7-azaindole photoacoustic imaging probe for detecting superoxide anions as well as preparation method and application of 7-azaindole photoacoustic imaging probe

Abstract

The invention discloses a 7-azaindole photoacoustic imaging probe for detecting superoxide anions, a preparation method and application, the photoacoustic imaging probe is named as AIH-OTF and 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 anions of living cells, in-situ evaluation of occurrence and repair of acetaminophen-induced liver injury is also determined by non-invasive monitoring of PA reaction of the AIH-OTF to superoxide anions, and the AIH-OTF probe can be used as a useful tool for accurately diagnosing drug-induced liver injury in vivo. The photoacoustic imaging probe disclosed by the invention can be used for detecting superoxide anions in vivo and diagnosing the DILI capability in real time through AIH-OTF; the cell toxicity is small, the biocompatibility is good, and the compound can be used for imaging of in-vivo DILI generation and repair.

Description

Technical Field

[0001] The invention belongs to the technical field of imaging probe detection, and in particular relates to a 7-azaindole photoacoustic imaging probe for detecting superoxide anions, a preparation method and an application thereof. Background Art

[0002] Drug-induced liver injury (DILI) is caused by the direct toxic effects of drugs and their metabolites on the liver, or by the specific response of the individual body to drugs. It has become the third most common liver disease with a lower incidence than viral hepatitis and fatty liver disease. DILI is irreparable and can further develop into liver failure or liver cancer. Due to the nonspecificity, concealment and severity of DILI in clinical practice, accurate diagnosis and timely treatment remain a great challenge. Recent studies have shown that intracellular oxidative and nitrosative stress are closely related to the degree of DILI. The metabolism of acetaminophen is the main cause of DILI, which can induce the production of superoxide anions in the liver. Superoxide anions are considered to be one of the most important reactive oxygen species and can be converted into other reactive oxygen species, including hydroxyl radicals, hypochlorous acid and hydrogen peroxide, by superoxide dismutase, leading to the destruction of cellular redox balance and damage to liver cells. On the other hand, as the main source of reactive nitrogen species, it can also react with endogenous nitric oxide to produce peroxynitrite in hepatocytes. The interaction of excessive peroxynitrite with biomacromolecules such as proteins, nucleic acids, and lipids can lead to dysfunction of cell physiology and structure, further exacerbating liver damage. Therefore, superoxide anion has become an important biomarker in the progression of drug-induced liver injury, and the establishment of an effective superoxide anion detection method is very important for the accurate diagnosis of drug-induced liver injury.

[0003] Photoacoustic (PA) imaging is a powerful biomedical imaging modality that enables noninvasive visualization of biological processes at the molecular and cellular levels in deep tissues with high spatial resolution. Using the near-infrared (NIR) operating window, PA provides 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 red shifts, and high activation multiples for the detection of superoxide anions is of great value for the diagnosis of drug-induced liver injury. Summary of the invention

[0004] The technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and provide a 7-azaindole photoacoustic imaging probe for detecting superoxide anions, a preparation method and an application thereof, so as to solve the detection problem of the photoacoustic probe with a long absorption wavelength, a large red shift and a high activation multiple for detecting superoxide anions for the diagnosis of drug-induced liver injury.

[0005] In order to solve the above technical problems, the basic concept of the technical solution adopted by the present invention is:

[0006] In one aspect, the present invention provides a 7-azaindole photoacoustic imaging probe for detecting superoxide anions. The photoacoustic imaging probe is named AIH-OTF, and its chemical structure is shown in Formula I:

[0007]

[0008] Formula Ⅰ.

[0009] Furthermore, the

[0010] Another aspect of the present invention protects the application of the above-mentioned photoacoustic imaging probe, which can be used for quantitative detection in vitro and can realize the ability to diagnose DILI at the in vivo level.

[0011] On the other hand, the present 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 the mixture is stirred at 30° C. for reaction for 2 hours; vacuum concentration is performed, and dichloromethane and methanol are used as eluents to purify the obtained residue by column chromatography to obtain a blue-green solid product, which is named AIH-OTF.

[0012] Furthermore, the preparation method of the dye AIH-OH is as follows: 7-chloro-6-hydroxy-2,3-dihydro-1H-xanthene-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]pyridin-7-ium is added, and the mixture is stirred at 100° C. for 12 hours to obtain a dark blue solution. The solvent is removed by rotary evaporation, and the obtained residue is purified by column chromatography to obtain a blue solid product named AIH-OH, whose chemical structure 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 the present invention is composed of a triflate moiety as a recognition unit and a semi-cyanine dye (AIH-OH) as a photoacoustic signal group. Since the esterification of the phenolic hydroxyl group blocks the intramolecular charge transfer effect of AIH-OH, AIH-OTF shows negligible absorption in the wavelength range of 680nm to 950nm. After the triflate group is nucleophilically attacked by superoxide anions, AIH-OTF is converted to AIH-OH, restoring the intramolecular charge transfer effect and generating a large Δλ (203nm), resulting in an activated PA response with high contrast (9.7 times). The results show that AIH-OTF achieves high-contrast PA imaging of endogenous superoxide anions in living cells. More importantly, the in situ assessment of the occurrence and repair of acetaminophen-induced liver damage is also determined by non-invasive monitoring of the PA response of AIH-OTF to superoxide anions. The AIH-OTF probe of the present invention can be used as a useful tool for accurate diagnosis of drug-induced liver injury in vivo.

[0018] After adopting the above technical scheme, 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 into the structure instead of indole, which makes the dye have a longer absorption wavelength, a larger molar extinction coefficient and a lower fluorescence quantum yield.

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

[0021] The PA imaging probe responds rapidly to superoxide anions, completing the response within 4 minutes; the in vitro photoacoustic intensity increases by 9.7 times when responding to superoxide anions; the photoacoustic detection limit is 33.2nM.

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

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

[0024] The specific implementation modes of the present invention are further described in detail below in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The accompanying drawings are part of this application and are used to provide a further understanding of the present invention. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention, but do not constitute an improper limitation of the present invention. Obviously, the drawings described below are only some embodiments. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work. In the drawings:

[0026] Figure 1 1HNMR spectrum of hemicyanine-like dye AIH-OH in dimethyl sulfoxide-d6 (DMSO-d6);

[0027] Figure 2 is the 13C NMR spectrum of the hemicyanine-like dye AIH-OH in dimethyl sulfoxide-d6 (DMSO-d6);

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

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

[0030] Figure 5 is the 13C NMR spectrum of the hemicyanine-like dye AIH-OTF in dimethyl sulfoxide-d6 (DMSO-d6);

[0031] Figure 6 This is the ESI-MS spectrum of the hemicyanine-like dye AIH-OTF;

[0032] Figure 7 The invention discloses ultraviolet absorption spectra and PA spectra of the probe AIH-OTF and superoxide anions, liquid chromatograms of different reaction systems, PA spectra of the probe to different concentrations of KO2, and PA spectra of the probe to different related substances.

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

[0034] Fig. 9 These are the images of HepG2 cells treated with the probe AIH-OTF of the present invention under APAP induction and the corresponding PA images.

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

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

[0037] In order to make the purpose, 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 in conjunction with the drawings in the embodiments of the present invention. The following embodiments are used to illustrate the present invention but are not used to limit the scope of the present invention.

[0038] Accurate diagnosis of drug-induced liver injury (DILI) plays a crucial role in effective treatment and avoiding the deterioration of liver injury. Molecular probes that achieve high-contrast photoacoustic (PA) imaging of superoxide anions (O2·-) are valuable tools for accurate diagnosis of DILI. However, existing PA probes for O2·- show relatively short absorption wavelengths and small red shifts, limiting their PA activation multiples. Here, we constructed a new O2·-activatable 7-azaindole-derived probe (AIH-OTF) for PA imaging of DILI in vivo. 7-azaindole-modified hemicyanine dye (AIH-OH) was used as a PA reporter for AIH-OTF due to its long absorption wavelength, large molar extinction coefficient, and low fluorescence quantum yield. The results showed that AIH-OTF exhibited an activated PA response to O2·- with a large red shift and high signal-to-background ratio. In vitro assays also confirmed the fast response speed, high sensitivity, and high selectivity of AIH-OTF for O2·- detection. Cell studies have shown that AIH-OTF can achieve high-contrast PA imaging of dynamic changes in endogenous O2·- in living cells. AIH-OTF has been successfully applied to detect the occurrence of acetaminophen-induced liver damage and to evaluate the therapeutic effects of liver-protective drugs through its PA response to O2·-. The AIH-OTF probe of the present invention may be a useful imaging tool for DILI diagnosis and pathogenesis research.

[0039] Example 1

[0040] Preparation process of hemicyanine dye AIH-OH, refer to route 1,

[0041]

[0042] Route 1

[0043] The specific steps are as follows:

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

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

[0046] in, Figure 1 The hydrogen spectrum of 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 of is: 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 spectrum analysis (negative ion mode in water) is: the theoretical molecular weight of AIH-OH is 419.15; the actual detection: the molecular weight is 419.24.

[0049] The above results prove that the present embodiment has successfully synthesized a hemicyanine-like dye AIH-OH, whose structural formula is:

[0050]

[0051] Example 2

[0052] The preparation process of dual-mode imaging probe AIH-OTF, refer to route 2,

[0053]

[0054] Route 2

[0055] The specific steps are as follows:

[0056] In an ice bath, 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), and the solution was stirred at 30 ° C for 2 h. After washing with saturated NaHCO3 solution and brine, the solution was concentrated in vacuo. MeOH / CH2Cl2 (1 / 60, v / v) was used as eluent to purify by column chromatography to obtain a blue-purple solid product named AIH-OTF with a yield of 79%.

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

[0058] in, Figure 4 The hydrogen spectrum of 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 of 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 spectrum analysis (negative ion mode in water) is: the theoretical molecular weight of AIH-OTF is 551.10; the actual detection: the molecular weight is 551.15.

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

[0062]

[0063] Example 3

[0064] Analysis of the performance of photoacoustic probe AIH-OTF in detecting superoxide anions in vitro:

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

[0066] In order to study the ultraviolet absorption change response of the probe AIH-OTF to superoxide anions, AIH-OTF (10 μM) without or with KO2 (50 μM) was quickly mixed in PBS / DMSO solution (10 mM, pH 7.4, v / v, 1 / 1). The UV-visible spectral response was measured, and the results are shown in Figure 2. Figure 7 As shown in Figure a, it can be found that after the probe is incubated with superoxide anions, the absorption peak of the probe AIH-OTF at 532nm disappears, and a new absorption peak appears at 730nm. This change proves that AIH-OTF can produce a significant response to superoxide anions.

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

[0068] In order to study the change of the photoacoustic intensity of the probe AIH-OTF, AIH-OTF (10 μM) without or with KO2 (50 μM) was quickly mixed in PBS / DMSO solution (10 mM, pH 7.4, v / v, 1 / 1). The photoacoustic performance was measured, and the results are shown in Figure 2. Figure 7 As shown in Figure B, it can be found that after the probe is incubated with superoxide anions, the maximum photoacoustic intensity appears at 740 nm. This change proves 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] In order to study the reaction mechanism of AIH-OTF to superoxide anion, HPLC chromatograms of AIH-OTF (10 μM), AIH-OH (10 μM) and the product of AIH-OTF (10 μM) reacting with KO2 (20 μM) at 37°C for 10 minutes were performed on a system 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. In order to further prove the reaction mechanism, ESI-MS was introduced to analyze the product of AIH-OTF (10 μM) reacting with KO2 (20 μM) at 37°C in positive mode for 10 minutes. The results are shown in Figure 2. Figure 7 c. These spectral changes in the absorption, fluorescence, and photoacoustic properties of AIH-OTF on KO2 can be attributed to the removal of O2 on the triflate group of AIH-OH. ·- Release during nucleophilic attack. In the HPLC spectrum of the reaction product, there are two peaks with retention times of 6.71 minutes and 8.66 minutes, corresponding to AIH-OH (6.68 minutes) and AIH-OTF (8.65 minutes), respectively. The above results well verify the proposed reaction mechanism.

[0071] (4) Investigating the ability of the prepared probe AIH-OTF to respond to superoxide anions in real-time fluorescence;

[0072] To investigate the response kinetics of AIH-OTF to O2·-, AIH-OTF (10 μM) was rapidly mixed with KO2 (0-100 μM) in a PBS / DMSO solution (10 mM, pH 7.4, v / v, 1 / 1). The photoacoustic (PA) intensity (PA740 intensity) was then recorded to verify the feasibility of AIH-OTF for quantitative analysis of superoxide anions by PA mode. For in vitro photoacoustic (PA) detection, wells (200 μL) filled with AIH-OTF and analytes were subjected to PA imaging with an MSOT imaging system, scanning from 680 nm to 850 nm at 10 nm intervals. Wells filled with PBS were used as controls for background inference. All MSOT data were acquired with 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 delivered to the sample via a ring-shaped fiber bundle. The acoustic signal generated was collected using a 256-element transducer array. The resulting photoacoustic image is shown in the figure below. Figure 7 With the concentration of superoxide anion as the horizontal axis and the corresponding PA intensity at 740nm as the vertical axis, the PA linear spectrum of the probe AIH-OTF detecting sulfatase was obtained. Figure 7 As shown in e.

[0073] like Figure 7 As shown in Figures 7d and 7e, the PA740 intensity also gradually increased with the increase of KO2 concentration, and a robust linear correlation was achieved in the concentration range of 2.5-40 μM. The detection limit was calculated to be 33.2 nM, indicating that the PA mode can detect O2 ·- The good sensitivity and high PA contrast are mainly attributed to the incorporation of 7-azaindole-modified hemicyanine as a reporter molecule, which is beneficial for the probe to O2 ·- These findings indicate that AIH-OTF is capable of detecting 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 different 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), peroxynitrous acid (0.1 mM), nitrate (0.1 mM), hydroxyl radical (0.1 mM), hydroxyl radical (0.1 mM), GSH (0.1 mM), potassium superoxide (50 μM). Then, the mixture was incubated at 37 °C for 10 min. Photoacoustic imaging was performed in a Vision256-TF MSOT imaging system. With different targets as the horizontal axis and the corresponding PA intensity at 740nm as the vertical axis, the photoacoustic selectivity diagram of the probe for detecting superoxide anions was obtained. The results are as follows: Figure 7 f.

[0076] from Figure 7 f It can be seen that 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 has a significant effect on O2 ·- High specificity.

[0077] Example 4

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

[0079] (1) Evaluation of the prepared probe AIH-OTF for endogenous O2 ·- PA imaging capabilities;

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

[0081] like Figure 8 As shown, HepG2 cells treated with AIH-OTF alone showed very weak PA740 signals. When cells were treated with lipopolysaccharide (LPS) and interferon-γ (INF-γ) and then incubated with AIH-OTF, strong PA740 intensity was observed. ·- The relative spectral changes showed that the PA740 intensity in HepG2 cells treated with LPS / INF-γ was enhanced 8.6-fold compared with cells incubated with the probe alone, while the scavenger caused a significant decrease in PA740 intensity, demonstrating that AIH-OTF can be used to detect endogenous O2 in living cells. ·- High contrast and selective PA imaging.

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

[0083] HepG2 cells were seeded on 10 cm dishes in 5 ml of medium containing 10% FBS and 1% penicillin / streptomycin at 37 °C for 24 h. The probe concentration used for cell imaging was 20 μM. Three experimental groups were designed to study the effect of AIH-OTF on the detection of cellular hepatotoxicity during O2 ·-Dynamic change ability. The first group of cells was incubated with AIH-OTF at 37°C for 1 hour. The second group of HepG2 cells were pretreated with APAP (200 μM) for 12 hours and incubated with AIH-OTF for 1 hour. The third group of HepG2 cells were pretreated with APAP (500 μM) for 12 hours and incubated with AIH-OTF for 1 hour. The fourth group of HepG2 cells were pretreated with APAP (500 μM) and NAC (300 μM) for 12 hours and incubated with AIH-OTF for 1 hour. Before PA imaging, the cells were washed three times with fresh PBS buffer (pH 7.4, 10 mM) and then centrifuged to obtain cell pellets. All cell PA data were collected with the 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 APAP and then incubated with AIH-OTF showed significantly increased PA signals ( Fig. 9 ), proving that APAP can induce O2 ·- In contrast, incubation of N-acetylcysteine ​​(NAC) with APAP-treated cells resulted in a significant attenuation of the PA signal, which was attributed to the alleviation of APAP-induced oxidative stress by NAC. These results suggest that AIH-OTF can be used to detect O2 in living cells. ·- Providing a PA platform for dynamic changes.

[0085] Example 5

[0086] Investigation of the prepared probe AIH-OTF for detecting O2 at the in vivo level ·- PA imaging performance:

[0087] (1) Evaluation of the prepared probe AIH-OTF for detecting O2 at the in vivo level ·- PA imaging capabilities;

[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, each including three mice. The mice in the control group were injected intraperitoneally with AIH-OTF. For the sample group, the mice were treated with APAP (500 mg / kg) for 6 hours and then injected intraperitoneally with AIH-OTF. The mice were anesthetized using 1% isoflurane in oxygen for PA imaging tests. PA images were obtained from the InVision 256-TF imaging system. Each mouse was placed in a prone position and placed in a 35°C water bath. Data were collected at intervals of 10 nm in wavelengths from 680 nm to 850 nm, and the entire liver was scanned at intervals of 0.3 mm with each wavelength. In vivo PA images were also collected before injection (0 hour) and at different time points (0.5, 1, 2, 3, 6 hours) after intraperitoneal injection of the AIH-OTF probe (100 μM, 50 μL). As Fig.10 As shown in a and 10b, the PA signal in the liver region gradually increased over time and reached a peak approximately 3 hours after intraperitoneal injection of AIH-OTF.

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

[0090] To explore the ability of AIH-OTF to diagnose DILI. The probe concentration used for in vivo PA imaging was 100 μM (50 μL). Mice (28-30 g) were randomly divided into three groups, each including three mice. The mice in the control group were injected intraperitoneally with AIH-OTF. For the sample group, the mice were treated with APAP (500 mg / kg) for 6 hours and then injected intraperitoneally with 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, and then injected intraperitoneally with AIH-OTF.

[0091] like Fig.10 c and 10d, the DILI group injected with AIH-OTF showed significant PA signal enhancement in the liver area compared with the normal group. Quantitative data showed that the PA740 intensity of the AIH-OTF-treated DILI group was measured to be 9.3 times that of the AIH-OTF-treated normal group, indicating high-contrast PA imaging of DILI in vivo. In contrast, the PA signal of the DILI group after NAC pretreatment was significantly weakened 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 DILI diagnosis.

[0092] Further, the degree of liver injury was assessed by tissue staining. Hematoxylin and eosin (H&E) staining analysis of major organs. Representative histological features of major organs (heart, liver, spleen, lung, kidney) removed from normal mice or DILI mice. The removed organs and tumor tissues were fixed with 4% formaldehyde solution, embedded in paraffin, and cut into 5 μm thick sections. The sections were dewaxed, hydrated, and stained with hematoxylin and eosin in sequence. The sections were rinsed with water and dehydrated for imaging acquisition. Fluorescent images were collected by fluorescence microscopy at 400 times magnification.

[0093] like Fig.10 As shown in Figure e, the liver area of ​​the APAP group showed obvious sinusoidal congestion and hemorrhage, as well as fatty degeneration of a large number of hepatocytes with dense vacuoles in the cytoplasm. In addition, hepatocyte necrosis with partial loss of nuclear dissolution and mild inflammatory infiltration and single inflammatory lesions were also seen. These symptoms confirmed the occurrence of DILI. As expected, the above symptoms in the liver area of ​​the DILI group were eliminated under NAC pretreatment, indicating that hepatoprotective drugs have a good therapeutic effect on liver injury. In summary, these results demonstrate that AIH-OTF can be used to image the occurrence and repair of DILI in vivo.

[0094] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment, it is not used to limit the present invention. Any technician familiar with this patent can make some changes or modify the technical contents suggested above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the solution of the present invention.

Claims

1. A 7-azaindole photoacoustic imaging probe for detecting superoxide anions, characterized in that: The photoacoustic imaging probe is named AIH-OTF, and its chemical structure is shown in Formula I:

2. Use of the 7-azaindole photoacoustic imaging probe for detecting superoxide anions as claimed in claim 1 in diagnosing DILI at the in vivo level.

3. The method for preparing the 7-azaindole photoacoustic imaging probe for detecting superoxide anions according to claim 1, characterized in that: The preparation process is: After mixing 2 equivalents of methyl trifluoromethanesulfonate, 1 equivalent of dye AIH-OH, anhydrous dichloromethane and 4 equivalents of triethylamine, the mixture was stirred at 30°C for 2 hours. After vacuum concentration, the residue was purified by column chromatography using dichloromethane and methanol as eluents to obtain a blue-green solid product named AIH-OTF.

4. The preparation method according to claim 3, characterized in that: The preparation method of the dye AIH-OH is: One equivalent of 7-chloro-6-hydroxy-2,3-dihydro-1H-xanthene-4-carboxaldehyde is dissolved in anhydrous ethanol containing one equivalent of piperidine, and then the solution is mixed with 1.25 equivalents of 2,3,3,7-tetramethyl-3H-pyrrolo[2,3-b]pyridin-7-ium, followed by stirring and reacting at 80° C. for 12 hours to obtain a dark blue solution; the solvent is removed by rotary evaporation, and the obtained residue is purified by column chromatography to obtain a blue solid product, named AIH-OH, whose chemical structure is shown in Formula II:

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

2.

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

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