7-azaindole photoacoustic imaging probes for detecting superoxide anion, methods of preparation and use

Abstract

The application discloses a 7-azaindole photoacoustic imaging probe for detecting superoxide anion, a preparation method and application, and the photoacoustic imaging probe is named AIH-OTF, which is a blue-green solid prepared from methyl trifluoromethanesulfonate, dye AIH-OH and the like; the AIH-OTF realizes high-contrast PA imaging of endogenous superoxide anion in living cells, and in-situ evaluation of occurrence and repair of acetaminophen-induced liver injury is also determined through non-invasive monitoring of the PA reaction of AIH-OTF to superoxide anion; the AIH-OTF probe can be used as a useful tool for accurately diagnosing drug-induced liver injury in vivo. The photoacoustic imaging probe can realize real-time detection of in-vivo superoxide anion through AIH-OTF and the ability of diagnosing DILI; the cell toxicity is small, the biological compatibility is good, and the photoacoustic imaging probe can be used for in-vivo imaging of occurrence and repair of DILI.

Description

Technical Field

[0001] This invention belongs to the field of imaging probe detection technology, specifically, it relates to a 7-azaindole photoacoustic imaging probe for detecting superoxide anions, its preparation method, and its application. Background Technology

[0002] Drug-induced liver injury (DILI) is caused by the direct toxic effects of drugs and their metabolites on the liver, or by an individual's specific response to drugs. It has become the third most common liver disease, with an incidence rate lower than viral hepatitis and fatty liver disease. DILI is irreversible and can progress to liver failure or liver cancer. Due to the non-specific, insidious, and severe nature of DILI in clinical practice, accurate diagnosis and timely treatment remain significant challenges. Recent studies have shown that intracellular oxidative and nitrosogenic stress are closely related to the severity of DILI. Acetaminophen metabolism is a major cause of DILI, inducing the production of superoxide anions in the liver. Superoxide anions are considered one of the most important reactive oxygen species (ROS), which can be converted into other ROS, including hydroxyl radicals, hypochlorous acid, and hydrogen peroxide, by superoxide dismutase, leading to disruption of cellular redox balance and hepatocyte damage. Furthermore, as a major source of reactive nitrogen species, it can also react with endogenous nitric oxide to produce peroxynitrite in hepatocytes. Excessive superoxide nitrite interacts with biomolecules such as proteins, nucleic acids, and lipids, leading to cellular physiological and structural dysfunction and further exacerbating liver damage. Therefore, superoxide anion has become an important biomarker in the progression of drug-induced liver injury, and establishing effective superoxide anion detection methods is crucial for the accurate diagnosis of drug-induced liver injury.

[0003] Photoacoustic (PA) imaging is a powerful biomedical imaging modality that enables non-invasive visualization of biological processes at the molecular and cellular levels in deep tissues with high spatial resolution. Utilizing a near-infrared (NIR) operating window, PA can provide a penetration depth of several centimeters with a resolution of approximately 100 μm. Due to its advantages, it provides a useful tool for clinical imaging of various diseases, including cancer diagnosis, metastasis assessment, and treatment monitoring. Therefore, the development of photoacoustic probes with long absorption wavelengths, large redshifts, and high activation folds for detecting superoxide anions is of significant value for the diagnosis of drug-induced liver injury. Summary of the Invention

[0004] The technical problem to be solved by this invention is to overcome the shortcomings of the prior art and provide a 7-azaindole photoacoustic imaging probe for detecting superoxide anions, its preparation method and application, so as to solve the detection problem of drug-induced liver injury by photoacoustic probes with long absorption wavelength, large redshift and high activation factor for detecting superoxide anions.

[0005] To solve the above-mentioned technical problems, the basic concept of the technical solution adopted by the present invention is as follows:

[0006] This invention provides a 7-azaindole photoacoustic imaging probe for detecting superoxide anions, named AIH-OTF, whose chemical structure is shown in Formula I:

[0007]

[0008] Formula I.

[0009] Furthermore, the aforementioned

[0010] In another aspect, this invention protects the application of the aforementioned photoacoustic imaging probe, which can perform quantitative detection in vitro and enable the diagnosis of DILI at the in vivo level.

[0011] In another aspect, this invention provides a method for preparing a 7-azaindole photoacoustic imaging probe for detecting superoxide anions, comprising the following steps: methyl trifluoromethanesulfonate, dye AIH-OH, anhydrous dichloromethane and triethylamine are mixed and stirred at 30°C for 2 hours; the mixture is then concentrated under vacuum, and the residue is purified by column chromatography using dichloromethane and methanol as eluents to obtain a blue-green solid product, named AIH-OTF.

[0012] Further, the preparation method of the dye AIH-OH is as follows: 7-chloro-6-hydroxy-2,3-dihydro1H-oxanthracene-4-carboxaldehyde is added to a solution of anhydrous EtOH containing a catalytic amount of piperidine, and 2,3,3,7-tetramethyl-3H-pyrrolo[2,3-b]pyridine-7-onium is added. The mixture is stirred at 100°C for 12 h to obtain a deep blue solution. The solvent is removed by rotary evaporation, and the resulting residue is purified by column chromatography to obtain a blue solid product, named AIH-OH, whose chemical structural formula is shown in Formula II:

[0013]

[0014] Formula II.

[0015] Furthermore, the molar ratio of the dye AIH-OH to methyl trifluoromethanesulfonate is 1:2.

[0016] Furthermore, the volume ratio of dichloromethane to methanol in the eluent is 10:1.

[0017] The 7-azaindole photoacoustic imaging probe of this invention consists of a trifluoromethanesulfonate moiety as the recognition unit and a hemicyanine dye (AIH-OH) as the photoacoustic signaling group. Due to the esterification of the phenolic hydroxyl group blocking the intramolecular charge transfer effect of AIH-OH, AIH-OTF exhibits negligible absorption in the wavelength range of 680 nm to 950 nm. Upon nucleophilic attack of the trifluoromethanesulfonate group by superoxide anion, AIH-OTF is converted to AIH-OH, restoring the intramolecular charge transfer effect and generating a large Δλ (203 nm), resulting in a high-contrast (9.7-fold) activated PA response. The results indicate that AIH-OTF achieves high-contrast PA imaging of endogenous superoxide anion in living cells. More importantly, the in situ assessment of the occurrence and repair of acetaminophen-induced liver injury was also determined by non-invasively monitoring the PA response of AIH-OTF to superoxide anion. The AIH-OTF probe of this invention can serve as a useful tool for the accurate in vivo diagnosis of drug-induced liver injury.

[0018] By adopting the above technical solution, the present invention has the following beneficial effects compared with the prior art.

[0019] (1) A new semicyanine dye (AIH-OH) was designed by introducing 7-azaindole to replace indole in the structure, which resulted in the dye having a longer absorption wavelength, a larger molar extinction coefficient and a lower fluorescence quantum yield.

[0020] (2) Rapid and sensitive response with low detection limit

[0021] The PA imaging probe responds rapidly to superoxide anions, completing the response within 4 minutes; the in vitro photoacoustic intensity shows a 9.7-fold increase in intensity upon response to superoxide anions; the photoacoustic detection limit is 33.2 nM.

[0022] (3) In situ diagnosis of acetaminophen-induced liver injury and repair using responsive PA signals

[0023] The photoacoustic imaging probe of this invention has the ability to detect superoxide anions in vivo in real time and diagnose DILI via AIH-OTF; it has low cytotoxicity, good biocompatibility, and can be used for imaging the occurrence and repair of DILI in vivo.

[0024] The specific embodiments of the present invention will now be described in further detail with reference to the accompanying drawings. Attached Figure Description

[0025] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments and descriptions of the invention are used to explain the invention, but do not constitute an undue limitation of the invention. Obviously, the drawings described below are merely some embodiments, and those skilled in the art can obtain other drawings based on these drawings without creative effort. In the drawings:

[0026] Figure 1 The 1H NMR spectrum of AIH-OH, a hemicyanine dye in dimethyl sulfoxide-d6 (DMSO-d6), is shown.

[0027] Figure 2 The 13C NMR spectrum of AIH-OH, a hemicyanine dye in dimethyl sulfoxide-d6 (DMSO-d6);

[0028] Figure 3 ESI-MS spectrum of the hemicyanine dye AIH-OH;

[0029] Figure 4 The 1H NMR spectrum of AIH-OTF, a hemicyanine dye in dimethyl sulfoxide-d6 (DMSO-d6);

[0030] Figure 5 The 13C NMR spectrum of AIH-OTF, a hemicyanine dye in dimethyl sulfoxide-d6 (DMSO-d6);

[0031] Figure 6 ESI-MS spectrum of AIH-OTF, a hemicyanine dye;

[0032] Figure 7 The present invention includes the ultraviolet absorption spectrum and PA spectrum of the probe AIH-OTF with superoxide anion, liquid chromatograms of different reaction systems, PA spectra of the probe with different concentrations of KO2, and PA spectra of the probe with different related substances.

[0033] Figure 8 This is a PA image of the probe AIH-OTF of this invention in HepG2 cells under stimulation with interferon and LPS.

[0034] Figure 9 Images of HepG2 cells treated with the probe AIH-OTF of this invention under APAP induction and corresponding PA images.

[0035] Figure 10 Representative PA images of DILI mice at different time points after intraperitoneal injection of AIH-OTF in in vivo experiments, representative PA images of DILI mice pretreated with NAC, and hematoxylin and eosin (H&E) staining images of treated liver tissue.

[0036] It should be noted that these accompanying drawings and textual descriptions are not intended to limit the scope of the invention in any way, but rather to illustrate the concept of the invention to those skilled in the art by referring to specific embodiments. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below with reference to the accompanying drawings. The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0038] Accurate diagnosis of drug-induced liver injury (DILI) is crucial for effective treatment and preventing further deterioration of liver damage. Molecular probes enabling high-contrast photoacoustic (PA) imaging of superoxide anion (O2·-) are valuable tools for accurate DILI diagnosis. However, existing O2·- PA probes exhibit relatively short absorption wavelengths and small redshifts, limiting their PA activation folds. Here, we constructed a novel O2·-activated 7-azaindole-derived probe (AIH-OTF) for in vivo PA imaging of DILI. The 7-azaindole-modified hemicyanine dye (AIH-OH) was used as the PA reporter gene for AIH-OTF due to its long absorption wavelength, high molar extinction coefficient, and low fluorescence quantum yield. Results showed that AIH-OTF exhibited an activated PA response to O2·-, with a large redshift and high signal-to-background ratio. In vitro assays also confirmed the rapid response, high sensitivity, and high selectivity of AIH-OTF for O2·- detection. Cellular studies have demonstrated that AIH-OTF can achieve high-contrast PA imaging of dynamic changes in endogenous O2·- in live cells. AIH-OTF has been successfully applied to detect acetaminophen-induced liver injury and to assess the therapeutic efficacy of hepatoprotective drugs through its PA response to O2·-. The AIH-OTF probe of this invention may serve as a useful imaging tool for the diagnosis and pathogenesis research of DILI.

[0039] Example 1

[0040] Preparation process of the semi-cyanine dye AIH-OH, refer to route 1.

[0041]

[0042] Route 1

[0043] The specific steps are as follows:

[0044] 1.05 g (4 mmol) of 7-chloro-6-hydroxy-2,3-dihydro1H-oxanthracene-4-carboxaldehyde was added to a solution of 2,3,3,7-tetramethyl-3H-pyrrolo[2,3-b]pyridine-7-onium (0.88 g (5 mmol)) in anhydrous EtOH containing a catalytic amount of piperidine. After 12 hours at 100 °C, a deep blue solution was obtained. The solvent was removed by rotary evaporation, and the residue was purified by column chromatography using DCM / MeOH (20 / 1, v / v) as the eluent to give a blue solid, AIH-OH, in 38% yield.

[0045] The semi-cyanine dye AIH-OH prepared in this embodiment was tested, and the results are as follows: Figures 1 to 3 As shown, they are respectively: Figure 1 AIH-OH is a hemicyanine dye in dimethyl sulfoxide-d6 (DMSO-d6). 1 HNMR spectrum; Figure 2 For AIH-OH in CD3OD 13 C NMR spectrum; Figure 3 The image shows the ESI-MS spectrum of the semi-cyanine dye AIH-OH.

[0046] in, Figure 1 The hydrogen spectrum was analyzed as follows 1 H NMR(400MHz,DMSO-d6)δ(ppm):8.58-8.61(1H,m,Ar-H),8.38-8.40(1H,m,Ar-H),8 .24-8.25(1H,m,Ar-H),7.92-7.93(1H,m,Ar-H),7.42(1H,s,Ar-H),6.97(1H,s,Ar -H),6.83(1H,s,=CH),6.47(1H,d,J=14.7Hz,=CH),6.02(1H,d,J=11.8Hz,=CH),4. 24(3H,s,CH3),2.51-2.57(4H,m,CH2),1.67-1.77(2H,m,CH2),1.41(6H,s,2×CH3).

[0047] Figure 2 The carbon spectrum analysis is as follows: 13C NMR(100MHz,DMSO-d6)δ(ppm):162.19,156.85,152.92,146.52,144.28,142.86,138.52,135.08,130.73,127.63, 121.31,119.42,118.97,114.50,112.26,109.37,101.57,98.91,95.19,53.10,28.60,23.12,20.37,19.60,9.13.

[0048] Figure 3 The ESI-MS mass spectrometry analysis (anion mode in water) showed that the theoretical molecular weight of AIH-OH was 419.15; the actual measured molecular weight was 419.24.

[0049] The above results prove that this embodiment has successfully synthesized the semi-cyanine dye AIH-OH, whose structural formula is:

[0050]

[0051] Example 2

[0052] Fabrication process of the dual-mode imaging probe AIH-OTF, refer to route 2.

[0053]

[0054] Route 2

[0055] The specific steps are as follows:

[0056] Trifluoromethanesulfonic anhydride (0.14 g, 0.5 mmol) was slowly added to a solution of AlH-OH (0.11 g, 0.25 mmol) and triethylamine (0.13 mL, 1 mmol) in CH2Cl2 (5.0 mL) in an ice bath, and the solution was stirred at 30 °C for 2 h. After washing with saturated NaHCO3 solution and brine, the solution was concentrated under vacuum. Purification was performed by column chromatography using MeOH / CH2Cl2 (1 / 60, v / v) as the eluent to obtain a blue-purple solid product, named AIH-OTF, in 79% yield.

[0057] The probe AIH-OTF prepared in this embodiment was detected, and the results are as follows: Figures 4 to 6 As shown, they are respectively: Figure 4 For the probe AIH-OTF in dimethyl sulfoxide-d6 (DMSO-d6) 1 H NMR spectrum; Figure 5 For the probe AIH-OTF in dimethyl sulfoxide-d6 (DMSO-d6) 13C NMR spectrum; Figure 6 The image shows the ESI-MS spectrum of the probe AIH-OTF.

[0058] in, Figure 4 The hydrogen spectrum was analyzed as follows 1 HNMR(400MHz,DMSO-d6)δ(ppm):8.65-8.69(1H,m,Ar-H),8.45-8.47(1H,m,Ar-H) ,8.30-8.32(1H,m,Ar-H),7.49(1H,s,Ar-H),7.05(1H,s,Ar-H),6.91(1H,s,Ar-H) ,6.54(1H,d,J=14.7Hz,=CH),5.86(1H,s,=CH),5.32(1H,t,J=4.7Hz,=CH),4.30( 3H,s,CH3),2.58-2.63(4H,m,2×CH2),1.75-1.80(2H,m,CH2),1.47(6H,s,2×CH3).

[0059] Figure 5 The carbon spectrum was analyzed as follows 13 C NMR (100MHz, DMSO-d6) δ (ppm): 162.56, 156.64, 152.92, 150.88, 148.98, 144.07, 141.03, 140.58, 139.89, 139.48, 135. 28,127.81,119.75,119.55,115.03,112.74,111.25,103.37,100.15,94.92,53.02,29.03,24.52,22.86,20.64,9.02.

[0060] Figure 6 The ESI-MS mass spectrometry analysis (anion mode in water) showed that the theoretical molecular weight of AIH-OTF was 551.10; the actual molecular weight was 551.15.

[0061] The above results prove that the probe AIH-OTF has been successfully synthesized in this embodiment, and its structural formula is as follows:

[0062]

[0063] Example 3

[0064] Performance analysis of the photoacoustic probe AIH-OTF in in vitro detection of superoxide anions:

[0065] (1) Investigate the changes in ultraviolet absorption of the prepared probe AIH-OTF in response to superoxide anions;

[0066] To investigate the UV absorption response of the probe AIH-OTF to superoxide anion, AIH-OTF (10 μM) with or without KO2 (50 μM) was rapidly mixed in PBS / DMSO solution (10 mM, pH 7.4, v / v, 1 / 1). Its UV-Vis spectral response was measured, and the results are as follows: Figure 7 As shown in Figure a, it can be observed that after incubation with superoxide anions, the absorption peak of the AIH-OTF probe at 532 nm disappears, while a new absorption peak appears at 730 nm. This change demonstrates that AIH-OTF can produce a significant response to superoxide anions.

[0067] (2) Investigate the photoacoustic intensity changes in response to superoxide anions by the prepared probe AIH-OTF;

[0068] To investigate the effect of the probe AIH-OTF on photoacoustic intensity changes, AIH-OTF (10 μM) with or without KO2 (50 μM) was rapidly mixed in PBS / DMSO solution (10 mM, pH 7.4, v / v, 1 / 1). Its photoacoustic properties were measured, and the results are as follows: Figure 7 As shown in Figure B, the probe exhibits maximum photoacoustic intensity at 740 nm after incubation with superoxide anions. This change demonstrates that AIH-OTF can produce a significant response to superoxide anions.

[0069] (3) Investigate and verify the reaction mechanism of the prepared probe AIH-OTF to superoxide anions;

[0070] To investigate the reaction mechanism of AIH-OTF with superoxide anion, HPLC chromatograms of the products from the reaction of AIH-OTF (10 μM), AIH-OH (10 μM), and AIH-OTF (10 μM) with KO2 (20 μM) at 37 °C for 10 min were obtained and analyzed on a system equipped with a C18 column (250 nm × 4.6 mm, 5 μM) under the following conditions: methanol / H2O = 90 / 10 (v / v); flow rate: 1.0 mL / min; detection wavelength: 650 nm. To further demonstrate the reaction mechanism, ESI-MS was used to analyze the products of the reaction of AIH-OTF (10 μM) with KO2 (20 μM) at 37 °C in positive mode for 10 min. The results are as follows: Figure 7 As shown in c, these spectral changes in the absorption, fluorescence, and photoacoustic properties of AIH-OTF to KO2 can be attributed to the oxidation of AIH-OH to the trifluoromethanesulfonate group by O2. ·- Release during nucleophilic attack. Two peaks with retention times of 6.71 min and 8.66 min were observed in the HPLC spectrum of the reaction product, corresponding to AIH-OH (6.68 min) and AIH-OTF (8.65 min), respectively. These results strongly validate the proposed reaction mechanism.

[0071] (4) Investigate the ability of the prepared probe AIH-OTF to exhibit real-time fluorescence response to superoxide anions;

[0072] To investigate the response kinetics of AIH-OTF to O2·-, AIH-OTF (10 μM) and KO2 (0–100 μM) were rapidly mixed in PBS / DMSO solution (10 mM, pH 7.4, v / v, 1 / 1). Photoacoustic (PA) intensity (PA740 intensity) was then recorded to verify the feasibility of quantitative analysis of superoxide anions via PA mode using AIH-OTF. For in vitro photoacoustic (PA) detection, wells (200 μL) filled with AIH-OTF and analyte were imaged using an MSOT imaging system, scanned from 680 nm to 850 nm at 10 nm intervals. Wells filled with PBS were used as a control for background inference. All MSOT data were acquired using an MSOT imaging system with the following parameters: pulse frequency (10 Hz), pulse length (8 ns), and maximum optical parameter oscillator energy (120 mJ at 740 nm). Light was transmitted to the sample via a loop fiber bundle. The generated acoustic signals were collected using a transducer array of 256 elements. The resulting photoacoustic image is shown below. Figure 7 As shown in d. Plotting the superoxide anion concentration on the x-axis and the corresponding PA intensity at 740 nm on the y-axis, a linear PA spectrum for the AIH-OTF probe in detecting sulfatase was obtained. The results are shown in d. Figure 7 As shown in e.

[0073] like Figure 7 As shown in d and 7e, the PA740 intensity also gradually increases with increasing KO2 concentration, and a robust linear correlation is achieved in the concentration range of 2.5–40 μM. The calculated detection limit is 33.2 nM, indicating that O2 detection via PA mode is feasible. ·- It exhibits excellent sensitivity. The good sensitivity and high PA contrast are mainly attributed to the incorporation of 7-azaindole-modified hemicyanine as a reporter molecule, which benefits the probe's response to O2. ·- The optical response. These findings indicate that AIH-OTF can detect O2 in PA mode. ·- .

[0074] (5) Investigate the selectivity of the prepared probe AIH-OTF for superoxide anions;

[0075] To explore the selectivity of the probe AIH-OTF, AIH-OTF (10 μM) was first mixed with various biologically relevant substances in PBS / DMSO solution (10 mM, pH 7.4, v / v, 1 / 1), including sodium sulfite (1 mM), sodium nitrate (1 mM), sodium hydrogen phosphate (1 mM), potassium chloride (1 mM), zinc chloride (1 mM), ferric chloride (1 mM), magnesium chloride (1 mM), sodium sulfide (1 mM), hydrogen peroxide (0.1 mM), hypochlorous acid (0.1 mM), pernitrite (0.1 mM), nitrate (0.1 mM), hydroxyl radical (0.1 mM), GSH (0.1 mM), and potassium superoxide (50 μM). The mixture was then incubated at 37 °C for 10 min. Photoacoustic imaging was performed using the Vision256-TF MSOT imaging system. Using different target analytes as the x-axis and the corresponding PA intensity at 740 nm as the y-axis, a photoacoustic selectivity map for probe detection of superoxide anions was obtained, as shown below. Figure 7 As shown in f.

[0076] from Figure 7 As can be seen from f, when AIH-OTF was incubated with potential interfering substances (including ROS, RNS, biothiols, and anions), the PA740 intensity did not increase significantly, while AIH-OTF treated with KO2 showed a significant increase in PA740 intensity, indicating that AIH-OTF is resistant to O2. ·- High specificity.

[0077] Example 4

[0078] The performance of the prepared probe AIH-OTF in PA imaging with superoxide anions at the cellular level was investigated.

[0079] (1) Evaluate the effect of the prepared probe AIH-OTF on endogenous O2. ·- PA imaging capability;

[0080] HepG2 cells were seeded in 5 ml of medium containing 10% FBS and 1% penicillin / streptomycin on 10 cm culture dishes at 37°C for 24 hours. The probe concentration for cell imaging was 20 μM. Three experimental groups were designed to investigate the effect of AIH-OTF on endogenous O2. ·-PA imaging capability was assessed. The first group of cells was incubated with AIH-OTF at 37°C for 1 hour. The second group of HepG2 cells was pretreated with lipopolysaccharide (LPS, 2 μg / mL) and interferon-γ (INF-γ, 100 ng / mL) for 12 hours, and then incubated with AIH-OTF for 1 hour. Before incubation with AIH-OTF, the third group of HepG2 cells was pretreated with LPS (2 μg / mL) and INF-γ (100 ng / mL) in the presence of TEMPO (500 μM) for 12 hours. Before PA imaging, cells were washed three times with fresh PBS buffer (pH 7.4, 10 mM) and then centrifuged to obtain cell pellet. All cellular PA data were acquired using an InVision 256-TF imaging system (iTheraMedical GmbH) with an excitation range of 680 nm to 850 nm, a pulse length of 8 ns, and a pulse frequency of 10 Hz.

[0081] like Figure 8 As shown, HepG2 cells treated with AIH-OTF alone exhibited a very weak PA740 signal. A strong PA740 intensity was observed when cells were treated with lipopolysaccharide (LPS) and interferon-γ (INF-γ) followed by incubation with AIH-OTF. LPS / INF-γ-induced O2 was scavenged using 2,2,6,6-tetramethylpiperidin-n-oxygen (TEMPO). ·- Subsequently, cells exhibited a significantly reduced PA740 intensity. Relative spectral changes showed that HepG2 cells treated with LPS / INF-γ exhibited an 8.6-fold increase in PA740 intensity compared to cells incubated with the probe alone, while the scavenger caused a significant decrease in PA740 intensity, demonstrating that AIH-OTF can be used for endogenous O2 in living cells. ·- High-contrast and selective PA imaging.

[0082] (2) Evaluation of O2 during the detection of cellular hepatotoxicity using the prepared probe AIH-OTF ·- Dynamic changes;

[0083] HepG2 cells were seeded in 5 ml of medium containing 10% FBS and 1% penicillin / streptomycin on 10 cm culture dishes at 37°C for 24 hours. The probe concentration for cell imaging was 20 μM. Three experimental groups were designed to investigate O2 during cellular hepatotoxicity detection using AIH-OTF. ·-The dynamic changes of cells were observed. The first group of cells was incubated with AIH-OTF at 37°C for 1 hour. The second group of HepG2 cells was pretreated with APAP (200 μM) for 12 hours and then incubated with AIH-OTF for 1 hour. The third group of HepG2 cells was pretreated with APAP (500 μM) for 12 hours and then incubated with AIH-OTF for 1 hour. The fourth group of HepG2 cells was pretreated with APAP (500 μM) and NAC (300 μM) for 12 hours and then incubated with AIH-OTF for 1 hour. Before PA imaging, cells were washed three times with fresh PBS buffer (pH 7.4, 10 mM) and then centrifuged to obtain cell pellet. All cell PA data were acquired using an InVision 256-TF imaging system (iThera Medical GmbH) with an excitation range of 680 nm to 850 nm, a pulse length of 8 ns, and a pulse frequency of 10 Hz.

[0084] HepG2 cells treated with 200 μM and 500 μM MAPAP, followed by incubation with AIH-OTF, showed significantly increased PA signaling. Figure 9 This demonstrates that APAP can induce O2 in cells. ·- The production of [unclear - likely referring to a specific type of protein]. In contrast, incubation of cells treated with N-acetylcysteine ​​(NAC) and APAP resulted in a significant attenuation of PA signaling, attributed to NAC mitigating APAP-induced oxidative stress. These results suggest that AIH-OTF can be used for detecting O2 in living cells. ·- The dynamic changes provide a PA platform.

[0085] Example 5

[0086] The prepared probe AIH-OTF was investigated for use in in vivo detection of O2. ·- PA imaging performance:

[0087] (1) Evaluation of the prepared probe AIH-OTF for in vivo detection of O2. ·- PA imaging capability;

[0088] The probe concentration used for in vivo PA imaging was 100 μM (50 μL). Mice (28–30 g) were randomly divided into two groups of three mice each. Control group mice received intraperitoneal injection of AIH-OTF. For the sample group, mice were treated with APAP (500 mg / kg) for 6 hours, followed by intraperitoneal injection of AIH-OTF. Mice were anesthetized with 1% isoflurane in oxygen for PA imaging. PA images were acquired using an InVision 256-TF imaging system. Each mouse was placed in a prone position and in a 35°C water bath. Data were acquired at 10 nm intervals in the wavelength range of 680 nm to 850 nm, and the entire liver was scanned at 0.3 mm intervals for each wavelength. In vivo PA images were also acquired before injection (0 h) and at different time points (0.5, 1, 2, 3, 6 h) after intraperitoneal injection of the AIH-OTF probe (100 μM, 50 μL). Figure 10 As shown in a and 10b, the PA signal in the liver region gradually increased over time and reached its peak approximately 3 hours after intraperitoneal injection of AIH-OTF.

[0089] (2) Evaluate the ability of the prepared probe AIH-OTF to diagnose DILI at the in vivo level;

[0090] To explore the diagnostic ability of AIH-OTF for DILI, the probe concentration for in vivo PA imaging was 100 μM (50 μL). Mice (28–30 g) were randomly divided into three groups, with three mice in each group. Control group mice received intraperitoneal injection of AIH-OTF. For the sample group, mice were treated with APAP (500 mg / kg) for 6 hours, followed by intraperitoneal injection of AIH-OTF. The third group of mice were pretreated with NAC (400 mg / kg) for 2 hours, then incubated with APAP (500 mg / kg) for 6 hours, followed by intraperitoneal injection of AIH-OTF.

[0091] like Figure 10 At days c and 10, compared with the normal group, the DILI group injected with AIH-OTF showed significant enhancement of PA signal in the liver region. Quantitative data showed that the PA740 intensity of the AIH-OTF-treated DILI group was 9.3 times that of the AIH-OTF-treated normal group, indicating high-contrast PA imaging of DILI in vivo. Conversely, the PA signal of the DILI group after NAC pretreatment was significantly attenuated to trigger the antioxidant defense of DILI, indicating that the PA response of AIH-OTF is related to the degree of oxidative stress in DILI. In addition, H&E staining experiments were performed to examine the morphology of liver tissue to evaluate the reliability of AIH-OTF for the diagnosis of DILI.

[0092] Furthermore, the degree of liver injury was assessed through tissue staining. Hematoxylin and eosin (H&E) staining analysis of major organs was performed. Representative histological features of major organs (heart, liver, spleen, lung, and kidney) were obtained from excised organs from normal mice or DILI mice. Excised organs and tumor tissues were fixed in 4% formaldehyde solution, embedded in paraffin, and cut into 5 μm thick sections. Sections were dewaxed, water-retained, and stained sequentially with hematoxylin and eosin. Sections were rinsed with water and dehydrated for imaging acquisition. Fluorescence images were collected using a fluorescence microscope at 400x magnification.

[0093] like Figure 10 As shown in Figure e, the liver region of the APAP group exhibited significant sinusoidal congestion and hemorrhage, as well as extensive hepatocyte steatosis and dense vacuolation in the cytoplasm. Furthermore, hepatocyte necrosis with partial nuclear loss, mild inflammatory infiltration, and single inflammatory lesions were also observed. These symptoms confirmed the occurrence of DILI. As expected, the aforementioned symptoms in the liver region of the DILI group were eliminated under NAC pretreatment, indicating that hepatoprotective drugs have a good therapeutic effect on liver injury. In conclusion, these results demonstrate that AIH-OTF can be used to image the occurrence and repair of DILI in vivo.

[0094] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A 7-azaindole photoacoustic imaging probe for detecting superoxide anion, characterized in that, The photoacoustic imaging probe is named AIH-OTF, and the chemical structural formula is shown as formula I. ； Formula I.

2. The use of the 7-azaindole photoacoustic imaging probe for detecting superoxide anion in the preparation of a probe for diagnosing DILI at a living body level.

3. The method for preparing the 7-azaindole photoacoustic imaging probe for detecting superoxide anions as described in claim 1, characterized in that, The preparation process is as follows: After mixing 2 equivalent amounts of methyl triflate, 1 equivalent amount of dye AIH-OH, anhydrous dichloromethane and 4 equivalent amounts of triethylamine, stirring at 30℃ for 2h, vacuum concentration, using dichloromethane and methanol as eluent, the obtained residue is purified by column chromatography to obtain the product as a blue-green solid, named AIH-OTF; wherein the chemical structural formula of AIH-OH is shown as formula II. ； Formula II.

4. The production method according to claim 3, characterized by, The preparation method of the dye AIH-OH is as follows: Dissolve 1 equivalent amount of 7-chloro-6-hydroxy-2,3-dihydro-1H-xanthene-4-carboxaldehyde in anhydrous ethanol containing 1 equivalent amount of piperidine, then mix the solution with 1.25 equivalent amounts of 2,3,3,7-tetramethyl-3H-pyrrolo[2,3-b]pyridine-7-ium, then stir at 80℃ for 12h to obtain a dark blue solution; remove the solvent by rotary evaporation, and purify the obtained residue by column chromatography to obtain the product as a blue solid, named AIH-OH.

5. The preparation method according to claim 3, characterized in that, The molar ratio of the dye AIH-OH to methyl triflate is 1:

2.

6. The preparation method according to claim 3, characterized in that, The volume ratio of dichloromethane to methanol in the eluent is 10:1.

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