Photoacoustic probe as well as preparation method and application thereof

Abstract

The invention discloses a photoacoustic probe as well as a preparation method and application thereof, and relates to the technical field of biomedicine. The invention provides an application of a photoacoustic molecular probe connected with targeted unwound collagen in preparation of a product for detection or auxiliary detection of cardiovascular diseases, or in preparation of a product for prediction or auxiliary prediction of aneurysm rupture risk. Experiments prove that the photoacoustic probe connected with the targeted unwinding collagen can be used for non-invasive photoacoustic ultrasonic imaging detection of vascular structure dysfunction diseases such as aneurysm and the like, and non-invasive early detection and rupture risk assessment of abdominal aortic aneurysm, coronary arterial aneurysm, cervical aneurysm and the like can be realized; and a powerful tool is provided for early intervention and precise treatment of cardiovascular diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a photoacoustic probe and a preparation method and application thereof. Background Art

[0002] Abdominal aortic aneurysm (AAA) is one of the most common aortic diseases. Its most serious consequence comes from rupture. The mortality rate of patients with AAA rupture is close to 90%. Up to 50% of patients with AAA rupture have died when they arrive at the operating room, and the perioperative mortality rate of patients with rupture is also as high as 50%. Since most AAAs have no symptoms before rupture, many patients are already in a very serious condition when they discover AAA. There is currently no drug that can reverse or stop the progression of AAA disease. If AAA is not discovered early and intervened in time, once AAA reaches the late stage, the risk of emergency surgery will greatly increase.

[0003] At present, arterial diameter is used as a diagnostic indicator of AAA and a standard for assessing rupture risk in clinical practice. Ultrasound and computed tomography angiography are often used to continuously evaluate the aortic anatomical structure, and the diameter and morphology of the aneurysm are relied on to stratify the severity of the disease to achieve AAA monitoring. However, since the diameter expansion of AAA does not progress linearly, ultrasound imaging is not sufficient to provide timely and accurate AAA prognosis, and predicting AAA rupture is still very challenging. In addition, since most AAAs are asymptomatic before rupture, there is an urgent need for effective detection technology to accurately assess the integrity of deep blood vessels and monitor the progression and rupture risk of AAA, so as to stratify the disease risk and intervene in high-risk individuals in a timely and effective manner. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes the use of a photoacoustic probe connected with an unwinding collagen targeting molecule in the preparation of a product for detecting or assisting in the detection of cardiovascular diseases or in the preparation of a product for predicting or assisting in predicting the risk of aneurysm rupture.

[0005] The present application also proposes a method for preparing a photoacoustic probe that can identify the integrity of vascular wall collagen tissue.

[0006] The invention also provides a photoacoustic probe prepared by the preparation method.

[0007] The invention also provides a kit.

[0008] In a first aspect of the present invention, a photoacoustic probe connected with an unwinding collagen targeting molecule is provided for use in any one of A1) to A2);

[0009] A1) Preparation of products for detecting or assisting in the detection of cardiovascular diseases;

[0010] A2) Prepare a product that predicts or assists in predicting the risk of aneurysm rupture.

[0011] The present invention has at least the following beneficial effects:

[0012] The photoacoustic probe connected to the unhelical collagen targeting molecule can specifically bind to the vascular disease tissue where the collagen molecules are unhelicaled, and can be used for non-invasive photoacoustic ultrasound imaging detection of vascular structural and functional disorders such as aneurysms. It can achieve non-invasive early detection and rupture risk assessment of AAA, providing a powerful tool for early intervention and precise treatment of clinical AAA, and is expected to play a role in the precise detection of various other cardiovascular diseases with collagen tissue damage (including but not limited to abdominal aortic aneurysm, intracranial aneurysm, aortic dissection, myocardial infarction, atherosclerosis, valvular disease, etc.), and can provide in vivo warning of the risk of aneurysm rupture.

[0013] The photoacoustic probe of the embodiment can deeply penetrate biological tissues, has high resolution and high sensitivity, and can detect arterial dilatation in the early stage of aneurysm (diameter thickening less than 50%) in vivo, etc., and has great potential application value in early and accurate tracing of deep tissues such as AAA.

[0014] In some embodiments of the present invention, the photoacoustic detection product has at least one of the functions of detecting cardiovascular disease and predicting the risk of aneurysm rupture.

[0015] In some embodiments of the present invention, the cardiovascular disease includes at least one of abdominal aortic aneurysm, intracranial aneurysm, carotid artery aneurysm, aortic dissection, myocardial infarction, atherosclerosis, and valvular disease.

[0016] In some embodiments of the present invention, the aneurysm includes, but is not limited to, an abdominal aortic aneurysm, an intracranial aneurysm, or a carotid artery aneurysm.

[0017] A second aspect of the present invention provides a method for preparing a photoacoustic probe, comprising the following steps:

[0018] S1, preparing a mixture of a serum albumin aqueous solution, a cyanine dye, a dehydrating solvent, and a cross-linking agent, and reacting the mixture to obtain serum albumin-cyanine dye nanoparticles;

[0019] The dehydration solvent includes at least one of acetone, methanol, acetonitrile, dichloromethane and tetrahydrofuran;

[0020] S2. The serum albumin-cyanine dye nanoparticles are connected to the unwinding collagen targeting molecules to obtain the photoacoustic probe.

[0021] The preparation method according to the embodiment of the present invention has at least the following beneficial effects:

[0022] Previous studies have shown that when using indocyanine green (ICG) for near-infrared zone II fluorescence in vivo imaging of small animals, the penetration ability is insufficient and only superficial blood vessels can be visualized in vivo. The visualization effect of deep blood vessels such as the abdominal aorta is poor, making it difficult to achieve in vivo visualization (such as Figure 1 The preparation method of the embodiment has simple steps and can prepare a photoacoustic probe that can deeply penetrate biological tissues, has high resolution, and has good in vivo imaging effect and specifically detects the unwinding of vascular collagen molecules.

[0023] The term "dehydrating solvent" refers to a solvent used to remove water.

[0024] In some embodiments of the present invention, the cyanine dye includes at least one of Cy7, IR825, IR780, IR806, IR820, IR783, ICG, IR1061, and FD1080.

[0025] In some embodiments of the present invention, the unwinding collagen targeting molecule comprises at least one of a polypeptide, an antibody, and an aptamer.

[0026] In some embodiments of the present invention, the untwisted collagen targeting molecule comprises a polypeptide that hybridizes and binds to untwisted collagen.

[0027] In some embodiments of the present invention, the unwinding collagen targeting molecule comprises an amino acid sequence of (GfO) n wherein n is a positive integer between 6 and 12, G is glycine, f is fluoroproline, and O is hydroxyproline. For example, n can be 6, 7, 8, 9, 10, 11 or 12.

[0028] In some embodiments of the present invention, the unwinding collagen targeting molecule comprises a polypeptide having an amino acid sequence of (GfO)9.

[0029] In some embodiments of the present invention, the cross-linking agent comprises 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC).

[0030] In some embodiments of the present invention, the serum albumin includes at least one of bovine serum albumin and human serum albumin.

[0031] In some embodiments of the present invention, the pH of the serum albumin aqueous solution is 7.5 to 9.5, for example, 7.5, 8, 8.5, 9 or 9.5.

[0032] In some embodiments of the present invention, the serum albumin concentration in the serum albumin aqueous solution is 5 g / L to 50 g / L, for example, 5 g / L, 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, 45 g / L or 50 g / L.

[0033] In some embodiments of the present invention, the volume ratio of the serum albumin aqueous solution to the dehydration solvent is 1:1 to 5. For example, it can be 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5 or 1:5.

[0034] In some embodiments of the present invention, the mass ratio of the cyanine dye to serum albumin is 1:1 to 10. For example, it can be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10.

[0035] In some embodiments of the present invention, the amount of the cross-linking agent is 0.05-0.5 mg / mg serum albumin, for example, 0.05 mg / mg serum albumin, 0.1 mg / mg serum albumin, 0.2 mg / mg serum albumin, 0.3 mg / mg serum albumin, 0.4 mg / mg serum albumin or 0.5 mg / mg serum albumin.

[0036] In some embodiments of the present invention, the reaction time of step S1 is 2 h to 8 h, for example, 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, 5 h, 5.5 h, 6 h, 6.5 h, 7 h, 7.5 h or 8 h.

[0037] In some embodiments of the present invention, the reaction temperature of step S1 is 20°C to 40°C. For example, it can be 20°C, 25°C, 30°C, 35°C or 40°C.

[0038] In some embodiments of the present invention, the step S1 includes a first post-reaction treatment, which includes at least one of removing the dehydration solvent and a first purification treatment.

[0039] In some embodiments of the invention, the removing the dehydrating solvent is performed by rotary evaporation.

[0040] In some embodiments of the present invention, the first purification process comprises ultrafiltration using an ultrafiltration membrane having a molecular weight cutoff of 10 kDa to 100 kDa, for example, 10 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa or 100 kDa.

[0041] In some embodiments of the present invention, in step S2, the ratio of serum albumin in the serum albumin-cyanine dye nanoparticles to the unwinding collagen targeting molecule is 1:1 to 10, in terms of molar equivalents. For example, it may be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10.

[0042] In some embodiments of the present invention, the reaction temperature of step S2 is 20°C to 50°C. For example, it can be 20°C, 25°C, 30°C, 35°C, 40°C, 45°C or 50°C.

[0043] In some embodiments of the present invention, step S2 includes: after the serum albumin-cyanine dye nanoparticles react with DBCO-Sulfo-NHS ester, they react with an untwisted collagen targeting molecule modified with an azide group to connect the serum albumin-cyanine dye nanoparticles and the untwisted collagen targeting molecule to obtain the photoacoustic probe.

[0044] In some embodiments of the present invention, the ratio of albumin in the serum albumin-cyanine dye nanoparticles: DBCO-Sulfo-NHS ester: unwinding collagen targeting molecule is 1:1-10:1-10 in terms of molar equivalents.

[0045] In some embodiments of the present invention, the ratio of albumin in the serum albumin-cyanine dye nanoparticles: DBCO-Sulfo-NHS ester: unwinding collagen targeting molecule is 1:3-5:3-5 in terms of molar equivalents.

[0046] In some embodiments of the present invention, the reaction time of the serum albumin-cyanine dye nanoparticles and DBCO-Sulfo-NHS ester is 0.5 h to 2 h, for example, 0.5 h, 1 h, 1.5 h or 2 h.

[0047] In some embodiments of the present invention, the serum albumin-cyanine dye nanoparticles further include a second purification treatment after reacting with DBCO-Sulfo-NHS ester. The second purification treatment includes ultrafiltration using an ultrafiltration membrane. The ultrafiltration membrane has a molecular weight cutoff of 10 to 100 kDa. For example, it can be 10 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa or 100 kDa.

[0048] In some embodiments of the present invention, the reaction time with the unwinding collagen targeting molecule modified with an azide group is 4 to 12 hours, for example, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, 10.5 hours, 11 hours, 11.5 hours or 12 hours.

[0049] In some embodiments of the present invention, after reacting with the unwinding collagen targeting molecule modified with an azide group, a third purification treatment is further included. The third purification treatment includes ultrafiltration using an ultrafiltration membrane. The ultrafiltration membrane has a molecular weight cutoff of 10 to 100 kDa X. For example, it can be 10 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa or 100 kDa.

[0050] In some embodiments of the present invention, the unwinding collagen targeting molecule modified with an azide group further comprises a linker for connecting the azide group and the unwinding collagen targeting molecule. The linker comprises lysine and / or 6-aminohexanoic acid.

[0051] The third aspect of the present invention provides a photoacoustic probe prepared by any preparation method described in the first aspect.

[0052] The photoacoustic probe according to the embodiment of the present invention has at least the following beneficial effects:

[0053] The photoacoustic probe of the embodiment can deeply penetrate biological tissues, specifically bind to vascular diseased tissues where collagen molecules are unwound, have high resolution and high sensitivity, and can detect arterial dilatation in the early stages of aneurysms (diameter thickening less than 50%) in vivo. It has great potential application value in the early and accurate tracing of deep tissues such as AAA, and in the accurate detection of various cardiovascular diseases with collagen tissue damage (including abdominal aortic aneurysm, intracranial aneurysm, aortic dissection, myocardial infarction, atherosclerosis, valvular disease, etc.).

[0054] In some embodiments of the present invention, the application of the photoacoustic probe in the field of cell imaging, tissue imaging, biomarker or photoacoustic imaging is also provided. The application is for non-disease treatment and diagnosis.

[0055] In some embodiments of the present invention, the use of the photoacoustic probe in the preparation of a photoacoustic detection product is also provided.

[0056] In some embodiments of the present invention, the average particle size of the photoacoustic probe is 100 nm to 300 nm, for example, 100 nm, 120 nm, 140 nm, 160 nm, 180 nm, 200 nm, 220 nm, 240 nm, 260 nm, 280 nm or 300 nm.

[0057] The fourth aspect of the present invention provides a kit, comprising the photoacoustic probe described in the embodiment of the third aspect.

[0058] Other features and advantages of the present invention will be set forth in the description which follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 It is the in vivo fluorescence development effect of ICG;

[0060] Figure 2 The results of the detection of collagen matrix destruction in the diseased vascular tissue of patients with aortic dissection (AD); wherein, Figure a is a CTA three-dimensional reconstruction image of a patient with aortic dissection (scale bar: 5 cm); Figures b and c are global images of H&E staining of frozen sections of aortic dissection tissue (scale bar: 1 mm) and local magnified images of representative areas (scale bar: 100 μm), respectively, i-iv represent areas gradually away from the dissection lesions; Figure d is a representative image of the magnified area (i-iv) tissue stained with a fluorescently labeled unwinding collagen targeting molecule - collagen hybrid peptide Cy3-CHP (scale bar: 100 μm); Figure e is a representative image of second harmonic generation (SHG) imaging of the magnified area (i-iv) (scale bar: 100 μm);

[0061] Figure 3 The results of detecting damaged collagen in diseased arterial tissues of human body; a and d are respectively the global image of the diseased vascular tissue of aortic dissection and the local magnified image of the representative area, b and e are respectively the global image of the diseased vascular tissue of another aortic dissection and the local magnified image of the representative area; c and f are respectively the global image of the diseased vascular tissue of coronary artery aneurysm and the local magnified image of the representative area; the scale bars are all 100μm;

[0062] Figure 4 The dissolution of IR1061 in different dehydration solvents;

[0063] Figure 5 The dissolution of ICG in different dehydration solvents;

[0064] Figure 6 The effect of different ratios of dehydrating solvent to serum albumin aqueous solution on the preparation of ICG nanoparticles;

[0065] Figure 7 The photoacoustic imaging results of the photoacoustic probes prepared with different dehydration solvents; Figure a is the photoacoustic imaging results of acetonitrile, dichloromethane, acetone, tetrahydrofuran, and ethyl acetate, and Figure b is the photoacoustic signal spectra of the corresponding groups;

[0066] Figure 8 Schematic diagram of photoacoustic imaging of IBN-CHP and synthesis of the photoacoustic probe; wherein, Figure a is a schematic diagram of photoacoustic imaging, and Figure b is a schematic diagram of synthesis;

[0067] Fig. 9 The fluorescence absorption and emission wavelength detection results of IBN;

[0068] Fig.10 For IBN-CHP and IBN- S Physical size characterization results of CHP; Figure a is a representative SEM image (scale: 1 μm), and Figure b is the particle size test result;

[0069] Fig.11 For IBN-CHP and IBN- S Photoacoustic excitation spectrum of CHP;

[0070] Fig.12 The corresponding relationship between different concentrations of IBN-CHP (measured in ICG molar concentration) and its photoacoustic signal value; wherein, Figure a is the correlation analysis between different concentrations of IBN-CHP and the corresponding photoacoustic value under 710nm excitation light; Figure b is the photoacoustic spectrum corresponding to different concentrations of IBN-CHP under different excitation lights;

[0071] Fig.13 The distribution results of IBN-CHP and IBN in mouse organs; Figure a is a representative fluorescence imaging image of the heart, liver, spleen, lung, and kidney of each group of mice, and Figure b is a quantitative statistical graph of the fluorescence signals of the heart, liver, spleen, lung, and kidney of each group of mice (n=3);

[0072] Fig.14 The biosafety test results of IBN-CHP;

[0073] Fig.15 Representative results of IBN-CHP in photoacoustic in vivo imaging of AAA (scale bar: 2 mm);

[0074] Fig.16 The representative results of IBN-CHP in vitro fluorescence imaging and light sheet three-dimensional imaging of abdominal aorta in AAA mice; Figure a is a representative image of in vitro fluorescence imaging of abdominal aorta; Figure b is a representative image of light sheet fluorescence microscopy of abdominal aorta in AAA mice (scale bar: 1 mm);

[0075] Fig.17The results of IBN-CHP predicting the risk of AAA rupture in mice; Figure a is the survival analysis results of mice in each group, Figure b is the representative image of the photoacoustic imaging results of mice in each group on the 14th day after modeling, Figure c is the representative image of the gross pathology of mice in each group, Figure d is the representative image of H&E pathological staining of mice in each group, and Figure e is the quantitative statistical results of the photoacoustic signals of mice in each group;

[0076] Fig.18 Figure 2 shows the imaging effects of different photoacoustic probes in mice with carotid aneurysms; Figure a shows the photoacoustic imaging of each group of mice with carotid aneurysms and the corresponding representative images of in vitro fluorescence imaging (scale bar: 1 mm); Figure b shows the representative images of light sheet fluorescence microscopy imaging based on IBN-CHP molecular probe for each group of mice with carotid aneurysms (scale bar: 1 mm); and Figure c shows the photoacoustic quantitative statistical graph of each group of mice with carotid aneurysms models. DETAILED DESCRIPTION

[0077] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0078] When a numerical range is disclosed herein, the above range is considered to be continuous and includes the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0079] In the description of the present invention, if there is a description of first, second, third, etc., it is only for the purpose of distinguishing technical features, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features or implicitly indicating the sequence of the indicated technical features.

[0080] In the description of the present invention, the reference term "and / or" includes all and any combinations of one or more of the associated listed items.

[0081] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0082] Unless otherwise specified, "room temperature" in the present invention means (25±5)°C.

[0083] In the description of the present invention, the unwinding collagen targeting molecule is a collagen hybrid peptide CHP, whose sequence is GfOGfOGfOGfOGfOGfOGfOGfOGfO (hereinafter referred to as (GfO)9); the control probe without targeting S CHP was obtained by randomly shuffling the target sequence (GfO)9, and the sequence was OfGGOfGfGfOfOGOfGOOfGGOOffG (hereinafter referred to as S (GfO)9);

[0084] Among them, G refers to glycine; f refers to fluoroproline; and O refers to hydroxyproline.

[0085] In the embodiments of the present invention, unless otherwise specified, Cy3-CHP refers to CHP labeled with Cy3 fluorescent dye; S CHP refers to Cy3 fluorescent dye labeled S CHP.

[0086] In the embodiment of the present invention, in -(GfO)9, - S The azido group (-N3) was introduced into the (GfO)9 sequence to obtain the peptides K(N3)-Ahx-(GfO)9 and K(N3)-Ahx- S (GfO)9 (K refers to lysine, Ahx refers to 6-aminohexanoic acid) to facilitate the subsequent click chemistry reaction with the -DBCO group. S The synthesis steps of (GfO)9 are as follows:

[0087] (1) According to the ratio of resin: amino acid: HATU: HOAT: DIEA = 1:5:5:5:7.5 (in equivalent ratio), weigh the required amino acid Fmoc-Lys(N3)-OH, HOAT and HATU, add chromatographic grade DMF and shake to fully dissolve the reagents; then add DIEA, mix well and the liquid turns light yellow and transfer to (GfO)9-resin or S The peptide synthesis tube of (GfO)9-resin was sealed with a sealing film, placed on a shaker, and reacted at room temperature for 3 hours. The empirical color reaction was negative; after 20% piperidine deprotection for 30 minutes, the color reaction was performed again, and the result was positive.

[0088] (2) TFA, TIS, and ddH2O were mixed in a volume ratio of 95:2.5:2.5 to obtain a shearing solution. 10 mL of the shearing solution was prepared for every 0.1 mmol of resin. The shearing solution was added to the peptide synthesis tube in step (1). After shearing for 3 h under magnetic stirring at room temperature, the reaction solution was transferred to a 50 mL centrifuge tube. The TFA in the centrifuge tube was blown away using flowing nitrogen in a fume hood, leaving the resin and a small amount of liquid.

[0089] (3) Ice ether precipitation: Add 5 mL of -80°C precooled ice ether to the 50 mL centrifuge tube in step (2) for precipitation, centrifuge at 4°C and 4000 rpm for 5 min, slowly pour off the ether supernatant, and retain the precipitate; repeat twice. After the residual ether evaporates completely, add 5 mL of ultrapure water to dissolve the precipitate, filter through a 0.22 μm microporous filter membrane, and collect the crude polypeptide aqueous solution.

[0090] (4) Product purification and molecular weight identification: The crude peptide product was purified by HPLC using a Shimadzu high performance liquid chromatograph, and the molecular weight of the purified target product was verified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS).

[0091] (5) The verified correct product (K(N3)-Ahx-(GfO)9 or K(N3)-Ahx- S (GfO)9) After freeze drying in a freeze dryer, it will be connected to "IBN-Sulfo-DBCO" later.

[0092] In the embodiments of the present invention, unless otherwise specified, the coupling agent is a Guanggong brand coupling agent with a product number of ZJ2773;

[0093] Porcine Pancreas Elastase (PPE) was purchased from Sigma Aldrich, 10.3 mg protein / mL, 5.9 units / mg protein.

[0094] CHP targets damaged collagen in diseased arterial tissue ex vivo

[0095] 1. The frozen sections of diseased arterial tissues of AD patients were subjected to H&E staining, Cy3-CHP immunofluorescence staining, and SHG imaging, and the normal blood vessels next to the tear were used as negative controls.

[0096] The results are as follows Figure 2 shown.

[0097] H&E staining showed that the aneurysm tissue of AD patients was disordered, with fibrous tissue proliferation, extensive collagen deposition, and a large number of inflammatory cells infiltrating the wall. Fluorescence imaging showed that collagen was significantly denatured and bound to Cy3-CHP, especially in the ruptured area of ​​AD samples; in contrast, the CHP fluorescence signal was limited in areas away from the tear area (i.e., relatively normal aortic tissue adjacent to the ruptured lesion that was surgically removed). SHG results showed that areas away from the dissection had strong SHG signals but low CHP signals, while the ruptured area had high CHP signals and low SHG signals.

[0098] SEM images showed that the collagen fibers in the tear area of ​​AD tissue were more disordered, while the collagen fibers away from the tear area were more regular.

[0099] 2. Use Cy3-CHP, Cy3- S CHP performed immunofluorescence staining on frozen sections of vascular tissues from 2 cases of aortic dissection and 1 case of coronary artery aneurysm.

[0100] The results are as follows Figure 3 shown.

[0101] The results showed that strong CHP signals were observed in the CHP staining group. S There was almost no CHP signal in the CHP staining group, which suggested that CHP could specifically target and bind to damaged collagen molecules in aortic dissection and coronary artery aneurysm.

[0102] ICG for photoacoustic imaging

[0103] 9 volumes of different dehydrating solvents were added to 20 mM IR1061 stock solution (solvent: DMSO) or 20 mM ICG stock solution (solvent: DMSO), mixed evenly, and photographed to evaluate the dissolution of each sample, and the photoacoustic imaging effect was tested respectively. Water was used as a blank control.

[0104] The dissolution and photoacoustic imaging effects of IR1061 are shown in Figure 2. Figure 4 , as shown in Table 1.

[0105] Table 1

[0106]

[0107] ICG-related dissolution and photoacoustic imaging effects are shown in Figure 5 , as shown in Table 2.

[0108] Table 2

[0109]

[0110] ICG has good solubility in several organic dehydrated solvents and can be completely dissolved; under the same conditions, IR-1061 can only be completely dissolved in dichloromethane, and cannot be completely dissolved in other organic dehydrated solvents tried, and a small amount of solid still remains.

[0111] Compared with IR-1061, the solubility and corresponding photoacoustic signal of ICG solution prepared under the same conditions are significantly better. When the dehydration solvent is acetonitrile, dichloromethane, acetone, and ethyl acetate, the photoacoustic signal of ICG is relatively high.

[0112] Preparation of ICG nanoparticles

[0113] 1. Add an appropriate volume of dehydration solvent to 5 μL of 10 mM ICG mother solution (solvent is DMSO), and add an appropriate volume of 1 g / L bovine serum albumin (BSA) aqueous solution (pH 8.5) under stirring at room temperature. After stirring at room temperature for 2 hours, take pictures and evaluate the preparation of nanoparticles in each group. Among them, the total volume of dehydration solvent and BSA aqueous solution is 500 μL; the volume ratio of BSA aqueous solution to dehydration solvent is set to 4:1, 3:2, 1:1, 2:3, and 1:4, respectively.

[0114] The results are as follows Figure 6 And as shown in Table 3.

[0115] Table 3

[0116]

[0117] Acetonitrile and acetone are more suitable for encapsulating and preparing ICG nanoparticles.

[0118] 2. Add 300 μL of dehydration solvent (acetonitrile, dichloromethane, acetone, tetrahydrofuran or ethyl acetate) to 5 μL of 10 mM ICG mother solution (solvent is DMSO), add 200 μL of 10 g / L BSA aqueous solution (pH 8.5) under stirring at room temperature, and add 200 μg of EDC for cross-linking. After stirring at room temperature for 2 hours, an ICG nanoparticle suspension is obtained, and the dehydration solvent in the ICG nanoparticle suspension is removed by rotary evaporation, and ultrafiltration is performed 3 times through a 50 kDa ultrafiltration tube to obtain an ICG nanoparticle solution. The prepared ICG nanoparticle solution (concentration of 1 mM) is subjected to in vitro photoacoustic imaging to test its photoacoustic signal.

[0119] The results are as follows Figure 7 shown.

[0120] The photoacoustic signal of the acetone group was the highest, with the average photoacoustic signal in the region of interest being 5.67 at most, and the wavelength corresponding to the peak being around 790 nm.

[0121] Example 1

[0122] This example provides a photoacoustic probe (IBN-CHP), the synthesis diagram is as follows Figure 8 The preparation method is as follows:

[0123] S1. Add 40 mg BSA to 4 mL deionized water, add 8 μL of 1 mol / L NaOH aqueous solution to it until the pH value of the BSA aqueous solution reaches 8.5, and obtain a BSA aqueous solution. Dissolve 15.5 mg ICG in 2 mL DMSO to obtain an ICG mother solution (ICG concentration is 10 mM). Add 6 mL acetone (preheated at 40°C) to 2 mL of ICG mother solution, continue to add 4 mL of BSA aqueous solution at one time while stirring at room temperature, and add 1 mg EDC for cross-linking to stabilize the nanoparticles. Stir at room temperature for 2 hours to obtain an IBN suspension. Remove acetone from the IBN suspension by rotary evaporation, ultrafilter 3 times through a 50 kDa ultrafiltration tube until the filtrate becomes colorless, and obtain a retentate (i.e., an IBN solution containing BSA-ICG nanoparticles (ICG-BN)).

[0124] S2. Dissolve an appropriate amount of DBCO-Sulfo-NHS ester (sulfonated diphenylcyclooctyne-succinimide ester, purchased from ClickChemistry Tools, item number A124-100) in 1×PBS to obtain a DBCO-Sulfo-NHS ester solution with a concentration of 10 mM. Mix the DBCO-Sulfo-NHS ester solution with the IBN solution obtained in step S1 and react on a shaker at room temperature for 30 minutes. Ultrafilter the reaction solution through a 50 kDa ultrafiltration tube to remove unreacted and excess DBCO-Sulfo-NHS ester. The liquid in the retentate tube is the IBN-Sulfo-DBCO solution. K(N3)-Ahx-(GfO)9(N3-CHP) was mixed with IBN-Sulfo-DBCO solution, reacted on a shaker overnight (8-12 h) at room temperature, and then ultrafiltered through a 50 kDa ultrafiltration tube. The liquid in the retention tube was the IBN-CHP solution containing the photoacoustic probe (ICG-BN-CHP).

[0125] Wherein, in terms of molar equivalent, IBN (quantitatively based on the amount of BSA substance as 1): DBCO-Sulfo-NHS ester: CHP=1:3:3.

[0126] The fluorescence properties of IBN solution were tested using a fluorescence spectrometer. Fig. 9 As shown in Figure 2, the fluorescence properties of IBN and ICG are similar, with the maximum emission wavelength near 814nm and the maximum absorption wavelength near 779nm.

[0127] Comparative Example 1

[0128] This example provides a photoacoustic probe (IBN- S CHP), the preparation method is basically the same as that in Example 1, except that K(N3)-Ahx-(GfO)9 is replaced by K(N3)-Ahx- S (GfO)9.

[0129] Test Example 1

[0130] In this detection example, the photoacoustic probes prepared in the above-mentioned Example 1 and Comparative Example 1 are characterized by using a scanning electron microscope and a dynamic light scattering instrument to detect their particle sizes.

[0131] The results are as follows Fig.10 shown.

[0132] The results showed that IBN-CHP and IBN- S The average particle size of CHP is about 145 nm.

[0133] Test Example 2

[0134] This test example is to measure the in vitro photoacoustic signal spectrum of the photoacoustic probes prepared in the above-mentioned Example 1 and Comparative Example 1. The specific test method is as follows:

[0135] IBN-CHP or IBN- S CHP solution (solvent is pure water) was injected into a polyethylene (PE) 10 tube to simulate the material in blood. The PE10 tube was then placed in a coupling agent without bubbles. The ultrasound probe was aimed at the center of the PE10 tube for imaging at the largest cross-section, and the transducer was fixed by an integrated small animal track system (Vevo integrated track system, VisualSonics). The PE10 tube was initially imaged using B mode to determine the acquisition position, and then switched to PA mode for PA imaging, using the same ultrasound transducer to detect the resulting acoustic emission. The Spectrum mode was selected to perform a full-band photoacoustic scan of the material in the PE10 tube. Vevo LAB 5.7.1 software (FUJIFILM VisualSonics Inc) was then used to store, process, and analyze the images to obtain the PA spectrum of the material.

[0136] The results are as follows Fig.11 As shown. IBN-CHP and IBN- S The photoacoustic spectra of CHP are basically consistent, with a spectral platform at 750nm-850nm and a maximum photoacoustic signal value at around 800nm.

[0137] Test Example 3

[0138] This test example explores the relationship between the in vitro photoacoustic signal intensity and concentration of the photoacoustic probe prepared in Example 1. The specific test method is as follows:

[0139] IBN-CHP solutions (solvent is pure water) with concentrations of 0.1mM, 0.2mM, 0.4mM, 0.6mM, 0.8mM, 1mM, 1.5mM, and 2mM were injected into the PE10 tube to simulate the material in the blood. Then the PE10 tube was placed in a coupling agent without bubbles. The ultrasound probe was aimed at the center of the PE10 tube at the largest cross-section for imaging. PA imaging was performed using the PA mode, and the acoustic emission generated was detected using the same ultrasound transducer. Selecting the Spectrum mode can perform a full-band photoacoustic scan of the material in the PE10 tube (focusing on recording the photoacoustic signal peak corresponding to the wavelength of 790nm). Vevo LAB 5.7.1 software (FUJIFILMVisual Sonics Inc) was then used to store, process, and analyze the images, and a graph of the change in the photoacoustic signal peak and concentration was drawn.

[0140] The photoacoustic signal intensity of IBN-CHP changes linearly with its concentration, e.g. Fig.12 shown.

[0141] Test Example 4

[0142] This detection example detects the biological safety of the photoacoustic probe prepared in the above-mentioned Example 1.

[0143] 1. After 8-week-old male C57BL / 6 mice (n=6) were anesthetized with 3% (vol / vol) isoflurane, 10 μL / g of 4% sodium pentobarbital was injected into the mouse intraperitoneally. The hair on the mouse abdomen was shaved, and the skin was incised along the midline of the abdomen with a length of about 1.0 cm. The infrarenal abdominal aorta was exposed under a stereomicroscope, and the infrarenal abdominal aorta was wrapped with a gauze block soaked with 10 μL PPE for 10 minutes, then the gauze block was removed, rinsed three times with saline, and the skin layer was sutured interruptedly to obtain an AAA mouse model.

[0144] Six AAA mouse models were randomly divided into two groups. On the 14th day after modeling, the photoacoustic probe IBN-CHP prepared in Example 1 and the IBN prepared in step S1 of Example 1 were injected into the two groups of mice through the tail vein. The injection dose was 100 nmol / mouse (in terms of ICG, 100 μL of aqueous solution). One hour after the tail vein injection, each tissue and organ was sampled and NIRF imaging was performed, and then the fluorescence intensity of each organ tissue was quantitatively analyzed, and the average value of the ICG fluorescence signal per unit area of ​​the heart, liver, spleen, lung, and kidney was calculated.

[0145] The results are as follows Fig.13 shown.

[0146] The results showed that in the comparison of fluorescence signals of heart, liver, spleen, lung and kidney, the fluorescence signal intensity of liver was the highest, followed by kidney, indicating that the two molecular probes were mainly metabolized by liver and kidney.

[0147] 2. After 8-week-old male C57BL / 6 mice (n=3 per group) were anesthetized with 3% (vol / vol) isoflurane, 10 μL / g of 4% sodium pentobarbital was injected into the mouse intraperitoneally. The hair on the mouse abdomen was shaved, and the skin was incised along the midline of the abdomen, with a length of about 1.0 cm. The infrarenal abdominal aorta was exposed under a stereomicroscope, and the infrarenal abdominal aorta was wrapped with a gauze block soaked with 10 μL PPE for 10 minutes, then the gauze block was removed, rinsed three times with saline, and the skin layer was sutured interruptedly to obtain an AAA mouse model.

[0148] The AAA mouse model was randomly divided into two groups (n=3 in each group). On the 14th day after modeling, one group of mice was injected with the photoacoustic probe IBN-CHP prepared in Example 1 through the tail vein, with an injection dose of 100 μL, an aqueous solution with an ICG concentration of 1 mM, and the administration frequency was once every 4 days, for a total of 3 times, as the IBN-CHP group. Another group of mice was injected with 100 μL PBS under the same conditions, as the PBS group. After the experiment, the heart, liver, spleen, lung, and kidney were taken for H&E staining.

[0149] The results are as follows Fig.14 shown.

[0150] Compared with the PBS group, the pathological structures of the heart, liver, spleen, lung and kidney in the IBN-CHP group did not undergo obvious pathological damage. IBN-CHP did not cause substantial damage to the organs of mice and had good biosafety.

[0151] Application Example 1

[0152] This application example provides the application of the photoacoustic probe IBN-CHP prepared in the above Example 1 in AAA imaging. The specific test method is as follows:

[0153] (1) Construction of mouse model:

[0154] Male C57BL / 6 mice aged 8 weeks, weighing 20-25g (n=3 per group) were anesthetized with 3% (vol / vol) isoflurane, and 10 μL / g of 4% sodium pentobarbital was injected into the mouse peritoneum. The hair of the mouse abdomen was shaved, and the skin was incised along the midline of the abdomen, with a length of about 1.0 cm. The infrarenal abdominal aorta was exposed under a stereomicroscope, and the infrarenal abdominal aorta was wrapped with a gauze block soaked with 10 μL PPE for 10 minutes, then the gauze block was removed, rinsed three times with saline, and the skin layer was sutured interruptedly. Thus, an AAA mouse model (AAA group) was obtained.

[0155] The construction method of the sham-operated control group (Sham group) was basically the same as that of the AAA mouse model, with the only difference being that the gauze soaked in porcine pancreatic elastase was replaced with a gauze soaked in physiological saline.

[0156] (2) Photoacoustic in vivo imaging:

[0157] On the 14th day after modeling of mice in the Sham group and AAA group, IBN-CHP aqueous solution with an ICG content of 100 nmol was injected into the tail vein of the two groups of mice, and photoacoustic imaging was performed 15 minutes after the injection to measure the photoacoustic signals of the infrarenal abdominal aortic aneurysm and its distal normal segment in different groups.

[0158] (3) In vitro fluorescence imaging and light sheet 3D imaging:

[0159] The abdominal aortas of mice in the Sham group and AAA group were sampled for in vitro fluorescence imaging, tissue clearing and light sheet fluorescence microscopy imaging.

[0160] Test results such as Figure 15-16 shown.

[0161] The results showed that under in vivo photoacoustic imaging, the photoacoustic signal of the infrarenal abdominal aorta of the Sham group mice injected with IBN-CHP was not significantly enhanced; while the photoacoustic signal of the infrarenal abdominal aorta aneurysm segment injected with IBN-CHP was significantly higher than that of the distal normal segment. Under in vitro fluorescence and light sheet three-dimensional imaging, the fluorescence signal of the lesion part of the abdominal aorta in the AAA group was significantly higher than that of the Sham, and there was a high fluorescence signal on the outside of the wall of the AAA lesion. This shows that IBN-CHP can target the untwisted collagen in the abdominal aortic aneurysm in vivo and produce a high photoacoustic signal.

[0162] Application Example 2

[0163] This application example provides the application of the photoacoustic probe IBN-CHP prepared in Example 1 above in predicting the risk of rupture in AAA mice. The mice used were 8-week-old male C57BL / 6 mice (n=3 per group). The specific test method is as follows:

[0164] Four batches were constructed to construct Sham group mice, BAPN (beta-aminopropionitrile, fumarate 3-aminopropionitrile fumarate, collagen cross-linking inhibitor) group mice, PPE group mice, and BAPN+PPE group mice. On the 14th day after surgery, the photoacoustic probe aqueous solution prepared in Example 1 was used to perform photoacoustic imaging on each group of mice, and the survival of the mice within 100 days after surgery was observed. After the experiment, the mice were dissected, the infrarenal abdominal aorta was taken, the pathological condition was observed, and H&E staining was performed.

[0165] The methods for constructing mouse models in each group are as follows:

[0166] Sham group: mice were provided with regular drinking water. 3% (vol / vol) isoflurane was used for general anesthesia. The mice underwent laparotomy to expose the infrarenal abdominal aorta and separate it from the vena cava. A gauze block soaked with 10 μL of saline was wrapped around the exposed infrarenal abdominal aorta. After 10 minutes, the exposed area was gently cleaned with saline and wiped dry with a cotton swab. The abdominal wound was sutured layer by layer, and the mice were returned to the cage.

[0167] BAPN (beta-aminopropionitrile, fumarate 3-aminopropionitrile fumarate) group: 3-aminopropionitrile fumarate (BAPN, Sigma Aldrich) was dissolved in drinking water to a final concentration of 2 mg / mL and provided to mice two days before surgery until the end of the experiment. During surgery, 3% (vol / vol) isoflurane was used for general anesthesia. The mice underwent laparotomy, and the infrarenal abdominal aorta was exposed and separated from the vena cava. A gauze block soaked in 10 μL of saline was wrapped around the exposed infrarenal abdominal aorta. After 10 minutes, the exposed area was gently cleaned with saline and dried with a cotton swab. The abdominal wound was sutured layer by layer, and the mice were returned to the breeding cage.

[0168] PPE group: mice were provided with regular drinking water. 3% (vol / vol) isoflurane was used for general anesthesia. The mice underwent laparotomy to expose the infrarenal abdominal aorta and separate it from the vena cava. A gauze block soaked with 10 μL PPE was wrapped around the exposed infrarenal abdominal aorta. After 10 minutes, the exposed area was gently cleaned with saline and wiped dry with a cotton swab. The abdominal wound was sutured layer by layer, and the mice were returned to the cage.

[0169] BAPN+PPE group: BAPN at a concentration of 0.2% was dissolved in drinking water and provided to mice two days before surgery until the end of the study. General anesthesia was performed with 3% (vol / vol) isoflurane. The mice underwent laparotomy to expose the infrarenal abdominal aorta and separate it from the vena cava. A gauze block soaked with 10 μL PPE was wrapped around the exposed infrarenal abdominal aorta. After 10 minutes, the exposed area was gently cleaned with saline and dried with a cotton swab. The abdominal wound was sutured layer by layer, and the mice were returned to the cage.

[0170] The results are as follows Fig.17 And as shown in Table 4.

[0171] Table 4

[0172]

[0173] Note: Rupture time refers to the number of days after the mouse modeling when the aneurysm ruptures and the mouse dies. Unruptured means that the mouse abdominal aortic aneurysm has not ruptured at the end of the observation time of 100 days. The photoacoustic signal and diameter of each row represent the data of the abdominal aortic aneurysm lesion segment and the normal abdominal aorta segment of the same mouse with the corresponding ear tag. The photoacoustic signal ratio represents the ratio of the photoacoustic signal of the abdominal aortic aneurysm lesion segment to the normal abdominal aorta segment of the same mouse. The diameter ratio represents the ratio of the diameter of the abdominal aortic aneurysm lesion segment to the normal abdominal aorta segment of the same mouse in the ultrasound image. A photoacoustic signal ratio higher than 1 represents the specific targeting of the photoacoustic probe to the AAA lesion.

[0174] There was no AAA rupture death in the BAPN and PPE groups, while more than half of the mice in the BAPN+PPE group (n=8) had fatal rupture within 100 days. Compared with the BAPN, PPE, and BAPN+PPE unruptured groups, the photoacoustic signal of the lesion wall in the BAPN+PPE ruptured group was significantly stronger and had the highest signal-to-noise ratio. Compared with the BAPN and PPE groups, the photoacoustic signal background noise ratio of the mice in the BAPN+PPE group was higher. Compared with the Sham group, the characteristic wavy structure of the wall of the mice in the BAPN group disappeared and the wall became thinner. The wall integrity of the abdominal aorta of the mice in the PPE group was lost, and irreversible AAA dilation occurred. The aneurysm wall of the mice in the BAPN+PPE group was more obviously dilated, and a false lumen or even a rupture appeared in the wall.

[0175] Application Example 3

[0176] This application example provides the application of the photoacoustic probe prepared in the above Example 1 in a carotid aneurysm model mouse. The specific test method is as follows:

[0177] The mice used were 8-week-old male C57BL / 6 mice (n=3 per group), weighing 20-25 g. The mice were anesthetized with 3% (vol / vol) isoflurane. After the mice had difficulty standing, 10 μL / g of 4% (wt / vol) sodium pentobarbital was injected intraperitoneally. The hair on the neck of the mice was shaved, and the skin was incised along the midline of the neck, with a length of about 1.0 cm. The submandibular gland was bluntly separated under a stereomicroscope to expose the right common carotid artery (RCCA) of the mice, and the right common carotid artery was wrapped with a gauze block soaked with 10 μL porcine pancreatic elastase (PPE) for 10 minutes, then the gauze block was removed and rinsed three times with saline. Afterwards, the left common carotid artery (LCCA) was exposed and wrapped with a gauze soaked in saline for 10 minutes. The gauze was then removed and rinsed with saline three times. The skin layer was sutured intermittently to construct a common carotid artery aneurysm mouse model. In order to simulate different time points in the early development of aneurysms, the photoacoustic probe (IBN-CHP) prepared in Example 1, IBN (prepared in step S1 of the preparation method in Example 1), and the photoacoustic probe (IBN-CHP) prepared in Comparative Example 1 were injected into the mice of each group on the 3rd, 7th, and 14th days after the common carotid artery aneurysm mouse model was established. S After CHP), 100 μL of an aqueous solution with an ICG concentration of 1 mM was injected for photoacoustic in vivo imaging of the common carotid artery.

[0178] The results are as follows Fig.18 shown.

[0179] Only the lesion wall of the common carotid artery aneurysm mice injected with IBN-CHP showed a strong photoacoustic signal, while the lesion wall of the common carotid artery aneurysm mice injected with IBN or IBN- S No obvious photoacoustic signals were observed in the lesion wall of the carotid aneurysm mice injected with CHP or in the normal carotid artery wall of the mice injected with IBN-CHP. This suggests that the IBN-CHP molecular probe can specifically target and trace damaged collagen in the aneurysm. On the third day after modeling, photoacoustic imaging was able to identify damaged collagen in the lesion wall of the common carotid artery aneurysm. This suggests that in the early stage of right common carotid artery aneurysm lesions, when collagen destruction reaches a certain extent, although no obvious aneurysmal lesion bulge is observed under ultrasound at this time, the IBN-CHP molecular probe can already visualize the damaged collagen. Therefore, photoacoustic imaging based on the IBN-CHP molecular probe can visualize the lesions of the aneurysm earlier than ultrasound, and photoacoustics can intuitively display the diseased collagen in the aneurysm by observing the "signal presence or absence".

[0180] The embodiments of the present invention are described in detail above in conjunction with the embodiments, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge scope of ordinary technicians in the relevant technical field without departing from the purpose of the present invention.

Claims

1. A photoacoustic probe connected with an unwinding collagen targeting molecule is used in any one of A1) to A2); A1) Preparation of products for detecting or assisting in the detection of cardiovascular diseases; A2) Prepare a product that predicts or assists in predicting the risk of aneurysm rupture.

2. The use according to claim 1, characterized in that: The cardiovascular disease includes at least one of abdominal aortic aneurysm, intracranial aneurysm, carotid artery aneurysm, aortic dissection, myocardial infarction, atherosclerosis, and valvular disease; And / or, the aneurysm includes but is not limited to a coronary artery aneurysm, an abdominal aortic aneurysm, an intracranial aneurysm or a carotid artery aneurysm.

3. A method for preparing a photoacoustic probe, characterized in that: The steps include: S1, preparing a mixture of a serum albumin aqueous solution, a cyanine dye, a dehydrating solvent, and a cross-linking agent, and reacting the mixture to obtain serum albumin-cyanine dye nanoparticles; The dehydration solvent includes at least one of acetone, methanol, acetonitrile, dichloromethane and tetrahydrofuran; S2. The serum albumin-cyanine dye nanoparticles are connected to the unwinding collagen targeting molecules to obtain the photoacoustic probe.

4. The preparation method according to claim 3, characterized in that: The cyanine dye includes at least one of Cy7, IR825, IR780, IR806, IR820, IR783, ICG, IR1061, and FD1080; And / or, the unwinding collagen targeting molecule comprises an amino acid sequence of (GfO) n A polypeptide, n is a positive integer between 6 and 12, G is glycine, f is fluoroproline, and O is hydroxyproline; And / or, the cross-linking agent comprises EDC.

5. The preparation method according to claim 3, characterized in that: The pH of the serum albumin aqueous solution is 7.5 to 9.5; and / or, the volume ratio of the serum albumin aqueous solution to the dehydration solvent is 1:1 to 5; And / or, the mass ratio of the cyanine dye to serum albumin is 1:1 to 10; And / or, the amount of the cross-linking agent is 0.05-0.5 mg / mg serum albumin.

6. The preparation method according to claim 3, characterized in that: The reaction time of step S1 is 2 h to 8 h.

7. The preparation method according to claim 3, characterized in that: The step S2 comprises: after the serum albumin-cyanine dye nanoparticles react with DBCO-Sulfo-NHS ester, they react with an untwisting collagen targeting molecule modified with an azide group to connect the serum albumin-cyanine dye nanoparticles and the untwisting collagen targeting molecule to obtain the photoacoustic probe.

8. The preparation method according to claim 7, characterized in that: The reaction time of the serum albumin-cyanine dye nanoparticles and DBCO-Sulfo-NHS ester is 0.5h to 2h; and / or, the reaction time with the unwinding collagen targeting molecule modified with an azide group is 4 hours to 12 hours; And / or, in terms of molar equivalents, serum albumin in the serum albumin-cyanine dye nanoparticles: DBCO-Sulfo-NHS ester: unwinding collagen targeting molecule = 1:1-10:1-10.

9. A photoacoustic probe, characterized in that: Prepared by the preparation method according to any one of claims 3 to 9.

10. A kit, characterized in that: Comprising the photoacoustic probe according to claim 9.

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