Ultrasonic molecular probe, preparation method and application of ultrasonic molecular probe in preparation of medicine for diagnosing and treating atherosclerotic plaque

Abstract

The invention discloses an ultrasonic molecular probe. The ultrasonic molecular probe comprises a targeted contrast agent and siRNA of fibronectin, wherein the targeted contrast agent and the siRNA are connected through G0-C14; the targeted contrast agent comprises gas vesicles covalently linked with annular polypeptide, and the annular polypeptide targets an atherosclerotic plaque region. The cyclic polypeptide can be combined with the outer structural domain B of fibronectin with high specificity, and the atherosclerotic plaque extracellular matrix is rich in the outer structural domain B of the fibronectin, so that enough targeted ultrasonic contrast agent can be quickly combined in the plaque region to generate strong signal enhancement in ultrasonic imaging, and on the basis, the targeted ultrasonic contrast agent can be quickly combined with the plaque region to generate strong signal enhancement in ultrasonic imaging. The ultrasonic molecular probe releases siRNA for atherosclerotic plaque treatment, and atherosclerotic plaque diagnosis and treatment integration is achieved. The invention further discloses a preparation method of the ultrasonic molecular probe and application of the ultrasonic molecular probe in preparation of atherosclerotic plaque diagnostic agents, drugs and drug delivery agents.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to an ultrasonic molecular probe, a preparation method and application thereof in the preparation of drugs for diagnosing and treating atherosclerotic plaques. Background Art

[0002] Ultrasound molecular imaging is widely used in the diagnosis of diseases such as tumors, inflammation, and atherosclerosis due to its non-invasive, radiation-free, and high tissue penetration. However, conventional ultrasound molecular imaging probes rely heavily on targeted microbubbles with an average particle size of 1 to 10 μm. These gas-containing microbubbles can only remain in the vascular lumen, which limits their potential for application in extravascular lesions. In recent years, targeted or non-targeted nanobubbles composed of various phospholipid or polymer shells have been developed for ultrasound contrast agents. Several studies have demonstrated the effectiveness of chemically synthesized nanocontrast agents in exhibiting favorable acoustic properties; however, obstacles such as complex preparation procedures, insufficient biocompatibility, and structural fragility remain. Gas vesicles (GVs) from marine bacteria and archaea provide a promising alternative to traditional acoustic contrast agents. These biosynthesized nanobubbles have an average particle size of approximately 200 nanometers, which is smaller than traditional chemically synthesized nanocontrast agents, thereby improving tissue penetration. Unlike synthetic ultrasound contrast agents, GVs are produced in cells that possess genes encoding GV proteins, allowing structural modification through genetic engineering.

[0003] The external domain B of fibronectin (FN-EDB) is a splice variant of fibronectin (FN) with 91 amino acids. FN lacking the FN-EDB domain is usually expressed in adult tissues, while FN-EDB is mainly present in fetal tissues. However, FN-EDB can be expressed during active tissue remodeling processes such as tumor angiogenesis and wound healing. FN-EDB was originally considered a tumor-specific biomarker, leading to the development of a variety of antibodies against it for imaging and treatment. The cyclic polypeptide CTVRTSADC (ZD2) is a newly developed small peptide sequence that binds to FN-EDB with high specificity. Compared with specific antibodies that have been developed in the past, small peptides can be used as ultrasonic molecular imaging probes targeting malignant tumors due to their lack of specificity, high cost-effectiveness and easier transformation. The applicant previously provided a tumor-targeting ultrasound contrast agent and its preparation method and application (CN202310218086.4), the ultrasound contrast agent comprising nanobubbles covalently linked to a cyclic polypeptide, and the cyclic polypeptide targets the tumor area. When the cyclic polypeptide is CTVRTSADC, it can bind to FN-EDB with high specificity, and FN-EDB is rich in the extracellular matrix of tumor cells. Therefore, the prepared ultrasound contrast agent can specifically gather and image in the tumor area, overcoming the shortcomings of traditional ultrasound contrast agents that are not tumor-specific and have poor sensitivity in detecting tumors. The ultrasound contrast agent can be used to prepare tumor diagnostic agents, anti-tumor drugs, and drug delivery agents.

[0004] However, their potential applications in other disease settings remain unexplored. Atherosclerosis refers to the deposition of blood components such as lipids, smooth muscle cell proliferation, and increased collagen fibers in the arterial intima, forming atherosclerotic lipid-containing necrotic lesions and vascular wall hardening. Current imaging strategies for atherosclerosis, such as ligand targeting and biomimetic coatings on nanoparticles, still need to be improved due to problems such as poor targeting and contrast. Existing clinical contrast agents are not specific for atherosclerotic plaques and have poor sensitivity in detecting atherosclerotic plaques. Summary of the invention

[0005] Based on the above background, the first purpose of the present application is to propose a use of a targeted contrast agent in the preparation of an atherosclerotic plaque diagnostic agent; or, a use of a targeted contrast agent in the preparation of an atherosclerotic plaque drug; or, a use of a targeted contrast agent in the preparation of an atherosclerotic plaque drug delivery agent; the targeted contrast agent includes a gas vesicle covalently linked to a cyclic polypeptide, and the cyclic polypeptide targets the atherosclerotic plaque area to solve the problem that existing clinical contrast agents are not specific to atherosclerotic plaques.

[0006] Furthermore, the cyclic polypeptide targets the extracellular domain B of fibronectin in the area of ​​atherosclerotic plaques.

[0007] Furthermore, the nucleotide sequence of the cyclic polypeptide is shown in SEQ ID NO.1.

[0008] Furthermore, gas vesicles were isolated from Halobacteria NRC-1.

[0009] Furthermore, the targeted contrast agent is prepared by the following method, comprising the following steps:

[0010] S1: Isolation of gas vesicles from marine bacteria or archaea;

[0011] S2: Preparation of cyclic peptides targeting atherosclerotic plaques;

[0012] S3: adding the cyclic polypeptide to a buffer, and adding a coupling activator to the buffer containing the cyclic polypeptide to obtain a mixed solution;

[0013] S4: adding the mixed solution into the gas vesicles for incubation, and the cyclic polypeptide is covalently linked to the surface of the gas vesicles;

[0014] S5: separation and purification to obtain the targeted contrast agent.

[0015] Furthermore, the gas vesicles in step S1 are gas vesicles separated from Holabacteria NRC-1.

[0016] Furthermore, step S1 includes the following steps:

[0017] S1-1: Add Halobacteria NRC-1 to ATCC culture medium, place in a shaker at 37°C and culture at 220 rpm / min for 7-9 days to isolate Halobacteria NRC-1;

[0018] S1-2: Add the isolated Halobacteria NRC-1 bacteria to an equal volume of TMC lysis buffer, centrifuge, wash with PBS, and continue centrifugation to obtain white gas vesicles.

[0019] Furthermore, in step S3, the addition ratio of the cyclic polypeptide to the buffer is 0.3-0.7 μg / ml.

[0020] Furthermore, the coupling activators in step S3 are 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, and the mixture is incubated on a horizontal shaker at room temperature for 2-3 hours.

[0021] Further, in step S4, gas vesicles with an OD500 of 3.5 are screened out and added to a sodium bicarbonate solution with a pH of 8.3, and the volume ratio of the gas vesicles to the sodium bicarbonate solution is 1:4-6; and the gas vesicle solution is added to the mixed solution in step S3, and incubated overnight on a shaker at 4°C.

[0022] Furthermore, the separation method in step S5 is centrifugation, and the centrifugation conditions are centrifugation at a centrifugal force of 200-300g for 2-3h, repeated 3-4 times.

[0023] The second purpose of the present application is to propose an ultrasonic molecular probe, comprising a targeted contrast agent and siRNA of fibronectin connected by G0-C14; the targeted contrast agent comprises a gas vesicle covalently connected to a cyclic polypeptide, and the cyclic polypeptide targets the atherosclerotic plaque area, so as to realize the integrated diagnosis and treatment of atherosclerotic plaques.

[0024] Furthermore, the cyclic polypeptide targets the extracellular domain B of fibronectin in the area of ​​atherosclerotic plaques.

[0025] Furthermore, the nucleotide sequence of the cyclic polypeptide is shown in SEQ ID NO.1.

[0026] Furthermore, gas vesicles were isolated from Halobacteria NRC-1.

[0027] The third object of the present application is to provide a method for preparing an ultrasonic molecular probe, comprising the following steps:

[0028] S1: Isolation of gas vesicles from marine bacteria or archaea;

[0029] S2: Preparation of cyclic peptides targeting atherosclerotic plaques;

[0030] S3: adding the cyclic polypeptide to a buffer, and adding a coupling activator to the buffer containing the cyclic polypeptide to obtain a mixed solution;

[0031] S4: adding the mixed solution into the gas vesicles for incubation, wherein the cyclic polypeptide is covalently linked to the surface of the gas vesicles;

[0032] S5: separation and purification to obtain targeted contrast agent;

[0033] S6: dissolving G0-C14 in acetone to prepare a G0-C14 solution, and diluting the fibronectin siRNA with pure water to obtain a siRNA solution;

[0034] S7: Mix the G0-C14 solution and the siRNA solution, add the targeted contrast agent, and incubate to obtain the ultrasound molecular probe.

[0035] Among them, steps S1-S5 refer to the technical solution of the targeted contrast agent in the first object of the invention. On this basis, steps S6-S7 are performed to connect the siRNA of fibronectin using cationic G0-C14.

[0036] Furthermore, in step S6, the siRNA is obtained by PCR amplification; the upstream primer sequence is shown as SEQ ID NO.2; and the downstream primer sequence is shown as SEQ ID NO.3.

[0037] Furthermore, in step S6, the concentration of the siRNA solution is 100 μM.

[0038] Further, in step S6, the concentration of the G0-C14 solution is 2.5 mg / ml.

[0039] Furthermore, in step S7, the volume ratio of the siRNA solution to the G0-C14 solution is 136:284; the volume ratio of the siRNA solution to the targeted contrast agent is 136:360; wherein the OD500 of the targeted contrast agent is 3.5.

[0040] The fourth object of the present application is to propose the use of the above-mentioned ultrasonic molecular probe in the preparation of an atherosclerotic plaque diagnostic agent;

[0041] or, use of ultrasonic molecular probes in the preparation of atherosclerotic plaque drugs;

[0042] Or, use of ultrasound molecular probe in preparing drug delivery agent for atherosclerotic plaque.

[0043] The present application first covalently links the cyclic polypeptide to the surface of the gas vesicle, and then uses the cationic G0-C14 to link the siRNA of the external domain B of fibronectin, thereby realizing the integrated diagnosis and treatment of atherosclerotic plaques.

[0044] The advantages of the present invention are:

[0045] (1) The targeted contrast agent for targeted preparation of atherosclerotic plaques provided by the present invention has a small particle size, strong specificity, low antigenicity, low preparation difficulty, low cost, and low toxicity.

[0046] (2) The targeted contrast agent for targeted preparation of atherosclerotic plaques provided by the present invention has high targeting properties both in vitro and in vivo.

[0047] (3) The ultrasonic molecular probe targeting atherosclerotic plaques provided by the present invention can be used for the diagnosis and treatment of atherosclerotic plaques with high expression of the external domain B of fibronectin.

[0048] (4) The present invention is the first to combine siRNA of the external domain B of fibronectin with gas vesicles for the treatment of atherosclerotic plaques. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 : absorbance of GVs and ZD2-GVs at OD500;

[0050] Figure 2 : Transmission electron microscopy images of the targeted contrast agent ZD2-GVs (scale bars are 100 nm (left) and 50 nm (right));

[0051] Figure 3 : In vitro ultrasound imaging of simple GVs contrast agent, targeted contrast agent ZD2-GVs, and non-targeted contrast agent Con-GVs;

[0052] Figure 4 : In vitro targeting of the contrast agent ZD2-GVs to the ox-LDL-induced A7R5 cell line (blue represents the cell nucleus and green represents the contrast agent);

[0053] Figure 5 : The aggregation of ZD2-GVs and Con-GVs in atherosclerotic plaques;

[0054] Figure 6 :Quantitative images of in vivo ultrasound imaging of targeted contrast agent ZD2-GVs and non-targeted contrast agent Con-GVs;

[0055] Figure 7 : Representative fluorescence images of FITC-labeled Con-GVs or FITC-labeled ZD2-GVs in atherosclerotic plaques (blue represents cell nuclei, green represents contrast agents, and red represents blood vessels);

[0056] Figure 8 :Pathological images of atherosclerotic plaques treated with ZD2-GVs-G0-C14@EDBsiRNA. DETAILED DESCRIPTION

[0057] The present application is further described below with reference to embodiments, but is not limited thereto.

[0058] The experimental methods in the following examples, unless otherwise specified, are all conventional methods or carried out according to the instructions of the kit. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0059] In the description of the embodiments and the accompanying drawings, for the sake of convenience, brevity, professional practice and the like, the terms or abbreviations and their sources are explained as follows.

[0060] Halobacteria NRC-1: Abbreviated as Halo bacteria, the original bacteria Halobacteria;

[0061] ATCC medium: The formula is as follows: 250 g sodium chloride, 20 g magnesium sulfate heptahydrate, 3 g trisodium citrate, 2 g potassium chloride, 3 g yeast extract, 5 g tryptone, 1 L pure water. The prepared solution was autoclaved at 121°C, 18 psi for 30 min.

[0062] Gas Vesicle: Gas Vesicle, referred to as GVs;

[0063] ZD2: cyclic polypeptide, the nucleotide sequence is shown in SEQ ID NO.1;

[0064] Fibronectin extracellular domain B: FN-EDB;

[0065] TMC lysis solution: The formula is as follows: Tris-HCl 2.42g, magnesium chloride 1.01g, calcium chloride 0.58g, pure water 1L. The prepared solution is adjusted to pH 7.5 with concentrated HCl, filtered with a 0.22μm filter, and can be stored at room temperature for a long time.

[0066] EDC: 1-ethyl-(3-dimethylaminopropyl)carbodiimide;

[0067] NHS: N-hydroxysuccinimide.

[0068] Embodiment 1:

[0069] A method for preparing an ultrasonic molecular probe comprises the following steps:

[0070] S1-1: Cultivation of Halo bacteria

[0071] Add Halo bacteria to ATCC culture medium and culture in a shaker at 37°C at 220 rpm / min for 7-9 days. Then place in a separatory funnel and let stand until Halo floats to the surface of the liquid, remove the lower culture medium, and separate the Halo bacteria.

[0072] S1-2: Isolation of gas vesicles (GVs)

[0073] The isolated Halo bacteria were added to an equal volume of TMC lysis solution and centrifuged at 300g for 4 hours, repeated 3 times, to separate GVs from the Halo bacteria. The GVs were then washed with PBS and centrifuged at 250g for 4 hours, repeated 3 times, to obtain white GVs, which were stored in a 4°C refrigerator for future use.

[0074] S2: Synthesize a control peptide and a cyclic peptide ZD2 peptide targeting ED-B FN, wherein the control peptide has no targeting to ED-B FN in the atherosclerotic plaque area, and the nucleotide sequences of the control peptide and ZD2 are shown in SEQ ID NO.4 and SEQ ID NO.1, respectively. Simultaneously synthesize the control peptide and ZD2 peptide labeled with FITC.

[0075] S3: Dissolve the ZD2 peptide in PBS solution, the addition ratio is generally 0.3-0.7 μg / ml; in this embodiment, it is 0.5 μg / ml. Specifically, weigh 1 mg of ZD2 peptide and dissolve it in 2 ml of PBS solution, add coupling activator (8 mg EDC and 12 mg NHS), and incubate on a horizontal shaker at room temperature for 2-3 hours.

[0076] S4: At the same time, the absorbance of GVs at OD500 was measured by using a microplate reader. 1 ml of gas vesicles with an OD500 of 3.5 was added with 4-6 ml of sodium bicarbonate solution at pH 8.3, and the ZD2 peptide solution in step S3 was slowly added, and the mixture was incubated overnight at 4°C on a shaker.

[0077] S5: The obtained mixture was centrifuged at 250 g for 2-3 h, and repeated 3-4 times to remove free EDC, NHS and ZD2 peptide. The obtained ZD2-GVs were stored in a refrigerator at 4°C. This is the desired targeted contrast agent.

[0078] In this example, the method of coupling the control peptide, FITC-labeled control peptide and FITC-labeled ZD2 to GVs was the same as above.

[0079] S6: Dilute FN siRNA (upstream primer sequence: SEQ ID NO.2; downstream primer sequence: SEQ ID NO.3) with nuclease-free pure water to a 100 μM siRNA solution. Prepare a G0-C14 solution at a ratio of 250 μg G0-C14 to 100 μL acetone (2.5 mg / ml).

[0080] S7: Take 136 μL siRNA solution and gently mix it with 284 μL G0-C14 solution, add 300 μL ZD2-GVs (OD500=3.5), incubate at 4°C for 40 min, centrifuge at 250 g for 3 h to obtain the ultrasonic molecular probe ZD2-GVs-G0-C14@FN-EDBsiRNA, wash and set aside.

[0081] In order to verify the physicochemical properties, in vitro ultrasound imaging performance, in vivo ultrasound imaging performance, targeting and histological evaluation of the targeted contrast agent (ZD2-GVs) and ultrasound molecular probe in Example 1, the following series of verifications were performed.

[0082] Verification 1: Verification of ZD2-GVs

[0083] The absorbance of GVs and ZD2-GVs at OD500 was measured by an ELISA instrument. Figure 1 As shown by Figure 1 It can be seen that FITC-labeled ZD2 was successfully coupled to the surface of GVs, generating an absorption peak of FITC at 488 nm.

[0084] The targeted contrast agent ZD2-GVs was observed by transmission electron microscope (TEM). Figure 2 As shown, it is monodisperse, has a rugby-shaped structure, has a relatively regular shape, and is uniform in size. Take 1 ml of ZD2-GVs solution and place it in a special dish of a laser particle size analyzer to detect its particle size, distribution, and zeta potential. The Zetasizer measures that its particle size distribution is relatively uniform, about 205.30 ± 4.02 nm, and the potential is about -32.47 ± 0.94 mV mV. This shows that the targeted contrast agent ZD2-GVs of the present invention has a small particle size and good stability.

[0085] Validation 2: Evaluation of in vitro ultrasound imaging performance

[0086] GVs of different concentrations (OD500 of 0.5, 1.0, 1.5, and 2.0) were placed in a 1% agarose model well, and the linear array probe (3-11 MHz) of the clinical ultrasound diagnostic equipment Mindray Reson 7 was placed on one side of the agarose model and imaged in ultrasound contrast imaging mode. The imaging results are shown in Figure 2. Figure 3 As shown, it is proved that GVs can produce strong and stable contrast ultrasound signals. As the concentration of GVs increases, its ultrasound signal becomes stronger.

[0087] Validation 3: Evaluation of in vitro targeting ability

[0088] Rat thoracic artery smooth muscle cells (A7R5 cells) were placed at 37°C and 5% CO2 and cultured in a high-glucose medium supplemented with 10% FBS and 1% PS. A7R5 cells in the logarithmic growth phase were inoculated in a 24-well plate (1×10 5 / well), incubated overnight, oxidized low-density lipoprotein (ox-LDL) was incubated with A7R5 cells at a working concentration of 80μg / ml for 72h, and after inducing A7R5 cells into foam cells, PBS was washed three times, and the cells were fixed with 4% paraformaldehyde for 15min. After washing with PBS, the cell nuclei were stained with DAPI staining solution, and after washing with PBS again, FITC-labeled targeted contrast agent ZD2-GVs and control contrast agent Con-GVs were added and incubated with A7R5 cell-derived foam cells at room temperature for 5 minutes. Observed by inverted microscope, such as Figure 4As shown, compared with the control contrast agent Con-GVs, the targeted contrast agent ZD2-GVs could accumulate more around the A7R5 cell-derived foam cells.

[0089] Validation 4: Evaluation of in vivo targeting capability

[0090] Healthy male SD rats were fed with high-fat diet for one week and then underwent left carotid artery endothelial injury via balloon catheter. After the surgery, the rats were fed with high-fat diet until an atherosclerotic plaque model was formed and then underwent in vivo ultrasound imaging. 300ul of targeted contrast agent ZD2-GVs and control contrast agent Con-GVs with an OD500 of 3.5 were injected through the tail vein, with an interval of more than half an hour. The imaging was simultaneously video-collected until the ultrasound contrast signal disappeared. Subsequently, the built-in analysis software of the Mindray Reson 7 device was used for quantitative analysis to compare the aggregation of ZD2-GVs and Con-GVs in the tumor, such as: peak time, peak intensity, metabolic time, area under the curve and other parameters, such as Figure 5 As shown in the figure, it was found that ZD2-GVs had a longer circulation time in the plaque and a relatively slow decay curve compared with Con-GVs. At 10 minutes after injection, the ultrasound signal intensities of ZD2-GVs and Con-GVs were 66.65±25.52au and 28.53±25.59au, respectively, confirming that ZD2-GVs have good ultrasound molecular imaging capabilities in plaques ( Figure 6 ).

[0091] Verification 5: Histological examination

[0092] In order to determine whether GVs can penetrate the endothelial gap of plaque neovascularization, the present invention sliced ​​the plaque after injecting FITC-labeled ZD2-GVs and Con-GVs through the tail vein. The slices were incubated with rat anti-mouse CD31 antibody at 4°C overnight, then incubated with Cy5-labeled goat anti-rabbit secondary antibody, and the cell nuclei were stained with DAPI. Finally, the slices were observed under a confocal fluorescence microscope. Figure 7 As shown, ZD2-GVs were found not only in the vasculature but also in the neovascular extravascular space of plaques, which provided strong evidence that ZD2-GVs could extravasate from the vasculature and enter the plaque matrix space.

[0093] Verification 6: Evaluation of the therapeutic effect of ZD2-GVs-G0-C14@FN-EDBsiRNA

[0094] The rats with atherosclerosis were randomly divided into 5 groups, 3 rats in each group. The PBS group was injected with 300μL PBS, the GVs group was injected with 300μL OD5003.5 GVs, the GVs-siRNA group was injected with 113.6μL siRNA+284μL G0-C14+360μL GVs-siRNA (OD5003.5), the ZD2-GVs-siRNA group was injected with 113.6μL siRNA+284μL G0-C14+360μL ZD2-GVs (OD5003.5), and the ZD2-GVs-siRNA+ultrasound burst group was injected with 113.6μL siRNA+284μL G0-C14+360μL ZD2-GVs (OD5003.5), and the ultrasound burst was performed every 5s until the ultrasound signal disappeared. The treatment interval was twice a week, the treatment cycle was 8 weeks, and a total of 16 times. After the treatment, the carotid artery was extracted from the rats, fixed with paraformaldehyde (4% PBS), and then frozen and sectioned. Hematoxylin-eosin (H&E), oil red O (ORO), and Masson staining were used to quantitatively evaluate the area of ​​atherosclerotic plaques and lipid and collagen fibers. Immunohistochemical analysis was performed using anti-CD68 antibody to evaluate macrophage density. New blood vessels and smooth muscle cells were identified by anti-VEGF and α-SMA antibody staining, respectively. The results are shown in Figure 8 As shown, the results indicate that ZD2-GVs-G0-C14@EDBsiRNA treatment can preserve smooth muscle cells and collagen fibers on the plaque surface, reduce plaque area, and form a thick fibrous cap, thereby stabilizing the plaque and reducing the risk of thrombosis due to plaque damage.

[0095] Although the present application has been described with reference to illustrative embodiments, it will be appreciated by those skilled in the art that various other changes, omissions, and / or additions may be made and that elements of the described embodiments may be substituted with substantial equivalents without departing from the spirit and scope of the present application. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the present application without departing from the scope of the present application. Therefore, it is not intended herein to limit the present application to the disclosed specific embodiments for carrying out the present application, but rather it is intended that the present application will include all embodiments within the scope of the appended claims.

[0096]

Claims

1. Use of a targeted contrast agent in the preparation of an atherosclerotic plaque diagnostic agent; Or, use of a targeted contrast agent in the preparation of a drug for treating atherosclerotic plaques; Or, use of a targeted contrast agent in the preparation of a drug delivery agent for atherosclerotic plaques; The targeted contrast agent includes a gas vesicle covalently linked to a cyclic polypeptide, and the cyclic polypeptide is targeted to an atherosclerotic plaque area.

2. The use according to claim 1, characterized in that: The cyclic polypeptide targets the extracellular domain B of fibronectin in areas of atherosclerotic plaques.

3. The use according to claim 1, characterized in that: The nucleotide sequence of the cyclic polypeptide is shown in SEQ ID NO.

1.

4. The use according to claim 1, characterized in that: The gas vesicles were isolated from Halobacteria NRC-1.

5. The use according to claim 1, characterized in that: The targeted contrast agent is prepared by the following method, comprising the following steps: S1: Isolation of gas vesicles from marine bacteria or archaea; S2: preparing the cyclic polypeptide targeting atherosclerotic plaques; S3: adding the cyclic polypeptide to a buffer, and adding a coupling activator to the buffer containing the cyclic polypeptide to obtain a mixed solution; S4: adding the mixed solution into the gas vesicles for incubation, wherein the cyclic polypeptide is covalently linked to the surface of the gas vesicles; S5: separating and purifying to obtain the targeted contrast agent.

6. An ultrasonic molecular probe, characterized in that: The invention comprises a targeting contrast agent and siRNA of fibronectin linked by G0-C14; the targeting contrast agent comprises a gas vesicle covalently linked to a cyclic polypeptide, and the cyclic polypeptide targets an atherosclerotic plaque area.

7. The ultrasonic molecular probe according to claim 6, characterized in that: The cyclic polypeptide targets the extracellular domain B of fibronectin in areas of atherosclerotic plaques.

8. The ultrasonic molecular probe as claimed in claim 6, characterized in that: The nucleotide sequence of the cyclic polypeptide is shown in SEQ ID NO.

1.

9. The ultrasonic molecular probe according to claim 6, characterized in that: The gas vesicles were isolated from Halobacteria NRC-1.

10. A method for preparing an ultrasonic molecular probe according to any one of claims 6 to 9, characterized in that: The steps include: S1: Isolation of gas vesicles from marine bacteria or archaea; S2: preparing the cyclic polypeptide targeting atherosclerotic plaques; S3: adding the cyclic polypeptide to a buffer, and adding a coupling activator to the buffer containing the cyclic polypeptide to obtain a mixed solution; S4: adding the mixed solution into the gas vesicles for incubation, wherein the cyclic polypeptide is covalently linked to the surface of the gas vesicles; S5: separating and purifying to obtain the targeted contrast agent; S6: dissolving G0-C14 in acetone to prepare a G0-C14 solution, and diluting the siRNA of the external domain B of fibronectin with pure water to obtain a siRNA solution; S7: mixing the G0-C14 solution and the siRNA solution, adding the targeted contrast agent, and incubating to obtain the ultrasonic molecular probe.

11. A method for preparing an ultrasonic molecular probe as claimed in claim 10, characterized in that: In the step S6, the siRNA is obtained by PCR amplification; the upstream primer sequence is shown as SEQ ID NO.2; and the downstream primer sequence is shown as SEQ ID NO.

3.

12. A method for preparing an ultrasonic molecular probe as claimed in claim 10, characterized in that: In step S6, the concentration of the G0-C14 solution is 2.5 mg / ml.

13. A method for preparing an ultrasonic molecular probe as claimed in claim 10, characterized in that: In step S7, the volume ratio of the siRNA solution to the G0-C14 solution is 136:284; the volume ratio of the siRNA solution to the targeted contrast agent is 136:360; wherein the OD of the targeted contrast agent is 500 is 3.

5.

14. Use of the ultrasonic molecular probe according to any one of claims 6 to 9 in the preparation of an atherosclerotic plaque diagnostic agent; or, use of ultrasonic molecular probes in the preparation of atherosclerotic plaque drugs; Or, use of ultrasound molecular probe in preparing drug delivery agent for atherosclerotic plaque.

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