Diagnosing Drug-induced Liver Injury by Multispectral Optoacoustic

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Diagnosing Drug-induced Liver Injury by Multispectral Optoacoustic Tomography and Fluorescence Imaging Using a Leucine-Aminopeptidase-Activated Probe Yong Huang, Yu Qi, Chenyue Zhan, Fang Zeng, and Shuizhu Wu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00107 • Publication Date (Web): 30 May 2019 Downloaded from http://pubs.acs.org on May 30, 2019

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Analytical Chemistry

Diagnosing Drug-induced Liver Injury by Multispectral Optoacoustic Tomography and Fluorescence Imaging Using a Leucine-Aminopeptidase-Activated Probe Yong Huang, Yu Qi, Chenyue Zhan, Fang Zeng,* Shuizhu Wu* State Key Laboratory of Luminescent Materials and Devices, College of Materials Science and Engineering, South China University of Technology, Guangzhou 510640, China. ABSTRACT: Drug-induced liver injury (DILI) is a frequent cause of hepatic dysfunction as well as the single most frequent reason for removing approved medications from the market, and the multispectral optoacoustic tomography (MSOT) is an emerging and non-invasive imaging modality for diagnosing and monitoring diseases. Herein, we report an activatable optoacoustic probe for imaging DILI through detecting the activity of leucine aminopeptidase (LAP). In this probe, an N-terminal leucyl moiety serving as the LAP recognition element is linked with a chromene-benzoindolium chromophore via 4-aminobenzylalcohol group. The elevated expression of hepatic LAP as a result of DILI cleaves the leucyl moiety and causes the red-shift of the probe’s absorption band, thereby generating prominent optoacoustic signals for MSOT imaging. During this process, the probe also exhibits prominent NIR fluorescence, which can be utilized for fluorescent imaging. More importantly, by rendering stacks of crosssectional images as maximal intensity projection (MIP) images, we could precisely locate the focus of drug-induced liver injury in mice. This probe is expected to serve a powerful tool for studying physiological and pathological processes related to LAP.

Drug-induced liver injury (DILI), which is caused by therapeutic medicines or other chemicals, has become a growing medical, scientific and public health problem.1-5 DILI is a common side effect of a number of medicines and poses a serious threat to human health and has an enormous economic burden on health care expenditures. Liver injury has been linked to nearly 1000 drugs with acetaminophen (APAP) being the principal offending one, and it is also a leading cause of attrition of compounds in drug development and the single most frequent reason for drug withdrawals from the market.5 The early diagnosis for drug-induced liver injury is therefor of high significance. In general, DILI is difficult to identify since many cases are less symptomatic or even asymptomatic.5,6 For the symptomatic patients, blood tests are commonly performed to assess liver functions or liver injury via determining the level of certain hepatic enzymes.7 However, sometimes the serum tests are not reliable, since some hepatic enzymes exist in multiple organs or tissues, the elevated serum level does not necessarily mean liver injury. On the other hand, the liver biopsy tests are also performed to aid the diagnosis and assess the severity of liver injuries.8 Nonetheless, results from these invasive tests rely on the representativity of the punctured sample and thereby show remarkable variability.9 Leucine aminopeptidases (LAP) are a class of enzymes capable of catalyzing the hydrolysis of leucine residues from the amino termini of protein or peptide substrates.10 It is generally presented in liver, lung, kidney, serum and some tumors.11,12 In liver tissue, LAP exists as a cytosolic enzyme, and liver diseases such as liver necrosis/injury, liver cirrhosis

and hepatitis generally produce an increase in serum LAP activity,13,14 which may be caused by the infiltration of hepatic LAP into bloodstream. Determination of LAP activity in serum is therefore of clinical significance for liver disorder diagonsis.15,16 In particular, it has long been found that druginduced liver injury (drug-induced hepatitis) also induces serum LAP elevation.17 Obviously, LAP can serve as a new diagnostic biomarker for drug-induced liver injury. Indeed, some analytical approaches such as high-performance liquid chromatography (HPLC) and electrophoretically mediated microanalysis (EMMA) have been employed to detect LAP level;18-20 and several fluorescent probes have been developed for monitoring LAP activity.21-26 The optical imaging is a non-invasive, sensitive and inexpensive modality with wide applicability.27-31 However, the conventional optical imaging provides limited anatomical information and suffers reduced sensitivity with increased imaging depth.32,33 Recently, optoacoustic tomography (OAT), also known as photoacoustic tomography (PAT), has emerged as a promising biomedical imaging modality in which both the optical and ultrasound techniques are employed to obtain realtime and in-situ images of biological tissue/structures without excising tissue.34,35 This technique can provide molecular information at clinically relevant depths with higher spatial resolution than conventional optical imaging techniques.36-39 In particular, multispectral optoacoustic tomography (MSOT),4042 which is a spectral optoacoustic technique, has been utilized in a wide range of biological imaging applications. A MSOT system operates by irradiating a sample with multiple wavelengths, allowing it to detect ultrasound waves from different photoabsorbing substances (contrast agents) in the

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tissue. Moreover, three-dimensional (3D) images can be obtained by using MSOT, enabling precise localization of disease foci. OAT signals originate from optical absorption in biological tissues, which can be generated by endogenous molecules such as hemoglobin or melanin, or exogenously delivered contrast agents.43,44 A variety of materials with their absorption in the near-infrared region have been developed as exogenous OAT imaging probes,45-58 but most of them were used as the inert contrast agents. For biological detection and imaging, the activatable (or smart) probes which produce significant signal outputs in response to a specific biomarker or stimuli are highly desirable and have generated great attention in the imaging community. Activatable probes can provide dynamic information directly reflecting disease progression and mechanisms, and to date, a variety of activatable probes have been developed to detect and image biological processes and diseases. For examples, Bhatia and coworkers utilized light, magnet and other means to activate sensors for disease detection and profiling.59,60 On the other hand, Ma’s group has developed a number of activatable fluorescence probes which can specifically respond to enzymatic biomarkers for imaging disease foci.21, 22 Recently, a few activatable molecular OAT probes have been developed.61-69 However, to our best knowledge, the activatable OAT probe that can monitor LAP has not yet been reported. Herein we report a molecular LAP probe (DLP) for diagnosing DILI by imaging elevated LAP level in liver region of mice both optoacoustically and fluorescently. As illustrated in Scheme 1, for this LAP probe, a chromenebenzoindolium chromophore serves as the reporter, 4aminobenzylalcohol as the linker and an N-terminal leucyl group as the recognition element. The hepatic LAP cleaves the N-terminal leucyl group and induces a cascade of reaction, thus activating the probe and realizing the sensitive detection of LAP. The activated probe exhibits high extinction coefficient at 705 nm and strong fluorescence at 733 nm, affording it a robust molecular probe for imaging LAP in vivo. Owing to the fluorogenic nature of the probe upon activation by LAP, we were able to evaluate the detection specificity of the probe, observe the fluorescence generation in live cells, and fluorescently image the liver injury. Moreover, on a MSOT facility, we could not only achieve the non-invasive evaluation of LAP level elevation as a result of liver injury, but also identify and locate the injury foci by obtaining threedimensional images.

Scheme 1. (a) Schematic illustration of the LAP-activatable MSOT imaging and NIR fluorescence imaging probe (DLP) in vivo. (b) The chemical structure and the proposed mechanism of DLP for LAP imaging.

EXPERIMENTAL SECTION Synthesis of 2-(2-(6-((4-(2-((tert-butoxycarbonyl)amino)-4methylpentanamido) benzyl)oxy)-1,2-dihydrocyclopenta[b]chromen-3-yl)vinyl)-3-ethyl-1,1-dimethyl-1H-benzo[e]indol3-ium iodide (DLP-Boc). A mixture of NIR-OH (112 mg, 0.2 mmol) (see Supporting Information) and K2CO3 (55 mg, 0.4 mmol) in DMF(5 mL) was stirred for 30 min at room temperature. Then compound 6 (80 mg, 0.2 mmol) was added to the reaction mixture and further reaction was continued for 10 hours. When the reaction was completed, it was diluted with CH2Cl2, washed with water, followed by saturated brine solution. The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The obtained residue was purified by silica gel chromatography (DCM: MeOH = 50: 1 to 10: 1 in v/v) to give a dark green solid (106 mg, 60%). 1H NMR (600 MHz, CDCl ) δ 8.81 (d, J = 22.9 Hz, 1H), 8.29 3 (d, J = 14.4 Hz, 1H), 8.20 (d, J = 8.5 Hz, 1H), 8.02 (d, J = 8.3 Hz, 1H), 7.99 (d, J = 8.0 Hz, 1H), 7.70-7.53 (m, 5H), 7.397.33 (m, 2H), 7.30 (d, J = 8.6 Hz, 1H), 7.13 (s, 1H), 7.11 (s, 1H), 6.91 (d, J = 8.4 Hz, 1H), 6.47-6.36 (m, 1H), 5.25 (d, J = 14.7 Hz, 1H), 5.14 (s, 2H), 4.73-4.59 (m, 2H), 4.32 (s, 1H), 3.11 (d, J = 5.3 Hz, 2H), 2.97 (d, J = 15.8 Hz, 2H), 2.06 (s, 6H), 1.81-1.69 (m, 3H), 1.57 (t, J = 6.3 Hz, 3H), 1.45 (s, 9H), 1.00-0.91 (m, 6H). HR-MS (ESI): calcd for C48H54N3O5+ [MI]+ 752.4058, found 752.4089. Synthesis of the probe (DLP) [2-(2-(6-((4-(2-amino-4methylpentanamido)benzyl)oxy)-1,2- dihydrocyclopenta[b] chromen-3-yl) vinyl)-3-ethyl-1,1-dimethyl-1Hbenzo[e]indol-3-ium iodide]. A solution of trifluoroacetic acid (1.5 mL) in CH2Cl2 (1.5 mL) was added dropwise to the DLP-Boc (88 mg, 0.1 mmol) in 3 mL CH2Cl2 at 0 °C, and then the reaction mixture was stirred at room temperature for 2 hours. The solvent was removed by evaporation under reduced pressure, and the crude product was purified by silica gel chromatography (DCM: MeOH = 8: 1 in v/v), affording DLP as a dark green solid (77 mg, 87%). 1H NMR (600 MHz, CD3OD) δ 8.48-8.40 (m, 1H), 8.32 (d, J = 8.4 Hz, 1H), 8.13 (d, J = 8.8 Hz, 1H), 8.08 (d, J = 8.2 Hz, 1H), 7.77 (d, J = 8.8


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Analytical Chemistry Hz, 1H), 7.73 (t, J = 7.6 Hz, 1H), 7.66 (d, J = 7.9 Hz, 2H), 7.60 (t, J = 7.5 Hz, 1H), 7.44 (dd, J = 15.3, 6.6 Hz, 3H), 7.24 (d, J = 23.8 Hz, 2H), 7.03 (d, J = 8.3 Hz, 1H), 6.37 (s, 1H), 5.20 (s, 2H), 4.49 (d, J = 4.7 Hz, 2H), 3.62 – 3.56 (m, 1H), 3.01 (d, J = 20.8 Hz, 4H), 2.05 (s, 6H), 1.77 (dt, J = 20.5, 6.6 Hz, 1H), 1.67 (dd, J = 13.7, 6.2 Hz, 1H), 1.53 (t, J = 7.0 Hz, 4H), 0.99 (dd, J = 10.8, 6.6 Hz, 6H). HR-MS (ESI): calcd for C43H46N3O3+ [M-I]+ 652.3534, found 652.3542. In vivo and ex vivo MSOT imaging. The drug-induced liver injury mice were chosen as an animal model to investigate the potential of the probe for MSOT imaging the revels of drug-induced liver injury. After treating the mice with different doses of APAP for six hours, the mice were anesthetized using 2% isoﬂurane in oxygen. A thin plastic tube was used to connect a syringe needle and a syringe body, and the needle was inserted into a tail vein of the mouse after anesthesia, and the mouse with the needle was then carefully placed into the chamber, leaving the syringe body outside the chamber, thus the probe solution could be pumped into the mouse via the plastic tube. The needle and the plastic tube were pre-filled with saline to prevent air from being injected into the tail vein. During the imaging, the mice were placed in the prone position in a water bath maintained at 34 °C, and anesthesia and oxygen are supplied through a breathing mask. Data were acquired along the animal with a 0.1 mm step through the liver region, with the imaging wavelengths of 680, 705, 730, 755, 780 and 800 nm. Images were acquired before and after tail vein injection of 3.9 mg kg-1 DLP for different time points. Images were reconstructed using backprojection followed by linear regression multispectral un-mixing. The OA spectrum of NIR-OH (red curve in Fig. 1c) was used as the spectrum reference for un-mixing NIR-OH signal. Orthogonal views were also created using the software of the instrument. For ex vivo MSOT imaging of excised organs, nine mice were randomly distributed into three groups (three mice in each group). One group was treated with PBS and used as the control, and the other two groups were respectively treated with 150 mg kg-1 or 300 mg kg-1 of APAP. 6 hours later, the mice were i.v. injected with 3.9 mg kg-1 DLP (in PBS buffer). After 30 min, the mice were humanely sacrificed with CO2 and major organs (heart, liver, spleen, lung and kidney) were harvested, fixed with formalin and embedded in 20 mm diameter agar gel phantoms for ex vivo MSOT imaging. OA data were acquired at the imaging wavelengths of 680, 705, 730, 755, 780 and 800 nm. Images were reconstructed using backprojection approach followed by linear regression multispectral unmixing. RESULTS AND DISCUSSION Design and Synthesis of DLP. The LAP-activatable probe was synthesized by conjugating chromene-benzoindolium chromophore with LAP substrate through a self-immolative linker. The detailed synthetic route of the DLP is depicted in Schemes S1 and S2, and the probe and some intermediate products were characterized using 1H NMR and highresolution mass spectroscopies (Fig. S1-S16). The detection mechanism of the probe towards LAP is shown in Scheme 1b. LAP cleaves the amide linkage between the substrate and the self-immolative linker, and eventually releases the active probe for dual-mode imaging.

Spectral Responses of the Probe towards LAP. The spectral properties of the probe in the presence or absence of LAP were investigated systematically in PBS buffer (10 mM, pH=7.4, with 5% of DMSO, v/v) at 37 °C. As shown in Fig. S17a, the probe DLP (10 μM) showed an absorption band with two peaks (at 616 nm and 666 nm) and a shoulder. Upon addition of LAP (50 U/L) to DLP solution, a red-shift of nearly 40 nm of absorption peaks was observed, and the redmost absorption peak shifted to 705 nm, an absorption wavelength well within the operation range of the MSOT system, and this makes DLP an ideal OAT imaging probe for LAP. Upon LAP treatment, significant NIR fluorescence enhancement at 733 nm can be observed in Fig. S17b, which suggests that DLP can also serve as a sensitive turn-on NIR fluorescent probe for LAP. The absolute fluorescence quantum yield for activated probe NIR-OH in pH 7.4 PBS with 5% DMSO was determined as 0.011 with the excitation wavelength of 705 nm at 28 °C. This quantum yield value is moderate among fluorescent NIR dyes but enough for fluorescence imaging. During the deactivation process, most absorbed energy will be converted into heat, and very small portion of the excitation energy is used for fluorescent emission. The time- and dose-dependent spectral measurements were performed after incubating the probe with LAP for varied time or at varied activities. As shown in Fig. 1, with the prolonged time of reaction between the probe DLP (10 μM) and LAP (50 U/L), the absorption for the probe increased gradually at 705 nm (Fig. 1a), and the fluorescence increased at 733 nm over time and reached the plateau at 30 min (Fig. 1b), indicating the fast response of the probe towards the LAP. We also recorded the optical response of DLP (10 μM) to LAP (varied activities) at the optimized conditions (reaction in pH 7.4 PBS buffer at 37 °C for 30 min), as shown in Fig. S18. With the increasing LAP activity from 0-30 U/L, the absorption band at 705 nm (Fig. S18a) and emission band at 733 nm (Fig. S18b) increased until they reached their maximums respectively.

Fig. 1 Optical responses of DLP to LAP in PBS (10 mM, pH=7.4, with 5% of DMSO, v/v). (a) Absorption spectra for the probe (10 μM) upon incubation with 50 U/L LAP for different time. (b) Time course of fluorescence spectra for the DLP (10 µM) in the presence of 50 U/L LAP at 37 °C in PBS buffer. The insets show



the plots of the fluorescence intensity at 733 nm versus reaction time. (c) The normalized optoacoustic signal intensities of the probe DLP (10 μM) (black line) and the activated probe NIR-OH (10 μM) (red line) determined in phantom. The MSOT values were normalized to the maximum optoacoustic intensity for activated probe NIR-OH at 680 nm. (d) The plot of mean optoacoustic intensity of the probe DLP (10 μM) upon reaction with LAP of varied activities for 30 min (n=3). On top of the figured is the representative optoacoustic image of the probe in phantom at varied LAP concentrations.

Moreover, the absorbance and fluorescence intensity of the probe in the presence of low levels of LAP (0-2 U/L) were determined, as shown in Fig. S19, which indicates that in this LAP activity range, both the absorbance and fluorescence basically increased linearly with LAP activity. In addition, the detection limit of fluorescence was determined to be 0.047 U/L for LAP (Fig. S19b). Also, the enzyme catalysis kinetics for DLP with LAP was determined (Fig. S20), and the Michaelis constant (Km) and maximum of initial reaction rate (Vmax) were found to be 13.176 µM and 1.185 µM/min respectively, indicating the strong affinity between the probe and LAP. The relationship between excitation wavelength and the optoacoustic signal intensity for the probe and activated probe in solutions were recorded on the MSOT system and presented in Fig. 1c. It can be seen from the figure that, without LAP treatment, the probe exhibited low optoacoustic intensity; while addition of LAP triggered remarkable increase in optoacoustic intensity. LAP can cleave the amide bond and then induce a cascade of subsequent reactions, eventually generating a NIR dye with a hydroxyl group (namely the activated probe NIR-OH). It is the strong absorption at 705 nm of NIR-OH that produces prominent OA signals upon excitation. The excitation wavelength dependence of optoacoustic signal intensities for the probe DLP (black line) and the activated probe NIR-OH (the red line) are similar to the corresponding absorption spectra shown in Fig. 1a, which further verifies that upon activation, the probe displays strong optoacoustic signal at around 705 nm. Moreover, the dosedependent optoacoustic signal intensities for the probe upon incubation with LAP of varied activities were given in Fig. 1d. The upper panel of the figure shows the MSOT images for the probe solutions in the presence of LAP of varied activities, and the lower panel displays the readout intensities for the corresponding images in the upper panel. We can find in the figure that, with the increasing LAP activity, the acquired optoacoustic intensities for the probe at 705 nm gradually increased and reached the maximum at the LAP activity of 30 U/L. These results suggest that the probe may be suitable for OAT imaging of LAP in vivo. To confirm the activation mechanism of the probe in the presence of LAP, high-resolution mass spectrometry (HR-MS) and high-performance liquid chromatography (HPLC) were used to analyze the reaction products of the probe with LAP. As depicted in Fig. S21a, as the probe DLP (10 μM) was treated with LAP (50 U/L) for 20 min, the MS peaks for NIROH (the reaction product) and DLP (the probe) could all be observed in MS spectrum. In addition, the HPLC profiles in Fig. S21b indicate that upon LAP treatment, the peak for NIROH (at 3.56 min) emerged, while that for the probe (at 4.77 min) decreased accordingly. These investigations all

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demonstrate that the chromophore NIR-OH was generated after incubation with LAP. Next, to investigate the detection specificity of the probe to LAP, we examined the fluorescent response of the probe to LAP as well as some potential interferents, such as some biologically essential metal ions that may form complexes with the probe or LAP, reactive oxygen species that may oxidize the probe, some biomolecules and related enzyme species (including some hepatic enzymes) that may interfere with the detection. As shown in Fig. S22a, only incubation with LAP generated prominent fluorescence response, whereas the addition of some metal ions (Na+, K+, Ca2+, Zn2+, Mg2+ and Cu2+), reactive oxygen species (H2O2 and ClO-), biomolecules (glucose, GSH, Cys and Hcy), some other enzymes (such as alkaline phosphatase, γglutamyltranspeptidase, nitroreductase, carboxylesterase, βgalactosidase, α-amylase and etc.) at much higher concentration than their physiological levels displayed a negligible response. On the other hand, the examination on the co-existing potential interferents also revealed that these interferents could not induce obvious influence on the fluorescence signal (Fig. S22b). To further verify the specificity of the probe to LAP, we added a specific LAP inhibitor (bestatin) to the LAP solution before incubation with the probe. As depicted in Fig. S23, bestatin could effectively decrease the fluorescence response in a dose-dependent manner compared to the control group, proving that the probe’s fluorescence signal was caused by LAP. We also evaluated the stability of the probe in healthy mice serum by recording the fluorescence intensities over a period of 120 min. As shown in Fig. S24, the probe is relatively stable in rodent serum. We also recorded the fluorescence intensities at 733 nm in the presence or absence of LAP under varied pHs (Fig. S25a). The fluorescence intensities were found rather stable at 733 nm in pH values ranging from 4 to 10 in the absence of LAP. While in the presence of LAP, the emission intensities slightly fluctuated under physiological pHs, indicating the applicability of the probe under physiological conditions. The fluorescence intensity at 733 nm for NIR-OH in DMSO/PBS mixture with varied PBS contents were recorded and presented in Fig. S25b, which reveals that the activated probe is fluorescent in BMSO/PBS mixtures with wide range of DMSO to PBS ratio. The photostability of the DLP and NIR-OH in PBS and fetal bovine serum were determined by continuous irradiating them with the pulsed laser of the MSOT system (at 705 nm with the energy density close to 20 mJ/cm2) and recording their MSOT signal intensities every 10 min. As shown in Fig. S26, the MSOT signal intensities for the probe and activated probe did not undergo remarkable reduction upon 120 min of irradiation at 705 nm, verifying the fairly good photostability of the probe. Optoacoustic and Fluorescence Imaging of LAP in Living Cells. We next investigated the capability of the probe for monitoring LAP activity in live cells. Before cellular imaging, MTT assays were performed to evaluate the probe’s toxicity towards the HepG2 cells. As shown in Fig. S27, the probe displayed low cytotoxicity towards the cells, and the cell viability remained nearly 80% as the probe’s concentration


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Analytical Chemistry reached 50 μM. For optoacoustic imaging of activated probe in live cells, the overall OA intensities were recorded for cell suspensions at varied excitation wavelengths (Fig. S28), and the MSOT images for the cells were reconstructed at a single wavelength of 705 nm. Fig. 2 and Fig. S29 show the imaging of the endogenous LAP activity in HepG2 cell cells. Upon incubation with 10 μM DLP, the MSOT signal (Fig. 2a and 2c) and red fluorescence (Fig. S29a and S29c) gradually increased over time and reached maximum in about 1 h, indicating the probe displays good cell permeability and can be activated by the endogenous LAP. To verify the MSOT signal and fluorescence was indeed caused by LAP, a LAP inhibitor (bestatin, 50 μM) was used to pre-incubate the cells before imaging. As shown in Fig. 2b and S29b, the bestatin-pretreated HepG2 cells generated very weak MSOT signal and intracellular fluorescence. Fig. 2d and S29d shows that the relative MSOT signal and fluorescence intensity for the bestatin-pretreated cells is much lower than that for non-pretreated cells. These results suggest that the probe can be activated by the intracellular LAP, and produce enhanced optoacoustic and fluorescence signals.

by these chromophores, part of the absorption energy is transferred into heat and then into ultrasound signals, which can be detected by an array of ultrasound transducers in the system to produce an optoacoustic tomographic image with both the signal from the activated probe and background signal from tissue-intrinsic chromophores. The in vivo OAT imaging was performed on the MSOT system using APAPtreated male mice. Fig. 3a and Fig. S30 shows the crosssectional images of the mouse pretreated with 300 mg kg-1 APAP before (0 min) and after intravenous injection of 3.9 mg kg-1 probe for varied time periods. In each image, the signal from the activated probe (represented in colour) is merged with the grayscale image of background signals (such as hemoglobin signals).

Fig. 2 Optoacoustic imaging of intracellular LAP in cells. (a) Typical MSOT images for HepG2 cells incubated with DLP (10 μM) for different times. The suspension (ca. 1×106 cell/mL) of the test cells and the untreated cells (reference) were respectively injected into two 3 mm diameter quartz tubes pre-embedded in a 20 mm phantom. The regions of interest are marked by dashed circles. The references were labeled with “1” and the test cells were labeled with “2”. Scale bar: 3 mm. (b) MSOT images for HepG2 cells pretreated with or without the LAP inhibitor bestatin (50 μM) for 1 h and then incubated with DLP (10 μM) for 1 h. Scale bar: 3 mm. (c) Relative MSOT intensity of the corresponding MSOT images in (a). (d) Relative MSOT intensity of the corresponding MSOT images in (b). The values of (c) and (d) are the mean value (± S.D.) from three separate measurements.

Diagnosis and Location of Drug-induced Liver Injury via Multispectral Optoacoustic Tomography Imaging. We then employed multispectral optoacoustic tomography (MSOT) to image the drug-induced liver injury (hepatotoxicity). In this study, acetaminophen (APAP, paracetamol) was intraperitoneally injected into mice to induce liver injury.70 As the liver-injured mouse is injected with the probe and illuminated with pulsed NIR laser light at different wavelengths generated by the MSOT system, the light can be absorbed by tissue-intrinsic chromophores (such as hemoglobin) as well as the exogenous chromophore (the activated probe NIR-OH in this study). Upon light absorption

Fig. 3 MSOT imaging for APAP-induced liver injury. (a) Crosssectional MSOT images for the liver-injured mice (pre-treated with 300 mg kg-1 of APAP) at different time points upon intravenous injection of DLP. The multispectral resolved signal for NIR-OH (in color) is overlaid with the grayscale singlewavelength (800 nm) background image. Organ labelling: 1. spinal cord; 2. aorta; 3. vena cava; 4. liver. Scale bar: 5 mm (b) Mean MSOT signal intensity in the region of interest in the liver at different time points post injection of DLP. (c) A simulated cryosection image of a male mouse with the cross section’s location comparable to those shown in a. The simulated cryosection image is chosen from the simulated image set which were created by software and stored in MSOT system. (d) Serum



levels of AST and ALT for the mice at 6 hours upon treatment with varied dose of APAP. (e) z-stack MIP images for the mice treated with different doses of APAP. Scale bar: 3 mm (f) Crosssectional MSOT images for excised organs (heart, liver, spleen, lung and kidney) of mice after pre-treatment with 0 (control), 150 and 300 mg kg-1 APAP for 6 h and then injection of the probe for 30 min, followed by immediate formalin fixation and embedment in phantom. Scale bar: 5 mm (g) Relative (normalized) MSOT signal intensity in the region of interest for excised organs. (h) Histological sections (Hematoxylin and eosin (H&E) staining) of the liver tissue of the mice treated with PBS, 150 or 300 mg kg-1 of APAP. Scale bar: 100 μm.

As shown in Fig. 3a and Fig. S30, the control (0 min) shows the anatomical background in which some organs, such as spinal cord (1), aorta (2), vena cava (3) and liver (4), can be identified. At 10 min after probe injection, the signals from activated probe clearly appear and gradually increase over time, and we can find that most of the signals reside in the liver area by referring to the background image. At 30 min after probe injection, the signal intensity reaches its peak level, and then gradually fades out due to metabolism by the liver. The mean signal intensity of activated probe signals in mice was obtained by defining the region of interest (ROI) and the results are given in Fig. 3b, which provides quantified evidence for the change in MSOT signals after probe injection. Moreover, the simulated cryosection image of a male mouse (Fig. 3c) can provide further evidence that most signals from the activated probe is in the liver area. Moreover, the z-stack MIP images which are able to reflect three-dimensional (3D) information of the liver injury are presented in Fig. 3e. An illustration on how the 3D images were rendered is given in Fig. S31. The mice under imaging were pretreated with 0, 150 or 300 mg kg-1 of APAP and then injected with the probe. Fig. 3e indicates that, as for the mouse treated only with PBS (the control), no OAT signal could be observed. In contrast, weak OAT signals were detected in the liver area of the mouse without APAP treatment but injected with the probe, indicating the low LAP level in the liver region in healthy mice. On the other hand, for the mice treated with over-dosed APAP (150 or 300 mg kg-1), strong MSOT signals can be clearly visualized in a large area of the liver, and the mouse treated with higher APAP dose suffered more severe liver damage. More importantly, these MIP images are like three-view images, and one can accurately locate the focus of the liver injury in abdominal cavity of the mice. These results indicate that the elevation of hepatic LAP level as a result of liver injury can be detected by MSOT in a temporal and spatial manner. To verify that APAP did induce the liver injury in mice, the aspartate transaminase (AST) and alanine aminotransferase (ALT) levels in serum were assayed by using the ELISA Kit assay. We can see in Fig. 3d that, after APAP treatment, the mice show much higher levels of ALT and AST; and higher dose of APAP can cause much more remarkable increase in serum ALT and AST level. This suggests that the successful establishment of the mouse model of liver injury; and the degree of injury is in correlation with the dose of APAP. Next, the mice treated with 0, 150 and 300 mg kg-1 of APAP were euthanized and their major organs were collected for ex vivo imaging. The overall optoacoustic intensities for the organs were recorded at 6 wavelengths (Fig. S32), and the

MSOT images were obtained after reconstruction and signal unmixing. As shown in Fig. 3f and 3g, the liver exhibits much stronger MSOT signal than other major organs (heart, spleen, lung, and kidney). This further proves the in vivo imaging result. The liver tissue sections (H&E staining) were also examined to evaluate the liver injury, as shown in Fig. 3f. The section for the control group and the mouse pretreated with PBS show no obvious pathological changes; while that for the mouse treated with APAP displays central necrosis with infiltration of inflammatory cells, as usually seen after APAP intoxication. Fluorescence Imaging of Drug-induced Liver Injury. DLP is an optoacoustic/fluorescent dual-responsive probe for LAP, to confirm the results of MSOT imaging, we performed fluorescent imaging using the probe. The time-dependent fluorescent images for the mice pretreated with PBS (the control), 150 mg kg-1 or 300 mg kg-1 APAP and then injected with the probe 6 h later are presented in Fig. 4a. It can be seen from the figure that, at 10 min upon probe injection the fluorescence for three groups emerged, then reached maximums at about 30 min and gradually decreased. The groups pretreated with APAP exhibited stronger abdominal fluorescence than the control group, and higher dose (300 mg kg-1) of APAP caused more prominent fluorescence. The mean fluorescent intensities obtained from the region of interest for three groups can provide quantified fluorescence change after probe injection (Fig. 4b). To further verify that the increased fluorescence intensity was induced by the elevated LAP level, the LAP activities in serum (Fig. S33a) and liver tissue (Fig. S33b) for healthy mice and APAP-treated mice were determined by a Mouse ELISA kit. For healthy mice, the serum and hepatic LAP activity were determined as ca. 45 U/L and 20 ng/mg, respectively; while upon treatment with 150 mg/kg or 300 mg/kg of APAP, both the serum and hepatic LAP levels greatly increased. Ex vivo imaging was performed for the dissected organs after the APAP-treated mice were used for fluorescence imaging and euthanized. As shown in Fig. 4c and S34, the liver exhibits much stronger fluorescence than other major organs such as heart, spleen, lung, and kidney, hence verifying the in vivo imaging result that only the liver displays strong fluorescence. To evaluate the biosafety of the probe, H&E staining analysis of histological sections of major organs from the control group and the group treated with PBS solution were performed, and the results are given in Fig. S35, which demonstrate that the injection of the probe did not cause obvious histological changes in comparison to the control and PBS group, suggesting the probe is of good biosafety. The fluorescence imaging experiments provide further evidence that the probe is capable of detecting and imaging LAP activity in vivo and the probe is a dual-mode imaging agent for LAP.


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Analytical Chemistry studies on Crohn’s disease patients, and the results suggest that MSOT can improve the diagnostic accuracy and reduce the frequency of colonoscopy procedures compared to several other modalities.71 However, to our best knowledge, this technology has not yet been used in human hepatic disease diagnosis. For use in human liver, the penetration depth is the main obstacle. Currently, a penetration depth of 4~5 cm has been achieved in vivo in the human breast.74,75 The penetration depth of 4~5 cm may not be enough for liver imaging, but development of more robust exogenous contrast agent (recently a penetration depth of 12 cm using phosphorus phthalocyanine has been reported76), more advanced excitation laser and other optoacoustic techniques, will further enhance the applications of MSOT in clinical imaging, including human liver imaging.

Fig. 4 Fluorescence imaging of APAP-induced liver injury. (a)Time course for fluorescence imaging in vivo. The mice were pretreated with PBS, 100 mg kg-1 or 300 mg kg-1 APAP for 6 h, followed by tail intravenous injection of 3.9 mg kg-1 DLP in PBS buffer. (b) The bar graphs show the mean fluorescence intensity at ROI of the liver region in mice of (a). (c) The fluorescence images for major organs harvested from the mice treated with varied dose of APAP.

By comparing the MSOT images shown in Fig. 3 and the fluorescence ones in Fig. 4, one can find that both the imaging modalities exhibit their own distinctive features. As an emission-based modality, fluorescence imaging offers superb molecular sensitivity and the resultant images are more compelling. However, a fluorescence imaging system detects emitted photons which propagate through animal tissue; and thus they are strongly scattered by tissue. As a result, precisely determining the origin of fluorescence signals from tissue at depth is impossible, and hence the spatial resolution is unsatisfactory. In addition, the fluorescence images are the projection-based planar ones, from which it is hard to determine whether the signals are from deeper tissue or from superficial tissue. Thus, the fluorescence is more suitable for low depth imaging. In contrast, as an absorption-based imaging modality, the optoacoustic imaging is less sensitive. However, ultrasound waves scatter much less in tissue compared to photons; and MSOT can thus achieve high spatial resolution at depth. Moreover, it can also obtain label-free contrast by acquiring the signals from endogenous substances and uses them as the background, as shown in Fig. 3. In addition, the z-stack image derived from plenty of cross-sectional signals enables threedimensional representation of the biodistribution of a specific exogenous contrast agent, and thereby allows us to locate the disease foci. These features make MSOT particularly advantageous for imaging biological events at depth. Owing to its advantages, the MSOT technology has been used into clinical trials such as diagnosis and test of inflammation, oncology, cardiology and etc.71-73 For example, MSOT has recently been successfully employed in clinical

CONCLUSIONS In conclusion, an activatable probe has been successfully developed for sensitive and specific detection and imaging of LAP by multispectral optoacoustic tomography. For this detection, the presence of LAP mediates the cleavage of the amide linkage in the probe and triggers a cascade of reactions, thereby releasing the NIR chromophore with red-shifted absorption and producing both the optoacoustic and fluorescent signals. The probe is able to track the endogenous LAP in living cells, and could also be used to diagnose and monitor the drug-induced liver injury in vivo by detecting the elevated LAP level. Moreover, the MIP images through zstack rendering can provide 3D information and allow us to locate the foci of the liver injury. We suppose this strategy may serve as a promising tool for studying of physiological and pathological processes related to LAP, and this strategy can also be used to design other activatable optoacoustic probes for other biomarkers.

ASSOCIATED CONTENT Supporting Information Supporting Information includes some of the experimental section, 1H NMR spectra, mass spectra, absorption spectra, fluorescence spectra, fluorescence images, selectivity and specificity evaluation, MTT assays, photostability and H&E staining histological analysis. The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] *E-mail: [email protected]

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We acknowledge the financial support by NSFC (21788102, 21875069, 21574044 and 51673066), the Science and Technology Planning Project of Guangzhou (Project No. 201607020015) and the Natural Science Foundation of Guangdong Province (2016A030312002). We are very grateful to Dr. Y. Qiu for her



kind helps in MSOT data processing as well as valuable suggestions in design of some MSOT experiments.

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Diagnosing Drug-induced Liver Injury by Multispectral Optoacoustic

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