Photoacoustic probe for detecting noradrenaline as well as preparation and application of photoacoustic probe

Abstract

The invention discloses a photoacoustic probe for detecting noradrenaline and preparation and application of the photoacoustic probe, the probe is named as QSH-NE, the probe takes 4-(bromomethyl)-2-formyl phenyl pyrrolidine-1-carboxylate as a skeleton to construct a recognition-release bifunctional unit, and sulfur-substituted hemicyanine QSH-OH is selected as a photoacoustic signal report group. The photoacoustic signal activation mechanism depends on that an aldehyde group captures an NE amino group to initiate intramolecular cyclization, then ortho-carbamate is triggered to be cracked to generate 2-hydroxyl, and finally reporter group release and ICT effect recovery are realized through a 1, 6-elimination reaction, so that accurate detection of NE is realized. The probe has the characteristics of high specificity, high sensitivity and quick response to NE, and realizes detection, imaging and drug treatment evaluation of noradrenaline in living cells and living animal brains. According to the technology, the comprehensive performance of NE detection is remarkably improved, and powerful tool support is provided for mechanism research, clinical diagnosis and drug development of depression.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biochemistry, and particularly relates to a photoacoustic probe for detecting norepinephrine and preparation and application thereof. BACKGROUND

[0002] Depression, as a high-incidence and pathogenesis-unknown coexisting mental illness, has the characteristics of chronic persistence and easy recurrence, and has become one of the four major diseases in the world. According to the estimate of the World Health Organization, about 280 million people are deeply troubled by it. Although the pathophysiological mechanism of depression has yet to be fully elucidated, more and more evidence shows that norepinephrine (NE) neurotransmission deficiency is one of the key neurobiological factors of depressive state. As an important neuroregulator in the central nervous system, NE exerts physiological functions by binding to target receptors after being released by neurons. Existing NE detection technologies such as capillary electrophoresis, chromatography, mass spectrometry and electrochemical methods are complex to operate and difficult to realize rapid and convenient detection of NE in biological systems. Although fluorescent probes can effectively overcome the above limitations and realize accurate detection of NE in life systems, the limitations of fluorescence imaging technology in penetration depth and spatial resolution restrict its wide application in deep tissue imaging.

[0003] As a new biomedical imaging technology, photoacoustic imaging can effectively reduce the interference of light absorption and scattering of biological tissues by detecting the ultrasonic waves generated by light absorption, realize non-invasive observation of deep tissue bioactive molecules (the maximum penetration depth is up to 7 centimeters), and has sub-millimeter level three-dimensional spatial resolution. The NE-responsive PA probes reported at present are mainly based on the "protection-deprotection" strategy, which restores the ICT process through cascade nucleophilic substitution reaction involving carbonate, so as to realize the activatable optical detection of NE. However, this strategy is limited by the slow kinetics of amino ester nucleophilic reaction, and is easily interfered by biological thiols such as cysteine. Therefore, developing new NE-responsive photoacoustic probes to realize rapid and high-specificity NE detection will help to deepen the understanding of the pathogenesis of depression and provide a more powerful tool for clinical diagnosis. SUMMARY

[0004] The present application aims at the deficiencies of existing norepinephrine detection technologies, especially the problems of limited probe strategy, low detection sensitivity and specificity faced by the photoacoustic probe method, and provides a novel NE-responsive photoacoustic probe (named QSH-NE) and its preparation method and biomedical application. The probe described in the present application takes 4-(bromomethyl)-2-formylphenyl pyrrolidine-1-carboxylate as the skeleton, constructs a "recognition-release" bifunctional unit, and selects a sulfur-substituted hemicyanine QSH-OH as a photoacoustic signal reporter group. The covalent connection is achieved by the nucleophilic substitution reaction of 4-bromomethyl and the hydroxyl group in the reporter group, which can destroy the intramolecular charge transfer effect, thereby quenching the photoacoustic signal. In the recognition stage, the aldehyde group located at the ortho position of the ester group can quickly "capture" the amino group of NE, and then form an intramolecular five-membered ring through nucleophilic reaction. Subsequently, the -NH- in the ring attacks the adjacent carbamate group to generate 2-hydroxyl; finally, the "release" process is started by the 1,6-elimination reaction driven by 2-hydroxyl, which promotes the dissociation of the hydroxyl group and restores the ICT effect, realizing the accurate detection of NE. The experimental results show that QSH-NE exhibits high sensitivity, excellent selectivity and significant photoacoustic signal activation multiple to NE in vitro and in vivo. By non-invasively monitoring the photoacoustic response of QSH-NE to NE, the present application successfully realizes the in-situ tracking of the occurrence and treatment process of depression. The present application constructs a norepinephrine-responsive photoacoustic probe technology platform, and the high-specificity NE detection capability provides a new tool for the pathological research and clinical diagnosis of depression.

[0005] To solve the above technical problems, the basic idea of the technical solution of the present application is:

[0006] In one aspect, the present application provides a photoacoustic probe for detecting norepinephrine, and the probe is named QSH-NE, and its chemical structural formula is shown as formula I:

[0007]

[0008] Formula I.

[0009] In another aspect, the present application provides a preparation method of a photoacoustic probe for detecting norepinephrine, and the preparation process is:

[0010] QSH-OH (0.05 g, 0.13 mmol) was added to a mixture of 4-(bromomethyl)-2- formylphenyl pyrrolidine-1-carboxylate (0.06 g, 0.16 mmol) and K2CO3 (0.05 g, 0.36 mmol) in N,N-dimethylformamide (10 mL), and the mixture was stirred at 80 °C for 5 h. The crude product was then purified by silica gel chromatography using CH2Cl2 / MeOH. The crude product was purified by silica gel column chromatography (40:1, v / v) as eluent to obtain a blue solid with a yield of 31% and named as QSH-NE.

[0011] In still another aspect, the application provides use of the norepinephrine-responsive photoacoustic probe as described above in the preparation of a product for detecting endogenous NE products in living cells.

[0012] In still another aspect, the application provides use of the norepinephrine-responsive photoacoustic probe as described above in the preparation of a product for non-invasively detecting depression progression in the brain of a living animal and evaluating the efficacy of drug intervention.

[0013] Compared with the prior art, the application has the following beneficial effects.

[0014] The probe has high specificity, high sensitivity and fast response characteristics for NE. The application uses 4-(bromomethyl)-2-formylphenyl pyrrolidine-1-carboxylate as a skeleton to construct a "recognition-release" bifunctional unit, and selects a sulfur-substituted hemicyanine QSH-OH as a photoacoustic signal reporter group, and designs and synthesizes a photoacoustic probe that can realize high specificity and high sensitivity detection of NE. The probe introduces an aldehyde group at the ortho position of the ester group, which can quickly "capture" the amino group, and then form an intramolecular five-membered ring through a nucleophilic reaction, and trigger a subsequent 1,6-elimination reaction, and finally release QSH-OH to activate the photoacoustic signal.

[0015] The probe realizes detection, imaging and drug treatment evaluation of norepinephrine in living cells and the brain of a living animal, and has good clinical application value.

[0016] The specific embodiments of the application will be further described in detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS

[0017] The accompanying drawings, which are part of this application, serve to further understand the application, and the illustrative embodiments of the application and their descriptions serve to explain the application, but do not constitute an improper limitation on the application. Obviously, the accompanying drawings in the following description are only some embodiments, and other drawings can be obtained by those skilled in the art without creative labor. In the drawings:

[0018] Figure 1Synthetic route of the probe QSH-NE described in the embodiments of the present application;

[0019] Figure 2 Principle diagram of the design of the probe QSH-NE described in the embodiments of the present application;

[0020] Figure 3 H NMR spectrum of the compound 5 in dimethyl sulfoxide-d6 (DMSO-d6); 1 H NMR spectrum;

[0021] Figure 4 C NMR spectrum of the compound 5 in dimethyl sulfoxide-d6 (DMSO-d6); 13 C NMR spectrum;

[0022] Figure 5 ESI-MS spectrum of the dye QSH-OH described in the embodiments of the present application;

[0023] Figure 6 H NMR spectrum of the probe QSH-NE described in the embodiments of the present application in dimethyl sulfoxide-d6 (DMSO-d6); 1 H NMR spectrum;

[0024] Figure 7 C NMR spectrum of the probe QSH-NE described in the embodiments of the present application in dimethyl sulfoxide-d6 (DMSO-d6); 13 C NMR spectrum;

[0025] Figure 8 ESI-MS spectrum of the probe QSH-NE described in the embodiments of the present application;

[0026] Figure 9 In vitro response of the probe QSH-NE described in the embodiments of the present application. (a) Schematic diagram of the reaction of the QSH-NE probe with NE, (b) UV spectrum, (c) fluorescence spectrum and (d) PA spectrum change, (e) PA imaging of the probe QSH-NE (10 μM) reacted with different concentrations of NE (0 - 8 mM) at 37 °C for 1 h, (f) Real-time UV absorption spectrum of the probe QSH-NE (10 μM) to NE (5 mM), (g) High performance liquid chromatogram of different reaction systems: 1. QSH-NE (5 μM) reacted with NE (2 mM); 2. QSH-NE (5 μM); 3. QSH-NE (5 μM); (h) Specific PA response of QSH-NE to NE (10 μM). Data are expressed as mean ± standard deviation (n=3).

[0027] Figure 10Live cell studies with the probe QSH-NE described in the examples of the present application. (a) Schematic of endogenous NE detection in cells, (b) PA images of PC12, HeLa and HepG2 cells with and without QSH-NE treatment, (c) PA 760 intensity of HeLa cells treated with QSH-NE (10 μΜ) and different concentrations of NE, (d) PA images of mice treated with PBS or QSH-NE (100 μΜ) and different concentrations of NE, (e) Corresponding PA 760 intensity of images of mice treated under the conditions described in (d). Scale bar is 1 mm, error bars represent standard deviation from 5 separate measurements.

[0028] Figure 11 The probe QSH-NE described in the examples of the present application is used for the detection of NE in vivo: (a) Schematic of in vivo PA imaging of depression in a mouse model; (b) H&E staining of DG, CA1 and CA3 regions of mice. Nuclear streaming and karyorrhexis (black arrows), vacuolar structures; Representative PA images of healthy mice at different time points after tail vein injection of QSH-NE. (d) Time-dependent PA 760 intensity changes of healthy mice after QSH-NE injection. (e) Representative PA images of brain regions of healthy and depressed mice after tail vein injection of QSH-NE or PBS. (f) Corresponding PA 760 intensity of mice under the conditions described in (e). (g) Representative PA images of brain regions of healthy mice, depressed mice and depressed mice treated with fluoxetine after tail vein injection of QSH-NE. (h) Corresponding PA 760 intensity of images of mice under the conditions described in (g). Error bars represent standard deviation from 5 separate measurements.

[0029] It should be noted that the drawings and detailed description are not intended to limit the scope of the present application in any way, but rather to illustrate the concept of the present application to those skilled in the art by referring to specific embodiments. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments will be described clearly and completely below with reference to the drawings in the embodiments of the present application. The following embodiments are used to illustrate the present application, but not to limit the scope of the present application.

[0031] Example 1

[0032] Preparation process of thiosemichrysamine dye QSH-OH, reference route 1,

[0033] ; Route 1

[0034] The specific steps are as follows:

[0035] Synthesis of compound 1 :

[0036] Dimethylthiocarbamoyl chloride (5.56 g, 4.5 mmol) was added to a solution of 2-hydroxy-4-methoxybenzaldehyde (4.51 g, 30 mmol) and K2CO3(8.29 g, 60 mmol) in acetonitrile (30 mL), the reaction mixture was stirred at 38°C for 24 hours, then filtered, the filtrate was concentrated under reduced pressure, the crude product was purified by silica gel column chromatography with PE / AcOEt (10 / 1, v / v) as eluent to obtain a white solid with a yield of 61%, named as compound 1;

[0037] Synthesis of compound 2:

[0038] Compound 1 (2.6 g, 11 mmol) was dissolved in toluene (10 mL) and refluxed at 175°C for 10 hours, then concentrated under reduced pressure, the crude product was purified by silica gel column chromatography with PE / CH2Cl2(10 / 1, v / v) as eluent to obtain a yellow solid with a yield of 32%, named as compound 2;

[0039] Synthesis of compound 3:

[0040] To a solution of compound 2 (0.80 g, 3 mmol) in MeOH (20 mL), NaOH was added and refluxed at room temperature for 2 hours, the reaction solution was acidified to pH = 5 with 10% aqueous HCl solution, then extracted with ethyl acetate (3 x 15 mL), dried with Na2SO4and concentrated to obtain colorless needles. Used directly in the next step without further purification. Named as compound 3;

[0041] Synthesis of compound 4:

[0042] Compound 3 (0.53 g, 3 mmol) and Cs2CO3(2.07 g, 6.3 mmol) were added to a solution of 2-bromocyclohex-1-en-1-carbaldehyde (0.60 g, 3.2 mmol) in N,N-dimethylformamide (20 mL), then the mixture was stirred at room temperature for 12 hours, the reaction was filtered and the filtrate was concentrated. Extracted with ethyl acetate (2 x 30 mL), the organic phase was dried with anhydrous sodium sulfate, filtered and concentrated, the crude product was purified by silica gel column chromatography with PE / AcOEt (8 / 1, v / v) as eluent to obtain a yellow solid with a yield of 65%, named as compound 4;

[0043] Synthesis of compound 5:

[0044] To a 20 mL ethanol (0.36 g, 1.5 mmol) solution of compound 4, piperidine (0.35 mL, 3.5 mmol) and 1,2-dimethylquinoline (0.21 g, 1.5 mmol) were added. After reflux at 80 °C for 12 hours under nitrogen protection, the solution turned blue. The solvent was removed by vacuum evaporation, and the residue was purified by column chromatography (eluent: DCM / MeOH = 100 / 1, v / v) to finally obtain a blue solid in 35% yield, which was named compound 5.

[0045] Synthesis of compound 6:

[0046] BBr3 (2.2 mL, 15 mmol) was slowly added to an anhydrous dichloride solution (10 mL) of compound 5 (0.72 g, 2.8 mmol). After stirring at room temperature for 12 hours, the reaction solution was quenched with saturated NaHCO3 solution (30 mL). The combined organic layers were dried with anhydrous Na2SO4, extracted with anhydrous CH2Cl2 (3 × 15 mL), and the solvent was evaporated to finally obtain a blue solid QSH-OH (yield 35%).

[0047] The thiocyanate hemicyanine dye QSH-OH prepared in this embodiment was tested, and the results are as follows: Figures 3 to 5 As shown: Figure 3 Compound 5 is a derivative of dimethyl sulfoxide-d6 (DMSO-d6). 1 H NMR spectrum; Figure 4 Compound 5 is a derivative of dimethyl sulfoxide-d6 (DMSO-d6). 13 C NMR spectrum; Figure 5 The above is the ESI-MS spectrum of the dye QSH-OH described in the embodiments of the present invention;

[0048] in, Figure 3 The hydrogen spectrum was analyzed as follows 1H NMR (400 MHz, DMSO-d6) δ (ppm): 8.73 (1H, d, J= 9.1 Hz, Ar-H), 8.54-8.58 (1H, t, J = 8.2 Hz, Ar-H), 8.38 (1H, d, J = 8.8Hz, Ar-H), 8.24 (1H, d, J = 7.9 Hz, Ar-H), 8.05 (1H, d, J = 7.6 Hz, Ar-H), 7.79-7.83 (1H, t, J = 7.6 Hz, Ar-H), 7.35 (1H, d, J = 8.5 Hz, Ar-H), 7.15(1H, d, J = 2.5 Hz, =CH), 7.07 (1H, s, Ar-H), 6.84 (1H, s, Ar-H), 6.81 (1H,d, J = 10.4 Hz, =CH), 5.76 (1H, s, =CH), 4.36 (3H, s, CH3), 3.87 (3H, s, CH3), 1.99 (2H, s, CH2), 1.79 (2H, s, CH2), 1.17 (2H, m, CH2).

[0049] Figure 4 The carbon spectrum analysis is as follows: 13 C NMR (100 MHz, DMSO-d6) δ (ppm): 160.31, 156.79,155.88, 153.21, 141.34, 139.58, 136.91, 133.33, 130.38, 129.19, 127.43,127.71, 127.29, 126.52, 119.70, 115.28, 112.89, 111.79, 110.85, 103.13,49.74, 29.01, 24.52, 20.65, 13.28.

[0050] Figure 5 The ESI-MS mass spectrometry resolution was: QSH-OH [MI] + The theoretical molecular weight is 384.52; the actual measured molecular weight is 384.49.

[0051] The above results prove that this embodiment has successfully synthesized the thiocyanate dye QSH-OH, whose structural formula is:

[0052] .

[0053] Example 2

[0054] The fabrication process of the probe QSH-NE is described in reference route 2.

[0055]

[0056] Route 2

[0057] The specific steps are as follows:

[0058] Synthesis of compound 6:

[0059] Pyrrolidine-1-carbonyl chloride (0.17 mL, 3 mmol) was added to a solution of 2-hydroxy-5-(hydroxymethyl)benzaldehyde (0.304 g, 2 mmol) and K₂CO₃ (0.414 g, 6 mmol) in acetonitrile (15 mL). The solution was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography with PE / AcOEt (4 / 1, v / v) as the eluent to give a colorless oily liquid. The yield was 59%, and the compound was named compound 6.

[0060] Synthesis of compound 7:

[0061] PBr3 was slowly added to a CH2Cl2 (6 mL) solution of compound 6 (0.348 g, 1.3 mmol) at 0 °C. After stirring at room temperature for 4 hours, the reaction solution was quenched with a saturated NaHCO3 solution (30 mL). The organic layer was dried with Na2SO4 and the solvent was removed by evaporation to obtain a white solid, which was named compound 7.

[0062] Synthesis of QSH-NE:

[0063] QSH-OH (0.05 g, 0.13 mmol) was added to a solution of compound 7 (0.06 g, 0.16 mmol) and K2CO3 (0.05 g, 0.36 mmol) in N,N-dimethylformamide (10 mL), and the mixture was stirred at 80 °C for 5 hours. The crude product was then purified by silica gel chromatography using CH2Cl2 / MeOH (v / v, 40:1) as the eluent to give a blue solid, named QSH-NE (yield 31%).

[0064] The probe QSH-NE prepared in this embodiment was detected, and the results are as follows: Figures 6 to 8 As shown, they are respectively: Figure 6 The probe QSH-NE described in dimethyl sulfoxide-d6 (DMSO-d6) 1 H NMR spectrum; Figure 7 The probe QSH-NE described in dimethyl sulfoxide-d6 (DMSO-d6) 13 C NMR spectrum;Figure 8 The image shows the ESI-MS spectrum of the probe QSH-NE described in this embodiment of the invention.

[0065] in, Figure 6 The hydrogen spectrum analysis is as follows: 1 H NMR (400 MHz, CD3OD) δ (ppm): 10.13(1H, s,CHO), 8.73 (1H, d, J = 4 Hz, Ar-H), 8.56 (1H, s, Ar-H), 8.38 (1H, d, J = 8.8Hz, Ar-H), 8.22 (1H, d, J = 7.9 Hz, Ar-H), 8.04 (1H, t, J = 7.6 Hz, Ar-H), 7.76-7.85 (3H, m, Ar-H), 7.61-7.65 (2H, m, Ar-H), 7.36 (1H, d, J = 8.4 Hz, Ar-H), 7.16 (1H, d, J = 2.5 Hz, =CH), 7.07 (1H, s, =CH), 6.84 (1H, d, J =10.4 Hz, =CH), 5.41 (2H, s, CH2), 4.33 (2H, t, J = 7.6 Hz, CH2), 3.83-3.96(2H, t, J = 12.8 Hz, CH2), 2.60-2.66 (4H, m, 2 × CH2), 1.69-1.75 (4H, m, 2 × CH2), 1.08-1.29 (2H, m, CH2).

[0066] Figure 7 The carbon spectrum analysis is as follows: 13 C NMR (100 MHz, DMSO-d6) δ (ppm): 190.93, 161.71,155.46, 154.18, 152.44, 150.75, 142.32, 141.62, 140.81, 139.37, 138.39,134.37, 132.70, 131.09, 130.66, 128.90, 127.24, 124.11, 121.10, 121.08,118.50, 115.57, 112.79, 112.79, 111.69, 101.22, 62.32, 56.32, 47.84, 46.86, 46.61, 31.57, 30.33, 29.54, 25.78, 25.55, 24.96.

[0067] Figure 8 The ESI-MS mass spectrometry resolution was: SH-NAL [MI] + The theoretical molecular weight is 615.77; the actual measured molecular weight is 615.35.

[0068] The above results prove that the photoacoustic probe QSH-NE has been successfully synthesized in this embodiment, and its structural formula is as follows:

[0069] .

[0070] Example 3

[0071] Performance analysis of the photoacoustic probe QSH-NE in in vitro detection of NE:

[0072] (1) Investigate the spectral characteristics of the prepared probe QSH-NE and its response to NE;

[0073] Specific experimental procedures: QSH-NE (10 μM) was incubated with different concentrations of NE (0-8 mM) in PBS / DMSO mixed solutions (10 mM, pH=7.4, v / v=1:1) at 37°C for 1 hour for photoacoustic detection. All UV-vis and fluorescence measurements were performed in PBS / DMSO (10 mM, pH=7.4, v / v=1:1) systems. The fluorescence spectral recording range was 780-890 nm, and the excitation wavelength was 760 nm. The excitation and emission slit widths were both 5 nm. PA imaging was captured on a multispectral photoacoustic tomography (MSOT) imaging system (inVision256 TF, iThera Medical GmbH) at wavelengths of 700-860 nm with 10 nm intervals.

[0074] Experimental results are as follows Figure 9 As shown. Figure 9 a. The PA signal of the QSH-NE detector is disrupted by the phenolic hydroxyl etherification reaction, causing the ICT process to be quenched. The specific reaction with NE reactivates the ICT process and releases the photoacoustic signal group QSH-OH. (e.g.) Figure 9 As shown in b, QSH-NE exhibits a maximum absorption wavelength at 585 nm. Upon addition of NE, the maximum absorption shifts to 760 nm, accompanied by a significant color change from deep blue to blue. The fluorescence spectrum shows a significant enhancement in response to NE in QSH-NE, exhibiting an 11.2-fold back ratio at 820 nm. Figure 9c). Further PA spectral analysis showed that QSH-NE initially produced very weak PA intensity, while NE incubation induced a significant increase in PA intensity at 760 nm, with a signal-to-background ratio of 6.2 times (c). Figure 9 d). These spectral changes can be attributed to a specific NE-mediated structural transformation from QSH-NE to QSH-OH. For example... Figure 9 As shown in e, the PA 760 intensity exhibits a concentration-dependent increase in response to NE. (R² = 0.991) The calculated detection limit is 0.035 mM between 0.8 and 6 mM concentrations.

[0075] (2) Investigate the time response characteristics of the prepared probe QSH-NE to NE;

[0076] Specific experimental procedures: QSH-NE (10 μM) and NE (5 mM) were rapidly mixed in a PBS / DMSO mixture (10 mM, pH 7.4, v / v = 1:1) and reacted at 37°C for 1 hour. The UV absorption intensity at 760 nm was recorded at different time points (0, 10, 20, 30, 40, and 60 minutes). Time-dependent measurements showed that the UV absorption intensity at 760 nm reached the reaction endpoint approximately 40 minutes after incubation of QSH-NE and NE. Figure 9 f).

[0077] (3) Investigate the response mechanism of the prepared probe QSH-NE to NE;

[0078] The response mechanism of QSH-NE to NE was then further explored.

[0079] Specific experimental procedures: QSH-NE (10 μM) and NE (5 mM) were reacted at 37℃ for 60 minutes, and the resulting product was subjected to high performance liquid chromatography (HPLC). A C18 column (250 mm × 4.6 mm, 5 μm) was used, with methanol / water (90:10 v / v) as the mobile phase, a flow rate of 1.0 mL / min, and a detection wavelength of 650 nm.

[0080] Experimental results are as follows Figure 9 As shown in g, after incubating QSH-NE with NE for 1 hour, a new chromatographic peak appeared at 3.925 min (consistent with the retention time of QSH-OH at 4.021 min), proving that QSH-OH was successfully released. These results fully validate the release mechanism of this NE-responsive photoacoustic probe under the action of NE.

[0081] Example 4

[0082] Investigate the photoacoustic imaging performance of the probe QSH-NE for intrinsic NE:

[0083] Based on the excellent responsiveness of the QSH-NE probe in vitro, this study further explored its application potential in endogenous NE in living cells and vivo. PC12 cells with high endogenous NE expression were selected as a positive model, and HeLa cells and HepG2 cells with low NE expression were used as negative controls.

[0084] Specific experimental steps:

[0085] (1) Photoacoustic imaging performance of probe QSH-NE on endogenous NE in cells;

[0086] Cell culture: PC12, HeLa, and HepG2 cells were cultured in DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin at 37 °C under humid conditions of 5% CO2. Cell density was determined using a TC20™ automated cell counter (BIO-RAD, USA).

[0087] Prior to photoacoustic imaging, cells were washed with PBS buffer and centrifuged to obtain a cell pellet. As expected, a significant PA 760 signal intensity was detected in PC12 cells incubated with QSH-NE. Figure 10 b). Negative control experiments with low NE expression further confirmed that the altered intracellular PA 760 signaling did indeed originate from NE activation. Notably, introducing different concentrations of NE into HeLa cells could trigger a PA response in a dose-dependent manner. Figure 10 c).

[0088] (2) Photoacoustic imaging performance of probe QSH-NE on in vivo endogenous NE;

[0089] A cervical cancer-bearing mouse model was constructed by subcutaneously injecting HeLa cells into BALB / c-nude mice. First, different doses of NE were added to the tumor, and then QSH-NE or PBS was injected into the tumor before in vivo PA imaging was performed. Figure 10 As shown in Figure d, compared with the PBS control group, the PA signal in the PBS group showed no significant change in the intratumoral injection QSH-NE experimental group, while in the experimental group, the PA signal in the intratumoral injection QSH-NE region gradually increased with increasing NE concentration. These results indicate that QSH-NE can serve as an effective tool for detecting NE in biological systems.

[0090] Example 5

[0091] SH-NAL probe used in in vivo diagnostic experiments for depression

[0092] (1) Probe safety assessment

[0093] Specific experimental procedures: Mice were euthanized 24 hours after tail vein injection of the probe QSH-NE, and major organs (including heart, liver, spleen, lungs, and kidneys) were harvested. H&E analysis procedure: Tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned; sections were dewaxed, hydrated, and stained with hematoxylin and eosin in sequence; after staining, the sections were washed with water, mounted, and images were acquired.

[0094] The safety of the probe was assessed using a hematoxylin-eosin (H&E) staining assay. No abnormal cell structures or signs of lesions were found in the heart, liver, spleen, lungs, and kidneys, confirming that the designed probe has low toxicity to normal organs and good biosafety.

[0095] (2) Construction of a corticosterone-induced depression model

[0096] This study used subcutaneous injection of corticosteroids, a classic drug that induces depression, to induce a mouse model of depression. This drug causes depression in more than half of clinical cases.

[0097] Specific experimental steps: C57 / B6 N mice were randomly divided into a control group and a model group. The control group was injected with physiological saline, while the model group was injected subcutaneously with corticosterone (10 mg / (kg·d)) for 21 consecutive days. Before imaging, the sucrose preference test (SPT), tail suspension test (TST), and forced swimming test (FST) were performed, and the hippocampus was stained with HE to verify the successful establishment of the depression model.

[0098] Compared with the normal group, the model mice in the depressed group showed significant behavioral changes, specifically a marked decrease in sucrose preference and a significantly prolonged immobility time. H&E staining showed significant morphological changes in the hippocampus of the model group mice. Figure 11 (b) The CA3 region shows disordered cell arrangement, while the dentate gyrus (DG) shows structural deformation, atrophy, and narrowing. As indicated by the black arrows, neurons in the DG and CA1 exhibit cytoplasmic hyperstaining and loss of internal structure, consistent with nuclear pyknosis and division. Furthermore, the red arrows indicate vacuolation near neurons, reflecting damage associated with neuroinflammation. These behavioral and pathological changes strongly support the successful construction of a corticosterone-induced depression model.

[0099] (3) Investigation on the photoacoustic imaging capability of QSH-NE to NE.

[0100] Specific experimental procedures: For in vivo PA imaging, all PA images of mice were captured using a multispectral photoacoustic tomography (MSOT) imaging system (inVision256 TF, iThera Medical GmbH). Each mouse was fixed with a two-dimensional slide, its brain was coated with medical gel, and it was placed in a 34°C water bath. The scanning wavelength was 700–860 nm with an interval of 10 nm. The entire brain was scanned at an interval of 0.3 mm. Guided ICA spectral unmixing was introduced to separate the signal from the activation probe and the signal from light absorbers in the tissue (such as hemoglobin). The average PA intensity was obtained from five different regions of interest (ROIs) in the brain using View MSOT.

[0101] like Figure 11 c and Figure 11 As shown in Figure d, the PA signal in healthy mice increases over time, peaks at approximately 2 hours post-injection, and then declines. It is noteworthy that the PA signal (PA) in the brains of healthy mice... QSH-NE / PA PBS = 5.1) significantly higher than depressed mice (PA) QSH-NE / PA PBS = 2.2)( Figure 11 e and Figure 11 (f) Changes in PA signaling can be explained by the different expression levels of NE in the two models, suggesting a correlation between decreased NE levels and depression. These results indicate that the probe can visually monitor the occurrence of depression through its specific PA response to NE in the brain.

[0102] (4) Exploration of the ability of QSH-NE to monitor drug therapy

[0103] C57 / B6 N mice were randomly divided into a healthy group, a depressed group, and a drug intervention group. The healthy and depressed groups received a tail vein injection of QSH-NE, while the drug intervention group received the selective serotonin reuptake inhibitor fluoxetine to improve depressive symptoms. Two hours after an intraperitoneal injection of fluoxetine (10 or 20 mg / kg) followed by a tail vein injection of QSH-NE, brain region PA imaging was performed in the mice. Figure 11 As shown in g, the PA signal was strongest in the healthy group and weakest in the depressed group. Compared with the depression control group, the PA signal in the fluoxetine treatment group was dose-dependently enhanced, suggesting that the brain NE level recovered after antidepressant intervention. Quantitative analysis confirmed that both the fluoxetine 10 mg / kg treatment group and the fluoxetine 20 mg / kg treatment group could enhance the PA760 intensity relative to the depression control group. Figure 11Specifically, the recovery rate of PA 760 intensity in depressed mice was 47% in the fluoxetine 10 mg / kg treatment group and 68% in the fluoxetine 20 mg / kg treatment group. These findings suggest that the effects of drug intervention on depression can be assessed by identifying specific PAs, and QSH-NE response signals to NE.

[0104] 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 photoacoustic probe for detecting norepinephrine, characterized in that, The probe was named QSH-NE, and its chemical structure is as follows: 。 2. A method for preparing a photoacoustic probe for detecting norepinephrine as described in claim 1, characterized in that, The preparation process is as follows: QSH-OH was added to a mixed solution of 4-(bromomethyl)-2-carboxyphenylpyrrolidine-1-carboxylate and K2CO3 in N,N-dimethylformamide, and the mixture was stirred at 80°C for 5 hours. The crude product was then purified by silica gel chromatography to obtain a blue solid, which was named QSH-NE.

3. According to the claims 2 The preparation method described above is characterized in that, The molar ratio of QSH-OH: 4-(bromomethyl)-2-carboxyphenylpyrrolidine-1-carboxylate: K2CO3 is 1.0:1.2:2.

8.

4. The preparation method according to claim 2, characterized in that, The purification process used CH2Cl2 / MeOH as the eluent, with a volume ratio of CH2Cl2 to MeOH of 40:

1.

5. The application of the photoacoustic probe for detecting norepinephrine as described in claim 1 in the preparation of products for detecting endogenous NE in live cells.

6. The use of the photoacoustic probe for detecting norepinephrine as described in claim 1 in the preparation of a product for non-invasive detection of the progression of depression in the brain of live animals and evaluation of the efficacy of drug intervention.

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