A ratiometric photoacoustic probe responsive to peroxynitrite and its preparation method and application

Abstract

The present invention belongs to the technical field of biochemistry, and relates to a ratiometric photoacoustic probe responsive to peroxynitrite, a preparation method thereof and an application. The structural formula of the probe is: . The ratiometric photoacoustic probe provided by the present invention has high selectivity and sensitivity; after reacting with ONOO−, the absorption wavelength of the formed hydroxyl anion redshifts by about 100 nm, showing obvious characteristics of ratiometric response; it has good stability under physiological pH conditions, which is helpful for real-time in-situ imaging of ONOO−; the probe itself has a strong photoacoustic signal. The preparation method of the ratiometric photoacoustic probe provided by the present invention uses easily obtainable raw materials, has mild and easily controllable reaction conditions, saves reaction costs, and ensures the yield of the target product. The ratiometric photoacoustic probe BXos4 provided by the present invention has high-efficient tumor targeting ability, provides a new detection means for the detection of ONOO−, and provides a potential tool for tumor diagnosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of biochemistry, and specifically, relates to a ratiometric photoacoustic probe responsive to peroxynitrite, a preparation method thereof, and an application thereof. Background Art

[0002] Peroxynitrite (ONOO - ) is a multifunctional reactive nitrogen species (RNS) that plays a crucial role in both physiological and pathological processes. Endogenous ONOO - is formed through the reaction between nitric oxide (•NO) and superoxide radical anion ( ), and exhibits strong oxidative and nitrative capabilities. Normal levels of ONOO - contribute to the immune system's fight against microbial threats and participate in signal transduction. In contrast, excessive ONOO - can cause damage to important biomolecules such as lipids, nucleic acids, and proteins, disrupt their normal functions, and lead to diseases such as inflammation, diabetes, neurodegenerative diseases, and cancer. It is worth noting that continuously high levels of ONOO - are closely related to the occurrence and development of tumors. Therefore, the visualization of ONOO - in tumors is crucial for elucidating the pathological mechanisms involving ONOO - and advancing tumor diagnosis and treatment.

[0003] In recent years, due to the advantages of high sensitivity and high contrast, fluorescent probes have made significant progress in the imaging of ONOO - in living cells and in vivo, and have become reliable visualization tools for studying the physiological and pathological roles of ONOO - . However, currently available fluorescent probes for ONOO - visualization have short emission wavelengths, mostly less than 800 nm, which pose challenges to achieving high-resolution in vivo imaging due to disadvantages such as shallow tissue penetration depth and background interference from tissue autofluorescence. Compared with fluorescence imaging, photoacoustic (PA) imaging detects ultrasonic waves instead of photons after light excitation, breaking through the limitations of optical imaging. PA imaging has received extensive attention due to its characteristics such as high sensitivity, high spatial resolution, and deep tissue penetration, and has been widely applied in the biomedical field. Although a large number of PA probes for visualizing biomarkers have been reported, there are few examples of PA probes for in vivo ONOO - imaging. Pu and Zhou et al. reported two examples of PA probes in the far-infrared region for ONOO -Imaging was performed, and its PA signal showed "off-on" and "on-off" response modes, respectively. Since visible light can be strongly absorbed and scattered by biological tissues, and the single-channel mode is easily interfered by background signals, it inevitably affects the accuracy of the resulting PA signal. To this end, researchers have developed PA probes with efficient near-infrared (NIR, 700-1700 nm) absorption and dual-channel ratiometric response behavior with self-calibration function to meet the challenge. For example, Lin et al. reported a ratiometric nanoprobe composed of a naphthalocyanine dye (775 nm) as a sensing component and a heptamethine dye (860 nm) as an internal standard for ONOO - Pu et al. reported a bulky borane-doped PA nanoprobe based on an aza-BODIPY structure with a ratiometric PA signal (PA 750 / PA 680 ), for ONOO in tumors - Imaging. Compared with the potential low reproducibility and leakage risk of nanoprobes using internal standard methods to achieve ratiometric imaging, the use of ratiometric response probes has been shown to be a more reliable approach. This requires the use of ONOO - The molecular electronic structure before and after the response produces a clear difference to prevent serious spectral crosstalk. In addition, the PA signal of the probe is in the NIR region greater than 800 nm before and after the response, which is more conducive to high-resolution imaging, which requires the PA chromophore to have efficient and adjustable NIR absorption ability. Therefore, the development of ratiometric PA probes with PA signals exceeding 800 nm is very important in realizing ONOO - Although bioremediation plays a key role in the in vivo high-resolution imaging of mitochondria, major challenges remain, such as the lack of molecular scaffolds and inadequate design strategies. Summary of the invention

[0004] The purpose of the present invention is to solve the above problems existing in the prior art and provide a ratiometric photoacoustic probe responsive to peroxynitrosyl and its preparation method and application. The probe of the present invention has both tumor targeting capability and ONOO - Combined with 3D photoacoustic imaging, the probe has ONOO in living tumors - The potential of high spatiotemporal resolution imaging to elucidate ONOO - It provides a reliable research tool for the regulation and intervention mechanisms of tumors and the diagnosis and treatment of related diseases.

[0005] In order to achieve the above object, the first aspect of the present invention provides a ratiometric photoacoustic probe responsive to peroxynitrosyl, the structural formula of the probe being:

[0006] .

[0007] The second aspect of the present invention provides a method for preparing the ratiometric photoacoustic probe, comprising the following steps:

[0008] (1) adding compound (I), compound (II), glacial acetic acid and piperidine to a first solvent to carry out a first reaction to obtain compound (III);

[0009] (2) adding compound (III), compound (IV), glacial acetic acid and piperidine obtained in step (1) to a second solvent to carry out a second reaction to obtain compound (V);

[0010] (3) Compound (V) obtained in step (2) and Biotin-mPEG- 3 , copper sulfate, and sodium ascorbate are added to a third solvent to carry out a third reaction to obtain a compound BXos4;

[0011] The reaction process is:

[0012] .

[0013] The third aspect of the present invention provides the ratiometric photoacoustic probe for preparing ONOO in vivo tumors. - Application in imaging agents.

[0014] The fourth aspect of the present invention provides the ratiometric photoacoustic probe for detecting ONOO in live mouse tumors. - Application in imaging; preferably, in live mouse tumor ONOO - Application in dynamic tracing.

[0015] The present invention has the following beneficial effects:

[0016] 1. The ratiometric photoacoustic probe provided by the present invention has high selectivity and sensitivity; - After the response, the absorption wavelength of the formed hydroxyl anion red-shifts by about 100 nm, which has the characteristics of obvious ratiometric response; it has good stability under physiological pH conditions, which is helpful for real-time in situ imaging of ONOO - The probe itself has a strong photoacoustic signal. At the same time, ratiometric photoacoustic imaging can significantly reduce the interference of photoacoustic signals of biological molecules (such as hemoglobin, melanin, etc.) in the body, which is conducive to high-resolution imaging in vivo.

[0017] 2. The preparation method of the ratiometric photoacoustic probe provided by the present invention uses easily available raw materials, mild reaction conditions, and is easy to control, thereby saving reaction costs and ensuring the yield of the target product.

[0018] 3. The ratiometric photoacoustic probe BXos4 provided by the present invention has efficient tumor targeting ability and can be used to detect ONOO in tumors. -Dynamic tracing. Combined with high-resolution three-dimensional photoacoustic imaging technology, the probe BXos4 can track ONOO in tumors. - High-resolution photoacoustic imaging of the dynamics of ONOO - The detection of gliomas provides a new detection method and a potential tool for the diagnosis of tumors.

[0019] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The above and other objects, features and advantages of the present invention will become more apparent through a more detailed description of exemplary embodiments of the present invention with reference to the accompanying drawings.

[0021] Figure 1 The NMR spectrum of BXos4 in the present invention is shown.

[0022] Figure 2 The present invention shows that BXos4 reacts with ONOO in phosphate buffered saline. - Absorption spectra before and after response.

[0023] Figure 3 The present invention shows that BXos4 and ONOO - Response photoacoustic imaging diagram, where Figure 3 Figure a shows the reaction of BXos3 with ONOO in solution. - Photoacoustic imaging, Figure 3 Figure b shows the action of BXos4 on ONOO in solution. - Photoacoustic imaging, Figure 3 Figure c shows the action of BXos4 on ONOO in solution. - The photoacoustic spectrum of the response, Figure 3 (d) shows the linear relationship between the photoacoustic signal ratio of BXos3 and BXos4 and their concentration.

[0024] Figure 4 The two-dimensional photoacoustic imaging of BXos4 in the tumor model mouse of the present invention is shown, wherein: Figure 4 a shows photoacoustic imaging of 4T1 mouse tumors after treatment with PBS, BXos3, BXos4, and NAC; Figure 4 Middle b shows the photoacoustic signal at 950 nm; Figure 4 Figure c shows the PA 950 / 850 Ratio signal at the tumor.

[0025] Figure 5 The three-dimensional photoacoustic imaging of BXos4 in the tumor model mouse of the present invention is shown, wherein: Figure 5a shows photoacoustic imaging of 4T1 mouse tumors after PBS, BXos4, and NAC treatment; Figure 5 b shows the 3D photoacoustic imaging image resolution; Figure 5 Figure c shows Figure 5 PA 950 / 850 Ratio signal at the tumor. DETAILED DESCRIPTION

[0026] The preferred embodiments of the present invention will be described in more detail below. Although the preferred embodiments of the present invention are described below, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein.

[0027] In order to achieve the above object, the first aspect of the present invention provides a ratiometric photoacoustic probe responsive to peroxynitrosyl, the structural formula of the probe being:

[0028] .

[0029] The second aspect of the present invention provides a method for preparing the ratiometric photoacoustic probe, comprising the following steps:

[0030] (1) adding compound (I), compound (II), glacial acetic acid and piperidine to a first solvent to carry out a first reaction to obtain compound (III);

[0031] (2) adding compound (III), compound (IV), glacial acetic acid and piperidine obtained in step (1) to a second solvent to carry out a second reaction to obtain compound (V);

[0032] (3) Compound (V) obtained in step (2) and Biotin-mPEG- 3 , copper sulfate, and sodium ascorbate are added to a third solvent to carry out a third reaction to obtain a compound BXos4;

[0033] The reaction process is:

[0034] .

[0035] According to the present invention, preferably, the first solvent and the second solvent are each independently anhydrous dioxane and / or toluene.

[0036] According to the present invention, preferably, in step (1), the molar ratio of compound (I) to compound (II) is 1:0.5-0.7; based on 1 mol of compound (I), the volume of glacial acetic acid is 5-15 mL, and the volume of piperidine is 10-30 mL.

[0037] According to the present invention, preferably, in step (1), the conditions of the first reaction include: a reaction temperature of 70° C. to 100° C., and a reaction time of 0.5 to 30 min.

[0038] According to the present invention, preferably, in step (2), the molar ratio of compound (III) to compound (IV) is 1:1.0-1.2; based on 1 mol of compound (III), the volume of glacial acetic acid is 10-30 mL, and the volume of piperidine is 30-50 mL.

[0039] According to the present invention, preferably, in step (2), the conditions of the second reaction include: a reaction temperature of 100° C. to 130° C., and a reaction time of 0.5 to 2 h.

[0040] According to the present invention, preferably, in step (3), the third solvent is a mixed solvent of dimethyl sulfoxide, tetrahydrofuran and water, wherein the volume ratio of dimethyl sulfoxide, tetrahydrofuran and water is 1-3:1-3:1;

[0041] According to the present invention, preferably, in step (3), the compound (V), Biotin-mPEG-N 3 , the molar ratio of copper sulfate and sodium ascorbate is 1:1.0~1.2:5~7:1.0~1.2;

[0042] The conditions of the third reaction include: reaction temperature of 20°C to 30°C, and reaction time of 5 to 24 hours.

[0043] According to the present invention, preferably, the compound (V) is first dissolved in the first part of the third solvent, the reaction temperature is lowered to -10~5°C, and Biotin-mPEG-N is added. 3 , copper sulfate, sodium ascorbate and a second part of a third solvent, and then raising the reaction temperature to carry out a third reaction.

[0044] The third aspect of the present invention provides the ratiometric photoacoustic probe for preparing ONOO in vivo tumors. - Application in imaging agents.

[0045] The fourth aspect of the present invention provides the ratiometric photoacoustic probe for detecting ONOO in live mouse tumors. - Application in imaging; preferably, in live mouse tumor ONOO - Application in dynamic tracing.

[0046] The present invention will be further described below in conjunction with embodiments, but the scope of the present invention is not limited to these embodiments.

[0047] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0048] The device used for two-dimensional photoacoustic imaging in the embodiment is Vevo F2 LAZR-X photoacoustic imaging platform, and the device used for three-dimensional photoacoustic imaging is LOIS-3D, Tomo Wave Laboratories, US. Example 1

[0049] Synthesis method of compound III: Under nitrogen protection, add compound I (2 mmol), compound II (1 mmol), acetic acid (20 mL) and piperidine (40 mL) into the reactor. React at 80°C for 3 minutes in 15 mL toluene solvent. After the reaction is completed, extract with dichloromethane, dry with anhydrous magnesium sulfate, filter, and spin dry. Use a developing solvent of petroleum ether / dichloromethane with a volume ratio of 1: 1 for silica gel column chromatography to obtain the corresponding compound III. The yield is 19%.

[0050] Synthesis method of compound V: Under nitrogen protection, compound III (1 mmol), compound IV (1 mmol), acetic acid (15 mL) and piperidine (40 mL) were added to the reactor, dissolved with anhydrous toluene (10 mL), and refluxed at 120°C for 1 hour. After the reaction was completed, dichloromethane was extracted, dried over anhydrous magnesium sulfate, filtered, and dried. Silica gel column chromatography was performed with a developing solvent of petroleum ether / dichloromethane in a volume ratio of 2: 3 to obtain the corresponding compound V. The yield was 48%.

[0051] The synthesis method of compound BXos3 or BXos4 is as follows: Compound V (0.02 mmol) is dissolved in 4 mL of a tetrahydrofuran / dimethyl sulfoxide mixed solvent, the reaction is cooled to 0°C in an ice bath, and mPEG-N is added. 3 or Biotin-mPEG-N 3 (average relative molecular mass = 5000, 0.02 mmol), then deionized water (1 mL) was added, sodium ascorbate (0.10 mmol) and copper sulfate pentahydrate (0.02 mmol) were added to the reaction bottle, and the reaction was completed after returning to room temperature for 12 hours. The tetrahydrofuran was removed by evaporation under reduced pressure and the system was transferred to a dialysis bag, which was placed in a 500 mL beaker and 300 mL of deionized water was added. The water was replaced every 8 hours, and after 48 hours, the solvent was removed and removed to obtain a black-green solid powder product BXos3 or BXos4 (yield: 99%).

[0052] The NMR image of compound BXos4 is shown in Figure 1 As shown in the figure, 1 H NMR (600 MHz, CDCl 3 ) δ / ppm =7.82 (s, 2H), 7.52–7.43 (m, 6H), 7.18 (d, J = 7.7 Hz, 2H), 7.17–7.10 (m, 2H),6.98 (s, 2H), 6.88 (s, 1H), 6.78–6.71 (m, 3H), 6.65 (s, 1H), 6.44 (s, 1H),5.60–5.37, 4.95, 4.55–3.12, 2.30–2.20 (m, PEG 5000 -Biotin), 5.23–5.11 (m, 4H),2.72–2.53 (m, 9H), 1.82–1.22 (m, 13 H). The structure of BXos4 can be verified.

[0053] The probe BXos4 was dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mM stock solution. The test solution was ethanol / phosphate buffered saline (10 / 90, 10 mM, pH 7.4, containing 0.4% Tween 80), and the probe test concentration was 10 μM. The absorption spectrum test results are shown in Figure 2 As shown, the absorption peak of probe BXos4 is around 846nm. - After the reaction, the absorption intensity at 846 nm gradually decreased, a new absorption peak appeared at 957 nm, and the absorption intensity gradually increased. The UV absorption spectrum showed that the probe BXos4 had obvious ratiometric response characteristics. Example 2

[0054] The probe BXos4 interacts with ONOO in vitro - Photoacoustic imaging of responses.

[0055] Different concentrations of ONOO - After reacting with BXos4, it was added to the photoacoustic tube and subjected to photoacoustic imaging test. Figure 3 As shown, with ONOO - As the concentration increases, the photoacoustic signal of the 850 nm channel gradually weakens, while the photoacoustic signal of the 950 nm channel gradually increases. Proportional processing of the photoacoustic imaging images of the two channels can obtain photoacoustic imaging images with higher resolution. Example 3

[0056] Photoacoustic imaging of mouse tumors using the probe BXos4.

[0057] BXos4 phosphate buffered saline solution (100 mM, 100 mL) was injected into wild mice and 4T1 tumor-bearing mice by tail vein injection. Two-dimensional photoacoustic imaging of the tumor sites was performed 1 hour later. Figure 4 As shown in the figure, in the PBS group, the photoacoustic signal was weak. In the BXos3 group without biotin group, the photoacoustic signal was slightly enhanced because BXos3 did not have targeting ability. In the BXos4 group, the probe had efficient targeting ability and the photoacoustic signal was significantly enhanced. The NAC group was a group with added reactive oxygen scavengers to remove reactive oxygen in tumors. This experiment proved that the probe BXos4 can be used to remove ONOO in tumors. - Dynamic imaging. Ratio-type photoacoustic imaging images were obtained through dual-channel photoacoustic imaging analysis.

[0058] Next, three-dimensional photoacoustic imaging was used to identify ONOO in the tumor. - Further research, such as Figure 5 As shown, a significantly enhanced ratiometric photoacoustic signal can be observed in the tumor, and the ratiometric photoacoustic signal is significantly decreased in the tumor added with NAC reactive oxygen species scavenger.

[0059] The above experimental results show that the probe BXos4 has efficient tumor targeting ability and can be used for ONOO in living tumors in combination with photoacoustic imaging. - Dynamic tracing has great application prospects in the biomedical field.

[0060] The embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A ratiometric photoacoustic probe responsive to peroxynitrosyl, characterized in that: The structural formula of the probe is: 。 2. The method for preparing a ratiometric photoacoustic probe according to claim 1, characterized in that: The steps include: (1) adding compound (I), compound (II), glacial acetic acid and piperidine to a first solvent to carry out a first reaction to obtain compound (III); (2) adding compound (III), compound (IV), glacial acetic acid and piperidine obtained in step (1) to a second solvent to carry out a second reaction to obtain compound (V); (3) adding the compound (V) obtained in step (2), Biotin-mPEG-N3, copper sulfate and sodium ascorbate to a third solvent to carry out a third reaction to obtain a compound BXos4; The reaction process is: 。 3. The preparation method according to claim 2, wherein The first solvent and the second solvent are each independently anhydrous dioxane and / or toluene.

4. The preparation method according to claim 2, wherein In step (1), the molar ratio of compound (I) to compound (II) is 1:0.5-0.7; based on 1 mol of compound (I), the volume of glacial acetic acid is 5-15 mL, and the volume of piperidine is 10-30 mL; The conditions of the first reaction include: reaction temperature of 70°C to 100°C, and reaction time of 0.5 to 30 min.

5. The preparation method according to claim 2, wherein: In step (2), the molar ratio of compound (III) to compound (IV) is 1:1.0-1.2; based on 1 mol of compound (III), the volume of glacial acetic acid is 10-30 mL, and the volume of piperidine is 30-50 mL; The conditions of the second reaction include: reaction temperature of 100° C. to 130° C., and reaction time of 0.5 to 2 h.

6. The preparation method according to claim 2, wherein: In step (3), the third solvent is a mixed solvent of dimethyl sulfoxide, tetrahydrofuran and water, wherein the volume ratio of dimethyl sulfoxide, tetrahydrofuran and water is 1-3:1-3:1; The molar ratio of the compound (V), Biotin-mPEG-N3, copper sulfate and sodium ascorbate is 1:1.0-1.2:5-7:1.0-1.2; The conditions of the third reaction include: reaction temperature of 20°C to 30°C, and reaction time of 5 to 24 hours.

7. The preparation method according to claim 6, wherein: Firstly, the compound (V) is dissolved in the first part of the third solvent, the reaction temperature is lowered to -10~5°C, Biotin-mPEG-N3, copper sulfate, sodium ascorbate and the second part of the third solvent are added, and then the reaction temperature is increased to carry out the third reaction.

8. The ratiometric photoacoustic probe of claim 1 is used to prepare ONOO in living tumors - Application in imaging agents.

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