Design and synthesis of a ratiometric photoacoustic imaging probe activated by selenol for visual monitoring of pathological progression of autoimmune hepatitis

Photoacoustic (PA) imaging with both the high contrast of optical imaging and the high spatial resolution of ultrasound imaging has been regarded as a robust biomedical imaging technique. Autoimmune hepatitis (AIH) is the second largest liver inflammatory disease after viral hepatitis, but its pathogenesis is not fully understood probably due to the lack of an effective in vivo monitoring approach. In this work, an innovative selenol-activated ratiometric PA imaging probe APSel was developed for visual monitoring of pathological progress of AIH. Selenols including selenocysteine (Sec, the major form of Se-containing species in vivo) have been demonstrated to have an effective antioxidant role in inflammation. The reaction of APSel with selenol results in a blue shift of the PA spectrum peak from 860 nm to 690 nm, which enables the ratiometric PA imaging. The APSel probe displays high sensitivity and selectivity to Sec and other selenols. The APSel probe was then employed for ratiometric PA imaging of selenol in cells, and for monitoring the development of AIH in a murine model by tracking the changes of selenol level. The results revealed that the level of selenol was closely correlated with the development of AIH. The proposed APSel, as the first example of a selenol-responsive PA imaging probe, provides a new tool and approach to study and diagnose AIH diseases.


Instruments
The 1 H nuclear magnetic resonance ( 1 H NMR) and 13 C NMR spectra were obtained using a 500 MHz Avance II DRX-500 NMR spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany). The mass spectra were obtained from an exactive ultra-high resolution liquid chromatography-mass spectrometry (LC-MS) system (Thermo Fisher Scientific Inc., Waltham, MA, USA). The Multiskan Mk3 microplate reader (photometer) was employed for enzyme labeling assays (Thermo Fisher Scientific Inc., Waltham, MA, USA). The Cary 60 UV−vis spectrophotometer was used for recording the absorption spectrum (Agilent Technologies Inc., Santa Clara, CA, USA). All optoacoustic measurements were performed by using a MSOT inVision 256-TF multispectral photoacoustic imaging system was (iThera Medical GmbH, Munich, Germany).

Synthesis and characterization of the probe Synthetic route of the compound Cy:
Synthesis of compound 1: 2,3,3-trimethylindolenine (3.18 g, 20 mmol), iodoethane (4.67 g, 30 mmol) and 15 mL of acetonitrile were added into a 50-mL flask. The mixture was heated to 55 °C, and the reaction was performed for 2 h. After cooling to room temperature, the crude product was collected by filtration and purified by S4 recrystallization in ethanol to afford compound 1 as white crystal (2.73 g, yield 73%). mmol) and dichloromethane (17.5 mL) was slowly added into the mixture of dichloromethane (20 mL) and N,N-dimethylformamide (20 mL) under ice bath condition, and then cyclohexanone (5.29 mL, 51 mmol) was added slowly. After removal from the ice bath, the reaction solution was heated to reflux. After stirring the reaction for 4 h, the reaction solution was cooled to room temperature, and then the reaction mixture was poured into 100 g of crushed ice four times. After allowing to stand overnight, the reaction product solution was filtered, and washed with frozen acetone to ultimately obtain a bright yellow crystal (5.39 g, yield 61%). Synthesis of compound 3: a mixed solution (150 mL) of n-butanol and cyclohexane (7:3, v/v) was added to a 250-mL flask, then compound 1 (1.50 g, 8 mmol) and compound 2 (0.69 g, 4 mmol) were added into the above flask under a nitrogen atmosphere. The mixture was heated to 117 °C, and subjected to reflux reaction for 6 h. Then, the n-butanol and cyclohexane were removed by rotating evaporation. A mixed solution of DCM/MeOH (100:1), v/v) was used as eluate to purify the crude product by column chromatography to give compound 3 as gold-green crystal (1.35 g, yield 66%).

Synthetic route of compound Cy-2:
Synthesis of compound 6: the synthesis method was similar to that of compound 2.
Using cyclopentanone (4.51 mL, 51 mmol) as raw material, we obtained a bright yellow crystal (3.48 g, yield 43%). The product was not characterized and directly used in the S6 next reaction.
Synthesis of compound 7: the synthesis method was similar to that of compound 3.

Synthesis of compound APSel probe:
The synthesis method was similar to that reported previously [6]. Using compound Cy-3 (0.07 g, 0.10 mmol) as raw material, we obtained a red-brown crystal (0.05 g, yield 58%).

Absorption spectrum and in vitro PA response of APSel to Sec
The APSel was dissolved in DMSO to prepare the stock solution (1 mM), and was For in vitro PA tests, the sample was transferred to a transparent suction 3-mm diameter tube, and embedded in the agar phantom. The agar phantom was fixed to the bracket and placed in water for testing. The PA imaging signals at 690 and 860 nm were recorded, and the PA intensity ratio PA690/PA860 was used to quantify the concentration of Sec. The detection limit was determined according to 3σ/κ (where σ is the standard deviation for the blank APSel solution in 20 measurements, κ is the slope of the linear S8 regression curve for PA690/PA860 to the Sec concentration. In the selectivity teste, PA imaging was performed after the APSel was mixed with different analytes for 20 min.

Cell culture
The

Cytotoxicity test
The MTT (methyl thiazolyl tetrazolium) assay was used to determine the cytotoxicity of APSel. The cells were seeded on 96-well cell culture plates and incubated at 37 °C in 5% CO2 for 24 h. Then, the APSel solution at different final concentrations of 0, 5,10,15,30 μM was added into the corresponding well in the cell culture plates. The cells were cultured in an incubator at 37 °C with 5% CO2 for 24 h.
Subsequently, 15 μL of MTT solution (5 mg/mL) was added to each well, and the plate was incubated for another 4 h at 37 °C with 5% CO2. Then, 150 μL of DMSO was added into each well after removing the medium. Finally, the absorbance of each well was measured at 570 nm wavelength using a Multiskan Mk3 microplate reader.

PA imaging of cells
The four groups of HL-7702 cells were seeded into the T75 flask, and cultured for 24 h at 37 °C with 5% CO2. The medium in the first and second groups of cells was replaced with fresh medium. The medium in the third group of cells was replaced with a medium containing Na2SeO3 (5 μM). The medium in the fourth group of cells was replaced with a medium containing INF-γ (60 ng/mL). After culturing for 24 h, the medium from each flask was removed, and the cells were washed three times with PBS (pH 7.4), and then digested with trypsin. The first group of cells was suspended in pure PBS, and the remaining groups of cells were suspended in PBS containing APSel (5 μM, 2% DMSO, v/v). Then, the cells in each group were continued to culture for 1 h at room temperature. Finally, each group of cells was transferred to a 300-μL centrifuge tube and centrifuged at 1,500 rpm and 4 °C for 7 min to obtain the cell pellet. The S9 centrifuge tube was fixed to the support, and PA imaging was performed on the multispectral photoacoustic tomography imaging system in water at 25 °C with excitation at 690 nm and 860 nm.

Animal model
Twelve-week-old male BALB/c mice (24-27 g) were purchased from Hunan SJA Laboratory Animal Co., Ltd. (Changsha, China). All animals were given food and water

In vivo toxicity assessment of APSel probe
Two groups of BALB/c mice were injected with either normal saline (control group) or APSel (0.2 mg/kg, test group) via tail vein. After 24 h, the mice were euthanized and the main organs (heart, liver, spleen, lung, kidney) were harvested. Then, tissue samples from these organs were fixed in a 4% paraformaldehyde solution and embedded in paraffin to prepare 3-µm thick sections. The sections were stained with hematoxylin and eosin (H & E) and the images of the biological tissue sections were captured under a microscope.
For the chronic toxicity evaluation, two groups of BALB/c mice were injected with normal saline or APSel (0.1, 0.2 mg/kg) via tail vein. After 7 days, the blood samples were collected from the eyeball of mice (about 0.8 mL) and routine blood analysis was performed directly on a blood analyzer.
For the assessment of systemic toxicity, two groups of BALB/c mice were injected with either normal saline (control group) or APSel (0.2 mg/kg) via tail vein. Then, the body weight of the mice was measured daily for 14 days and checked for clinically relevant abnormalities. When the mice were unable to eat, injured or died, the experiment on the mice was suspended.

PA imaging of exogenous Sec in vivo
The mice were anesthetized by isoflurane (2%), and the hair on their back was shaved. Then, 100 μL of 15 μM APSel solution was injected subcutaneously at both sides of each mouse back into the A and B ROIs. The mice were then coated with ultrasound gel, and were fitted with a breathing mask with 2% isoflurane anesthesia. S10 Then, the mice were wrapped with a film and placed in water at 34 °C for PA imaging (with 690 and 860 nm wavelength excitation). After that, the mice were further injected with 100 μL of 0.9% of sterile saline in ROI A, and with 100 μL of 30 μM of Sec in ROI B. After 30 min, the PA imaging PA was performed.

PA imaging of liver from AIH disease model mice
Male BALB/c mice (24-27 g) were randomly divided into five groups. The first and second groups of mice were injected with normal saline (100 μL) via tail vein. The remaining groups of mice were injected with normal saline (100 μL) containing Con A (15 mg/kg) via the tail vein. After 10 h, the third group was injected with normal saline (150 μL) containing APSel (0.1 mg/kg, 5% DMSO, v/v) via tail vein. After 1 h, the mice were euthanized, and their liver was dissected. The liver was photographed and used for PA imaging (5 nm steps, using an excitation in the wavelength range of 680-960 nm), and then fixed in a 4% paraformaldehyde solution. After 24 h, the first, second and fourth groups of mice were subjected to the same procedure as the third group, but the first group of mice were injected with saline without APSel. After 72 h, the fifth group of mice was also subjected to the same procedure as the third group.

Distribution of probe in mouse organs
Two groups of BALB/c mice were injected with either normal saline (control group) or APSel (0.1 mg/kg, test group). After 1 h, the mice were euthanized, and their main organs (heart, liver, spleen, lung, kidney) were harvested. The organs were immediately coated with ultrasound gel. Then the organs were wrapped with a film and placed in water at 34 °C for PA imaging (5 nm steps, the excitation was in the wavelength range of 680-960 nm).

Biochemical analysis of normal and AIH disease model mice
The blood collected from normal and AIH disease model mice was kept static at room temperature for 1.5 h and then centrifuged for 10 min (3,000 rpm, 4 °C).
Subsequently, the supernatant was used to determine the content of AST and ALT using the automatic biochemical analyzer. The paraformaldehyde fixed liver was sliced into sections, and then the liver sections were stained by H&E, and images captured under a microscope.

S11
The in vivo and in vitro PA intensity were obtained by using the post-processing software ViewMSOT (iThera Medical GmbH) of the MSOT inVision 256-TF multispectral photoacoustic tomography imaging system. The GraphPad Prism v7.04 software (GtraphPad Software Inc., La Jolla, CA, USA) was used for statistical analysis.
The student's t-test method was used for all data analysis and data are expressed as the mean ±standard deviation. When the P value <0.05, the difference is considered to be statistically significant. Table S1. Photophysical parameters of APSel and its precursors in DMSO S13 FIGURES Fig. S1. LC-MS spectrum of Cy.            Representative images of entities of mouse liver in different conditions.