ALP-activated probe for diagnosis of liver injury by multispectral optoacoustic tomography

Article abstract of DOI:10.1016/bs.mie.2021.06.019

In this chapter, we highlight the advantages of multispectral optoacoustic tomography (MSOT) technique and the activatable photoacoustic probes in the biomedical field, and give a brief introduction to enzyme-activated probes for disease diagnosis and therapeutic outcome evaluation. We also present a detailed description of the procedures for the synthesis of an activatable small molecule probe C₁X-OR₁ and confirmation of its specific response to alkaline phosphatase in solution and cells. With MSOT, the liposomal C₁X-OR₁ can be utilized for detection of hepatic ALP as well as for in vivo diagnosis of drug-induced liver injury in a three-dimensional manner.

Full text of DOI:10.1016/bs.mie.2021.06.019

CHAPTER TWELVE
ALP-activated probe for diagnosis
of liver injury by multispectral
optoacoustic tomography
Yinglong Wu, Fang Zeng*, Lihe Sun, Junjie Chen, and Shuizhu Wu*
State Key Laboratory of Luminescent Materials and Devices, Guangdong Provincial Key Laboratory of
Luminescence from Molecular Aggregates, College of Materials Science and Engineering, South China
University of Technology, Guangzhou, China
*Corresponding authors: e-mail address: mcfzeng@scut.edu.cn; shzhwu@scut.edu.cn
Contents
1. Introduction
302
2. Construction of activatable photoacoustic probes for detection of specific
biomarkers
303
305
3. Design of the ALP-activated small molecule photoacoustic probe (C1X-OR1)
4. Synthesis of the probe C1X-OR1 and the hepatocyte-targeting phospholipid
DSPE-PEG2000-ChA
4.1 Equipment
4.2 Reagents
4.3 Procedures
305
306
307
307
310
310
311
311
318
318
319
319
5. Response of C1X-OR1 toward ALP in solution
5.1 Equipment
5.2 Reagents
5.3 Procedure
6. Response of C1X-OR1 toward ALP in cells
6.1 Equipment
6.2 Reagents
6.3 Procedures
7. In vivo detection of ALP for imaging drug-induced liver injury by use
of liposomal C1X-OR1
7.1 Equipment
7.2 Regents
320
322
322
323
328
329
329
7.3 Procedure
8. Summary
Acknowledgment
References
#
Methods in Enzymology, Volume 657
ISSN 0076-6879
https://doi.org/10.1016/bs.mie.2021.06.019
Copyright 2021 Elsevier Inc.
All rights reserved.
301

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Yinglong Wu et al.
Abstract
In this chapter, we highlight the advantages of multispectral optoacoustic tomography
(MSOT) technique and the activatable photoacoustic probes in the biomedical field, and
give a brief introduction to enzyme-activated probes for disease diagnosis and thera-
peutic outcome evaluation. We also present a detailed description of the procedures for
the synthesis of an activatable small molecule probe C1X-OR1 and confirmation of its
specific response to alkaline phosphatase in solution and cells. With MSOT, the liposo-
mal C1X-OR1 can be utilized for detection of hepatic ALP as well as for in vivo diagnosis
of drug-induced liver injury in a three-dimensional manner.
1. Introduction
Drug-related toxicity leads to millions of emergency department visits
and even hospitalizations every year, thus causing an increasing concern in
the field of modern medicine (Wang, Avorn, & Kesselheim, 2013). Liver is
the primary organ for concentrating and metabolizing drugs (Corsini &
Bortolini, 2013), and drug-induced liver injury (DILI) with clinical mani-
festations ranging from asymptomatic biochemical changes to liver failure
(Sakatis et al., 2012), becomes one of the most common complications and
the main reason of post-marketing drug withdrawal (Kola & Landis, 2004).
Therefore, a preclinical or clinical screening method for drug-induced liver
injury is urgently needed to improve patient safety and therapeutic outcomes.
It has been reported that several biomarkers especially the circulating
ones have been utilized for diagnosis of DILI in clinical trials and clinical
practices (Yang, Salminen, & Schnackenberg, 2012). Among these, alkaline
phosphatase (ALP) which can hydrolyze phosphate monoesters into
corresponding hydroxyl products and inorganic phosphates shows a vital
role in maintaining the balance of dephosphorylation and phosphorylation
processes in cells (Fedarko, Bianco, Vetter, & Robey, 1990). Typically, ele-
vation of ALP levels in serum is considered to be associated with liver disease
(Kaplan & Righetti, 1970). However, in addition to being present in the
liver, ALP is also expressed in other tissues and organs including intestine,
bone, placenta, etc. (Coleman, 1992). Only the elevated level of ALP in
the liver can sufficiently serve as the indicator for liver dysfunction.
Hence, spatiotemporal detection of ALP level in liver is critical to diagnosis
of drug-induced liver injury.
For in vivo identification and quantification of disease-specific bio-
markers at molecular levels, the nonionizing and noninvasive photoacoustic
(PA, also known as optoacoustic) imaging which combines the superb

ALP-activated probe for diagnosis of liver injury
303
contrast and high sensitivity of optical imaging with the deep-tissue penetra-
tion and high-resolution attainable in ultrasound imaging, is an advantageous
approach (Wang & Yao, 2016). Particularly, multispectral optoacoustic
tomography (MSOT), which is an emerging functional photoacoustic imag-
ing technique, can map the distribution of specific contrast agents with unique
spectra by acquiring multispectral data and subsequently performing spectral
unmixing using the preset algorithms (Taruttis & Ntziachristos, 2015).
Furthermore, MSOT can realize three-dimensional (3D) visualization using
volumetric imaging technique or through reconstruction of a maximum
intensity projection (MIP) image from a stack of two-dimensional cross-
sectional images (Gottschalk et al., 2019), which can achieve accurate location
of the biomarkers in tissues of interest and hence displays great potential for
preclinical research and clinical practice.
To achieve functional PA imaging, various contrast agents including
inorganic nanomaterials, semiconducting polymer nanoparticles and organic
small-molecule dyes have been developed and used in PA imaging for
disease diagnosis, therapy guidance, evaluation of treatment outcomes,
etc. ( Jeevarathinam, Lemaster, Chen, Zhao, & Jokerst, 2020; Wu, Sun,
Zeng, & Wu, 2019). Among them, some activatable contrast agents (probes)
with the ability of specifically respond to biological stimuli and subsequently
eliciting changes in photoacoustic signal are quite advantageous, as they can
sensitively and specifically locate disease foci and track its dynamic processes
with low background noise (Knox et al., 2017).
In this chapter, we highlight the strategy for designing activatable PA pro-
bes for specific biomarker, and briefly introduce enzyme-activated probes
through concentrating on their photophysical properties, specific response
to enzymes and biomedical applications in MSOT imaging. Also, we give
a detailed overview of a small-molecule PA probe C1X-OR1, which is devel-
oped by our group and able to specifically respond to the hepatic ALP level
and thus achieving in vivo diagnosis of drug-induced liver injury using MSOT
(Wu et al., 2018).
2. Construction of activatable photoacoustic probes
for detection of specific biomarkers
When constructing an activatable PA probe for detection of a specific
biomarker with MSOT imaging, several issues should be carefully consid-
ered. First, the signaling chromophore should have a characteristic absorp-
tion peak with a quite high molar extinction coefficient at near-infrared

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Yinglong Wu et al.
(NIR) wavelengths, which affords sufficient contrast to endogenous
absorbers (such as hemoglobin, melanin, etc.) upon spectral unmixing even
at diluted concentrations. Second, the recognition moiety of a PA probe
must exhibit high sensitivity and specificity to its corresponding biomarker
in tissues of interest, thereby reducing undesirable background noise and
false-positive signals. Third, once the signaling chromophore and recogni-
tion moiety are selected or constructed, the activation mechanism should be
reasonably designed. Typically, the activation mechanisms for eliciting
changes in photoacoustic signal are usually achieved by the variations in
photophysical properties of the probes, which are caused by a number of
changes in molecular structure, such as conversion of intramolecular elec-
tronic state, alteration of intramolecular movement/rotation degree, trans-
formation between aggregate and nonaggregate states, and so on.
In recent years, a great number of activatable photoacoustic probes have
been developed for specific identification of various biologically-relevant
species such as nucleic acids, ions, ROS/RNS, enzymes, pH, etc. (Zlitni,
Gowrishankar, Steinberg, Haywood, & Gambhir, 2020). In particular,
enzyme-activated probes are ideal ones because enzyme-mediated reactions
exhibit some attractive advantages such as high efficiency and substrate spec-
ificity, thus contributing to rapid and precise disease diagnosis (Cheng et al.,
2020). For instance, hyaluronidase (HAase), which can selectively degrade
its substrate hyaluronan (HA) into small fragments, has been used as a
diagnostic and prognostic indicator for multiple cancers. For example, by
employing a hyaluronidase (HAase)-activated PA probe fabricated by
self-assembly of a positively charged tricyanofuran-containing polyene
(TCHM) as the signaling chromophore and the negatively charged HA as
the response moiety, the lymphatic metastasis and orthotopic bladder tumors
can be spatiotemporally located and diagnosed using MSOT (Wu, Chen,
Sun, Zeng, & Wu, 2019). In addition, a nitroreductase-reponsive PA
probe has also been developed through conjugation of an electron donor
dihydroxanthene, an electron acceptor quinolinium and the recognition
element nitrobenzyloxydiphenylamino. The overexpressed nitroreductase
in tumor cells can specifically turn on the probe’s PA signal, thus achieving
visualization of metastases from the orthotopic breast tumors to lymph nodes
and then lung, and monitor of the chemotherapy efficacy (Ouyang et al.,
2020). Moreover, many other probes are designed to be activated by
enzymes in the tumor microenvironment for tumor-related applications.
Among these activatable PA probes, the hepatic enzyme-activated probe

ALP-activated probe for diagnosis of liver injury
305
for diagnosis of liver disease is still limited, and this prompted our group to
develop an ALP-activated photoacoustic probe for imaging of drug-induced
liver injury.
3. Design of the ALP-activated small molecule
photoacoustic probe (C1X-OR1)
The ALP-activated photoacoustic probe C1X-OR1 consists of an
identification moiety phosphate (R1) and a signaling chromophore
C1X-OH which is also the activated product of the ALP-mediated reaction.
Upon responding to ALP, the enzyme-mediated hydrolysis transforms the
electron-withdrawing phosphate (R1) into the electron-donating hydroxyl,
thus converting C1X-OR1 into its activated product (C1X-OH). Different
from C1X-OR1, the activated product C1X-OH displays a relatively high
extinction coefficient (7.7ꢀ104 Mꢁ1 cmꢁ1) at 684nm, which is suitable for
MSOT imaging. Such an ALP-mediated structural transformation leads to
variation in absorption followed by a significant change in PA signal, thereby
realizing diagnosis of drug-induced liver injury with MSOT imaging
(Scheme 1). To achieve higher water dispersibility and hepatocyte targetability,
a phospholipid DSPE-PEG2000-ChA was synthesized and formed into lipo-
somes with the probe.
Scheme 1 Illustration for response of C1X-OR1 to ALP and its activation mechanism.
4. Synthesis of the probe C1X-OR1 and the
hepatocyte-targeting phospholipid
DSPE-PEG2000-ChA
The probe C1X-OR1 and hepatocyte-targeting phospholipid DSPE-
PEG2000-ChA were synthesized according to the synthetic routes depicted
in Scheme 2, and well-characterized by 1H NMR and mass spectrometry.

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Yinglong Wu et al.
Scheme 2 Synthetic routes of the probe C1X-OR1 and the hepatocyte-targeting phos-
pholipid DSPE-PEG2000-ChA.
The familiarity with basic synthetic laboratory equipment and standard
organic chemistry techniques are needed to understand the following syn-
thetic procedures. All solvents for silica gel chromatography are defined as
the ratio of volume to volume.
4.1 Equipment
1. Magnetic stirrer
2. Vacuum pump
3. Rotary evaporator
4. Vacuum oven
5. Refrigerator
6. Freeze-dryer
7. An oil bath
8. Two-neck round flasks
9. Pressure equalizing dropping funnels
10. Condensers
11. Buchner funnels
12. Columns for chromatography

ALP-activated probe for diagnosis of liver injury
307
13. Thin layer chromatography (TLC) silica gel
14. Dialysis membranes (MwCO¼1000)
15. Rubber stoppers
16. Syringes
17. Magnetic PTFE stir bars
4.2 Reagents
1. Phosphorus oxychloride
2. Cyclohexanone
3. 1,2,3,3-tetramethyl-3H-indolium iodide
4. Resorcinol
5. Diethyl chlorophosphate
6. Bromotrimethylsilane
7. Cholic acid
8. N-(3-Dimethylaminopropyl)-N0-ethylcarbodiimide hydrochloride crys-
talline (EDCꢂHCl)
9. N-Hydroxysulfosuccinimide sodium salt (Sulfo-NHS)
10. 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino
(polyethylene glycol)-2000] (DSPE-PEG2000-NH2)
11. Acetic anhydride
12. Sodium acetate
13. Potassium carbonate
14. Organic solvents: dichloromethane, acetonitrile, dimethylformamide,
methanol
15. Deionized water
4.3 Procedures
4.3.1 2-Chloro-3-(hydroxymethylene)cyclohex-1-ene-1-carbaldehyde
(compound 1)
1. Add anhydrous dimethylformamide (20mL, 273mmol, 2.1 equiv.) into
a 250mL two-neck round flask equipped with a magnetic PTFE stir bar
2. Dissolve phosphorus oxychloride (20mL, 131mmol, 1.0 equiv.) with
the organic solvent dichloromethane (20mL) in a pressure equalizing
dropping funnel equipped with a rubber stopper
3. Place the dropping funnel onto the round flask
4. Cool the reaction mixture in an ice bath
5. Vacuum and purge the reaction vessel with nitrogen five times
6. Open the stopcock and add phosphorus oxychloride solution dropwise
within 20min at 0°C under nitrogen atmosphere

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Yinglong Wu et al.
7. Stir the reaction mixture at room temperature for another 30min
8. Add cyclohexanone (5.0g, 51mmol, 0.4 equiv.) drop by drop into the
mixture via a syringe
9. Reflux the reaction mixture with a condenser under vigorously stirring
in an oil bath for 1.5h at 80°C
10. Cool down the reaction mixture, pour it into ice-cold water, and then
allow it to stand in a refrigerator overnight
11. Filter the resulting suspension and collect the precipitate
12. Dry the product Compound 1 in a vacuum oven as a yellow solid
(5.2g, yield: 59%).
4.3.2 2-(-2-(-2-Chloro-3-(2-(-1,3,3-trimethylindolin-2-ylidene)
ethylidene)cyclohex-1-en-1-yl)vinyl)-1,3,3-trimethyl-3H-indol-
1-ium (compound 2)
1. Add Compound 1 (0.86g, 5mmol, 1.0 equiv.) and 1,2,3,3-tetramethyl-
3H-indolium iodide (3.01g, 10mmol, 2.0 equiv.) into a 100mL
two-neck round flask equipped with a magnetic PTFE stir bar
2. Dissolve the mixed solids with anhydrous acetic anhydride (40mL)
3. Add sodium acetate (0.82g, 10mmol, 2.0 equiv.) into the mixture
4. Vacuum and purge the reaction vessel with nitrogen five times
5. Reflux the mixture in an oil bath set at 130°C for 1h
6. Remove the solvent by vacuum-rotary evaporation
7. Purify the resulting green solid mixture by silica gel chromatography
with dichloromethane/methanol (v/v¼20:1) as the eluent
8. Dry the target product compound 2 in a vacuum oven as a dark green
solid (2.8g, yield: 91%).
4.3.3 2-(2-(6-Hydroxy-2,3-dihydro-1H-xanthen-4-yl)vinyl)-
1,3,3-trimethyl-3H-indol-1-ium (compound 3, C1X-OH)
1. Dissolve resorcinol (0.88g, 8mmol, 2.0 equiv.) with anhydrous aceto-
nitrile (30mL) in a 100mL two-neck round-bottom flask equipped
with a magnetic PTFE stir bar
2. Add potassium carbonate (1.1g, 8mmol, 2.0 equiv.) into the flask
3. Stir the mixture at room temperature for 30min
4. Dissolve Compound 2 (2.44g, 4mmol, 1.0 equiv.) with the organic
solvent acetonitrile (20mL) in a pressure equalizing dropping funnel
equipped with a rubber stopper
5. Place the dropping funnel onto the flask
6. Vacuum and purge the reaction vessel with nitrogen five times

ALP-activated probe for diagnosis of liver injury
309
7. Open the stopcock and add Compound 2 solution dropwise at room
temperature
8. Heat the mixture in an oil bath at 55°C for 4h
9. Remove the solvent by vacuum-rotary evaporation
10. Purify the crude product by silica gel column chromatography with
dichloromethane/methanol (v/v¼20:1) as eluent
11. Dry the target product compound 3 (C1X-OH) in a vacuum oven as a
greenish blue solid (1.44g, yield: 70%).
4.3.4 2-(2-(6-((Diethoxyphosphoryl)oxy)-2,3-dihydro-1H-xanthen-4-yl)
vinyl)-1,3,3-trimethyl-3H-indol-1-ium (compound 4)
1. Dissolve Compound 3 (1.0g, 1mmol, 1.0 equiv.) and potassium carbon-
ate (0.68g, 5mmol, 5.0 equiv.) with anhydrous acetonitrile (20mL) in a
50mL round-bottom flask equipped with a magnetic PTFE stir bar
2. Vacuum and purge the reaction vessel with nitrogen five times
3. Stir the mixture at room temperature for 30min
4. Add a solution of diethyl chlorophosphate (3.44g, 20mmol, 20 equiv.)
in acetonitrile (10mL) into the above mixture drop by drop via a syringe
5. Heat the reaction mixture at 40°C overnight
6. Remove the solvent by vacuum-rotary evaporation
7. Purify the crude product by silica gel column chromatography with dic-
hloromethane/methanol (v/v¼30:1) as eluent
8. Dry the target product compound 4 in a vacuum oven as dark blue solid
(1.08g, yield: 83%).
4.3.5 1,3,3-Trimethyl-2-(2-(6-(phosphonooxy)-2,3-dihydro-1H-xanthen-
4-yl)vinyl)-3H-indol-1-ium (compound 5, C1X-OR1)
1. Dissolve compound 4 (0.64g, 1mmol, 1.0 equiv.) with anhydrous ace-
tonitrile (15mL) in a 50mL round-bottom flask equipped with a mag-
netic PTFE stir bar
2. Vacuum and purge the reaction vessel with nitrogen five times
3. Inject bromotrimethylsilane (3.06g, 20mmol, 20 equiv.) into the above
solution via a syringe
4. Stir the mixture at room temperature for 2 days
5. Add methanol (6mL) into the mixture to quench the reaction
6. Stir the resulting mixture for another 1 day
7. Remove the solvent by vacuum-rotary evaporation
8. Purify the resulting residue by silica gel column chromatography with
dichloromethane/methanol (v/v¼15:1) as eluent

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Yinglong Wu et al.
9. Dry the target product compound 5 (C1X-OR1) in a vacuum oven as
dark blue solid (0.2g, yield: 45%).
4.3.6 DSPE-PEG2000-ChA (compound 6)
1. Dissolve cholic acid (41mg, 0.1mmol, 10 equiv.), EDCꢂHCl (38mg,
0.2mmol, 20 equiv.) and Sulfo-NHS (43mg, 0.2mmol, 20 equiv.) with
anhydrous dimethylformamide (2mL) in a 10mL round-bottom flask
equipped with a magnetic PTFE stir bar
2. Stir the mixture for 2h at room temperature
3. Add DSPE-PEG2000-NH2 (27.5mg, 0.01mmol, 1.0 equiv.) into the
flask
4. Vacuum and purge the reaction vessel with nitrogen five times
5. Stir the reaction mixture at room temperature for 2 days
6. Monitor the conjugation efficiency by thin-layer chromatography (TLC).
7. Dilute the reaction mixture with deionized water (8mL).
8. Dialyze the mixture against deionized water in the dark at room temper-
ature for 2 days
9. Lyophilize the above dialysate to obtain the target product Compound 6
(DSPE-PEG2000-ChA) as white powder (18.2mg, yield: 58%).
5. Response of C1X-OR1 toward ALP in solution
After completing the synthesis, it is necessary to (i) investigate the
changes in optical spectra and photoacoustic signal of C1X-OR1 in response
to ALP, (ii) evaluate the probe C1X-OR1’s selectivity toward ALP and
anti-interference to other possible interfering species, and (iii) verify the pro-
posed mechanism that C1X-OR1 produces the product C1X-OH upon
ALP treatment. Note that we prepared the stock solutions of the compounds
in advance and diluted them into solutions with appropriate concentrations
for tests to minimize experimental error caused by weighing trace amounts
of compound each time.
5.1 Equipment
1. Hitachi U-3010 UV–vis spectrophotometer
2. InVision128 multispectral optoacoustic tomographic (MSOT) imaging
system (iThera Medical GmbH) equipped with a phantom
3. Hitachi F-4600 fluorescence spectrophotometer
4. Agilent 1260 Infinity liquid chromatograph (with diode-array detector,
DAD)

ALP-activated probe for diagnosis of liver injury
311
5. Magnetic stirrer
6. Freeze-dryer
7. Refrigerator
8. Microbalance
9. Ultrasonic cleaner
10. Centrifuge
11. A water bath
12. Quartz cuvettes
13. NMR tubes
14. PTFE syringe filters (0.22μm)
15. Vials
16. Micropipettes and tips
17. Syringes
18. Spatulas
5.2 Reagents
1. Synthetic C1X-OR1 and C1X-OH
2. ALP
3. Acetylcholinesterase (AchE)
4. Glutamyl transferase (GGT)
5. Carboxylesterase (CaE)
6. Nitroreductase (NTR)
7. Cysteine (Cys)
8. Homocysteine (Hcy)
9. Glutathione (GSH)
10. Ascorbic acid (AA)
11. Hydrogen peroxide solution (H2O2)
12. Organic solvents: DMSO, methanol, acetonitrile (HPLC grade)
13. Aqueous buffer: 10mM Tris–HCl (pH¼8.0)
14. Deionized water
5.3 Procedure
5.3.1 Preparation of stock solutions of C1X-OR1 and C1X-OH
1. Label the vials with the name of compounds (C1X-OR1 or C1X-OH).
2. Place the vials onto the weighing platform and zero the microbalance
3. Weigh a certain amount of solid compound into its corresponding vial
with a spatula

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Yinglong Wu et al.
4. Add the calculated volume of DMSO to each vial with a micropipette
for preparation of a 1mM stock solution
5. Cap the vials and sonicate until the solid compounds are completely
dissolved
6. Store the prepared stock solutions in a refrigerator at ꢁ20°C. Note: The
stock solutions need to be fully thawed and shaken before use
5.3.2 Measurement of optical properties of C1X-OR1 and its proposed
activated product C1X-OH
The experiments give absorption spectra of C1X-OR1 as well as its proposed
activated product C1X-OH, and the extinction coefficient for C1X-OH at
the maximum absorption wavelength. This information is important, as the
photoacoustic performance of a signaling chromophore is closely associated
with its absorption properties. The absorption spectra of C1X-OR1 and its
proposed activated product C1X-OH are shown in Fig. 1A and the extinc-
tion coefficient at the maximum absorbance wavelength (684nm) for
C1X-OH is determined as 7.7ꢀ104 Mꢁ1 cmꢁ1. In addition, PA signal of
C1X-OR1 and C1X-OH at different wavelengths (Fig. 1B) are determined
using MSOT imaging system. The results are conducive to verification of
the possibility that C1X-OR1 generates PA signal after responding to
ALP and turning into C1X-OH.
Fig. 1 Optical properties of C1X-OR1 and its activated product C1X-OH. (A) Absorption
spectra and (B) Normalized PA signal intensities as a function of wavelength for
C1X-OR1 (5μM) and its activated product C1X-OH (3μM). Reproduced with permission
from Springer Nature: Wu, Y., Huang, S., Wang, J., Sun, L., Zeng, F., & Wu, S. (2018).
Activatable probes for diagnosing and positioning liver injury and metastatic tumors by
multispectral optoacoustic tomography. Nature Communications, 9, 3983. https:/doi.
org/10.1038/s41467-018-06499-1.

ALP-activated probe for diagnosis of liver injury
313
(A). Measurement of absorption spectra of C1X-OR1 and its proposed
activated product C1X-OH
1. Warm up the Hitachi U-3010 UV–vis spectrophotometer in
advance
2. Set up the parameters to collect absorbance spectra from 500 to
760nm, at 1nm increments
3. Correct the systemic baseline for the UV–vis spectrophotometer
without any sample
4. Correct the solvent baseline for the UV–vis spectrophotometer
by using two quartz cuvettes with Tris-HCl buffer (1mL) con-
taining 5% DMSO
5. Remove the cuvette from the sample holder while maintain the
cuvette in the reference holder still
6. Prepare the test solution of C1X-OR1 and C1X-OH by diluting
their stock solution with DMSO and Tris–HCl buffer under stir-
ring. The concentration of test solution (containing 5% DMSO)
of C1X-OR1 and C1X-OH is 5μM and 3μM, respectively
7. Pipette 1mL test solution of C1X-OR1 or C1X-OH into the
quartz cuvette, respectively, and then put it in the sample holder
8. Start measurement to collect the absorbance spectrum and record
the wavelength of maximum absorbance for C1X-OR1 and
C1X-OH
9. Measure four other test solutions of C1X-OH at different final
concentrations for determination of its extinction coefficient.
Noted that the peak absorbance value of all the test samples
should fall between 0.1 and 1.0a.u
10. Create a linear regression line by use of the peak absorbance value
of each sample and its corresponding concentration, and then
calculate the extinction coefficient of C1X-OH by the Beer-
Lambert Law
(B). Measurement of photoacoustic signal of C1X-OR1 and its proposed
activated product C1X-OH
1. Switch on the MSOT imaging system and fill the internal water
tank to the marking position with deionized water
2. Warm up the system to 34°C in advance of this experiment
3. Set up the parameters to collect photoacoustic signal from 680 to
900nm at 5nm increments, 3 frames per wavelengths
4. As described in Procedure 5.3.2-6, prepare test solutions of
C1X-OR1 and C1X-OH

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Yinglong Wu et al.
5. Fully fill commercial NMR tubes with test solutions of C1X-OR1
and C1X-OH, respectively, and then place the NMR tubes into
the phantom
6. Fix the phantom in the holder of the instrument and then put the
holder with samples into the instrument
7. Start measurement to acquire original data
8. Reconstruct MSOT images at each wavelength using a standard
back-projection algorithm within the ViewMSOT software
9. Draw an equal circle line on the sample position in the image and
select the circular area as region of interest (ROI).
10. Record the mean PA intensity at each wavelength of ROI
5.3.3 Optical and acoustic response of C1X-OR1 toward ALP
In order to investigate the response of C1X-OR1 toward ALP, the absor-
bance, photoacoustic and fluorescence change after incubating with varied
amount of ALP for 30min at 37°C are recorded. The results are shown in
Fig. 2.
(A). Measurement of changes in absorbance spectrum of C1X-OR1 upon
incubation with varied amounts of ALP at 37°C for 30min
1. Repeat Procedure 5.3.2 (A) steps 1–5
Fig. 2 Optical and acoustic response of C1X-OR1 toward ALP. (A) Absorption spectra for
C1X-OR1 upon incubation with varied concentrations of ALP. The insets display the plots
of absorbance ratio A684/A602 vs ALP concentration. (B) PA response of C1X-OR1 to ALP at
different concentrations. The insets display the representative PA images of C1X-OR1 at
684nm in phantom after reacting with different concentrations of ALP. (C) Fluorescent
spectra for C1X-OR1 upon incubation with varied concentration of ALP. The insets dis-
play the plots of fluorescence intensity at 712nm vs ALP concentration. Reproduced
with permission from Springer Nature: Wu, Y., Huang, S., Wang, J., Sun, L., Zeng, F., &
Wu, S. (2018). Activatable probes for diagnosing and positioning liver injury and metastatic
tumors by multispectral optoacoustic tomography. Nature Communications, 9, 3983.
https://doi.org/10.1038/s41467-018-06499-1.

ALP-activated probe for diagnosis of liver injury
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2. Prepare the test solution by incubating C1X-OR1 (5μM) with var-
ied amounts of ALP (0, 0.04, 0.08 0.12, 0.16, and 0.20U/mL) at
37°C in Tris–HCl buffer solution containing 5% DMSO for 30min
3. Pipette 1mL test solution into the quartz cuvette and then put it in
the sample holder
4. Start measurement to collect the absorbance spectrum and record
theabsorbanceintensityat602and684nm(themaximumabsorbance
wavelength of C1X-OR1 and its activated product C1X-OH,
respectively)
5. Analyze the data and plot absorbance ratio A684/A602 as a function
of ALP concentration
(B). Measurement of changes in photoacoustic signal of C1X-OR1 upon
reaction with varied amounts of ALP at 37°C for 30min
1. Repeat Procedure 5.3.2 (B) steps 1–2
2. Set up the parameters to collect optoacoustic signal at 684nm (the
maximum optoacoustic signal wavelength of the activated product
C1X-OH), 3 frames for each test
3. Prepare the test solution by incubating C1X-OR1 (5μM) with
varied concentrations of ALP (0, 0.04, 0.08 0.12, 0.16, and
0.20U/mL) at 37°C in Tris–Hcl buffer solution containing 5%
DMSO for 30min
4. Fully fill a commercial Wilmad NMR tube with the test solution,
place the tube into the phantom, and then fix the phantom on the
instrument holder
5. Repeat Procedure 5.3.2 (B) steps 5–9
6. Analyze the data and plot relative PA intensity as a function of ALP
concentration
(C). Measurement of fluorescence spectra of C1X-OR1 upon reaction
with varied concentrations of ALP at 37°C for 30min
1. Warm up the Hitachi F-4600 fluorescence spectrophotometer in
advance
2. Set up the parameters (PMT voltage¼600V, Ex¼680nm,
EM¼695–900nm at 1nm increasement, Ex slit¼5nm, Em
slit¼5nm, Response time¼4s, Scan speed¼240nm/min) to
collect fluorescence spectrum
3. Prepare the test solution by incubating C1X-OR1 (5μM) with
varied amounts of ALP (0, 0.04, 0.08 0.12, 0.16, and
0.20U/mL) at 37°C in Tris–Hcl buffer solution containing 5%
DMSO for 30min

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4. Pipette 1mL test solution into the quartz cuvette and then put it in
the sample holder
5. Start measurement to collect the fluorescence spectrum and record
the fluorescence intensity at 712nm (the maximum fluorescence
emission wavelength of the activated product C1X-OH)
6. Analyze the data and plot fluorescence intensity at 712nm as a
function of ALP concentration
5.3.4 Evaluation of C1X-OR1’s selectivity toward ALP
and anti-interference to other possible interfering species
To study the response selectivity, we parallelly examine various potential
interfering substances by MSOT imaging under the same test conditions.
As for anti-interference experiments, C1X-OR1 is incubated with ALP in
the presence of one potential interfering species. The results are displayed
in Fig. 3.
1. Repeat Procedure 5.3.3 (B) steps 1–2
2. Prepare the test solutions by mixing C1X-OR1 (5μM) with ALP
(0.20U/mL) and a potential interference substance at 37°C in Tris–
Hcl buffer solution containing 5% DMSO for 30min. The concentra-
tion of AchE, GGT, CaE or NTR is 0.20U/mL, and that of Cys, Hcy
or GSH is 1mM, and that of H2O2 or AA is 100μM
3. Repeat Procedure 5.3.3 (B) steps 4–5
4. Analyze the data
Fig. 3 Selectivity and anti-interference ability of C1X-OR1. (A) PA response of C1X-OR1 in
the presence of 0.2U/mL ALP or a potential interference substance (upper panel), and
(B) PA response of the probes in the presence of 0.2U/mL ALP and a potential interfer-
ence substance (lower panel). Reproduced with permission from Springer Nature: Wu, Y.,
Huang, S., Wang, J., Sun, L., Zeng, F., & Wu, S. (2018). Activatable probes for diagnosing and
positioning liver injury and metastatic tumors by multispectral optoacoustic tomography.
Nature Communications, 9, 3983. https:/doi.org/10.1038/s41467-018-06499-1.

ALP-activated probe for diagnosis of liver injury
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5.3.5 Verification of the proposed activation mechanism
The proposed response mechanism that C1X-OR1 produces the product
C1X-OH upon treatment with ALP is verified by HPLC, as shown in Fig. 4.
1. Prepare the sample of enzyme-mediated reaction by mixing
C1X-OR1 and ALP for 20min. The procedure is given as follows:
5μM C1X-OR1 and 0.2U/mL ALP is stirred at 37°C in 2mL Tris–
Hcl buffer solution containing 5% DMSO for 20min, and the reaction
mixture is lyophilized and re-dissolved in 2mL methanol. After that, the
solution is filtered by a syringe with a PTFE filter. The resultant solution
is used as the sample for HPLC test
2. Purge each solvent (acetonitrile, methanol and deionized water) channel
for 10min at a flow rate of 1mL/min
3. Set up the parameters (Mobile phase: methanol/acetonitrile/water¼
75/15/10, Flow rate: 1mL/min, Injection volume: 20μL, Stop time:
30min, DAD wavelength channel: 684nm, Temperature: 25°C).
4. Balance the baseline
Fig. 4 HPLC data of pure C1X-OR1, mixture of C1X-OR1 and ALP upon incubation for
20min and pure C1X-OH. Mobile phase: methanol/acetonitrile/water¼75/15/10.
Reproduced with permission from Springer Nature: Wu, Y., Huang, S., Wang, J., Sun, L.,
Zeng, F., & Wu, S. (2018). Activatable probes for diagnosing and positioning liver injury
and metastatic tumors by multispectral optoacoustic tomography. Nature communica-
tions, 9, 3983. https://doi.org/10.1038/s41467-018-06499-1.

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5. Add test solutions into 1mL sample vials, respectively. Sample 1:
C1X-OH (5μM) in methanol; Sample 2: mixture of C1X-OR1 (5μM)
and ALP (0.2U/mL) upon reaction for 20min in methanol; Sample 3:
C1X-OR1 (5μM) in methanol
6. Run samples and collect their data
6. Response of C1X-OR1 toward ALP in cells
Since C1X-OR1 is designed to be used as an activatable PA probe for
detection of ALP in a biological environment, it is vital to demonstrate that
C1X-OR1 is noncytotoxic to cell lines (Fig. 5). The murine aneuploid
fibrosarcoma cells (L929) and human hepatocarcinoma cells (HepG2) are
cultured in complete medium at 37°C under 5% of CO2. When the cell
density reaches 70–80% of confluence, subculturing is considered complete.
The medium is changed approximately every 1–2 days.
6.1 Equipment
1. Cell incubator
2. Biological safety cabinet
3. Thermo MK3 ELISA reader
4. BD CS6 flow cytometer
Fig. 5 Cell viabilities and flow cytometry profiles for activation of C1X-OR1 in ALP over-
expressed cells. (A) Viabilities of L929 and HepG2 cells upon 24h of incubation with
C1X-OR1 of different concentrations (0, 1, 5, 10 and 50μM). (B) Flow cytometry profiles
for HepG2 cells in the absence (the control) and presence of C1X-OR1 for 0.5 or 1h.
Reproduced with permission from Springer Nature: Wu, Y., Huang, S., Wang, J., Sun, L.,
Zeng, F., & Wu, S. (2018). Activatable probes for diagnosing and positioning liver injury
and metastatic tumors by multispectral optoacoustic tomography. Nature communica-
tions, 9, 3983. https://doi.org/10.1038/s41467-018-06499-1.

ALP-activated probe for diagnosis of liver injury
319
5. Olympus IX 71 fluorescence microscope with a DP72 color CCD
6. Centrifuge
7. 96-well plates
8. 6-well plates
9. 30-mm glass culture dishes
10. Polylysine-coated cell culture glass slides
11. Micropipettes and tips
6.2 Reagents
1. Stock solution (1mM) of C1X-OR1
2. L929 cells and HepG2 cells
3. RPMI 1640 Medium supplemented with 10% FBS and 1% penicillin
and streptomycin
4. Dulbecco’s modified eagle medium (DMEM) supplemented with 10%
FBS and 1% penicillin and streptomycin
5. Methylthiazolyldiphenyl-tetrazolium bromide (MTT)
6. Organic solvents: DMSO
7. Aqueous buffer: 1ꢀ PBS (pH 7.4)
6.3 Procedures
6.3.1 Validation that C1X-OR1 is noncytotoxic to cells
1. Culture L929 cells and HepG2 cells in RPMI1640 medium and DMEM,
respectively
2. Seed 25 wells of a 96-well plate with 100μL (5ꢀ104 cells/mL of media)
L929 or HepG2 cells
3. Incubate the plate at 37°C for 24h
4. Wash the cells with 1ꢀ PBS solution, and replace the PBS with fresh
culture medium (100μL) containing C1X-OR1 at different concentra-
tion (0, 1, 5, 10, and 50μM) and 0.2% v/v DMSO
5. Incubate the plate at 37°C for another 24h
6. Wash the cells with 1ꢀ PBS solution, and then replace the PBS with
fresh culture medium (100μL) containing 0.5mg/mL MTT
7. Incubate the plate at 37°C for 2h
8. Discard the culture medium, and then add 150μL of DMSO into each
well to dissolve the precipitates
9. Place the plate into the Thermo MK3 ELISA Reader, and measure the
absorbance value at 570nm. Note that the statistical mean and standard
deviation were employed to estimate cell viability

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6.3.2 Activation of C1X-OR1 in ALP overexpressed cells
1. Culture HepG2 cells in DMEM
2. Seed the wells of a 6-well plate with 1mL (2ꢀ105 cells/mL of media)
HepG2 cells
3. Incubate the plate at 37°C for 24h
4. Add 5μL stock solution of C1X-OR1 to each well
5. Incubate cells with C1X-OR1 at 37°C for another 0.5 or 1h
6. Wash the cells with 1ꢀ PBS solution for three times, and then discard
the PBS
7. Trypsinize the cells for 2min at 37°C and then add 1mL prewarmed
medium to each well for trypsin neutralization
8. Blow the cells gently with a pipette, and then centrifuge the cells at
1000rpm for 5min
9. Discard the supernatant and disperse the cell pellet into a single cell sus-
pension with prewarmed PBS
10. Analyze 1ꢀ104 cells with a BD CS6 flow cytometer and collet the sig-
nals in FL4 channel
6.3.3 Imaging endogenous ALP in cells by use of C1X-OR1
1. Culture L929 cells or HepG2 cells in RPMI1640 medium or DMEM,
respectively
2. Pass 2mLL929 and HepG2 cells (2ꢀ105 cells/mL of media) on the cell
culture glass slides modified with polylysine in the 30-mm petri dishes
3. Incubate the cells at 37°C under 5% CO2 for 24h
4. Add 10μL stock solution of C1X-OR1 to each dish. NOTE: In one dish,
HepG2 cells were inhibited with Na3VO4 (10mM) for 30min before
addition of C1X-OR1
5. Incubate cells with C1X-OR1 at 37°C for another 1h
6. Wash the cells with 1ꢀ PBS solution for three time and then take the
glass slides out of the dishes
7. Place the glass slides onto an Olympus IX71 inverted fluorescence
microscope with a DP72 color CCD and acquire fluorescence images.
(Excitation filter: 530–550nm, Emission filter: >590nm) (Fig. 6)
7. In vivo detection of ALP for imaging drug-induced
liver injury by use of liposomal C1X-OR1
Since the activation of C1X-OR1 by cellular ALP has been confirmed
in vitro, its ability for diagnosing drug-induced liver injury should also be
fully investigated in vivo. For in vivo experiments, to enhance the

ALP-activated probe for diagnosis of liver injury
321
Fig. 6 Fluorescent microscopic images for C1X-OR1. Bright field (top row) and fluores-
cence images (middle row) for L929 and HepG2 cells incubated with C1X-OR1 for 1h,
and images for HepG2 cells pretreated with Na3VO4 (an ALP inhibitor) and then incu-
bated with C1X-OR1 for 1h. Reproduced with permission from Springer Nature: Wu, Y.,
Huang, S., Wang, J., Sun, L., Zeng, F., & Wu, S. (2018). Activatable probes for diagnosing
and positioning liver injury and metastatic tumors by multispectral optoacoustic tomogra-
phy. Nature communications, 9, 3983. https://doi.org/10.1038/s41467-018-06499-1.
dispersibility in blood stream and the accumulation and retention of
C1X-OR1 in liver, we fabricate liposomal C1X-OR1 by encapsulating it into
the synthetic hepatocyte-targeting phospholipid DSPE-PEG2000-ChA
together with several other commercial phospholipids. We first confirmed
that liposomal C1X-OR1 is nontoxic in vivo, and then use it to image
Drug induced liver injury (DILI) via photoacoustic and fluorescent imaging.
We also investigate the relationship between dose of administrated drug and
liver injury by determining serum levels of some hepatic biomarkers, which
further indicates the increased hepatic ALP level as the result of liver injury can
be spatiotemporally detected by liposomal C1X-OR1 with MSOT imaging.
Before conducting any in vivo experiment, institutional approval of the
animal experiments must be obtained, and the training in the ethics and
practice of animal handling must be completed. All our animal experiments
were conducted under SPF environment in the Laboratory Animal Center
of South China Agricultural University. All the experimental protocols
have been approved by the Animal Ethics Committee of South China

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Yinglong Wu et al.
Agricultural University in accordance with the Guidelines for Care and Use
of Laboratory Animals of China, the Regulations on the Management of
Laboratory Animals of China, and the Regulations on the Administration
of Laboratory Animals of Guangdong Province. 7–8weeks old male
BALB/c nude mice (16–20g) are purchased from Guangdong Medical
Laboratory Animal Center (GDMLAC). Mice are housed in a specific
pathogen-free room with laminar airflow hoods and 12h light/12h dark
cycle. Every three mice are assigned in one sterile cage with bedding material
of sterilized wood chips and fed autoclaved chow and water ad libitum. Prior
to dissection operation, the mice are exposed to a rising concentration of
carbon dioxide gas for euthanasia.
7.1 Equipment
1. JEM-1400 transmission electron microscopy
2. Malvern Nano-ZS90 particle size analyzer
3. Hitachi U-3010 UV–vis spectrophotometer
4. InVision128 multispectral optoacoustic tomographic (MSOT) imaging
system (iThera Medical GmbH)
5. AMI small animal fluorescence imaging system
6. Thermo MK3 ELISA reader
7. Centrifuge
8. Refrigerator
9. Freeze-dryer
10. Microscope
11. Microbalance
12. Vacuum pump
13. Rotary evaporator
14. Single-neck round bottom flasks
15. Dialysis membranes (MwCO¼1000)
16. Rubber stoppers
17. Syringes
18. Magnetic stirrer
19. Magnetic PTFE stir bar
7.2 Regents
1. C1X-OR1 solid
2. Synthetic DSPE-PEG2000-ChA powder
3. 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)

ALP-activated probe for diagnosis of liver injury
323
4. 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC)
5. ELISA Kits of alkaline phosphatase (ALP), aspartate transaminase (AST),
alanine aminotransferase (ALT)
6. Hematoxylin and Eosin Stain Kit
7. Formalin solution (36.5–38%)
8. Isoflurane
9. Organic solvents: THF, DMSO
10. Aqueous buffer: 1ꢀPBS (pH 7.4)
11. Deionized water
7.3 Procedure
7.3.1 Preparation of liposomal C1X-OR1 for in vivo experiments
Liposomal C1X-OR1 is prepared according to a procedure stated below:
1. Dissolve DPPC (12.46mg), DSPC (2.8mg) and DSPE-PEG2000-ChA
(1.7mg) at a molar ratio of 85/10/5 with the organic solvent tetrahydro-
furan (1mL) in a 5mL single-neck round bottom flask equipped with a
magnetic PTFE stir bar
2. Add a solution of C1X-OR1 (20mg) in THF (0.5mL) into the flask
under continuous stirring
3. Inject the above mixture dropwise into the deionized water (10mL) in
50mL single-neck round bottom flask equipped with a magnetic PTFE
stir bar under continuous stirring
4. Remove the organic solvent was removed by vacuum-rotary evaporation
5. Dialyze the mixture against the deionized water in the dark at room tem-
perature for 24h
6. Centrifuge the suspension at 4°C to collect the nanoparticles
7. Wash the resulting solid with the deionized water for three times
8. Resuspend the nanoparticles in 1ꢀ PBS (or water for the concentration
determination) of designated volume (20mL) to obtain liposomal
C1X-OR1
After preparation of the liposomal C1X-OR1, its average diameter is deter-
mined to be ꢃ105nm by transmission electron microscopic (TEM) and
dynamic light scattering (DLS). For determination of concentration of liposo-
mal C1X-OR1 in water and C1X-OR1 loading, liposomal C1X-OR1 solution
is freeze-dried and weighed. After that, lyophilized liposomal C1X-OR1 is dis-
solved in 1mL of DMSO. The loading efficiency of the C1X-OR1 by phos-
pholipids is calculated based on the absorbance intensity at 684nm according to
a predetermined standard calibration curve. The concentration of liposomal

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Yinglong Wu et al.
C1X-OR1 in water is calculated to be 0.7mg/mL and C1X-OR1 loading effi-
ciency is determined as 42.7% based on the following two formulas:
(1) Concentration of liposomal probe¼(mass of nanoparticles)/(volume of
solution)
(2) Loading efficiency¼(mass of loaded C1X-OR1)/(mass of nanoparticles)
ꢀ100%
7.3.2 Confirmation of liposomal C1X-OR1’s nontoxicity in vivo
Before being used for animal experiments, it is necessary to verify that lipo-
somal C1X-OR1 is nontoxic in vivo by measuring changes in serum bio-
chemistry and histopathology, and the procedure is given below:
1. Anesthetize the mice in an isoflurane/oxygen chamber, and maintain an
anesthetized state by use of 1% isoflurane delivered via a breathing mask
2. Inject liposomal C1X-OR1 (8.75mg/kg) or saline intravenously into
mice using insulin syringe and feed them for another day as usual
3. Collect blood samples of those mice from their orbital sinus, and eutha-
nize them via exposure to carbon dioxide gas in a rising concentrations
and then excise their main organs including heart, liver, spleen, lung and
kidney
4. Centrifuge the blood samples and collect serum for ALT, AST and ALP
assay using ELISA kits
5. Flush the excised organs with sterile PBS solution, fix the organs with
formalin solution, and embed them in paraffin. Then use hematoxylin
and eosin stain the paraffin sections for fluorescence imaging under a
microscope
6. Analyze the data
7.3.3 MSOT imaging of drug-induced liver injury in mouse model using
liposomal C1X-OR1
This experiment is to demonstrate that the drug-induced liver injury in
mouse model can be diagnosed by the systemically administered liposomal
C1X-OR1 with MSOT. Herein, we utilize N-acetyl-p-aminophenol
(APAP) to establish mouse model of drug-induced liver injury. APAP is a
clinical medicine for fever and pain treatment, but overdose may cause hep-
atotoxicity accompanied by elevation in hepatic ALP (Simon & Sutherland,
1977). For APAP-induced liver injury, the mice are intraperitoneally
administrated with varied dosage of APAP predissolved in sterilized saline
solutions at a concentration of 15mg/mL (Fig. 7).

ALP-activated probe for diagnosis of liver injury
325
Fig. 7 In vivo PA imaging drug-induced liver injury by using C1X-OR1. (A) Schematic
illustration for PA imaging of DILI with liposomal C1X-OR1. (B) Typical MSOT images
of the mice administrated with different amount of APAP at 30min upon injection of
liposomal C1X-OR1. (C) Mean PA intensities at ROI in liver area for the mice administrated
with different amount of APAP. (D) A cryosection image of a male mouse with the cross
section’s location comparable to those shown in panel (B). (E) Representative 3-D MSOT
images for the mice pretreated 300mg/kg APAP after intravenous injection of liposomal
C1X-OR1 for 30min. Reproduced with permission from Springer Nature: Wu, Y., Huang, S.,
Wang, J., Sun, L., Zeng, F., & Wu, S. (2018). Activatable probes for diagnosing and position-
ing liver injury and metastatic tumors by multispectral optoacoustic tomography. Nature
Communications, 9, 3983. https://doi.org/10.1038/s41467-018-06499-1.
1. Repeat Procedure 5.3.2 (B) steps 1–2
2. Import the absorption spectra of C1X-OH into the system
3. Set up the parameters to collect optoacoustic signals at 680, 684, 700,
715, 730, 760, 800 (background), and 850nm (NOTE: These wave-
lengths are chosen for correspondence with main turning points in

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Yinglong Wu et al.
the absorption spectra of endogenous hemoglobin and activated prod-
uct C1X-OH), 10 frames per wavelength
4. Anesthetize the mice preadministered with varied dose of APAP in an
isoflurane/oxygen chamber, and maintain an anesthetized state by
using 1% isoflurane delivered through a breathing mask during the
experiments
5. Place the mice inside the sample holder and insert a catheter connected
with a syringe into the tail vein
6. Seal the holder with the ultrathin plastic membrane to prevent water
from leaking into the animal holder during experiments, and remove
air between mice and membrane with medical ultrasonic couplant
7. Place the holder with mice into the system and inject liposomal
C1X-OR1 (8.75mg/kg) into the mice using the syringe
8. Start measurement to acquire data 30min after probe injection
9. Reconstruct MSOT images using a standard back-projection algorithm
and separate PA signals of the activated C1X-OR1 from those of
endogenous absorbers such as hemoglobin using guided ICA spectral
unmixing protocol
10. Select a region of interest (ROI) volume consisting of transverse slices
with a step size of 0.2mm, spanning through the liver region
11. Render the z-stack cross-sectional images into an orthogonal MIP image
12. Record the mean PA intensity of ROI
13. Analyze the data
7.3.4 Fluorescence imaging of drug-induced liver injury in mouse model
using liposomal C1X-OR1
1. Switch on the AMI small animal fluorescence imaging system and start
AMIView software
2. Initialize the instrument and cool the CCD camera to operating temper-
ature at ꢁ90°C
3. Set up the parameters to collect fluorescence images. (excitation filter:
675nm; emission filter of 715nm; Power: 30%; exposure: 0.5s; Binning:
none; FOV: 20cm; Objective height: 1.5cm)
4. Anesthetize the mice preadministered with varied dose of APAP in an iso-
flurane/oxygen chamber and inject liposomal C1X-OR1 (8.75mg/kg)
into the mice via tail vein
5. Place the mice into the chamber of the imaging system, maintain an
anesthetized state by use of 1% isoflurane delivered via a breathing mask
and close the instrument door

ALP-activated probe for diagnosis of liver injury
327
Fig. 8 In vivo fluorescence imaging drug-induced liver injury by using C1X-OR1.
(A) Representative fluorescence images indicating biodistribution of the activated
C1X-OR1 for the mice administrated with varied dose of APAP. (B) Mean fluorescent inten-
sities at ROI of the mice administrated with different amount of APAP. Reproduced
with permission from Springer Nature: Wu, Y., Huang, S., Wang, J., Sun, L., Zeng, F., &
Wu, S. (2018). Activatable probes for diagnosing and positioning liver injury and metastatic
tumors by multispectral optoacoustic tomography. Nature Communications, 9, 3983. https:/
doi.org/10.1038/s41467-018-06499-1.
6. Start measurement to acquire images 30min after injection
7. Select a region of interest (ROI) spanning through the liver region
8. Record the mean FL intensity of ROI
9. Analyze the data (Fig. 8)
7.3.5 Verification that treatment with overdosed APAP causes liver
injury
To confirm the relationship between APAP overdose and liver injury, sev-
eral clinically representative parameters including serum ALT, AST and
ALP for liver dysfunctions are measured by using ELISA kits. APAP over-
dose leads to a significant increase in serum enzyme activity (Fig. 9), thus
further indicating that the mice administrated with overdose APAP in the
imaging experiments suffer liver damage. All these results suggest that,
drug-induced liver injury accompanied by the elevated hepatic ALP level
can be spatiotemporally detected by MSOT.
These mice are sacrificed, and their serum is collected 12h after APAP
administration. The procedures for measuring serum ALT/AST/ALP using
ELISA kits are strictly performed according to the instructions.
1. Dilute standard into five different concentrations for working curve
2. Add the five diluted standards (50μL) and fivefold diluted serum sam-
ples (50μL) into the wells of ELISA plate
3. Cover the plate and incubate it for 30min at 37°C after gentle shaking
4. Dilute concentrated wash solution 30 times with deionized water

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Yinglong Wu et al.
Fig. 9 Serum biochemistry evaluation. (A) Serum levels of AST as well as ALT and (B) Serum
ALP levels for the mice at 12h upon administration with different amount of APAP.
Columns represent means ꢄ SD. Error bars represent the standard deviation (SD).
Reproduced with permission from Springer Nature: Wu, Y., Huang, S., Wang, J., Sun, L.,
Zeng, F., & Wu, S. (2018). Activatable probes for diagnosing and positioning liver injury and
metastatic tumors by multispectral optoacoustic tomography. Nature communications, 9,
3983. https:/doi.org/10.1038/s41467-018-06499-1.
5. Discard the liquid in the wells and wash five times with 30-fold diluted
wash solution
6. Add 50μL of enzyme-labeled reagent to each well
7. Repeat the incubating procedure as in step 3
8. Repeat the wash procedure as in step 5
9. Add 50μL of Color Development Reagent A and 50μL of Color
Development Reagent B to each well successively. Cover the plate
and incubate it for 15min in the dark at 37°C after gentle shaking
10. Add 50μL of Stop Solution to each well. NOTE: The solution will
immediately change from blue to yellow once terminating the reaction
11. Zero the blank well. Read absorbance at 450nm within 15min by
using a Thermo MK3 ELISA Reader
8. Summary
Herein, we detail the synthesis and validation of C1X-OR1, an
ALP-activated PA probe that can be used for spatiotemporal detection and
imaging of liver injury in vivo. We believe this probe C1X-OR1 may be
applied in preclinical screening for hepatotoxicity to improve patient safety
and be utilized as a convenient and cost-effective tool in pharmaceutical
industry to avoid post-marketing drug withdrawal. More generally, we

ALP-activated probe for diagnosis of liver injury
329
demonstrate a systematic and comprehensive approach for design, preparation
as well as application of activatable PA probes for disease diagnosis with mul-
tispectral photoacoustic imaging in a 3D manner and hopefully this approach
could be used to accurately diagnose and locate various diseases by construc-
tion of other activatable probes.
Acknowledgment
This work was supported by the National Natural Science Foundation of China (21788102) and
the Fund of Guangdong Provincial Key Laboratory of Luminescence from Molecular
Aggregates (2019B030301003).
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