Application of type iv collagen as a target in early diagnosis of aortic aneurysm / dissection

Abstract

The present application relates to the four type collagen as the specific molecular target point in the targeted diagnosis and / or treatment of aortic dissection / aneurysm. The COLIV targeting peptide segment and its derivative are synthesized independently, the occurrence of aneurysm / dissection is monitored by monitoring the activation of endothelial cells, the monitoring mode is one or several of the following: ultrasonic detection, MRI detection, CTA detection, fluorescence imaging detection; in addition, the COLIV targeting peptide segment can also be used as a molecular target point of a targeted drug delivery system, to ensure that the disease site has a higher blood drug concentration and smaller toxic side effects, the drug is: a drug administered through the gastrointestinal tract, a drug administered by intravenous injection, a drug administered by subcutaneous embedding, preferably a drug administered by subcutaneous embedding.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of diagnosis and treatment of vascular diseases, in particular to a new aortic aneurysm targeting molecule and a method and reagent for early diagnosis and targeted treatment of thoracic aortic aneurysm dissection and abdominal aortic aneurysm. BACKGROUND

[0002] Aortic aneurysm refers to the local or diffuse abnormal expansion of the aortic wall beyond 50% of the normal diameter. Once it occurs, the rupture rate is as high as 80% or more. Aortic aneurysm is prone to occur in large arteries, including ascending aorta, aortic arch, descending thoracic aorta, thoraco-abdominal aorta and abdominal aorta. Due to the high blood pressure and flow rate of large arteries, it is easy to expand rapidly and even tear to form a dissection under stress, which threatens the life of the patient. Traditional diagnosis of aortic aneurysm relies on a combination of imaging and clinical symptoms, among which CTA and MRI are widely used as the gold standard for diagnosis in clinical practice.

[0003] In recent years, epidemiological data has found that in the very early stage of aortic aneurysm, the clinical symptoms are occult and the lumen does not appear obvious expansion; but at this time, the endothelium and the middle layer of the vessel wall have already undergone significant pathophysiological changes. At this time, if stress or sudden blood pressure rise occurs, it is easy to tear and form a dissection. Epidemiological data also confirms that there is no linear relationship between lumen diameter and rupture risk, and some aortic aneurysms with smaller diameters have a higher risk of rupture. Due to the high mortality rate after aortic dissection rupture and the forward shift of the age of onset, there is an urgent need for a specific and sensitive method to detect it in the early stage, so as to improve the survival rate and quality of life of patients.

[0004] With the in-depth study of the pathogenesis of aortic aneurysm, it is found that there are many pathological changes earlier and more specific than diameter changes in the formation process of aortic aneurysm, such as activation of endothelial cells, apoptosis of smooth muscle cells, degradation of medial elastic lamina and infiltration of inflammatory cells. In this process, the first to appear is the activation of endothelial cells, including the expansion of intercellular space, cell shrinkage and endothelial shedding. Type IV collagen is a major component of the subendothelial basement membrane, which can be exposed and detected after the activation of endothelial cells; in normal conditions, type IV collagen is mainly involved in the formation and stability of the basement membrane, while in pathological processes, it promotes cell migration. The development of molecular pathology provides the possibility for microscopic detection, which can select appropriate molecules in the pathological process as targets, specifically bind to them, and specifically monitor the occurrence and development of the pathological process; and through targeted drug loading, it can improve the blood drug concentration in the lesion part and reduce the toxicity to other organs.

[0005] Fluorescence imaging technology as an excellent detection and tracking means is widely used in the field of life science. Fluorescence imaging technology generally realizes the luminescence imaging in cells or animal bodies by designing reasonable and effective fluorescent molecules and assisting with functional modification. It has the characteristics of strong selectivity, high sensitivity, strong visualization effect and good repeatability, and is fully utilized in disease detection and diagnosis, nanomaterial tracking, new drug effect evaluation and the like. The general fluorescent probe needs to have certain excellent biocompatibility and optical properties, that is, it needs to have high fluorescence quantum yield and molar extinction coefficient, and at the same time, it needs to avoid the same excitation wavelength as the biological matrix to be effectively detected in cells or animal bodies. Most of the existing fluorescent probes are basically biological fluorescent probes and chemical synthesis probes, wherein the biological fluorescent probes are generally inherent biological proteins such as gene-encoded fluorescent proteins and the like, and the chemical fluorescent probes, including artificial synthesis of isothiocyanate fluorescein, rhodamine b, cyanine dye fluorescent markers and the like, are widely used in scientific research. SUMMARY

[0006] Therefore, the purpose of the present application is to study whether collagen IV can be used as a diagnostic marker for the precise diagnosis of aneurysm as a molecular target for the existing diagnosis and treatment technology.

[0007] The present application firstly relates to the application of collagen IV (COLIV) as a diagnostic detection target in the early diagnosis of aortic dissection / aneurysm.

[0008] The present application also relates to the application of collagen IV (COLIV) as a diagnostic detection target in the development / screening of early diagnostic markers for aortic dissection / aneurysm.

[0009] Further, the present application also relates to a polypeptide (COLIV-peptide) targeting collagen IV, wherein the sequence of the polypeptide is shown in SEQ ID NO. 1.

[0010] SEQ ID NO. 1: CGGGKPLVWLK.

[0011] The present application also relates to a nucleotide fragment encoding the polypeptide targeting collagen IV.

[0012] Further, the present application also relates to the application of the polypeptide (COLIV-peptide) targeting collagen IV or a derivative thereof in the preparation of an early diagnostic marker for aortic dissection / aneurysm, wherein the diagnosis includes but is not limited to any one or several of the following: detection under ultrasound, detection under MRI, detection under CTA, detection under fluorescence imaging.

[0013] The present application also relates to the use of the collagen IV-targeted peptide or its derivative in the preparation of a medicament for the treatment or prevention of aortic dissection / aneurysm, which medicament includes, but is not limited to, a medicament for gastrointestinal administration, a medicament for intravenous injection, and a medicament for subcutaneous embedding administration.

[0014] The derivative of the collagen IV-targeted peptide includes, but is not limited to, a product obtained by modifying / complexing / covalently coupling the collagen IV-targeted peptide with one or more of a fluorescent marker, an MRI marker, or other chemiluminescent marker or electrical signal marker.

[0015] Preferably, the derivative of the collagen IV-targeted peptide (COLIV-peptide) is COLIV-RhB obtained by covalently modifying the collagen IV-targeted peptide with rhodamine B, and the chemical structure of the derivative is shown in Formula 1 below:

[0016]

[0017] More preferably, the derivative of the collagen IV-targeted peptide (COLIV-peptide) is COLIV-Gb obtained by amide coupling of the collagen IV-targeted peptide with two molecules of gadolinium 1,4,7,10-tetraazacyclododecane-1,,4,7,10-tetracarboxylate, and the structure is shown in Formula 2 below:

[0018]

[0019] Most preferably, the derivative of the collagen IV-targeted peptide (COLIV-peptide) is a product (Collagen IV-targeted contrast agent, C4TCA) obtained by covalently modifying the collagen IV-targeted peptide with rhodamine B and then amide coupling of the product with two molecules of gadolinium 1,4,7,10-tetraazacyclododecane-1,,4,7,10-tetracarboxylate, and the structure is shown in Formula 2 below:

[0020]

[0021] Further, the present application also relates to the use of the COLIV-RhB, COLIV-Gb, and C4TCA in the preparation of a preparation for the early diagnosis of an aortic aneurysm.

[0022] Further, the present application also relates to a preparation method of the C4TCA, which comprises the steps of,

[0023] (1) fully salify the carboxyl group of N-hydroxysuccinimide activated 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (NHS-DOTA);

[0024] (2) complex N-hydroxysuccinimide modified 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (NHS-DOTA, compound 1) with gadolinium chloride hexahydrate to obtain N-hydroxysuccinimide modified 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid gadolinium (NHS-DOTA-Gd, compound 2);

[0025] (2) react COLIV-RhB (compound 3) with NHS-DOTA-Gd to obtain C4TCA (compound 4).

[0026] Preferably, the synthesis steps are:

[0027] (1) dissolve 12.7 mg of N-hydroxysuccinimide (NHS) activated 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (DOTA), i.e. NHS-DOTA, in 1 ml of deionized water, add 8 mg of sodium bicarbonate to it, and magnetically stir at room temperature for 10-20 minutes to fully salify the carboxyl group.

[0028] (2) dissolve 9.4 mg of gadolinium chloride hexahydrate in 200 μL of deionized water, slowly drop the obtained solution into the solution of example 4 (1), and magnetically stir at room temperature for 12 hours to fully complex.

[0029] (3) dissolve 20 mg of rhodamine b modified type IV collagen targeting polypeptide (RhB-COLIV) in 800 μL of water in the dark, add the obtained solution to the final solution of example 4 (2), magnetically stir at room temperature in the dark for 12 hours, purify the obtained mixture by ultrafiltration through 1000 MW dialysis membrane, and obtain a red flocculent solid by freeze-drying, and all post-treatment is in the dark.

[0030] The synthesis route is

[0031] BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 , time diagram of endothelial cell activation and medial elastic lamina rupture in aneurysm progression

[0033] Figure 2 , diagram showing the expression site of type IV collagen in normal / pathological blood vessels

[0034] Figure 3 Figure 2. The effect of C4TCA on early MRI and fluorescence imaging detection of mouse aortic aneurysm DETAILED DESCRIPTION

[0035] Experimental animals

[0036] Thoracic aortic dissection / aortic aneurysm model: 3-week-old male wild-type mice (C57BL / 6); ApoE- / - mice (C57BL / 6J) were purchased from Beijing Huafukang Biotechnology Co., Ltd. All animals were bred and raised in the SPF level environment animal room of Beijing Cardiovascular and Pulmonary Disease Research Institute. The wild-type mice should be male mice that reached 3 weeks of age and weighed about 10 g. The ApoE- / - mice should be male mice that were about 6 weeks old and weighed about 20-22 g. All experimental operations were performed according to the NIH Guide for the Care and Use of Laboratory Animals established in 1996 and the experimental procedures stipulated by the Capital Medical University Experimental Animal Management Committee. All experimental animals were grouped according to the random method.

[0037] Example 1, construction of thoracic aortic dissection / aortic aneurysm mouse model (mouse TAD model)

[0038] Construction and evaluation of BAPN water group mouse TAD model

[0039] Take 3-week-old C57BL / 6 background male mice and give them normal diet,

[0040] BAPN water group (n = 16): dissolve BAPN in drinking water at a dose of 1 g / kg / day.

[0041] On the 14th day of BAPN feeding, 5 mice were randomly selected for thoracic aortic ultrasound to determine whether there was widening / dissection of the thoracic aorta. After repeated experiments, some mice had formed aortic dissection / aortic aneurysm at 14 days, and began to die in succession.

[0042] Example 2, tissue section detection of mouse TAD model

[0043] 1. Frozen section:

[0044] At the end of the experiment, mice were sacrificed under anesthesia with pentobarbital (100 mg / kg) and the heart was perfused with heparinized saline to remove residual blood. The aortic arch and descending aorta were isolated and cut under a stereomicroscope and fixed in 4% paraformaldehyde for 2 hours, then transferred to 30% sucrose solution at 4°C overnight for adequate dehydration. The next day, the vessels were removed from the sucrose solution, the tissue water was absorbed with filter paper, and the vessels were embedded vertically in OCT embedding medium, wrapped in tin foil and slowly frozen in liquid nitrogen, and stored in a -80°C refrigerator. Continuous frozen tissue sections were prepared with a thickness of 5 μm and attached to polylysine-coated glass slides.

[0045] 2. Paraffin sections:

[0046] At the end of the experiment, mice were sacrificed under anesthesia with pentobarbital (100 mg / kg) and the heart was perfused with heparinized saline to remove residual blood. The aortic arch was isolated and cut under a stereomicroscope and placed in 10% formaldehyde solution for fixation; normal aorta and TAD tissues were placed in 10% formaldehyde solution for fixation. After at least 24 hours, the vessels were removed for dehydration and paraffin-embedded tissues (vessels vertically downward). Continuous sections were prepared with a thickness of 5 μm and attached to polylysine-coated glass slides.

[0047] Example 3, Histopathological examination of mouse TAD model:

[0048] I. Observation of elastic lamina by elastic fiber staining,

[0049] The specific operation is as follows:

[0050] 1.1. Frozen sections:

[0051] (1) Remove OCT from the frozen sections in PBS;

[0052] (2) Add 1 drop (100 μl) of Lugol's iodine solution (reagent A) and incubate for 5 minutes, then wash with water;

[0053] (3) Add 1 drop (100 μl) of sodium thioacid (reagent B) and treat for 5 minutes;

[0054] (4) Wash with running water for 5 minutes and wash with 70% alcohol;

[0055] (5) Add 1 drop (100 μl) of aldehyde fuchsin dye (reagent C) and stain for 10 minutes, then wash with 70% alcohol twice (about 30 seconds each time, until the section is no longer decolorized) and wash with water;

[0056] (6) Add 1 drop (100 μl) of orange G dye (reagent D) and stain for 10 seconds, then wash with water;

[0057] (7) 95% alcohol dehydration 5 min;

[0058] (8) Absolute alcohol dehydration 5 min;

[0059] (9) Xylene clearing 5 min, mounting;

[0060] 2. Paraffin section:

[0061] (1) Paraffin section was routinely dewaxed with xylene and then deparaffinized with different concentrations of ethanol: xylene I (20 min)→ xylene II (20 min)→ xylene III (20 min)→ 100% alcohol (5 min)→ 95% alcohol (5 min)→ 80% alcohol (5 min)→ tap water to remove alcohol.

[0062] (2) 1 drop (100 μl) of Lugol's iodine solution (reagent A) was added and incubated for 5 min, and then washed with water;

[0063] (3) 1 drop (100 μl) of sodium thioacid (reagent B) was added and incubated for 5 min;

[0064] (4) The section was washed with running water for 5 min and then washed with 70% alcohol;

[0065] (5) 1 drop (100 μl) of aldehyde fuchsin dye (reagent C) was added and incubated for 10 min, and then washed with 70% alcohol twice (about 30 s each time until the section was no longer decolorized), and then washed with water;

[0066] (6) 1 drop (100 μl) of orange G dye (reagent D) was added and incubated for 10 s, and then washed with water;

[0067] (7) 95% alcohol dehydration 5 min;

[0068] (8) Absolute alcohol dehydration 5 min;

[0069] (9) Xylene clearing 5 min, mounting;

[0070] The observation was performed using a Nikon ECLIPSE 90i microscope. The elastic fibers were blue-violet and the background was yellow to different degrees. The inner side of the elastic lamina was part of the neointima.

[0071] The results showed that after BAPN induction, the endothelial damage in the mouse TAD model appeared after 2 weeks, while the dissection appeared after 3 weeks, but there was no significant change in general. By comparing the general situation, elastic fibers and endothelial damage, we found that the endothelial damage appeared earliest and was the most sensitive compared to the former, which suggested that we should pay attention to the structure under the endothelium, thereby providing a potential target for early diagnosis. Figure 1

[0072] ​II. Observation of the structure of the blood vessel wall by immunofluorescence staining, as follows:

[0073] 1. Detection of the localization of different collagens in the vessel wall:

[0074] (1) The OCT was washed off the frozen section in PBS;

[0075] (2) 4% paraformaldehyde was used for fixation for 10 min, and PBS was used for washing three times.

[0076] (3) Serum blocking for 30 min

[0077] (4) Primary antibody fixation, overnight.

[0078] (COL I, rabbit, 1:500, Figure 2 Top, left 1)

[0079] (COL III, rabbit, 1:500, Figure 2 Top, left 2)

[0080] (COL IV, rabbit, 1:500, Figure 2 Top, left 3)

[0081] (EC, rat, 1:500, Figure 2 All top)

[0082] (5) Room temperature equilibration for 30 min, PBS was used for washing three times.

[0083] (6) Secondary antibody for 40 min, PBS was used for washing three times.

[0084] (Anti-rabbit, red, 1:1000)

[0085] (Anti-rat, green, 1:1000)

[0086] (7) DAPI mounting.

[0087] (8) Observation of the specimen morphology under a microscope.

[0088] 2. Detection of the change in the expression amount of COL IV at different periods of modeling:

[0089] (1) The OCT was washed off the frozen section in PBS;

[0090] (2) 4% paraformaldehyde was used for fixation for 10 min, and PBS was used for washing three times.

[0091] (3) Serum blocking for 30 min

[0092] (4) Primary antibody fixation, overnight.

[0093] (COL IV, rabbit, 1:500, Figure 2 Middle)

[0094] (a-SMA, Rat, 1:500, Figure 2 middle)

[0095] (5) Equilibrate at room temperature for 30 minutes and wash three times with PBS.

[0096] (6) Incubate with secondary antibody for 40 min and wash three times with PBS.

[0097] (anti-rabbit, green, 1:1000)

[0098] (anti-mouse, red, 1:1000)

[0099] (7) Cover the slides with DAPI.

[0100] (8) Observe the specimen morphology under a microscope.

[0101] 3. Sirius Red picric acid staining to assist in locating various types of collagen

[0102] (1) Paraffin sections were cut into 4-6 μm thick and dewaxed to remove water.

[0103] (2) Immerse in lapis lazuli blue solution for 5 to 10 minutes.

[0104] (3) Rinse with distilled water three times.

[0105] (4) Sirius red saturated picric acid solution for 15-30 minutes; (Harris hematoxylin solution can be used to lightly stain the cell nucleus).

[0106] (5) Directly use anhydrous ethanol for differentiation and dehydration.

[0107] (6) Xylene transparent, optical gum sealing.

[0108] Observation under polarized light microscope:

[0109] Type I collagen fibers: red or yellow, tightly arranged, and with strong birefringence.

[0110] Type II collagen fibers: various colors, loose reticular, weakly birefringent.

[0111] Type III collagen fibers: green, thin fibers, weakly birefringent.

[0112] Type IV collagen fibers: light yellow, weakly birefringent (basement membrane component).

[0113] The results showed that compared with the other two collagens (COLI and COLIII) that are abundant in blood vessels, COLIV is specifically located in the basement membrane under the endothelium of the blood vessels. It is the first layer exposed after vascular injury and can detect arterial damage at a very early stage. In addition, COLIV staining at SHAM, BAPN1W, 2W, 3W, and 4W time points revealed that the expression of COLIV in the subendothelium gradually increased with the model time ( Figure 2 ), the above proves that COLIV can be used as a molecular target for early thoracic aortic dissection / aortic aneurysm.

[0114] Example 4: Construction of Type IV Collagen Targeting Material C4TCA

[0115] To further detect early thoracic aortic dissection / aortic aneurysm in vivo, COLIV-RhB was further labeled with an MRI nuclear magnetic marker to synthesize C4TCA. The synthesis steps of C4TCA are as follows:

[0116] (1) Take 12.7 mg of NHS-activated 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (DOTA), i.e., NHS-DOTA, and dissolve it in 1 ml of deionized water. Add 8 mg of sodium bicarbonate and stir magnetically at room temperature for 10-20 minutes to fully salt the carboxyl groups.

[0117] (2) 9.4 mg of gadolinium chloride hexahydrate was dissolved in 200 μL of deionized water, and the resulting solution was slowly added dropwise to the solution of Example 4(1). The mixture was magnetically stirred at room temperature for 12 hours to allow for complete complexation.

[0118] (3) Take 20 mg of rhodamine b-modified type IV collagen targeting peptide (RhB-COLIV) and dissolve it in 800 μL of water in a dark environment. The resulting solution is added to the final solution obtained in Example 4 (2). The mixture is magnetically stirred at room temperature in a dark environment for 12 hours. The resulting mixture is purified by ultrafiltration through a 1000 MW dialysis membrane. The final solution is freeze-dried to obtain a red flocculent solid C4TCA. All post-treatments are carried out in a dark environment.

[0119] Example 5: Early diagnosis of aortic aneurysm using the targeted material C4TCA under in vivo MRI conditions and pathological sections

[0120] The experiment was performed using mice fed with BAPN water for 2 weeks as described above.

[0121] 1) 1.5 mg of the C4TCA material prepared in Example 4 was injected into the orbital vein of mice under suborbital anesthesia.

[0122] 2) Let it stand for 30 minutes to allow the material to fully circulate through the blood and then perform gas anesthesia again

[0123] 3) Connect to ECG and respiratory gating, and place in 7T MRI instrument

[0124] 4) Use the TI SE sequence to perform tomography of the entire descending aorta and the suprarenal part of the abdominal aorta.

[0125] 5) The lesioned part of the mouse blood vessels was extracted and made into frozen sections. After drying, the sections were directly sealed with DAPI and the fluorescence of the materials was observed under a microscope.

[0126] The results were compared with the MRI results to determine whether the enhanced MRI signal actually showed pathophysiological changes.

[0127] The results showed that on the 14th day of the aneurysm model, some mice showed enhanced signals in the descending aortic arch, and it was at this time that endothelial damage was observed in the pathological sections; while those vessels that did not show enhanced signals did not show any pathological physiological changes in the endothelium / media layer ( Figure 3 ), indicating that COLIV-targeted MRI and fluorescent dual probe C4TCA can specifically detect endothelial damage at an early stage, thereby monitoring the occurrence of TAD.

[0128] Example 6: Early diagnosis of aortic aneurysm using COLIV-Gb under in vivo MRI conditions

[0129] The experiment was performed using mice fed with BAPN water for 2 weeks as described above.

[0130] 1) 1.3 mg of the COLIV-Gb material prepared in Example 4 was injected into the orbital vein of mice under suborbital anesthesia.

[0131] 2) Let it stand for 30 minutes to allow the material to fully circulate through the blood and then perform gas anesthesia again

[0132] 3) Connect to ECG and respiratory gating, and place in 7T MRI instrument

[0133] 4) Use the TI SE sequence to perform tomography of the entire descending aorta and the suprarenal part of the abdominal aorta.

[0134] The results showed that when COLIV-Gb was used alone in the same mouse BAPN model, similar diagnostic results as C4TCA could be achieved under in vivo MRI diagnostic conditions, indicating that COLIV-Gb, like C4TCA, can also specifically detect endothelial damage at an early stage, thereby monitoring the occurrence of TAD.

[0135] Finally, it should be noted that the above embodiments are only used to help those skilled in the art understand the essence of the technical solution of the present invention and are not intended to limit the scope of protection of the present invention. SEQUENCE LISTING <110> Beijing Institute of Cardiovascular and Pulmonary Disease <120> Use of type IV collagen as a target for early diagnosis of aortic aneurysm / dissection <160> 1 <170> PatentIn version 3.3 <210> 1 <211> 11 <212> PRT <213> COLIV-peptide <400> 1 Cys Gly Gly Gly Lys Pro Leu Val Trp Leu Lys 1 5 10

Claims

1. Use of a polypeptide derivative specifically targeting type IV collagen in the preparation of an early diagnostic marker for aortic dissection and aortic aneurysm, characterized in that: The derivatives of the polypeptide targeting type IV collagen are specifically: The collagen IV-targeted contrast agent (C4TCA) is a product obtained by covalently modifying the type IV collagen peptide shown in SEQ ID NO. 1 with rhodamine B and then coupling two molecules of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid gadolinium via an amide bond. The structure of the product is shown in Formula 3. The preparation steps of the C4TCA are: (1) 12.7 mg of NHS-activated 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (DOTA) was dissolved in 1 ml of deionized water. 8 mg of sodium bicarbonate was added and the mixture was stirred magnetically at room temperature for 10-20 minutes to fully salt the carboxyl groups. (2) 9.4 mg of gadolinium chloride hexahydrate was dissolved in 200 μL of deionized water, and the resulting solution was slowly added dropwise to the solution of step (1), and magnetically stirred at room temperature for 12 hours to allow for full complexation; (3) 20 mg of rhodamine b-modified type IV collagen targeting peptide (RhB-COLIV) was dissolved in 800 μL of water in a dark environment, and the resulting solution was added to the solution obtained in step (2). The mixture was magnetically stirred at room temperature in a dark environment for 12 hours. The resulting mixture was purified by ultrafiltration through a 1000 MW dialysis membrane, and the final solution was lyophilized to obtain a red flocculent solid.

2. The use according to claim 1, characterized in that The diagnosis includes any one or more of ultrasound detection, MRI detection, CTA detection, and fluorescence imaging detection.

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