A photoacoustic probe and a preparation method and application thereof

Abstract

The application discloses a photoacoustic probe and a preparation method and application thereof, and relates to the technical field of biomedicine. The application provides application of the photoacoustic molecular probe connected with a target unwinding collagen in preparation of products for detecting or assisting in detecting cardiovascular diseases, or in preparation of products for predicting or assisting in predicting aneurysm rupture risk. Experimental results prove that the photoacoustic probe connected with the target unwinding collagen can be used for non-invasive photoacoustic ultrasonic imaging detection of vascular structure function disorder diseases such as aneurysm, and can realize non-invasive early detection and rupture risk assessment of abdominal aortic aneurysm, coronary aneurysm, carotid aneurysm and the like, thereby providing a powerful tool for early intervention and precise treatment of cardiovascular diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a photoacoustic probe, its preparation method, and its application. Background Technology

[0002] Abdominal aortic aneurysm (AAA) is one of the most common aortic diseases, with its most serious consequence being rupture. The mortality rate for ruptured AAA is close to 90%; up to 50% of ruptured AAA patients die by the time they reach the operating room, and the perioperative mortality rate is also as high as 50%. Because most AAA cases are asymptomatic before rupture, many patients are diagnosed when their condition is already extremely critical. Currently, there are no drugs to reverse or stop the progression of AAA. If AAA is not detected early and intervened in a timely manner, the risk of emergency surgery increases significantly once it reaches an advanced stage.

[0003] Currently, arterial diameter is used clinically as a diagnostic indicator for aortic aneurysm (AAA) and a standard for assessing rupture risk. Imaging techniques such as ultrasound and computed tomography angiography are used to continuously assess the aortic anatomy, relying on the diameter and morphology of the aneurysm to stratify disease severity and thus monitor AAA. However, because the diameter expansion of AAA does not progress linearly, ultrasound imaging is insufficient to provide timely and accurate prognostic information, and predicting AAA rupture remains a significant challenge. Furthermore, since most AAA cases are asymptomatic before rupture, there is an urgent need for effective detection technologies to accurately assess the integrity of deep vessels and monitor the progression and rupture risk of AAA, enabling disease risk stratification and timely and effective intervention for high-risk individuals. Summary of the Invention

[0004] This invention aims to at least address one of the technical problems existing in the prior art. To this end, this invention proposes the application of a photoacoustic probe linked to unwinding collagen targeting molecules in the preparation of products for detecting or assisting in the detection of cardiovascular diseases, or in the preparation of products for predicting or assisting in the prediction of aneurysm rupture risk.

[0005] This application also proposes a method for preparing a photoacoustic probe that can identify the integrity of collagen tissue in blood vessel walls.

[0006] The present invention also proposes a photoacoustic probe prepared by the above-described method.

[0007] The present invention also proposes a reagent kit.

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

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

[0010] A2) Prepare products that predict or assist in predicting the risk of aneurysm rupture.

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

[0012] Photoacoustic probes linked to unwinding collagen-targeting molecules can specifically bind to vascular lesions where collagen molecules have unwound. This allows for non-invasive photoacoustic ultrasound imaging detection of vascular structural and functional disorders such as aneurysms, enabling non-invasive early detection and rupture risk assessment of aneurysms (AAA). This provides a powerful tool for early intervention and precision treatment of AAA in clinical practice and is expected to play a role in the precise detection of various other cardiovascular diseases involving collagen tissue damage (including but not limited to abdominal aortic aneurysms, intracranial aneurysms, aortic dissection, myocardial infarction, atherosclerosis, and valvular diseases), providing in vivo early warning of aneurysm rupture risk.

[0013] The photoacoustic probe of the embodiment can penetrate biological tissues deeply, has high resolution and high sensitivity, and can detect arterial dilation in the early stage of aneurysm (diameter increase of less than 50%). It 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 function of detecting cardiovascular diseases 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 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, abdominal aortic aneurysm, intracranial aneurysm, or carotid aneurysm.

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

[0018] S1. Prepare a mixture of serum albumin aqueous solution, cyanine dye, dehydrating solvent, and crosslinking agent, and react 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 linked to unwind collagen targeting molecules to obtain the photoacoustic probe.

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

[0022] Previous studies have shown that indocyanine green (ICG) has insufficient penetration for near-infrared II fluorescence in vivo imaging of small animals, only able to visualize superficial blood vessels and poorly visualize deep vessels such as the abdominal aorta, making it difficult to achieve in vivo visualization (e.g. Figure 1 (As shown), and photoacoustic imaging is not possible. The preparation method of the embodiment is simple and can prepare a photoacoustic probe that can penetrate biological tissues deeply, has high resolution, and good in vivo imaging effect for the specific detection of vascular collagen molecule unwinding.

[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 unwound collagen targeting molecule includes at least one of a polypeptide, an antibody, and an aptamer.

[0026] In some embodiments of the present invention, the unwound collagen targeting molecule includes a polypeptide that hybridizes with unwound collagen.

[0027] In some embodiments of the present invention, the unwound collagen targeting molecule comprises the amino acid sequence (GfO). n The polypeptide, where 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 unwound collagen targeting molecule comprises a polypeptide with the amino acid sequence (GfO)9.

[0029] In some embodiments of the present invention, the crosslinking agent includes 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, it can be 7.5, 8, 8.5, 9, or 9.5.

[0032] In some embodiments of the present invention, the serum albumin concentration in the aqueous serum albumin solution is 5 g / L to 50 g / L. For example, it can be 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 dehydrating 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 crosslinking agent is 0.05 to 0.5 mg / mg serum albumin. For example, it can be 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 2h to 8h. For example, it can be 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, 5h, 5.5h, 6h, 6.5h, 7h, 7.5h or 8h.

[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, step S1 includes a first post-reaction treatment. The first post-reaction treatment includes at least one of the removal of the dehydrating solvent and a first purification treatment.

[0039] In some embodiments of the present invention, the removal of the dehydrating solvent is carried out by rotary evaporation.

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

[0041] In some embodiments of the present invention, in step S2, the ratio of serum albumin to unwinding collagen targeting molecules in the serum albumin-cyanine dye nanoparticles is 1:1 to 10, in molar equivalents. 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.

[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 unwinding collagen targeting molecules modified with azide groups to connect the serum albumin-cyanine dye nanoparticles and the unwinding collagen targeting molecules to obtain the photoacoustic probe.

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

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

[0046] In some embodiments of the present invention, the reaction time between the serum albumin-cyanine dye nanoparticles and DBCO-Sulfo-NHS ester is 0.5 h to 2 h. For example, it can be 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, after reacting with DBCO-Sulfo-NHS ester, further undergo a second purification process. The second purification process includes ultrafiltration using an ultrafiltration membrane. The ultrafiltration membrane has a molecular weight cutoff of 10–100 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 azide-modified unwinding collagen targeting molecule is 4 h to 12 h. For example, it can be 4 h, 4.5 h, 5 h, 5.5 h, 6 h, 6.5 h, 7 h, 7.5 h, 8 h, 8.5 h, 9 h, 9.5 h, 10 h, 10.5 h, 11 h, 11.5 h, or 12 h.

[0049] In some embodiments of the present invention, a third purification process is further included after the reaction with the azide-modified unwinding collagen targeting molecule. The third purification process includes ultrafiltration using an ultrafiltration membrane. The ultrafiltration membrane has a molecular weight cutoff of 10–100 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.

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

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

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

[0053] The photoacoustic probe of this embodiment can penetrate deep into biological tissues and specifically bind to vascular lesions caused by collagen molecules unwinding. It has high resolution and high sensitivity, and can detect arterial dilation in the early stage of aneurysms (diameter increase of less than 50%) in vivo. It has great potential application value in the early and accurate tracing of deep tissues such as AAA, as well as in the accurate detection of various cardiovascular diseases with collagen tissue damage (including abdominal aortic aneurysm, intracranial aneurysm, aortic dissection, myocardial infarction, atherosclerosis, valvular lesions, etc.).

[0054] In some embodiments of the present invention, the photoacoustic probe is also provided for use in cell imaging, tissue imaging, biomarking, or photoacoustic imaging. Such use is for non-disease treatment and diagnostic purposes.

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

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

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

[0058] Other features and advantages of the present invention will be set forth in the following description. Attached Figure Description

[0059] Figure 1 The in vivo fluorescence imaging effect of ICG;

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

[0061] Figure 3 The images show the detection results of damaged collagen in diseased arterial tissue in the human body. Among them, images a and d are the global map and a magnified local area of ​​a representative region of aortic dissection lesion tissue, respectively; images b and e are the global map and a magnified local area of ​​a representative region of another aortic dissection lesion tissue, respectively; images c and f are the global map and a magnified local area of ​​a representative region of coronary artery aneurysm lesion tissue, respectively; the scale bar is 100μm.

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

[0063] Figure 5 The solubility of ICG in different dehydrating 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 Photoacoustic imaging results of photoacoustic probes prepared with different dehydration solvents; Figure a shows the photoacoustic imaging results of acetonitrile, dichloromethane, acetone, tetrahydrofuran, and ethyl acetate, and Figure b shows the photoacoustic signal spectra of each group.

[0066] Figure 8 Figure a is a schematic diagram of the photoacoustic imaging of IBN-CHP and the 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] Figure 9 The results of fluorescence absorption and emission wavelength detection for IBN;

[0068] Figure 10 For IBN-CHP and IBN- S Physical size characterization results of CHP; where Figure a is a representative scanning electron microscope image (scale bar: 1 μm), and Figure b is the particle size test results;

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

[0070] Figure 12 The correlation between different concentrations of IBN-CHP (measured as ICG molar concentration) and their photoacoustic signal values ​​is shown in Figure a. Figure a shows the correlation analysis between different concentrations of IBN-CHP and their corresponding photoacoustic values ​​under 710 nm excitation light. Figure b shows the photoacoustic spectra of different concentrations of IBN-CHP under different excitation lights.

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

[0072] Figure 14 The results are for the biosafety testing of IBN-CHP.

[0073] Figure 15 A representative result of IBN-CHP in photoacoustic in vivo imaging at AAA (scale bar: 2mm);

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

[0075] Figure 17The results of IBN-CHP prediction of AAA mouse rupture risk are shown in Figure a. The survival analysis results of each group of mice are shown in Figure b. The representative photoacoustic imaging results of each group of mice on day 14 after modeling are shown in Figure c. The representative gross pathological images of each group of mice are shown in Figure d. The representative H&E pathological staining images of each group of mice are shown in Figure e. The quantitative statistical results of photoacoustic signal of each group of mice are shown in Figure e.

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

[0077] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0078] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0079] In the description of this invention, the use of terms such as first, second, third, etc., is only for the purpose of distinguishing technical features and should not be construed as indicating or implying relative importance, or implicitly indicating the number of technical features indicated, or implicitly indicating the order of the technical features indicated.

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

[0081] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0082] Unless otherwise specified, "room temperature" in this invention means (25±5)℃.

[0083] In the description of this invention, the unwound collagen targeting molecule is the collagen hybrid peptide CHP, with the sequence GfOGfOGfOGfOGfOGfOGfOGfOGfO (hereinafter referred to as (GfO)9); a control probe without targeting capability is also included. S CHP is obtained by randomly shuffling the target sequence (GfO)9, with the sequence being OfGGOfGfGfOfOGOfGOOfGGOOffG (hereinafter referred to as...). S (GfO)9);

[0084] In this context, 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; Cy3- S CHP refers to the labeling of Cy3 fluorescent dyes. S CHP.

[0086] In embodiments of the present invention, in -(GfO)9, - S Introducing an azide group (-N3) into the (GfO)9 sequence yields the peptides K(N3)-Ahx-(GfO)9 and K(N3)-Ahx- S (GfO)9 (K refers to lysine, Ahx refers to 6-aminohexanoic acid) is used to facilitate subsequent click chemistry reactions with the -DBCO group. The peptides K(N3)-Ahx-(GfO)9 and K(N3)-Ahx- S The synthesis steps of (GfO)9 are as follows:

[0087] (1) Weigh the required amino acids Fmoc-Lys(N3)-OH, HOAT, and HATU according to the ratio of resin: amino acid: HATU: HOAT: DIEA = 1:5:5:5:7.5 (on an equivalent basis). Add chromatographic grade DMF and shake to fully dissolve the reagents. Then add DIEA, mix well, and transfer the liquid to a container with (GfO)9-resin or... S The (GfO)9-resin peptide synthesis tube was sealed with sealing film and placed on a shaker for 3 hours at room temperature. A colorimetric reaction was performed, which was negative. After deprotection with 20% piperidine for 30 minutes, a second colorimetric reaction was performed, which 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. The shearing solution was added to the polypeptide synthesis tube in step (1) at a ratio of 10 mL of shearing solution per 0.1 mmol of resin. After shearing with magnetic stirring at room temperature for 3 h, the reaction solution was transferred to a 50 mL centrifuge tube. The TFA in the centrifuge tube was blown away with flowing nitrogen in a fume hood, leaving the resin and a small amount of liquid.

[0089] (3) Precipitation with ice-cold ether: Add 5 mL of pre-cooled ice-cold ether at -80℃ to the 50 mL centrifuge tube from step (2) for precipitation. Centrifuge at 4℃ and 4000 rpm for 5 min, slowly discard the ether supernatant, and retain the precipitate; repeat twice. After the residual ether has completely evaporated naturally, add 5 mL of ultrapure water to dissolve the precipitate, filter through a 0.22 μm microporous membrane, and collect the crude product of the 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 the freeze dryer, it is ready to be connected to “IBN-Sulfo-DBCO”.

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

[0093] Porcine pancreas elastase (PPE) was purchased from Sigma Aldrich, at a protein concentration of 10.3 mg / mL and a protein concentration of 5.9 units / mg.

[0094] CHP targeting damaged collagen in diseased arterial tissue

[0095] 1. Frozen sections of diseased arterial tissue from AD patients were subjected to H&E staining, Cy3-CHP immunofluorescence staining, and SHG imaging, with normal blood vessels adjacent to the tear used as negative controls.

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

[0097] H&E staining revealed disordered aneurysm tissue structure, fibrosis, extensive collagen deposition, and abundant inflammatory cell infiltration in the vessel wall of AD patients. Fluorescence imaging showed significant collagen degeneration and binding to Cy3-CHP, particularly in the rupture area of ​​AD samples; in contrast, CHP fluorescence signal was limited in areas far from the tear (i.e., relatively normal aortic tissue adjacent to the surgically resected ruptured lesion). SHG results showed strong SHG signal but low CHP signal in areas far from the dissection, while the ruptured area exhibited high CHP signal and low SHG signal.

[0098] SEM images show that collagen fibers in the torn areas of AD tissue are more disordered, while collagen fibers further away from the torn areas are more regular.

[0099] 2. Use Cy3-CHP, Cy3- S CHP was used to perform immunofluorescence staining on frozen sections of vascular tissue from two cases of aortic dissection and one case of coronary artery aneurysm.

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

[0101] The results showed that strong CHP signal was visible in the CHP staining group, while S The CHP staining group showed almost no CHP signal. This suggests that CHP can specifically target and bind to damaged collagen molecules in aortic dissections and coronary aneurysms.

[0102] ICG for photoacoustic imaging

[0103] Nine times the volume of different dehydrating solvents were added to either 20 mM IR1061 stock solution (solvent: DMSO) or 20 mM ICG stock solution (solvent: DMSO). After thorough mixing, the solubility of each sample was evaluated by photographing, and the photoacoustic imaging effect was detected. Water was used as a blank control.

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

[0105] Table 1

[0106]

[0107] The dissolution of ICG and the photoacoustic imaging effects are as follows: Figure 5 As shown in Table 2.

[0108] Table 2

[0109]

[0110] ICG showed good solubility in several organic dehydrating solvents and could dissolve completely; however, under the same conditions, IR-1061 could only dissolve completely in dichloromethane, and could not dissolve completely in other organic dehydrating solvents tested, leaving a small amount of solid residue.

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

[0112] Preparation of ICG nanoparticles

[0113] 1. Add an appropriate volume of dehydrating solvent to 5 μL of 10 mM ICG stock solution (solvent: DMSO), and add an appropriate volume of 1 g / L bovine serum albumin (BSA) aqueous solution (pH 8.5) while stirring at room temperature. After stirring at room temperature for 2 h, photograph and evaluate the nanoparticle preparation of each group. The total volume of dehydrating solvent and BSA aqueous solution was 500 μL; the volume ratio of BSA aqueous solution to dehydrating solvent was set to 4:1, 3:2, 1:1, 2:3, and 1:4, respectively.

[0114] The results are as follows Figure 6 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 dehydrating solvent (acetonitrile, dichloromethane, acetone, tetrahydrofuran, or ethyl acetate) to 5 μL of 10 mM ICG stock solution (solvent: DMSO). While stirring at room temperature, add 200 μL of 10 g / L BSA aqueous solution (pH 8.5) and 200 μg of EDC for crosslinking. After stirring at room temperature for 2 h, an ICG nanoparticle suspension is obtained. Remove the dehydrating solvent from the ICG nanoparticle suspension by rotary evaporation. Ultrafilter three times using a 50 kDa ultrafiltration tube to obtain an ICG nanoparticle solution. Perform in vitro photoacoustic imaging on the prepared ICG nanoparticle solution (concentration: 1 mM) to test its photoacoustic signal.

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

[0120] The acetone group had the highest photoacoustic signal, with the highest average photoacoustic signal value in the region of interest being 5.67, and the peak wavelength being around 790 nm.

[0121] Example 1

[0122] This example provides a photoacoustic probe (IBN-CHP), and the synthesis schematic is shown below. Figure 8 As shown. The preparation method is as follows:

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

[0124] S2. Dissolve an appropriate amount of DBCO-Sulfo-NHS ester (sulfonated diphenylcyclooctynyl succinimide ester, purchased from ClickChemistry Tools, catalog number A124-100) in 1×PBS to obtain a 10 mM DBCO-Sulfo-NHS ester solution. 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 min. Ultrafilter the reaction solution through a 50 kDa ultrafiltration tube to remove unreacted and excess DBCO-Sulfo-NHS ester; the liquid in the retrieval tube is the IBN-Sulfo-DBCO solution. K(N3)-Ahx-(GfO)9(N3-CHP) was mixed with IBN-Sulfo-DBCO solution and reacted overnight (8-12 h) on a shaker at room temperature. The mixture was then ultrafiltered through a 50 kDa ultrafiltration tube, and the liquid in the filter tube was the IBN-CHP solution containing the photoacoustic probe (ICG-BN-CHP).

[0125] In this context, based on molar equivalents, IBN (with BSA as the molar amount of 1): DBCO-Sulfo-NHS ester: CHP = 1:3:3.

[0126] The fluorescence properties of the IBN solution were detected using fluorescence spectroscopy. The results are as follows: Figure 9 As shown, IBN has similar fluorescence properties to ICG, with a maximum emission wavelength around 814 nm and a maximum absorption wavelength around 779 nm.

[0127] Comparative Example 1

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

[0129] Detection Example 1

[0130] This test example characterizes the photoacoustic probes prepared in Example 1 and Comparative Example 1 using scanning electron microscopy and dynamic light scattering instrument, and detects their particle size.

[0131] The results are as follows Figure 10 As shown.

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

[0133] Detection Example 2

[0134] This test example examines the in vitro photoacoustic signal spectra of the photoacoustic probes prepared in Example 1 and Comparative Example 1. The specific test method is as follows:

[0135] 1 mM concentration of IBN-CHP or IBN- S CHP solution (using pure water as solvent) was injected into a polyethylene (PE) 10 tube to simulate the material in blood. The PE 10 tube was then placed in a bubble-free coupling agent. An ultrasound probe was positioned to image the central, largest cross-section of the PE 10 tube, with the transducer fixed by an integrated small animal track system (Vevo Integrated Track System, VisualSonics). Initial imaging of the PE 10 tube was performed in B mode to determine the acquisition location, followed by PA imaging in PA mode. The resulting acoustic emission was detected using the same ultrasound transducer. Spectrum mode was selected for a full-spectrum photoacoustic scan of the material within the PE 10 tube. The images were then stored, processed, and analyzed using Vevo LAB 5.7.1 software (FUJIFILM VisualSonics Inc.) to obtain the PA spectrum of the material.

[0136] The results are as follows Figure 11 As shown. IBN-CHP and IBN- S The photoacoustic spectra of CHP are basically the same, with a spectral plateau in the 750nm-850nm range and the maximum photoacoustic signal value around 800nm.

[0137] Detection Example 3

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

[0139] IBN-CHP solutions (using pure water) at concentrations of 0.1 mM, 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM, 1 mM, 1.5 mM, and 2 mM were injected into PE10 tubes to simulate the material in blood. The PE10 tubes were then placed in a bubble-free coupling agent. An ultrasonic probe was positioned to image the central, largest cross-section of the PE10 tube. PA imaging was performed in PA mode, and the resulting acoustic emission was detected using the same ultrasonic transducer. Spectrum mode was selected to perform a full-spectrum photoacoustic scan of the material in the PE10 tube (with a focus on recording the photoacoustic signal peak at 790 nm). The images were then stored, processed, and analyzed using Vevo LAB 5.7.1 software (FUJIFILM Visual Sonics Inc.), and a graph showing the changes in photoacoustic signal peaks versus concentration was plotted.

[0140] The photoacoustic signal intensity of IBN-CHP exhibits a linear relationship with concentration, such as... Figure 12 As shown.

[0141] Detection Example 4

[0142] This test example examines the biosafety of the photoacoustic probe prepared in Example 1 above.

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

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

[0145] The results are as follows Figure 13 As shown.

[0146] The results showed that among the fluorescence signals of the heart, liver, spleen, lungs, and kidneys, the liver had the highest fluorescence signal intensity, followed by the kidneys, indicating that the two molecular probes are mainly metabolized by the liver and kidneys.

[0147] 2. Eight-week-old male C57BL / 6 mice (n=3 per group) were anesthetized with 3% (vol / vol) isoflurane and then injected intraperitoneally with 10 μL / g of 4% sodium pentobarbital. The fur 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 subrenal abdominal aorta was exposed under a stereomicroscope and wrapped with a gauze soaked in 10 μL of PPE for 10 minutes. The gauze was then removed, the mice were rinsed three times with physiological saline, and the skin layer was intermittently sutured to obtain the AAA mouse model.

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

[0149] The results are as follows Figure 14 As shown.

[0150] Compared with the PBS group, the IBN-CHP group did not show significant pathological damage to the heart, liver, spleen, lungs, and kidneys. IBN-CHP did not cause substantial damage to the organs of mice and demonstrated good biocompatibility.

[0151] Application Example 1

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

[0153] (1) Constructing a mouse model:

[0154] Eight-week-old male C57BL / 6 mice, weighing 20-25g (n=3 per group), were anesthetized with 3% (vol / vol) isoflurane and then injected intraperitoneally with 10 μL / g of 4% sodium pentobarbital. The fur on the mouse abdomen was shaved, and the skin was incised along the midline of the abdomen, approximately 1.0 cm in length. The subrenal abdominal aorta was exposed under a stereomicroscope and wrapped with gauze soaked in 10 μL of PPE for 10 minutes. The gauze was then removed, the mice were rinsed three times with physiological saline, and the skin was intermittently sutured. This resulted in the AAA mouse model (AAA group).

[0155] The sham control group mice were constructed in the same way as the AAA mouse model, except that the gauze soaked in porcine pancreatic elastase was replaced with gauze soaked in physiological saline.

[0156] (2) Photoacoustic in vivo imaging:

[0157] On day 14 after modeling in mice in the Sham and AAA groups, IBN-CHP aqueous solution with an ICG content of 100 nmol was injected into the tail vein of mice in both groups. Photoacoustic imaging was performed 15 minutes after injection to measure the photoacoustic signal at the aortic aneurysm in the lower abdominal region and the normal segment distal to it in different groups.

[0158] (3) In vitro fluorescence imaging and light-sheet three-dimensional imaging:

[0159] Abdominal aorta samples were taken from mice in the Sham and AAA groups for in vitro fluorescence imaging, tissue clearing, and light-slide fluorescence microscopy imaging.

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

[0161] The results showed that, under in vivo photoacoustic imaging, the photoacoustic signal in the subrenal abdominal aorta of Sham mice injected with IBN-CHP did not show significant enhancement; however, the photoacoustic signal in the subrenal abdominal aortic aneurysm segment injected with IBN-CHP was significantly higher than that in the distal normal segment. Under in vitro fluorescence and light-film three-dimensional imaging, the fluorescence signal of the lesion portion of the abdominal aorta in the AAA group was significantly higher than that in Sham, and high fluorescence signal was present on the outer side of the vessel wall at the AAA lesion site. This indicates that IBN-CHP can target unwinding collagen in abdominal aortic aneurysms in vivo and generate high photoacoustic signals.

[0162] Application Example 2

[0163] This application example demonstrates the use 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 testing method is as follows:

[0164] Four batches of mice were constructed, including the Sham group, the BAPN (beta-aminopropionitrile, fumarate 3-aminopropionitrile fumarate, collagen crosslinking inhibitor) group, the PPE group, and the BAPN+PPE group. On the 14th day after surgery, photoacoustic imaging was performed on each group of mice using the photoacoustic probe aqueous solution prepared in Example 1 above, and the survival of the mice was observed within 100 days after surgery. After the experiment, the mice were dissected, and the abdominal aorta below the kidney was harvested for pathological observation and H&E staining.

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

[0166] Sham group: Mice were provided with standard drinking water. General anesthesia was administered using 3% (vol / vol) isoflurane. An abdominal surgery was performed on the mice to expose the subrenal abdominal aorta and separate it from the vena cava. A gauze pad soaked in 10 μL of physiological saline was wrapped around the exposed subrenal abdominal aorta. After 10 minutes, the exposed area was gently cleaned with physiological saline and dried with cotton swabs. The abdominal wound was sutured layer by layer, and the mice were returned to their rearing cages.

[0167] BAPN (beta-aminopropionitrile, fumarate 3-aminopropionitrile fumarate) group: BAPN (Sigma Aldrich) was dissolved in drinking water to a final concentration of 2 mg / mL and administered to mice two days before surgery until the end of the experiment. During surgery, general anesthesia was administered using 3% (vol / vol) isoflurane. The mice underwent laparotomy to expose the subrenal abdominal aorta and separate it from the vena cava. A gauze pad soaked in 10 μL of physiological saline was wrapped around the exposed subrenal abdominal aorta. After 10 minutes, the exposed area was gently cleaned with physiological saline and dried with cotton swabs. The abdominal wound was sutured layer by layer, and the mice were returned to their housing cages.

[0168] PPE group: Mice were provided with standard drinking water. General anesthesia was administered using 3% (vol / vol) isoflurane. An abdominal surgery was performed on the mice to expose the subrenal abdominal aorta and separate it from the vena cava. A 10 μL gauze soaked in PPE was wrapped around the exposed subrenal abdominal aorta. After 10 minutes, the exposed area was gently cleaned with physiological saline and dried with cotton swabs. The abdominal wound was sutured layer by layer, and the mice were returned to their rearing cages.

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

[0170] The results are as follows Figure 17 As shown in Table 4.

[0171] Table 4

[0172]

[0173] Note: Rupture time refers to the day after mouse modeling where the aneurysm ruptured and the mouse died. Unruptured means that the abdominal aortic aneurysm in the mouse had not ruptured by the end of the observation period of 100 days. The photoacoustic signal and diameter in each row represent the data of the lesion segment and the normal segment of the abdominal aorta in the same mouse with the corresponding ear tag. The photoacoustic signal ratio represents the ratio of the photoacoustic signal of the lesion segment to the normal segment of the abdominal aorta in the same mouse, and the diameter ratio represents the ratio of the diameter of the lesion segment to the normal segment of the abdominal aorta in the ultrasound image of the same mouse. A photoacoustic signal ratio greater than 1 indicates that the photoacoustic probe is specifically targeted to the AAA lesion.

[0174] Mice in the BAPN and PPE groups died without AAA rupture, while more than half of the mice in the BAPN+PPE group (n=8) experienced lethal rupture within 100 days. Compared to the BAPN, PPE, and BAPN+PPE unruptured groups, the BAPN+PPE ruptured group showed significantly stronger photoacoustic signals and the highest signal-to-noise ratio in the lesion wall. Compared to the BAPN and PPE groups, the BAPN+PPE group mice had a higher photoacoustic signal-to-background-noise ratio. Compared to the Sham group, the BAPN group mice showed loss of characteristic wavy structure and thinning of the aortic wall, the PPE group mice showed loss of abdominal aortic wall integrity and irreversible AAA dilation, and the BAPN+PPE group mice showed more pronounced aneurysm wall dilation, false lumens, and even ruptures in the aortic wall.

[0175] Application Example 3

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

[0177] Eight-week-old male C57BL / 6 mice (n=3 per group), weighing 20-25g, were used. Mice were anesthetized with 3% (vol / vol) isoflurane. After the mice developed difficulty standing, they were intraperitoneally injected with 10 μL / g of 4% (wt / vol) sodium pentobarbital. The hair on the mice's necks was shaved, and the skin was incised approximately 1.0 cm long along the midline of the neck. Under a stereomicroscope, the submandibular gland was bluntly dissected to expose the right common carotid artery (RCCA). The RCCA was then wrapped with gauze soaked in 10 μL of porcine pancreatic elastase (PPE) for 10 minutes, after which the gauze was removed and the mice were rinsed three times with physiological saline. Subsequently, the left common carotid artery (LCCA) was exposed, wrapped with gauze soaked in physiological saline for 10 minutes, then the gauze was removed, the carotid artery was rinsed three times with physiological saline, and the skin layer was intermittently sutured to construct a mouse model of carotid artery aneurysm. To simulate different time points in the early development of the aneurysm, on days 3, 7, and 14 after modeling, the photoacoustic probe (IBN-CHP) prepared in Example 1, IBN (prepared in step S1 of Example 1), and the photoacoustic probe (IBN-CHP) prepared in Comparative Example 1 were injected into mice in each group. S After CHP, 100 μL of 1 mM ICG aqueous solution was injected to perform photoacoustic in vivo imaging of the common carotid artery.

[0178] The results are as follows Figure 18 As shown.

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

[0180] The embodiments of the present invention have been described in detail above with reference to the examples. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A method for preparing a photoacoustic probe, characterized in that, Includes the following steps: S1. Prepare a mixture of serum albumin aqueous solution, cyanine dye, dehydrating solvent, and crosslinking agent, and react to obtain serum albumin-cyanine dye nanoparticles. The dehydrating solvent is acetone; the volume ratio of the serum albumin aqueous solution to the dehydrating solvent is 1:1~5; the cyanine dye is ICG; the pH of the serum albumin aqueous solution is 7.5~9.5; the mass ratio of the cyanine dye to serum albumin is 1:1~10; Step S1 includes a first post-reaction treatment; the first post-reaction treatment includes removing the dehydrating solvent; S2. The serum albumin-cyanine dye nanoparticles are linked to unwind collagen targeting molecules to obtain the photoacoustic probe.

2. The preparation method according to claim 1, characterized in that, The unwound collagen targeting molecule includes the amino acid sequence (GfO). n The polypeptide, n is a positive integer between 6 and 12, G is glycine, f is fluoroproline, and O is hydroxyproline; And / or, the crosslinking agent includes EDC.

3. The preparation method according to claim 1, characterized in that, The amount of the cross-linking agent used is 0.05~0.5 mg / mg serum albumin.

4. The preparation method according to claim 1, characterized in that, The reaction time for step S1 is 2 h to 8 h.

5. The preparation method according to claim 1, characterized in that, Step S2 includes: after the serum albumin-cyanine dye nanoparticles react with DBCO-Sulfo-NHS ester, they react with unwinding collagen targeting molecules modified with azide groups to connect the serum albumin-cyanine dye nanoparticles and the unwinding collagen targeting molecules to obtain the photoacoustic probe.

6. The preparation method according to claim 5, characterized in that, The reaction time between the serum albumin-cyanine dye nanoparticles and DBCO-Sulfo-NHS ester is 0.5 h to 2 h. Alternatively, the reaction time with the azide-modified unwinding collagen targeting molecule is 4 h to 12 h; Alternatively, in molar equivalents, the ratio of serum albumin:DBCO-Sulfo-NHS ester:unconjugated collagen targeting molecule in serum albumin-cyanine dye nanoparticles is 1:1~10:1~10.

7. A photoacoustic probe, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 6.

8. A reagent kit, characterized in that, Includes the photoacoustic probe as described in claim 7.

9. The photoacoustic probe prepared by the method according to any one of claims 1 to 6 is used in any one of A1) to A2); A1) Prepare products for detecting or assisting in the detection of cardiovascular diseases; A2) Prepare products that predict or assist in predicting the risk of aneurysm rupture.

10. The application according to claim 9, characterized in that, The cardiovascular diseases mentioned include at least one of the following: abdominal aortic aneurysm, intracranial aneurysm, carotid aneurysm, aortic dissection, myocardial infarction, atherosclerosis, and valvular disease; And / or, the aneurysm includes a coronary aneurysm, an abdominal aortic aneurysm, an intracranial aneurysm, or a carotid aneurysm.

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