabilitation groupV.ieAwsArstihcloewOnnlinine
weak intracellular fluorescence can be observed. For optoacoustic Fig. 3E, upon treatment with higher dose oDf OalIc:o10h.o10l,3t9h/De0sCigCn0a4ls63a5rCe
imaging of CYP450 reductase under hypoxia in HepG2 cells, as shown stronger, indicating that the CYP450 reductase in the liver is
in Fig. S17, significant OA signals can be seen in the cells; while OA remarkably higher compared to that of the control group. While
signals are insignificant in the cells under normoxia. To verify the OA weak OA signal can be seen for the rehabilitation group.
signal and fluorescence was indeed caused by CYP450 reductase, an
inhibitor of P450 reductases (diphenyl iodonium chloride (DPI), 500
μM) was used to preincubate the cells before imaging.23,24 As shown
in Fig. S17, the DPI pretreated HepG2 cells generate very weak OA
signal and weak fluorescence compared to the cells without inhibitor
pretreatment. These results suggest that the probe can be activated
by CYP450 reductase in hypoxia and generate optoacoustic and
fluorescence signals and thus it could be used for detecting alcohol-
induced liver injury via responding to hepatic CYP450 in hypoxia.
To evaluate the biosafety of the probe, the histology sections
H&E staining) of organs collected from the control and the mice
(
intravenously injected with the probe were compared. As depicted
in Fig. S18, there is no obvious histopathological difference between
the sections of probe-treated mice and the control, indicating that
the probe NIR-NO is biocompatible. As alcohol-induced liver injury
occurs, hepatic CYP450 reductase is overexpressed with enhanced
3
hypoxia in the liver. We thus investigated the probe’s capability for
fluorescent imaging of alcohol-induced liver injury via responding to
hepatic CYP450 reductase in hypoxia. In this study, alcohol was
delivered into mice by oral gavage to induce alcoholic liver injury.
Then the mice were i.v. injected with the probe and fluorescent
imaging was performed for varied time. The time-dependent
fluorescent images for the mice pre-treated with PBS (control group),
-1
-1
4
g·kg or 8 g·kg alcohol and the rehabilitation group (treated with
metadoxine) are presented in Fig. 2. It can see that, for the alcohol-
treated mice groups, the fluorescent signals in the liver region are
quite prominent after the probe injection, and reaches the maximum
in about 10 min. The alcohol-treated group exhibits stronger
-1
fluorescence than the control group, and a higher dose (8 g·kg ) of
alcohol causes more prominent fluorescence, while the
rehabilitation group exhibits weaker fluorescence signal. It seems
that the activated probe is metabolized out of the body through the
intestines. The mean fluorescent intensities obtained from the Fig.2 Fluorescence imaging of alcohol-induced liver injury in mouse
region of interest (ROI) for the four groups provide quantified model. (A) Time course for fluorescence imaging in vivo. The mice
fluorescence changes (Fig. 2 B). In addition, ex vivo imaging was were pretreated with PBS, 4 g/kg EtOH, 8 g/kg EtOH or 8 g/kg EtOH
performed for the dissected organs of the mice (Fig. 2C). As shown in + 200 mg/kg metadoxine once a day for three consecutive days,
-1
Fig. 2C and Fig. S19, it is obvious that the fluorescence in liver is much followed by tail intravenous injection of 2.7 mg kg NIR-NO in PBS.
stronger than other organs such as heart, spleen, lung, and kidney.
(B) Mean fluorescence intensity at ROI (red-line circle) of the liver
Afterwards, multispectral optoacoustic tomography was region in mice of (A). (C) Fluorescence images for major organs
employed to image alcohol-induced liver injury in mice. Fig. 3A shows harvested from the mice treated with varied dose of EtOH and the
-
1
the cross-sectional images of the mice pretreated with 8 g·kg
rehabilitation group. (excitation filter: 710 nm, emission filter: 750
alcohol at different time points after probe injection. The lower panel nm)
in this figure displays the colour images representing biodistribution
2
of the activated probe NIR-NEt via spectral unmixing, while the
As alanine transaminase (ALT) level in blood is usually used to
assess liver functions, to verify that alcohol did induce the liver injury
in mice, the ALT activity in the blood of the mice was measured by
Elisa kit. As shown in Fig 3D, the mice groups treated with alcohol
show much higher levels of serum ALT than the control group; and
the blood chemistry test results indicate that the degree of liver
injury is in correlation with the dose of alcohol. Moreover, the organs
of the mice treated with PBS, 4 g·kg , and 8 g·kg of alcohol and the
rehabilitation group were collected for ex vivo imaging. As shown in
Fig. S22 and S23, the liver exhibits much stronger MSOT signal than
other major organs. Furthermore, the liver tissue sections (H&E
staining) from the mice were also examined to evaluate the liver
injury, as shown in Fig. 3F, we can see that the section for the control
group show no obvious pathological changes, while that for the mice
upper panel presents the overlay of activated probe’s image with the
background image (at 800 nm) as an anatomical reference in which
the spinal cord, thoracic aorta, and liver are labelled. At 10 min after
probe injection, the signal intensity reaches the maximum. The mean
signal intensities of the activated probe signal in the region of
interest (ROI) over time are shown in Fig. 3B, which provides
quantified evidence for the change in MSOT signals. The time-
dependent MSOT signals for the other three groups (pretreated with
-1
-1
-1
PBS or 4 g·kg alcohol and the rehabilitation group) are shown in Fig.
S20 and S21. Based on the simulated cryosection image of a male
mouse (Fig. 3C), it is clear that most signals from the activated probe
are in the liver area. In addition, we obtained orthogonal-view 3D
images for the control mice, the mice groups pretreated with
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J. Name., 2013, 00, 1-3 | 3
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treated with alcohol exhibits necrosis with infiltration of imaging the probe can locate liver injury precisely. ThVisiepwrAortbicelecOonulinlde
inflammatory cells.
serve as a promising tool for studyingDOthI:e10p.1h0y3s9i/oDlo0CgiCc0a4l 6a35nCd
pathological processes related to alcoholic liver diseases. And the
work would offer useful insight for the design of other probes for
MSOT imaging and fluorescent imaging applications in biology and
medicine.
This work was supported by NSFC (21875069 and 51673066),
the Natural Science Foundation of Guangdong Province
(
2016A030312002) and the Fund of Guangdong Provincial Key
Laboratory of Luminescence from Molecular Aggregates
2019B030301003).
(
Conflicts of interest
There are no conflicts to declare.
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Fig. 3 (A) Representative cross-sectional MSOT images of the mouse
upon injection of NIR-NO. The mouse was pre-treated with 8 g·kg-1
EtOH once a day for three consecutive days. Upper panel: overlay of
the activated probe NIR-NEt
wavelength (800 nm) background image. Lower panel:
multispectrally resolved signal for NIR-NEt (the activated probe).
Organ labelling: 1. Spinal cord; 2. Aorta; 3. Liver. Scale bar: 3 mm. (B)
2
signal with the grayscale single-
1
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liver; 7. portal vein; 8. stomach. (D) Serum levels of ALT for different
mice groups. (E) Representative orthogonal-view 3D MSOT images
for the mice at 10 min post i.v. injection of the probe (scale bar: 3
mm). (F) Representative H&E staining of liver sections from different
mice group (scale bar: 100 μm).
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In summary, a water-soluble activatable probe has been
developed for detecting and imaging alcoholic liver injury in mouse
model through near-infrared fluorescence and multi-spectral
optoacoustic tomography imaging. Via responding in-situ to the
overexpressed CYP450 reductase in hypoxia in hepatic region, the
0 Y. Wu, J. Chen, L. Sun, F. Zeng, S. Wu, Adv. Funct. Mater.,
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019, 29, 1807960.
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1 L. H. Patterson, Cancer. Metast. Rev., 1993, 12, 119-134.
2 Y. Lu and A. I. Cederbaum, Free. Radic. Biol. Med., 2008, 44,
723-738.
probe is activated with the N-oxide moiety being transformed into 23 H. J. Knox, T. W. Kim, Z. Zhu and J. Chan, ACS Chem. Biol.,
the aromatic tertiary amine group, thereby generating the NIR
chromophore with enhanced fluorescence and red-shifted
absorption which produce both the fluorescent and optoacoustic
signals. The probe is able to image and monitor alcohol-induced liver
injury and its rehabilitation in mouse model. Moreover, via MSOT
2018, 13, 1838-1843.
4 H. J. Knox, J. Hedhli, T. W. Kim, K. Khalili, L. W. Dobrucki and
J. Chan, Nat. Commun., 2017, 8, 1794.
2
4
| J. Name., 2012, 00, 1-3
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DOI: 10.1039/D0CC04635C
An activatable probe has been developed for imaging alcoholic liver injury through detecting the
overexpressed cytochrome p450 (cyp450) reductase in hypoxia in hepatic region.