A tumor-targeting ultrasound contrast agent and its preparation method and application

Abstract

The present invention provides a tumor-targeting ultrasound contrast agent and a preparation method and application thereof, wherein the ultrasound contrast agent comprises nanobubbles covalently linked to a cyclic polypeptide, wherein the cyclic polypeptide targets the tumor region. When the cyclic polypeptide is a cyclic polypeptide CTVRTSADC, it can bind to ED-B FN with high specificity, and the tumor cell extracellular matrix is ​​rich in ED-B FN, so the ultrasound contrast agent prepared by this method can specifically gather and image in the tumor region, overcoming the shortcomings of traditional ultrasound contrast agents that are not tumor-specific and have poor sensitivity when detecting tumors. The ultrasound contrast agent can be used to prepare tumor diagnostic agents, prepare anti-tumor drugs, and prepare drug delivery agents, and has extremely high application value. The manufacturing process of this ultrasound contrast agent is simple, the experimental equipment is cheap, the production cost is reduced, and it is conducive to large-scale production.

Description

Technical Field

[0001] The invention relates to the field of contrast agents, and in particular to an ultrasound contrast agent targeting a tumor, and a preparation method and application thereof. Background Art

[0002] Ultrasound imaging technology has the advantages of being non-invasive, radiation-free, and inexpensive. It is widely used in the field of clinical medicine and has become an effective means of disease diagnosis and treatment. Ultrasound imaging technology requires reagents that can enhance medical detection signals-ultrasound contrast agents. In recent years, ultrasound contrast agents have been widely used in the diagnosis of thrombosis, tumors, inflammation, and other diseases; at the same time, the application of ultrasound contrast agents allows ultrasound assistance and targeted release of the drugs or genes they carry, thereby improving the treatment effect. At present, most ultrasound contrast agents are micron-sized and cannot penetrate the endothelial gaps of tumor blood vessels for ultrasonic molecular imaging or focused delivery of drugs / genes. Therefore, it is necessary to explore ultrasound contrast agents with high penetration in tumor blood vessels.

[0003] Nanobubbles represent a common nanoscale ultrasound contrast agent with strong permeability and high stability, which can not only enhance the extravascular ultrasound imaging of tumors, but also deliver drugs or genes to tumor parenchymal cells with ultrasound assistance. Recently, gas vesicles (GVs) from some marine bacteria and archaea have been reported as new nanoscale contrast agents. However, most of the GVs reported in the previous literature did not show good contrast imaging capabilities due to their small size. Therefore, there is currently a lack of ultrasound contrast agents containing GVs with good imaging capabilities.

[0004] Traditional ultrasound contrast agents are microbubbles made of gas wrapped in membrane materials such as phospholipids and albumin. These gas-containing microbubbles can pass through the systemic circulation and pulmonary circulation, and are mostly used clinically to show the distribution and integrity of the blood pool system. Compared with ordinary contrast agents, targeted contrast agents can recognize specific antigen epitopes of target tissues at the molecular level and firmly bind to them, ultimately allowing the target tissue to be specifically visualized. In order to achieve this goal, contrast agents need to be targeted and modified. At present, there is an urgent need for ultrasound contrast agents with tumor targeting capabilities on the market.

[0005] The prior art contains an ultrasound contrast agent targeting a single disease, which adds phosphocerebroside to the microbubble outer membrane component of the contrast agent to obtain ultrasound contrast targeting tumor tissue. However, since the microbubbles do not specifically bind to tumor cells, the tumor targeting of the ultrasound contrast agent is poor, and therefore the sensitivity in detecting tumors is poor. At present, there is a need to explore ultrasound contrast agents with high specificity and good tumor targeting.

[0006] At the same time, in addition to the above problems, in order to extract pure microbubbles, the targeted ultrasound contrast agent in the prior art uses rotary evaporation to continuously distill volatile solvents under reduced pressure conditions; in order to crush solid particles or cell tissues in the liquid, ultrasonic crushing and other steps are required in the preparation process. In the preparation process, a variety of precision instruments are required, the experimental requirements are high, the preparation cost is high, and the preparation process is complicated.

[0007] In order to make ultrasound contrast agents have targeting functions, peptides or proteins with targeting functions can be added to them. However, due to the high antigenicity of peptides or proteins, adding peptides or proteins generally produces an immune response in the patient's body and is likely to cause harm to the human body. Therefore, there is a lack of targeted ultrasound contrast agents with low antigenicity and mild performance on the market.

[0008] Therefore, existing ultrasound contrast agents have the disadvantages of low specificity, low tumor targeting, poor imaging, low tumor penetration, high antigenicity, low safety, great difficulty in preparation, and high cost. Summary of the invention

[0009] The purpose of the present invention is to provide an ultrasound contrast agent with tumor targeting function, which solves the problems of low specificity, low tumor targeting, poor imaging, low tumor penetration, high antigenicity, low safety, great difficulty in preparation and high cost of traditional ultrasound contrast agents proposed in the above background technology.

[0010] To achieve the above object, the present invention provides the following technical solutions:

[0011] An ultrasound contrast agent includes nanobubbles covalently linked to a cyclic polypeptide, the cyclic polypeptide targeting a tumor. Since the cyclic polypeptide targets the tumor site, the ultrasound contrast agent accumulates in the tumor area, so the ultrasound contrast agent has the advantages of high specificity, high tumor targeting, and high tumor penetration.

[0012] Furthermore, the cyclic polypeptide is a cyclic polypeptide CTVRTSADC, and its amino acid sequence is: CTVRTSADC (SEQ ID NO.1), or a sequence with more than 90% homology thereto. The cyclic polypeptide CTVRTSADC is a small peptide sequence with low antigenicity, and can specifically bind to the tumor area without generating an immune response in the patient's normal tissues, and has good tumor targeting function, overcoming the disadvantage of high antigenicity of traditional ultrasound contrast agents containing polypeptides and proteins, and is therefore a targeted ultrasound contrast agent with low antigenicity and mild performance.

[0013] Furthermore, the nanobubbles are gas vesicles extracted from Holabacteria NRC-1. Most of the gas vesicles reported in previous literature did not show good contrast imaging capabilities due to their small size, while the gas vesicles extracted from Holabacteria NRC-1 in the present invention have good imaging capabilities, overcoming the disadvantage of poor imaging capabilities of traditional gas vesicles.

[0014] In another aspect, the present invention provides a method for preparing an ultrasound contrast agent, comprising the following steps:

[0015] S1. preparing a tumor-targeting cyclic polypeptide, and adding the tumor-targeting cyclic polypeptide into a buffer;

[0016] S2, adding a coupling activator to a buffer solution containing the cyclic polypeptide to obtain a mixed solution;

[0017] S3, preparing nanobubbles, adding the mixed solution into the nanobubbles for incubation, during which the cyclic polypeptide is covalently linked to the surface of the nanobubbles through an amide reaction;

[0018] S4. Extract the nanobubbles covalently linked to the cyclic polypeptide to obtain an ultrasound contrast agent.

[0019] The nanobubbles covalently linked to the cyclic polypeptide are manufactured through the above steps. Since the cyclic polypeptide has the function of targeting tumors, the ultrasound contrast agent can target tumors. The function of the ultrasound contrast agent targeting tumors enables it to play a role in tumor diagnosis, preparation and delivery of tumor drugs.

[0020] Furthermore, the coupling activator in step S2 is 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide. EDC and NHS have the advantages of being non-toxic and biocompatible, and are good materials for activating carboxyl groups for amide reaction.

[0021] Furthermore, the nanobubbles in step S1 are gas vesicles extracted from Holabacteria NRC-1, and the placement ratio of the cyclic polypeptide to the buffer is 150-300 μg / ml. Within this concentration range, the reaction between the cyclic polypeptide and the gas vesicles is more sensitive.

[0022] Further, after obtaining the mixed solution in step S2, the absorbance of the nanobubbles at OD500 is measured by a microplate reader, and the nanobubbles with OD500 of 2.5-3.0 are screened out for subsequent steps. This process extracts high-concentration nanobubbles, which helps to increase the intensity of the ultrasound signal of the ultrasound contrast agent.

[0023] Furthermore, in step S3, the ratio of the mixed solution to the gas vesicles is 1:1-2:3. Within this concentration range, the chemical coupling reaction between the cyclic polypeptide and the gas vesicles proceeds smoothly.

[0024] Furthermore, the extraction method in step S4 is centrifugation, and the centrifugation conditions are centrifugation at a centrifugal force of 200-300g for 2-3h, repeated 3-4 times. The free coupling activator in the mixed solution is removed by centrifugation to obtain an ultrasound contrast agent.

[0025] In another aspect, the present invention provides uses of the ultrasound contrast agent, including uses in preparing tumor diagnostic agents; and / or uses in preparing anti-tumor drugs; and / or uses in preparing drug delivery agents.

[0026] Since the ultrasound contrast agent manufactured by the present invention contains a tumor-targeting cyclic polypeptide, it can specifically aggregate in the tumor area, and the anti-tumor drug prepared by it can specifically act on the tumor site without causing damage to normal tissues, so it has the advantages of high specificity, high tumor targeting, good anti-tumor effect, and small sequelae; and the tumor diagnostic agent prepared using the ultrasound contrast agent has the advantage of high accuracy; since the ultrasound contrast agent has a small molecular weight and strong tumor penetration, it is a good anti-tumor drug delivery agent, so the drug delivery agent prepared by the ultrasound contrast agent manufactured by the present invention makes the delivery of anti-tumor drugs more accurate, shortens the drug action time, and enhances the efficacy of the drug.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention provides a tumor-targeting ultrasound contrast agent and a preparation method and application thereof. By covalently linking a tumor-targeting cyclic polypeptide on the surface of a nanobubble, the contrast agent has the function of targeting the tumor. For example, the cyclic polypeptide CTVRTSADC (ZD2 peptide) can target the tumor, resulting in the ultrasound contrast agent specifically targeting the tumor, so the ultrasound contrast agent has the advantages of high tumor penetration and high tumor targeting. The gas vesicles extracted from HolabacteriaNRC-1 are used as nanobubbles. The molecular weight of such gas vesicles is small, so the antigenicity is small, the safety is high, and it also has the advantage of good imaging. At the same time, the manufacturing method of the ultrasound contrast agent in this scheme is a simple experimental operation, which reduces the difficulty of manufacturing the ultrasound contrast agent and reduces the manufacturing cost. The ultrasound contrast agent manufactured by the present invention is helpful for the early diagnosis of tumor cells, the preparation of tumor drugs or the delivery of tumor drugs, and has a very high application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0030] Figure 1 A flow chart for preparing an ultrasound contrast agent;

[0031] Figure 2 In vitro ultrasound imaging of targeted ultrasound contrast agent ZD2-GVs and non-targeted ultrasound contrast agent CTR-GVs;

[0032] Figure 3 It is a line graph of the absorbance values ​​of FITC-labeled targeted ultrasound contrast agent ZD2-GVs and blank bubble GVs at different wavelengths;

[0033] Figure 4A This is a schematic diagram of the in vitro targeting of the targeted ultrasound contrast agent ZD2-GVs to the bladder cancer MB49 cell line observed under a fluorescence microscope. Figure 4B This is a bar graph comparing the fluorescence intensity of targeted ultrasound contrast agent ZD2-GVs and non-targeted ultrasound contrast agent CTR-GVs in vitro;

[0034] Figure 5 It is the quantitative image of in vivo ultrasound imaging of targeted ultrasound contrast agent ZD2-GVs and non-targeted ultrasound contrast agent CTR-GVs;

[0035] Fig. 6A Representative fluorescence images of FITC-labeled CTR-GVs or FITC-labeled ZD2-GVs in tumor sections of MB49 tumor-bearing mice. Figure 6B The figure is a bar graph comparing the fluorescence intensity of FITC-labeled ZD2-GVs and FITC-labeled CTR-GVs in tumor sections of MB49 tumor-bearing mice. DETAILED DESCRIPTION

[0036] The technical solution of this patent will be further described in detail below in conjunction with specific implementation methods. It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by ordinary technicians in the technical field to which this application belongs.

[0037] Existing clinical contrast agents are not tumor-specific and have poor sensitivity in detecting tumors. The development of tumor-specific contrast agents will improve the accumulation of contrast agents within tumors. Currently, there is a lack of ultrasound contrast agents on the market that are highly specific and have strong tumor penetration and can target tumors.

[0038] The present invention provides a tumor-targeting ultrasound contrast agent and a preparation method and application thereof. By covalently linking a cyclic polypeptide with tumor targeting function to the surface of nanobubbles, the ultrasound contrast agent can be specifically aggregated in the tumor area, thereby improving the tumor targeting of the ultrasound contrast agent. A small molecular weight cyclic polypeptide CTVRTSADC (ZD2 peptide) is prepared. The ZD2 peptide has the advantages of low antigenicity and good tumor targeting. At the same time, the extraction method of gas vesicles is optimized to obtain gas vesicles with good imaging properties. Moreover, the manufacturing process of this ultrasound contrast agent is simple, the experimental equipment is cheap, the production cost is reduced, and it is conducive to large-scale production.

[0039] Embodiment 1:

[0040] This embodiment introduces a tumor-targeting ultrasound contrast agent and its preparation method and application.

[0041] The unique microenvironment of malignant tumors is conducive to the survival, proliferation and metastasis of cancer cells. The expression of cancer-related proteins in the extracellular matrix (ECM) of malignant tumors is abnormally high. For example, the macromolecular glycoprotein fibronectin (FN), one of the most abundant components of the extracellular matrix, plays a key role in tumorigenesis, angiogenesis and metastasis. FN mainly exists in two different forms, one is cellular FN (cFN), which encodes additional domains A and B (ED-A and ED-B). The other form is plasma FN (pFN), which does not contain ED-A and ED-B. Studies have confirmed that ED-B FN is a known marker of angiogenesis and epithelial-mesenchymal transition, and plays an important role in the metastasis and invasion of cancer. Therefore, highly expressed ED-B FN is a promising biomarker in the detection and diagnosis of malignant tumors.

[0042] Existing clinical contrast agents are not tumor-specific and therefore have poor sensitivity in detecting tumors. The development of tumor-specific contrast agents will improve the accumulation of contrast agents in tumors. Cyclic peptide CTVRTSADC (ZD2 peptide) is a newly developed small peptide sequence that can specifically bind to ED-B FN. ZD2 peptide can bind to ED-B FN with high specificity, and ED-B FN is rich in tumor extracellular matrix. At the same time, ZD2 peptide has the advantages of low antigenicity, low cost and easy conversion.

[0043] The present invention provides a tumor-targeting ultrasound contrast agent to help early detection, accurate diagnosis and accurate treatment of tumors. Covalently linking the ZD2 peptide to the surface of GVs will effectively improve the tumor targeting efficacy of the ultrasound contrast agent and significantly increase the enrichment of the genes it carries in the target tissue. The following is further explained in conjunction with the specific scheme:

[0044] An ultrasound contrast agent comprises GVs covalently linked to a ZD2 peptide, wherein the amino acid sequence of the ZD2 peptide is CTVRTSADC (SEQ ID NO. 1), or a sequence having more than 90% homology thereto, preferably more than 92%, 95%, or 98% homology thereto.

[0045] ZD2 peptide is a small peptide sequence with low antigenicity and can specifically bind to the tumor area without producing an immune response in the patient's normal tissues. It has good tumor targeting function and overcomes the disadvantage of high antigenicity of traditional ultrasound contrast agents containing polypeptides and proteins. Therefore, this ultrasound contrast agent is a targeted ultrasound contrast agent with high specificity, high tumor targeting, low antigenicity and mild performance.

[0046] Furthermore, GVs are extracted from Holabacteria NRC-1. Most of the GVs reported in previous literature did not show good contrast imaging ability due to their small size, while the GVs extracted from Holabacteria NRC-1 in the present invention have good imaging ability, overcoming the disadvantage of poor imaging ability of traditional GVs.

[0047] In recent years, ultrasound contrast agents have been widely used in the diagnosis of thrombosis, tumors, inflammation and other diseases. These materials allow ultrasound-assisted, targeted release of the drugs or genes they carry, thereby improving the treatment effect. Existing clinical contrast agents are not tumor-specific and have poor sensitivity in detecting tumors.

[0048] The development of tumor-specific contrast agents will improve the accumulation of contrast agents in tumors. The present invention covalently links the ZD2 peptide to the surface of GVs for the first time to produce a tumor-targeted ultrasound contrast agent, which helps in the early detection, accurate diagnosis and accurate treatment of tumors.

[0049] Since the ultrasound contrast agent manufactured by the present invention has the advantages of high specificity and high tumor targeting, the tumor drug prepared by it can act specifically on the tumor site without causing damage to normal tissues, so the tumor diagnostic agent prepared by it has high accuracy, good anti-tumor effect and few sequelae. Since the ultrasound contrast agent also has the advantages of good tumor penetration and good imaging, and its molecular weight is small and tumor penetration is strong, it is a good drug delivery agent. The ultrasound contrast agent manufactured by the present invention makes drug delivery more accurate, shortens the drug action time, and enhances the efficacy of the drug.

[0050] This ultrasound contrast agent can be used to prepare a diagnostic agent used in the process of tumor diagnosis; this ultrasound contrast agent can be used in combination with an anti-tumor drug to prepare an anti-tumor drug; and this ultrasound contrast agent can be used to prepare a tumor delivery drug, which is targeted to the tumor site, thereby achieving remote control and localized drug release at the tumor site.

[0051] Embodiment 2:

[0052] This example describes in detail the preparation method of the tumor-targeting ultrasound contrast agent.

[0053] Please refer to the attached Figure 1 , Figure 1 The figure is a flow chart of preparing an ultrasound contrast agent. In this example, GVs extracted from Holabacteria NRC-1 and ZD2 peptide are selected to prepare a targeted ultrasound contrast agent ZD2-GVs targeting ED-B FN. The preparation method is as follows:

[0054] S1. Prepare ZD2 peptide. Dissolve 200 ug of ZD2 peptide in 1 ml of PBS buffer.

[0055] The preparation method of ZD2 peptide is as follows:

[0056] S13. Synthesize a ZD2 peptide targeting ED-B FN, whose amino acid sequence is: CTVRTSADC (SEQ ID NO. 1), and simultaneously synthesize a FITC-labeled ZD2 peptide.

[0057] The cyclic peptide CTVRTSADC is a small peptide sequence with low antigenicity, which overcomes the disadvantage of high antigenicity of traditional ultrasound contrast agents containing peptides and proteins, and can therefore reduce the immune response produced by the drug, with high safety. In addition, the ZD2 peptide can specifically bind to the tumor area and has good tumor targeting function, so it is a good material for preparing ultrasound contrast agents with tumor targeting. In this embodiment, the ZD2 peptide is selected as a cyclic peptide targeting tumors. It can be understood that, for example, other cyclic peptides with tumor targeting function, such as RGD peptide, can also be used in the present invention;

[0058] Specifically, step S1 includes preparing ZD2 peptide, and then weighing 200 ug of ZD2 peptide and dissolving it in 1 ml of PBS solution.

[0059] S2. Add a coupling activator to the PBS buffer containing the ZD2 peptide to obtain a mixed solution.

[0060] The coupling activators used in this embodiment are 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS). EDC and NHS have the advantages of being non-toxic and biocompatible, and are often used to activate carboxyl groups for amide bond reactions;

[0061] Specifically, step S2 includes adding 3 mg EDC and 3 mg NHS to a buffer containing a cyclic polypeptide to obtain a mixed solution, and incubating the mixed solution on a horizontal shaker at room temperature for 2-3 hours, thereby obtaining activated ZD2. The ultrasonic signal of GVs is stronger when the concentration range is OD500=2.5-3.0, so the absorbance of GVs at OD500 is measured by a microplate reader, and GVs with a concentration of OD500=2.5-3.0 are extracted for the next step. In this embodiment, GVs with OD500=3.0 are used.

[0062] S3. Prepare GVs, add the mixture to 1 ml of GVs, and incubate overnight on a shaker at 4° C. In the presence of EDC and NHS, the ZD2 peptide is covalently linked to the GVs via an amide reaction.

[0063] The steps for preparing GVs are as follows:

[0064] S31, extract Halobacteria NRC-1 bacteria: add Halobacteria NRC-1 (Halo) into ATCC culture medium, place in a shaker at 37°C and culture at 220 rpm / min for 7-9 days. Then, place the culture medium in a separatory funnel and let it stand until Halo floats to the surface of the liquid, remove the lower culture medium, and separate the Halo bacteria;

[0065] S32, extract GVs: add the separated Halo bacteria to an equal volume of TMC lysis solution, centrifuge at 300g for 4h, repeat 3 times, and obtain GVs. Then wash the GVs with PBS, centrifuge at 250g for 4h, repeat centrifugation 3 times, obtain white GVs, and store them in a 4℃ refrigerator for future use. In this step, pure GVs are obtained by two-step centrifugation.

[0066] As nanobubbles, GVs have the characteristics of small particle size and high tumor penetration. Most gas vesicles reported in previous literature did not show good contrast imaging ability due to their small size, while the gas vesicles extracted from Holabacteria NRC-1 in the present invention have good imaging ability, overcoming the disadvantage of poor imaging ability of traditional gas vesicles. It is understandable that other nanobubbles with good imaging ability are also suitable for the present invention.

[0067] The placement ratio of the mixed liquid to the GVs ranges from 1:1 to 2:3. In this embodiment, the placement ratio of the mixed liquid to the GVs is 1:1.

[0068] S4. Extract GVs covalently linked to ZD2 peptide to obtain targeted ultrasound contrast agent ZD2-GVs targeting ED-B FN.

[0069] In this embodiment, the method for extracting ZD2-GVs in step S4 is centrifugation, which includes centrifuging the obtained mixture at a centrifugal speed of 250g for 2-3h, repeating 3-4 times to remove free EDC, NHS and ZD2. The obtained targeted contrast agent is stored in a refrigerator at 4°C.

[0070] The above preparation method obtains an ultrasound contrast agent with ZD2 peptide covalently coupled to the surface of GVs. Since ZD2 peptide can bind ED-B FN with high specificity, and ED-B FN is rich in the extracellular matrix of tumor cells, it allows rapid binding of sufficient targeted contrast agents to produce a strong enhanced signal in ultrasound imaging. Therefore, the ultrasound contrast agent prepared by this method 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.

[0071] Moreover, ZD2 peptide is a newly developed small peptide sequence that can specifically bind to ED-B FN. Compared with the specific antibodies that have been developed in the past, the small peptide has the advantages of lack of specificity, high cost-effectiveness and easy conversion, and can be used to prepare ultrasonic molecular imaging probes targeting malignant tumors. Therefore, the ultrasonic contrast agent prepared by this method has the advantages of low antigenicity, good imaging, high tumor targeting, low toxicity, etc. At the same time, the preparation method is simple, does not require the use of complex precision instruments, and the cost of using ZD2 peptide as a raw material is low, which greatly reduces the production cost and is conducive to the large-scale production of ultrasonic contrast agents.

[0072] Embodiment 3:

[0073] This example verifies that the ultrasound contrast agent of the present invention has the advantages of small particle size and high stability.

[0074] Check the stability of the ultrasound contrast agent: Using a transmission electron microscope, it is found that the ultrasound contrast agent prepared by the present invention is a monodisperse rugby-shaped structure with a regular shape and uniform size. Take 1 ml of the ultrasound contrast agent solution and place it in a special dish for 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, with a particle size of about 236.70 ± 3.46 nm and a potential of about -40.17 ± 1.21 mV. The above experimental results show that the ultrasound contrast agent of the present invention has a small particle size and good stability.

[0075] Ultrasonic contrast agents in the prior art are mostly micrometer-sized and cannot penetrate the endothelial gap of tumor blood vessels for ultrasonic molecular imaging or focused delivery of drugs / genes. However, the ultrasonic contrast agent manufactured by the present invention is nanometer-sized, with a small particle size and high stability, which is conducive to penetrating the endothelial gap of tumor blood vessels for ultrasonic molecular imaging or focused delivery of drugs / genes. Therefore, the ultrasonic contrast agent has great potential in tumor diagnosis, tumor drug manufacturing and delivery.

[0076] Check the ultrasound contrast agent's in vitro ultrasound imaging effect: GVs of different concentrations were placed in a 1% agarose model hole, the concentration range of GVs was OD500 = 0.5-2.5, the linear array probe of the clinical ultrasound diagnostic equipment Mindray Reson 7 was placed on one side of the agarose model under the condition of 3-11MHz, and imaging was performed in ultrasound contrast imaging mode, and it was found that it produced a strong and stable contrast ultrasound signal. Please refer to Figure 2 , Figure 2 The in vitro ultrasound imaging diagram of the targeted ultrasound contrast agent ZD2-GVs and the non-targeted ultrasound contrast agent CTR-GVs. The horizontal axis shows that the concentration of OD500 increases from left to right, and the white area in the circle is the in vitro ultrasound imaging diagram of ZD2-GVs and CTR-GVs. The figure shows that as the concentration of GVs increases, the ultrasound signal of ZD2-GVs becomes stronger and stronger. This experimental result shows that the ultrasound contrast agent of the present invention has the advantage of good imaging.

[0077] Embodiment 4:

[0078] In this embodiment, the most suitable wavelength for distinguishing blank bubble GVs from targeted ultrasound contrast agent ZD2-GVs was found through experiments.

[0079] A control peptide and a ZD2 peptide targeting ED-B FN were synthesized, and their amino acid sequences were CERAK and CTVRTSADC, respectively. FITC-labeled control peptide and ZD2 peptide were also synthesized, and then the control peptide and ZD2 peptide were coupled to the surface of GVs, respectively.

[0080] Please see attached Figure 3 , Figure 3 The figure is a line graph of the absorbance values ​​of FITC-labeled targeted ultrasound contrast agent ZD2-GVs and blank bubble GVs at different wavelengths. The horizontal axis is the wavelength, and the vertical axis is the absorbance value. The horizontal line shows that the absorbance value of GVs decreases with the increase of wavelength in the wavelength range of 400-600nm. The horizontal line with dots shows that the absorbance value of ZD2-GVs is stable between 2.5-3.0 in the wavelength range of 400-500nm. The absorbance value of ZD2-GVs gradually decreases in the wavelength range of 500-600nm, and the decrease is similar to that of GVs. Figure 3 The results show that the absorbance value of ZD2-GVs at 488nm is higher than that of GVs, indicating that the FITC-labeled ZD2 peptide is successfully coupled to the surface of GVs at different wavelengths and produces the absorption peak of FITC at 488nm. The FITC-labeled control peptide, like the targeting peptide, produces the absorption peak of FITC at 488nm and can be successfully labeled on the surface of GVs. Therefore, the most suitable wavelength for distinguishing blank bubble GVs from the targeted ultrasound contrast agent ZD2-GVs is 488nm.

[0081] Embodiment 5:

[0082] In this example, the in vitro and in vitro targeting capabilities of ultrasound contrast agents for targeting tumors were verified through experiments.

[0083] Evaluation of targeting ability in vitro:

[0084] The bladder cancer cell line (MB49 cells) was cultured in a high-glucose medium supplemented with 10% FBS and 1% PS at 37°C and 5% CO2. MB49 cells in the logarithmic growth phase were seeded in 24-well plates (1×10 5 The cells were fixed with 4% paraformaldehyde for 15 min, washed with PBS, and then the cell nuclei were stained with DAPI staining solution; washed with PBS buffer again, and then FITC-labeled targeted ultrasound contrast agent ZD2-GVs and control non-targeted ultrasound contrast agent CTR-GVs were added to the MB49 cell line, incubated at room temperature for 5 min, and finally observed under an inverted microscope.

[0085] Please refer to Figure 4 for the experimental results. Figure 4A The figure is a schematic diagram of the in vitro targeting of the targeted ultrasound contrast agent ZD2-GVs to the bladder cancer MB49 cell line observed under a fluorescence microscope. The DAPI column in the figure shows the area where cells are observed under a microscope, and the white dots are cells; the Merge column shows the image obtained by superimposing the images of the DAPI column and the FITC column; the FITC column shows cells with FITC fluorescence labeling under a microscope, among which cells with FITC fluorescence labeling were only found in the ZD2-GVs group, indicating that the targeted ultrasound contrast agent ZD2-GVs has an in vitro targeting effect in the bladder cancer MB49 cell line. Figure 4B The present invention is a bar graph comparing the fluorescence intensity of the targeted ultrasound contrast agent ZD2-GVs and the non-targeted ultrasound contrast agent CTR-GVs in vitro. The horizontal axis is the name of the experimental group, the vertical axis is the fluorescence intensity, and the asterisk indicates a significant difference between the two groups. The bar graph shows that the fluorescence intensity of the targeted ultrasound contrast agent ZD2-GVs in vitro in the bladder cancer MB49 cell line is significantly higher than that of the non-targeted ultrasound contrast agent CTR-GVs, which indicates that compared with the non-targeted ultrasound contrast agent, the targeted ultrasound contrast agent ZD2-GVs can have more aggregation around the MB49 cell line, proving that the ultrasound contrast agent of the present invention has good in vitro targeting ability.

[0086] Evaluation of in vivo ultrasound imaging:

[0087] 1×10 6MB49 cells were injected subcutaneously into the right thigh of male C57BL / 6 mice and the tumors were grown to 100 cm 3 In vivo ultrasound imaging was performed at 1:100 ul of targeted ultrasound contrast agent ZD2-GVs with an OD500 of 3.0 and control non-targeted ultrasound contrast agent CTR-GVs were injected through the tail vein, with an interval of more than half an hour. Video acquisition was performed at the same time until the ultrasound contrast signal disappeared. Subsequently, quantitative analysis was performed using the built-in analysis software of the Mindray Reson 7 device to compare the aggregation of ultrasound contrast agents and non-targeted ultrasound contrast agents in the tumor, such as: peak time, peak intensity, metabolic time, area under the curve and other parameters. It was found that compared with the control group, the ultrasound contrast agent had a longer circulation time in the tumor and its attenuation curve was relatively slow.

[0088] Please see attached Figure 5 , In vivo ultrasound imaging quantification diagram of targeted ultrasound contrast agent ZD2-GVs and non-targeted ultrasound contrast agent CTR-GVs. The horizontal axis is the time of in vivo ultrasound quantification, and the vertical axis is the ultrasound signal intensity. The black line represents the non-targeted ultrasound contrast agent GVs-CERAK in the control group, and the gray line represents the ultrasound contrast agent GVs-ZD2 in the experimental group. One minute after ultrasound quantification, the ultrasound signal intensity of GVs-ZD2 and GVs-CERAK increased rapidly, and then the ultrasound signal intensity of the two continued to decrease, while the ultrasound signal intensity of GVs-ZD2 was always higher than that of GVs-CERAK. Figure 5 It was shown that 5 minutes after injection, the ultrasound signal intensities of the ultrasound contrast agent and the non-targeted ultrasound contrast agent were 67.949±9.953au and 18.508±6.944au, respectively. The imaging effect of the ultrasound contrast agent was much better than that of the non-targeted ultrasound contrast agent, confirming that the ultrasound contrast agent of the present invention has good ultrasound molecular imaging capability in tumors.

[0089] Embodiment 6:

[0090] This example demonstrates that ultrasound contrast agents can extravasate from the vascular system and enter the tumor stroma space.

[0091] Histological examination:

[0092] To verify that GVs can penetrate the endothelial gap of tumor blood vessels, MB49 tumor-bearing mice were divided into two groups and injected with FITC-labeled ZD2-GVs and Ctr-GVs through the tail vein, respectively, and then the tumors were sectioned. The sections 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, and finally observed under a confocal fluorescence microscope.

[0093] Please refer to Figure 6 for the experimental results. Fig. 6A The figures are representative fluorescence images of FITC-labeled CTR-GVs or FITC-labeled ZD2-GVs in tumor sections of MB49 tumor-bearing mice, where the DAPI column shows the cell nuclei of MB49 tumor-bearing mice observed under a microscope; the AF647-Anti-CD31 column shows the fluorescently labeled blood vessels, which can be observed in the FITC-labeled CTR-GVs group, while no fluorescence is shown in the FITC-labeled ZD2-GVs group, indicating that the experimental results are accurate and there is no fluorescence error; the Merge column shows the image obtained by superimposing the images of the DAPI column, AF647-Anti-CD31 column and FITC-GVs column; the FITC-GVs column shows the cells labeled with FITC fluorescence observed under a microscope. Fig. 6A FITC fluorescently labeled cells were only observed in the ZD2-GVs group, indicating that ZD2-GVs were distributed in the tumor sections of MB49 tumor-bearing mice. Figure 6B The fluorescence intensity comparison bar graph of FITC-labeled ZD2-GVs and FITC-labeled CTR-GVs in tumor sections of MB49 tumor-bearing mice. The horizontal axis is the grouping, the vertical axis is the fluorescence intensity, and the asterisk indicates a significant difference between the two groups. Figure 6 shows that ZD2-GVs exist in the extravascular space of the tumor. This shows that ultrasound contrast agents exist not only in the vascular system, but also in the extravascular space of the tumor, providing strong evidence that ultrasound contrast agents can extravasate from the vascular system and enter the tumor matrix space.

[0094] This application is the first to covalently link the ZD2 peptide to the surface of biological protein nanobubbles, and prepare an ultrasound molecular probe with GVs as a carrier and targeting tumor ED-B FN, which provides assistance for the early development, accurate diagnosis and precise treatment of tumors.

[0095] The invention has successfully prepared a novel targeted ultrasound contrast agent ZD2-GVs targeting tumor ED-B FN through in vitro imaging performance experiments, cell adhesion experiments, in vivo ultrasound experiments, and tumor immunofluorescence experiments. The novel targeted ultrasound contrast agent ZD2-GVs has a small particle size, good imaging effect, and good targeting ability. At the same time, the targeted ultrasound contrast agent ZD2-GVs can penetrate the tumor blood vessels to reach the tumor parenchyma area, providing a basis for targeted delivery as a drug or gene carrier.

[0096] In summary, the present invention provides a tumor-targeting ultrasound contrast agent, a preparation method and an application thereof. By covalently linking a cyclic polypeptide with a tumor targeting function to the surface of nanobubbles, the ultrasound contrast agent can be specifically aggregated in the tumor area, thereby improving the tumor targeting of the ultrasound contrast agent. For example, the cyclic polypeptide CTVRTSADC (ZD2) has the advantages of high specificity, good tumor targeting, and low antigenicity. At the same time, the extraction method of the gas vesicles is optimized, and gas vesicles with good imaging properties are obtained. Moreover, the manufacturing process of this ultrasound contrast agent is simple, the experimental equipment is affordable, the production cost is reduced, and it is conducive to large-scale production. This ultrasound contrast agent can be used for early diagnosis of tumor cells, preparation of tumor drugs or delivery of tumor drugs, and has extremely high application value.

[0097] The above description is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown herein, but will conform to the widest range consistent with the principles and novel features applied for herein.

Claims

1. Use of an ultrasound contrast agent in the preparation of a bladder cancer diagnostic agent, characterized in that: The ultrasound contrast agent comprises nanobubbles covalently linked to a cyclic polypeptide, wherein the cyclic polypeptide targets the tumor area; The nanobubbles are gas vesicles extracted from Holabacteria NRC-1, and the gas vesicles with OD500 of 2.5-3.0 are screened out; the cyclic polypeptide is the cyclic polypeptide CTVRTSADC, and its amino acid sequence is: CTVRTSADC (SEQ ID NO.1).

2. The use of the ultrasound contrast agent according to claim 1 in the preparation of a bladder cancer diagnostic agent, characterized in that: The preparation method of the ultrasonic contrast agent comprises the following steps: S1, preparing the tumor-targeting cyclic polypeptide, and adding the cyclic polypeptide into a buffer; S2, adding a coupling activator to a buffer containing the cyclic polypeptide to obtain a mixed solution; S3, preparing the nanobubble, adding the mixed solution into the nanobubble for incubation, and covalently connecting the cyclic polypeptide to the surface of the nanobubble; S4. Extracting the nanobubbles covalently linked to the cyclic polypeptide to obtain the ultrasound contrast agent.

3. The use of the ultrasound contrast agent according to claim 2 in the preparation of a bladder cancer diagnostic agent, characterized in that: The coupling activator in step S2 is 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide.

4. Use of the ultrasound contrast agent according to claim 2 or 3 in the preparation of a bladder cancer diagnostic agent, characterized in that: The placement ratio of the cyclic polypeptide to the buffer is 150-300 μg / ml.

5. The use of the ultrasound contrast agent according to claim 4 in the preparation of a bladder cancer diagnostic agent, characterized in that: After the gas vesicles are prepared in step S3, the absorbance of the gas vesicles at OD500 is measured using an ELISA instrument, and the gas vesicles with an OD500 of 2.5-3.0 are screened out for subsequent steps.

6. Use of the ultrasound contrast agent according to claim 5 in the preparation of a bladder cancer diagnostic agent, characterized in that: In the step S3, the placement ratio of the mixed liquid to the gas vesicles is in the range of 1:1-2:

3.

7. Use of the ultrasound contrast agent according to claim 2 or 3 or 5 or 6 in the preparation of a bladder cancer diagnostic agent, characterized in that: The extraction method in step S4 is centrifugation, and the centrifugation conditions are centrifugation at a centrifugal force of 200-300g for 2-3h, repeated 3-4 times.

8. Use of an ultrasound contrast agent in the preparation of an anti-bladder cancer drug, characterized in that: The ultrasound contrast agent is the ultrasound contrast agent described in claim 1.

9. Use of an ultrasound contrast agent in the preparation of a bladder cancer drug delivery agent, characterized in that: The ultrasound contrast agent is the ultrasound contrast agent described in claim 1.

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