A molecular probe based on amphiphilic BODIPY dye self-assembly to enhance photoacoustic intensity and its preparation method and application

Abstract

The present invention discloses a method for preparing a molecular probe based on amphiphilic BODIPY dye self-assembly to enhance photoacoustic intensity and its application in evaluating anticancer drugs, Chinese medicine and clinical contrast agent-induced nephropathy models. The photoacoustic molecular probe BDP-3 designed by the present invention is a fluoroboron dipyrrole derivative having self-assembly properties by introducing an amphiphilic group. The present invention also provides a method for preparing the molecular probe BDP-3. Since BDP-3 has a good photoacoustic imaging effect and a particle size of less than 5nm, it can pass through the glomerular filtration membrane, so it is used as a photoacoustic imaging contrast agent to evaluate the degree of renal damage in nephropathy models induced by anticancer drugs, Chinese medicine and clinical contrast agents. The advantages of the present invention are that BDP-3 not only has a high renal clearance efficiency and good biocompatibility, but also uses a photoacoustic imaging system to monitor the degree of renal damage in the kidneys of living mice in real time and non-invasively, and has broad application prospects in biological imaging.

Description

Technical Field

[0001] The invention belongs to photoacoustic molecular probe technology, and specifically relates to a method for preparing a molecular probe based on amphiphilic BODIPY dye self-assembly to enhance photoacoustic intensity, and its application in evaluating anticancer drugs, traditional Chinese medicine and clinical contrast agent-induced nephropathy models. Background Art

[0002] There are many causes of kidney damage. For example, cis-dichlorodiamineplatinum (CDDP) is a widely used anticancer drug that can cause renal epithelial cell proliferation and podocyte fusion, leading to the occurrence of acute kidney injury. Aristolochic acid (AA) belongs to the nitrophenanthrene class of compounds. The content of this ingredient is high in some Chinese herbal medicines such as Aristolochia. At present, Chinese medicines containing AA are mostly used in the clinical treatment of rheumatic and immunological diseases and certain cancers. In recent years, with the in-depth study of AA, it has been found that improper use of AA can lead to the occurrence of Chinese herbal medicine nephropathy, which is gradually called aristolochic acid nephropathy (AAN).

[0003] For acute kidney injury, urine microalbuminuria, estimated glomerular filtration rate (eGFR) and renal biopsy are commonly used clinically to diagnose the extent of kidney disease, but they all have limitations: urine microalbuminuria is the main means of early diagnosis of kidney injury, but factors such as infection, fever, exercise and genetics can also affect urine microalbuminuria; the commonly used indicator of eGFR is serum creatinine, but it can be interfered with by a variety of physiological and pathological factors; renal puncture biopsy is the gold standard for diagnosing kidney injury, but it is an invasive examination and is difficult to use as a routine examination method.

[0004] Photoacoustic imaging is a non-invasive, low-cost imaging technology that has developed rapidly in recent years and relies on light absorption in biological tissues. It can make up for the defects of existing imaging modes and provide diagnostic information that is currently unavailable, such as tissue oxygenation levels and high-resolution vascular network information. It has broad application prospects in the biomedical field. Photoacoustic imaging is based on the photoacoustic effect. When a substance is irradiated by a continuous electromagnetic wave with a pulse width of a few nanoseconds or intensity modulation, it absorbs the energy of the electromagnetic wave and converts it into heat energy. Ultrasound is generated after instantaneous thermal expansion effects and tiny vibration relaxation. The carrier of light absorption information is not an optical signal but ultrasound, and the scattering of ultrasound in tissues is 2-3 orders of magnitude weaker than the scattering of optical signals. Therefore, when the distance is greater than the mean free path of light propagation in tissues, photoacoustic imaging can obtain higher acoustic resolution, and compared with traditional optical imaging technology, it can achieve high-resolution imaging representing optical absorption in deep tissues. Therefore, photoacoustic imaging technology shows great potential in the field of biological diagnosis and treatment.

[0005] However, existing contrast agents are usually used for cardiac diagnosis or interventional surgery. There is a lack of contrast agents for evaluating renal injury, and existing contrast agents have safety issues. Contrast-induced nephropathy is one of the most common causes of acute kidney injury in hospitals, with significant morbidity and mortality in the short or long term.

[0006] Therefore, there is an urgent need to provide a photoacoustic contrast agent with strong photoacoustic response and high safety for the detection of renal injury. Summary of the invention

[0007] In response to the problems existing in the prior art, the present application provides a method for preparing a molecular probe based on the self-assembly of amphiphilic BODIPY dyes to enhance the photoacoustic intensity, and provides its application in the evaluation of anticancer drugs, traditional Chinese medicine and clinical contrast agent-induced kidney disease models.

[0008] The first object of the present invention is to provide a molecular probe based on amphiphilic BODIPY dye self-assembly to enhance the photoacoustic intensity.

[0009] The second object of the present invention is to provide a method for preparing a molecular probe based on the self-assembly of amphiphilic BODIPY dye to enhance the photoacoustic intensity.

[0010] The third object of the present invention is to provide a molecular probe based on amphiphilic BODIPY dye self-assembly to enhance photoacoustic intensity for use in evaluating anticancer drugs, traditional Chinese medicine and clinical contrast agent-induced renal disease models.

[0011] The technical solution of the present invention is as follows:

[0012] A molecular probe BDP-3, wherein the molecular probe BDP-3 has a molecular formula of C 63 H 69 BF2N4O8S2, the structural formula is as follows:

[0013]

[0014] The preparation method of the aforementioned molecular probe BDP-3 and the preparation method of the photoacoustic molecular probe are as follows:

[0015]

[0016] The preparation method comprises the following steps:

[0017] (1) Compound BDP-0 (Zhu; Zhang, Chem. Commun., 2011, 47, 3508-3510) and N-iodosuccinimide were dissolved in dichloromethane and stirred at room temperature. The obtained product was washed with a saturated sodium sulfate solution and extracted with dichloromethane. The organic phase obtained by extraction was subjected to rotary evaporation to remove the solvent to produce a crude product, which was further purified by silica gel column chromatography to obtain compound BDP-1;

[0018] The structural formula of BDP-0 is The structural formula of BDP-1 is

[0019] (2) dissolving the compound BDP-1 obtained in step (1) in toluene, adding p-dimethylaminobenzaldehyde, piperidine and p-toluenesulfonic acid crystals to obtain a mixture, heating the mixture with stirring for reaction, cooling the mixture to room temperature and evaporating under reduced pressure; washing the obtained product with ultrapure water and extracting with dichloromethane; the organic phase obtained by extraction is subjected to rotary evaporation to remove the solvent to produce a crude product, which is further purified by silica gel column chromatography to obtain compound BDP-2;

[0020] The structural formula of BDP-2 is

[0021] (3) After mixing dehydrated tetrahydrofuran and diisopropylamine, the mixture is continuously purged with nitrogen; the compound BDP-2 obtained in step (2), 2-ethynylthiophene, tetrakis(triphenylphosphine)palladium and copper iodide are added to obtain a mixture; after the mixture is stirred for reaction in nitrogen, the mixture is cooled to room temperature and the solvent is evaporated under reduced pressure; the crude product is purified by silica gel column chromatography to obtain compound BDP-3.

[0022] Furthermore, the eluent used for the silica gel column chromatography purification in step (1), step (2) and step (3) is dichloromethane / acetone / petroleum ether, with a volume ratio of 1:1:2.

[0023] Furthermore, the molar ratio of the compound BDP-0 (Zhu; Zhang, Chem. Commun., 2011, 47, 3508-3510) and N-iodosuccinimide in step (1) is 1:4-5; the stirring time is 3-5h; and the volume ratio of dichloromethane used for extraction in step (1) to the solution to be extracted is 1-2:1.

[0024] Furthermore, the concentration of the compound BDP-1 in step (2) in toluene is 15-17 μM, the molar ratio of the compound BDP-1, p-dimethylaminobenzaldehyde and toluenesulfonic acid crystals is 1:2-3:1, and the volume ratio of toluene and piperidine is 30:1.

[0025] Furthermore, the mixture in step (2) is heated and stirred at a temperature of 130-150° C. for 5-10 min, and then cooled to room temperature. The volume ratio of ultrapure water to dichloromethane and the mixture in step (2) is 1:1:1, and the number of dichloromethane extractions is 3 times.

[0026] Furthermore, in step (3), the nitrogen purge time is 15-20 minutes.

[0027] Furthermore, in step (3), the volume ratio of tetrahydrofuran to diisopropylamine is 2:1; the amount percentage of compound BDP-2, 2-ethynylthiophene, tetrakis(triphenylphosphine)palladium and copper iodide is 10:25:1:1.

[0028] Furthermore, the stirring in step (3) is stirring the mixture at 55-65° C. for 10-15 h.

[0029] The use of the aforementioned molecular probe BDP-3 in the preparation of a photoacoustic imaging contrast agent for detecting the changing trend of the degree of renal damage.

[0030] Furthermore, the kidney injury is drug-induced acute kidney injury.

[0031] In certain specific embodiments, the drug-induced acute kidney injury is drug-induced acute kidney injury induced by anticancer drugs, traditional Chinese medicine or clinical contrast agents.

[0032] Furthermore, the application is to intravenously inject the molecular probe BDP-3 into the organism to be evaluated, and use the photoacoustic imaging method to continuously observe the photoacoustic signal of the kidney of the organism to be evaluated at 720nm to detect the changing trend of the degree of kidney damage; when the photoacoustic signal increases during the continuous observation period, it indicates that the degree of kidney damage has worsened, and when the photoacoustic signal decreases during the continuous observation period, it indicates that the degree of kidney damage has decreased.

[0033] Furthermore, the molecular probe BDP-3 is prepared into a probe solution with a concentration of 80-100 μM of the molecular probe BDP-3 using DMSO / H2O with a water content of 85-95%, and then the solution is injected into the organism to be evaluated through the tail vein.

[0034] Furthermore, the molecular probe BDP-3 can undergo specific changes in solutions with different water contents. Specifically, the specific change refers to the size change of the amphiphilic BDP-3 in solutions with different water contents due to self-assembly into small molecular polymers due to J-aggregation. As the water content increases from 40% to 90%, the hydrodynamic diameter of the water-soluble BDP-3 probe increases from 0.64±0.11nm to 3.74±0.39nm.

[0035] Furthermore, the photoacoustic molecular probe BDP-3 exhibits different photoacoustic signals in solutions with different water contents due to aggregation and self-assembly into small molecule polymers. As the water content increases from 0% to 90%, the ultraviolet absorption peak of BDP-3 red-shifts from 768 nm to 800 nm, and the transition point is at a water content of 40%; the peak wavelength of the photoacoustic signal spectrum red-shifts from 680 nm to 720 nm, and the photoacoustic intensity increases by 2.06 times.

[0036] The molecular probe BDP-3 of the present application has the property of enhancing the photoacoustic signal after being dissolved in water, and the particle size is less than 5 nm. Based on this property, it is used in the preparation of a photoacoustic imaging contrast agent for detecting the changing trend of the degree of kidney damage.

[0037] Furthermore, a molecular probe BDP-3 with a water content of 90% and a concentration of 100 μM was intravenously injected into the individual to be evaluated, and the photoacoustic imaging method was immediately used to continuously observe the photoacoustic signal of the kidney of the individual to be evaluated, and the strength of the photoacoustic signal was used to evaluate the degree of kidney damage. The enhanced photoacoustic signal represents the enrichment of the photoacoustic molecular probe BDP-3 in the kidney. The main reason is that when the kidney is damaged, the renal blood flow slows down, so that BDP-3 cannot be excreted from the body and accumulates in the kidney lesions; the weakened photoacoustic signal represents that the photoacoustic molecular probe BDP-3 is cleared in the kidney, which means that the severity of kidney damage is reduced.

[0038] In a specific embodiment, the photoacoustic imaging method uses a photoacoustic imaging system, wherein the photoacoustic conditions are: frequency (Frequency): 21MHz, frame delay (Frame Delay): 4, photoacoustic parameter (PAMode Gain): 32dB; B-mode parameter (B-Mode Gain): 22dB, duration (Persisitence): 6, wavelength (Wavelength): 720nm, scanning depth (Depth): 12mm, photoacoustic acquisition mode (PAAcquisition): Single, response gate (Resp Gate): Off, energy (Correct Energy): on, and the photoacoustic imaging is performed immediately after the injection of the photoacoustic molecular probe BDP-3. The photoacoustic imaging duration includes but is not limited to 120min.

[0039] The beneficial effects of the present invention are:

[0040] Provided is a photoacoustic molecular probe BDP-3 with dipyrromethene boron difluoride (BODIPY) as a parent core, wherein the BDP-3 is a fluoroboron dipyrrole derivative having self-assembly properties by introducing an amphiphilic group; the photoacoustic molecular probe of the present invention will self-assemble into a small molecule polymer in solutions with different water contents to enhance the photoacoustic intensity at 720 nm, so that it can be used as a photoacoustic imaging contrast agent to evaluate the trend of changes in the degree of renal damage. The photoacoustic molecular probe of the present invention has a simple molecular preparation process and high yield, and is suitable for large-scale production; the photoacoustic molecular probe of the present invention is used as a photoacoustic imaging contrast agent to evaluate the degree of renal damage in a renal disease model induced by anticancer drugs, traditional Chinese medicine and clinical contrast agents, and the photoacoustic imaging effect is good. Since its particle size is less than 5nm, it can pass through the glomerular filtration membrane, has a high renal clearance rate, and can monitor renal damage in real time and non-invasively using a photoacoustic imaging system. The evaluation method is simple to operate and easy to promote, and has broad application prospects in biological imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 is the H NMR spectrum of BDP-1 obtained in the present invention;

[0042] Figure 2 is the nuclear magnetic hydrogen spectrum of BDP-2 obtained in the present invention;

[0043] Figure 3 is the HR-ESI mass spectrum of BDP-2 obtained in the present invention;

[0044] Figure 4 is the NMR carbon spectrum of BDP-3 obtained in the present invention;

[0045] Figure 5 HR-ESI mass spectrum of BDP-3 obtained in the present invention;

[0046] Figure 6 This is a graph showing the particle size variation of BDP-3 obtained in the present invention when the water content ranges from 40% to 90%;

[0047] Figure 7 The ultraviolet absorption variation diagram of BDP-3 obtained by the present invention when the water content ranges from 10% to 90%;

[0048] Figure 8 This is a photoacoustic signal change diagram of BDP-3 obtained in the present invention when the water content ranges from 10% to 90%;

[0049] Fig. 9 This is a graph showing the toxicity test results of BDP-3 obtained in the present invention on HK-2 cells;

[0050] Fig.10This is the in vivo biocompatibility test result of BDP-3 obtained by the present invention: Fig.10 a is a blank group, Fig.10 b: BDP-3 injection group;

[0051] Fig.11 This is the photoacoustic imaging of BDP-3 in the mouse kidney area obtained by the present invention: Fig.11 a is a blank group, Fig.11 d is the 24h cisplatin modeling group, Fig.11 e is the 48h cisplatin modeling group, Fig.11 f is the cisplatin modeling group for 72 hours, Fig.11 b is aristolochic acid nephropathy group, Fig.11 c, diatrizoate group;

[0052] Fig.12 This is a graph showing the change of the photoacoustic signal value of BDP-3 in the mouse kidney region obtained by the present invention over time: Fig.12 a is the blank group, cisplatin modeling 24h group, cisplatin modeling 48h group, cisplatin modeling 72h group, Fig.12 b is aristolochic acid nephropathy group, Fig.12 c, diatrizoate group;

[0053] Fig.13 This is the H&E staining image of BDP-3 obtained in the present invention in mouse kidney: Fig.13 a is the blank group, and Figure 13d is the cisplatin modeling 24h group, Fig.13 e is the 48h cisplatin modeling group, Fig.13 f is the cisplatin modeling 72h group, and Figure 13b is the aristolochic acid nephropathy group, Fig.13 c, diatrizoate group; DETAILED DESCRIPTION

[0054] The following embodiments of the present invention are described in further detail in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but should not be regarded as limiting the scope of the present invention.

[0055] In the embodiments of the present invention, "mM" refers to mmol / L.

[0056] Example 1 Preparation of BDP-1:

[0057] Compound BDP-0 (324.0 mg, 0.50 mM) (Zhu; Zhang, Chem. Commun., 2011, 47, 3508-3510) and N-iodosuccinimide (450.0 mg, 2 mM) were dissolved in dichloromethane (100 mL) and stirred at room temperature. The obtained product was washed with a saturated sodium sulfate solution and extracted with dichloromethane, wherein the volume ratio of dichloromethane to the solution to be extracted was 1:1. The extracted organic phase was evaporated by a rotary evaporator to produce a crude product, which was further purified by silica gel column chromatography, and the eluent was dichloromethane / acetone / petroleum ether in a ratio of 1:1:2 to obtain compound BDP-1 (375.0 mg, 84%).

[0058] The structural formula of BDP-0 is The structural formula of BDP-1 is

[0059] The H NMR spectrum of BDP-1 prepared in Example 1 is as follows: Figure 1 shown. 1 H NMR (CDCl3, 600MHz, ppm), δ7.02 (d, J=12Hz, 1H), 6.80 (d, J=3Hz, 1H), 6.76 (dd, J=3, 2.4Hz, 1H), 4.25-4.20 (m, 2H), 4.15-4.10 (m,2H),3.94-3.90(m,2H),3.88-3.83(m,2H),3.78(m,2H),3.74-3.54(m ,12H),3.52(m,2H),3.39(s,3H),3.36(s,3H),2.63(s,6H),1.47(s,6H).

[0060] Example 2 Preparation of BDP-2:

[0061] Compound BDP-1 (450.0 mg, 0.50 mM) was dissolved in 30 mL of toluene to make the concentration of compound BDP-1 in toluene 16.6 μM, and p-dimethylaminobenzaldehyde (178.9 mg, 1.2 mM), piperidine (1 mL) and p-toluenesulfonic acid crystals (10 mg) were added to obtain a mixture, wherein the molar ratio of compound BDP-1, p-dimethylaminobenzaldehyde and toluenesulfonic acid crystals was 1:2:1, and the volume ratio of toluene and piperidine was 30:1; the mixture was stirred at 140°C for 5 min, cooled to room temperature, and evaporated under reduced pressure; the product was washed with ultrapure water and extracted with dichloromethane, 100 mL each time, for 3 times; the organic phase obtained by extraction was rotary evaporated to remove the solvent to produce a crude product, which was further purified by silica gel column chromatography with dichloromethane / acetone / petroleum ether in a ratio of 1:1:2 to obtain compound BDP-2 (302.1 mg, 52%).

[0062] H NMR spectrum and HR-ESI mass spectrum of BDP-2 prepared in Example 2 Figure 2 , 3 As shown, Figure 2 is the result of H NMR spectrum, Figure 3 The results of HR-ESI mass spectrum. 1 H NMR (400MHz, DMSO-d6, ppm), 8.11 (d, J=16.4Hz, 2H), 7.46 (d, J=8.8Hz,4H),7.37(d,J=16.4Hz,2H),7.16(d,J=8.4Hz,1H),7.06(d,J=2Hz,1H),6.90(dd ,J=2,1.6Hz,1H),6.82(d,J=9.2Hz,4H),4.20-4.16(m,2H),3.83-3.77(m,2H),3.74-3.70 (m,2H),3.64(m,2H),3.59-3.43(m,12H),3.40-3.38(m,2H),3.25(s,3H),3.21(s,3H),3.02(s,12H),1.50(s,6H).HRMS(ESI + ):m / z calcd for C 51 H 63 BF2I2N4O8[M+H + ]:1162.2796; found:1162.2781.

[0063] Example 3 Synthesis of Photoacoustic Molecular Probe BDP-3:

[0064] After mixing 10 mL of dehydrated tetrahydrofuran and 5 mL of diisopropylamine, the mixture was purged with nitrogen for 15 min; the compound BDP-2 (232.4 mg, 0.2 mM) obtained in step (2), 2-ethynylthiophene (54.0 mg, 0.5 mM), tetrakis(triphenylphosphine)palladium (21.1 mg, 0.02 mM) and copper iodide (3.8 mg, 0.02 mM) were added to obtain a mixture; the mixture was stirred at 60° C. for 12 h in nitrogen, and then the mixture was cooled to room temperature and evaporated under reduced pressure to remove the solvent; the crude product was purified by silica gel column chromatography, the eluent was dichloromethane / acetone / petroleum ether, and the ratio was 1:1:2, to obtain compound BDP-3 (145.8 mg, 65%).

[0065] Example 3: H NMR spectrum and HR-ESI mass spectrum of BDP-3 prepared Figure 4 , 5 As shown, Figure 4 is the result of H NMR spectrum, Figure 5 The results of HR-ESI mass spectrum. 1 H NMR (400MHz, CDCl3, ppm), 8.44–8.27 (d, J=16.4Hz, 2H), 7.71 –7.64(m,2H),7.61(d,J=8.8Hz,4H),7.30(dd,J=1.2,1.2Hz,2H),7.25(d,J=0.8Hz,2H), 7.03(m,3H),6.90(d,J=2Hz,1H),6.87(dd,J=2,1.2Hz,1H),6.83–6.66(m,4H),4.25(t,J= 4.8Hz,2H),4.17(t,J=4.8Hz,2H),3.97–3.91(m,2H),3.90–3.84(m,2H),3.79(m,2H),3.75 –3.56(m,12H),3.51(m,2H),3.39(s,3H),3.34(s,3H),3.05(s,12H),1.62(s,6H).HRMS(ESI + ):m / z calcd for C 63 H 69 BF2N4O8S2[M+H + ]:1122.4618; found:1122.4586.

[0066] Example 4 Changes in particle size, UV and photoacoustic properties of photoacoustic molecular probe BDP-3 in solutions with different water contents:

[0067] The polymerization behavior of BDP-3 in solutions with different water contents was studied by adjusting the ratio of dimethyl sulfoxide and water in the solvent (0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% DMSO / H2O). The water content was prepared from 0% to 90%, and the probe BDP-3 was dissolved in the prepared solution at a concentration of 100μM. The experimental results are shown in Figure 6 , 7 and 8. The aggregation of BDP-3 probes was directly detected by dynamic light scattering. The hydrodynamic diameter of BDP-3 probes increased from 0.64±0.11nm to 3.74±0.39nm as the water content increased from 40% to 90% ( Figure 6 As the water content increases from 10% to 90%, the UV absorption peak of 100 μM BDP-3 red-shifts from 768 nm to 800 nm, with the transition point at 40% ( Figure 7 ). Then, the effect of BDP-3 aggregation on photoacoustic intensity was measured in vitro at water contents ranging from 10% to 90%. The peak wavelength of the spectrum red-shifted from 680nm to 720nm during aggregation. Importantly, the photoacoustic intensity increased by 2.06 times during the aggregation process when the water content increased from 40% to 90% ( Figure 8 ). Therefore, BDP-3 may self-assemble into small-molecule nanoaggregates, which can generate photothermal effects and amplify the amplitude of photoacoustic signals.

[0068] Example 5 Study on the Cytotoxicity of Photoacoustic Probe BDP-3:

[0069] Normal human kidney cells HK-2 cells (Jiangsu KeyGen Biotechnology Co., Ltd.) were cultured at 1×10 4 Cells were inoculated into a 96-well plate, with 200 μL of culture medium in each well, and the edge wells were filled with sterile phosphate buffer solution (PBS). The plates were cultured in a 37°C, 5% CO2 incubator for 24 hours, and the cell morphology was observed under an inverted microscope to ensure that the cells were in good growth condition. After the culture medium was removed and rinsed with PBS, 20 μL of BDP-3 aqueous solution with different concentration gradients (20, 40, 60, 80, 100 μM) and 180 μL of DMEM medium were added, and the plates were incubated at 37°C for 24 hours. After the incubation, 200 μL of MTT solution (0.5 mg / mL) was added to each well, and the plates were incubated at 37°C for 4 hours. The MTT solution was removed, and 150 μL of dimethyl sulfoxide (DMSO) was added to each well for dissolution, and the plates were shaken for 10 minutes. The 96-well plate was placed on a microplate reader, and the absorbance (OD) value of the solution in each well was read at a dual wavelength of 570 nm for detection and 650 nm for reference.

[0070] Cell viability (%) = (OD value of sample / OD value of blank) × 100%. The experimental results are as follows: Fig. 9 As shown, after BDP-3 and cells were co-cultured for 24 hours, the cell survival rate was greater than 80%, and the BDP-3 prepared by the present invention had low cytotoxicity.

[0071] Example 6 In vivo biocompatibility analysis of photoacoustic probe BDP-3:

[0072] Male ICR mice were selected and divided into a blank group and a model group. The blank group received a single tail vein injection of 100 μL of normal saline, and the model group received a single tail vein injection of an equal volume of BDP-3 aqueous solution (100 μM). 24 hours after the tail vein injection, the blank group and the model group mice were treated by neck extension, and the heart, liver, spleen, lung, kidney, and intestine were quickly removed and fixed with 4% paraformaldehyde. The fixed heart, liver, spleen, lung, kidney, and intestine were embedded, paraffin sections were made, and conventional H&E staining was performed. The pathological changes of the sliced ​​tissue were observed using a digital pathology section scanner. The results are as follows Fig.10 As shown, the heart, liver, spleen, lung, kidney, and intestinal tissues in the experimental group had good morphology and no obvious abnormalities, which further proved that BDP-3 has good biocompatibility.

[0073] Example 7 Animal grouping and construction of drug-induced acute kidney injury model:

[0074] Male ICR mice were selected and randomly divided into 6 groups:

[0075] 1) Blank group (n=3): continuous intragastric administration of 0.9% saline (0.1 mL / 10 g) for 14 days, and a single intraperitoneal injection of saline (0.1 mL / 10 g) on ​​days 11-14;

[0076] 2) Cisplatin 24h group (n=3): continuous intragastric administration of 0.9% saline (0.1 mL / 10 g) for 14 days, and a single intraperitoneal injection of cisplatin (20 mg / kg) on ​​the 13th day;

[0077] 3) Cisplatin 48h group (n=3): continuous intragastric administration of 0.9% saline (0.1 mL / 10 g) for 14 days, and a single intraperitoneal injection of cisplatin (20 mg / kg) on ​​the 12th day;

[0078] 4) Cisplatin 72h group (n=3): continuous intragastric administration of 0.9% saline (0.1 mL / 10 g) for 14 days, and a single intraperitoneal injection of cisplatin (20 mg / kg) on ​​the 11th day;

[0079] 5) Aristolochic acid nephropathy group (n=3), continuous gavage of 0.9% saline (0.1 mL / 10 g) for 14 days, and a single intraperitoneal injection of aristolochic acid (75 mg / kg) on ​​the 11th day;

[0080] 6) The diatrizoate group (n=3) was given 0.9% saline (0.1 mL / 10 g) by intragastric administration for 14 consecutive days, and a single intraperitoneal injection of diatrizoate (120 mg / kg) was given on the 11th day.

[0081] Example 8 Photoacoustic imaging of photoacoustic probe BDP-3 in mouse kidney:

[0082] After intraperitoneal injection of the modeling agent for different time periods, the mice were anesthetized, and the prepared BDP-3 aqueous solution (100 μM) was injected into the mice through the tail vein. Immediately, the photoacoustic imaging system was used to detect the kidney area for 120 min, and the photoacoustic conditions were: frequency (Frequency): 21 MHz, frame delay (Frame Delay): 4, photoacoustic parameter (PAMode Gain): 32 dB; B-mode parameter (B-Mode Gain): 22 dB, duration (Persisitence): 6, wavelength (Wavelength): 720 nm, scanning depth (Depth): 12 mm, photoacoustic acquisition mode (PA Acquisition): Single, response gate (RespGate): Off, energy cut (Correct Energy): on, and the test results are shown in Figure 2. Fig.11 , 12 As shown in the figure, with the increase of imaging time and modeling time of the cisplatin model group, the photoacoustic signal of the cisplatin model group became stronger and stronger, indicating that drug-induced acute kidney injury gradually worsened ( Fig.11 d, e, f, Fig.12 a); Aristolochic acid nephropathy model group ( Fig.11 b, Fig.12 b) and the diatrizoate group ( Fig.11 c, Fig.12 c) The photoacoustic signals were strongest at 15 min and then gradually weakened, and the imaging agent clearance efficiency of the two model groups was lower than that of the control group, indicating the accumulation of BDP-3 in the aristolochic acid nephropathy model group and the diatrizoate group and the gradual clearance process. This further proved that the filtration efficiency of the aristolochic acid nephropathy model group and the diatrizoate group was lower than that of the blank group, and more serious renal damage occurred. It also proved that BDP-3 has a good photoacoustic imaging effect and can be used as a photoacoustic imaging contrast agent to evaluate kidney damage in kidney disease models induced by anticancer drugs, traditional Chinese medicine and clinical contrast agents.

[0083] Example 9 H&E staining and changes in functional biochemical indicators of mouse kidneys:

[0084] Venous blood was drawn from the orbits of living ICR mice under isoflurane anesthesia; renal tissue samples were stained with H&E and then imaged on a digital slide scanner. Fig.13 As shown in the figure, with the increase of cisplatin modeling time, the renal injury was most severe at 72h of cisplatin modeling, with necrosis and degeneration of renal tubular epithelial cells and atrophy of glomeruli ( Fig.13 d, e, f); At the same time, the renal injury in the aristolochic acid nephropathy model group and the diatrizoate meglumine group was aggravated compared with the control group, with atrophy of the glomeruli and vacuolar changes in the renal tubular epithelial cells ( Fig.13 b, c); Renal functional indicators in cisplatin-induced acute kidney injury showed that the kidney injury was the most serious at 72 hours. The kidney functional indicators BUN and Scr in the aristolochic acid nephropathy model group and the diatrizoate group were higher than those in the blank group, indicating that kidney injury occurred. The same as the results of photoacoustic imaging, further verified that the photoacoustic imaging system can detect kidney injury in the kidney disease model induced by anticancer drugs, traditional Chinese medicine and clinical contrast agents in real time, in situ and sensitively.

[0085] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. It should be pointed out that for ordinary technicians in this technical field, improvements or changes can be made based on the above description, and these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A method for preparing a molecular probe BDP-3, characterized in that: The molecular probe BDP-3 structural formula is as follows: The method comprises the following steps: (1) Compound BDP-0 and N-iodosuccinimide were dissolved in dichloromethane and stirred at room temperature. The obtained product was washed with a saturated sodium sulfate solution and extracted with dichloromethane. The organic phase obtained by extraction was subjected to rotary evaporation to remove the solvent to produce a crude product, which was further purified by silica gel column chromatography to obtain compound BDP-1; The structural formula of BDP-0 is The structural formula of BDP-1 is (2) dissolving the compound BDP-1 obtained in step (1) in toluene, adding p-dimethylaminobenzaldehyde, piperidine and p-toluenesulfonic acid crystals to obtain a mixture, heating the mixture with stirring for reaction, cooling the mixture to room temperature and evaporating under reduced pressure; washing the obtained product with ultrapure water and extracting with dichloromethane; the organic phase obtained by extraction is subjected to rotary evaporation to remove the solvent to produce a crude product, which is further purified by silica gel column chromatography to obtain compound BDP-2; The structural formula of BDP-2 is (3) After mixing dehydrated tetrahydrofuran and diisopropylamine, the mixture is continuously purged with nitrogen; the compound BDP-2 obtained in step (2), 2-ethynylthiophene, tetrakis(triphenylphosphine)palladium and copper iodide are added to obtain a mixture; after the mixture is fully stirred in nitrogen for reaction, the mixture is cooled to room temperature and the solvent is evaporated under reduced pressure; the crude product is purified by silica gel column chromatography to obtain compound BDP-3.

2. The preparation method according to claim 1, characterized in that: The eluent used for the silica gel column chromatography purification in step (1), step (2) and step (3) is dichloromethane / acetone / petroleum ether, with a volume ratio of 1:1:

2.

3. The preparation method according to claim 1, characterized in that: The molar ratio of the compound BDP-0 and N-iodosuccinimide in step (1) is 1:4-5; the stirring time is 3-5h; and the volume ratio of the dichloromethane used for extraction in step (1) to the solution to be extracted is 1-2:

1.

4. The preparation method according to claim 1, characterized in that: The concentration of the compound BDP-1 in step (2) in toluene is 15-18 μM, the molar ratio of the compound BDP-1, p-dimethylaminobenzaldehyde and toluenesulfonic acid crystals is 1:2-3:1, and the volume ratio of toluene and piperidine is 30:

1.

5. The preparation method according to claim 1, characterized in that: The mixture in step (2) is heated and stirred at a temperature of 130-150° C. for 5-10 min, and then cooled to room temperature.

6. The preparation method according to claim 1, characterized in that: In step (3), the volume ratio of tetrahydrofuran to diisopropylamine is 2:1; the amount percentage of compound BDP-2, 2-ethynylthiophene, tetrakis(triphenylphosphine)palladium and copper iodide is 10:25:1:

1.

7. The preparation method according to claim 1, characterized in that: The stirring in step (3) is stirring the mixture at 55-65° C. for 10-15 h.

8. The preparation method according to claim 1, characterized in that: The nitrogen purge time in step (3) is 15-20 min.

9. Use of the molecular probe BDP-3 prepared according to claim 1 in the preparation of a photoacoustic imaging contrast agent for detecting the changing trend of the degree of renal damage, wherein the renal damage is drug-induced acute renal injury, and the molecular probe BDP-3 is prepared into a probe solution with a concentration of 80-100 μM by using DMSO / H2O with a water content of 85-95%, and then the solution is injected into the tail vein of the organism to be evaluated.

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