Ultrasound contrast agent, method for preparing same, and use thereof

Abstract

The present invention relates to the field of biopharmaceuticals, and in particular, to an ultrasound contrast agent, a method for preparing same, and use thereof. An antibody targeting the prostate-specific membrane antigen is covalently linked to the surface of a biological protein nanobubble for the first time, and an ultrasonic molecular probe using a gas vesicle as the carrier and targeting the prostate-specific membrane antigen is obtained. The present invention improves the imaging performance and the tumor targeting property of the ultrasound contrast agent, features a simplified preparation process, and aids in early drug development, precision diagnosis, and precision treatment of tumors such as prostate cancer.

Description

Ultrasound contrast agent, preparation method and application thereof Technical Field

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

[0002] In recent years, ultrasound contrast agents, as powerful tools in ultrasound imaging technology, have gained widespread application in the diagnosis of clinical conditions such as thrombosis, tumors, and inflammation, benefiting from their significant advantages such as non-invasiveness, radiation-free nature, and low cost. In addition to their imaging capabilities, some ultrasound contrast agents also have the ability to deliver targeted drugs to the affected area, achieving the goal of integrated diagnosis and treatment and providing new possibilities for improving disease treatment.

[0003] Conventional ultrasound contrast agents are primarily composed of gas encapsulated by membrane materials such as phospholipids and albumin. These gas-containing microbubbles can circulate through the systemic and pulmonary circulation, effectively demonstrating the distribution and integrity of the blood system. However, the particle size of conventional ultrasound contrast agents is primarily in the micrometer range, making them unable to penetrate the endothelial space of tumor blood vessels, thus limiting the efficiency of ultrasound molecular imaging and drug delivery. To overcome this challenge, the search for more penetrating ultrasound contrast agents has become an important research direction for improving the application of ultrasound technology in tumor diagnosis and treatment.

[0004] Among numerous research areas, nanobubbles are a highly sought-after nanoscale ultrasound contrast agent. For example, gas vesicles (GVs) produced by marine bacteria and archaeal cells offer the advantages of strong permeability and high stability. They can deliver drugs or genes to tumor parenchymal cells in a targeted manner through ultrasound, bringing new possibilities for tumor treatment. However, due to the small size of nanoscale ultrasound contrast agents, experiments have not yet demonstrated their ability to provide strong imaging contrast, and therefore, imaging performance still needs to be optimized.

[0005] In addition to focusing on imaging performance, researchers are also committed to developing targeted ultrasound contrast agents that can bind to target tissue-specific antigens or receptors at the molecular level to achieve tissue-specific imaging effects. For example, some studies have added lipid phosphoside to the outer membrane components of microbubbles and initially obtained ultrasound contrast agents that can target tumor tissues. However, since the microbubbles do not bind specifically to tumor cells, their targeting to tumor tissues is not high enough, and their sensitivity in tumor detection is relatively low. This echoes the extremely challenging task of designing ultrasound contrast agents that achieve highly unique binding with target tissues.

[0006] In addition, the existing preparation technology of ultrasound contrast agents relies on a variety of high-precision instruments and equipment, which has high requirements for experimental operation and is difficult to prepare, and also increases the production cost. For example, in order to extract high-purity microbubbles, it is necessary to use a reduced-pressure rotary evaporator to continuously distill volatile solvents under high temperature conditions; for example, in order to release solid particles or biological cell components in liquid preparations, it is necessary to introduce ultrasonic fragmentation and other operations. At the same time, the solvents and volatile substances involved also pose certain safety risks. Therefore, the process preparation optimization of ultrasound contrast agents remains an important aspect of future research.

[0007] In summary, the existing ultrasound contrast agents have problems such as poor imaging performance, low tumor targeting and difficulty in preparation, which need to be solved urgently.

[0008] Summary of the Invention

[0009] The purpose of the present invention is to overcome the shortcomings of the existing technology, mix nanobubbles and antibodies targeting prostate-specific membrane antigen, obtain an ultrasound contrast agent targeting prostate-specific membrane antigen-positive tumors, and its preparation method and application.

[0010] Specifically, the present invention provides the following technical solutions:

[0011] In a first aspect, the present invention provides a method for preparing an ultrasound contrast agent, comprising: S1, preparing gas vesicles; S2, activating antibodies targeting prostate-specific membrane antigens; S3, mixing the gas vesicles and the activated antibodies targeting prostate-specific membrane antigens to obtain a mixed solution; and S4, purifying the mixed solution to obtain the ultrasound contrast agent.

[0012] In some embodiments, step S1 comprises: adding lysis solution to NRC-1 halobacterium and centrifuging to obtain gas vesicles.

[0013] In some embodiments, in step S1, the lysis solution is N-trimethyl chitosan.

[0014] In some embodiments, step S2 comprises: dissolving the antibody targeting prostate-specific membrane antigen in a buffer solution, and then activating the antibody using a coupled activator.

[0015] In some embodiments, in step S2, the dissolved concentration of the antibody targeting prostate-specific membrane antigen is 150-300 μg / ml.

[0016] In some embodiments, in step S2, the coupling activator is 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide.

[0017] In some embodiments, in step S3, the gas vesicles having an OD500 of 2.5-3.0 are selected for mixing.

[0018] In some embodiments, the volume ratio of the gas vesicles to the activated prostate-specific membrane antigen-targeting antibody is 1:1-2:3.

[0019] The second aspect of the present invention provides an ultrasound contrast agent prepared by the above-mentioned method for preparing an ultrasound contrast agent.

[0020] The third aspect of the present invention provides a use of the above-mentioned ultrasound contrast agent in the preparation of a tumor diagnostic agent, a drug delivery agent and / or an anti-tumor drug.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) The ultrasound contrast agent provided by the present invention has a component that targets prostate-specific membrane antigen, can accumulate in tumor cell areas such as prostate cancer, specifically bind to tumor cells, exhibit good tumor targeting function, improve the sensitivity of tumor diagnosis and detection, and can play an application value in drug delivery and drug preparation.

[0023] (2) The ultrasound contrast agent provided by the present invention has antibodies targeting prostate-specific membrane antigens, which have low antigenicity and will not produce immune responses in the patient's normal tissues. It has relatively mild performance and reduces the impact of sequelae.

[0024] (3) The ultrasound contrast agent provided by the present invention uses gas vesicles extracted from NRC-1 salt bacteria as carriers, showing a higher ultrasound signal intensity and a slower attenuation curve, thus having good imaging performance.

[0025] (4) The preparation process of the ultrasound contrast agent provided by the present invention is simple, and the experimental equipment used is affordable, which reduces time and money costs and increases the possibility of large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG1 is a flow chart of preparing an ultrasound contrast agent in an embodiment;

[0027] FIG2 is a graph showing a stability test of an ultrasound contrast agent in an embodiment, wherein FIG2A is a graph showing particle size distribution, and FIG2B is a graph showing potential distribution;

[0028] FIG3 is an in vitro ultrasound imaging test diagram in one embodiment;

[0029] FIG4 is a diagram of an in vitro targeting ability test in one embodiment;

[0030] Figure 5 is an in vivo ultrasound imaging test diagram in an embodiment, wherein Figure 5A is an ultrasound imaging diagram, Figure 5B is a recording diagram of the ultrasound signal intensity within 0-8 minutes after injection of the ultrasound contrast agent, and Figure 5C is a statistical diagram of the ultrasound signal intensity at 30, 180, and 300 seconds after injection of the ultrasound contrast agent. DETAILED DESCRIPTION

[0031] The technical solution of this patent will be further described in detail below in conjunction with specific embodiments. It should be noted that the following detailed description is exemplary and is intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this application belongs.

[0032] Example 1: Preparation of ultrasound contrast agent PSMA-GVs

[0033] As shown in FIG1 , the method for preparing an ultrasound contrast agent provided in this embodiment includes: S1, preparing gas vesicles; S2, activating an antibody targeting prostate-specific membrane antigen; S3, mixing the gas vesicles and the activated antibody targeting prostate-specific membrane antigen to obtain a mixed solution; S4, purifying the mixed solution to obtain an ultrasound contrast agent. The details are as follows:

[0034] (1) Preparation of gas vesicles.

[0035] Inoculate Halobacteria NRC-1 (Halo) into ATCC medium and culture in a shaker at 37°C and 220 rpm / min for 7-9 days. After the culture is complete, transfer the culture medium to a separatory funnel and allow it to stand. Once the strain floats to the surface, remove the lower layer of culture medium and isolate the Halo bacteria.

[0036] The isolated Halo bacteria were added to an equal volume of N-trimethyl chitosan (TMC) lysate and centrifuged at 300g for 4 hours, repeated three times. The resulting gas vesicles were then washed with PBS buffer and centrifuged at 250g for 4 hours, repeated three times, to obtain pure white gas vesicles. These were then stored in a refrigerator at 4°C until further use.

[0037] In this embodiment, gas vesicles are extracted from NRC-1 halobacterium as raw materials for ultrasound contrast agents, which can improve the imaging capability of ultrasound contrast agents. In other embodiments, other microorganisms can be used.

[0038] (2) Activation of an antibody targeting prostate-specific membrane antigen (PSMA) (PSMA / GCPII Polyclonal antibody, proteintech, 13163-1-AP).

[0039] 200 μg of an antibody targeting prostate-specific membrane antigen was dissolved in 1 mL of PBS buffer, and then 3 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 3 mg of N-hydroxysuccinimide (NHS) were added. The mixture was incubated on a horizontal shaker at room temperature for 2-3 hours to obtain a mixed solution containing the activated antibody targeting prostate-specific membrane antigen.

[0040] In this example, EDC and NHS were used as coupling activators to activate carboxyl groups to form amide bonds. Both reagents are non-toxic and biocompatible. Furthermore, in this example, the ratio of the PSA-targeting antibody to PBS buffer was 200 μg / mL. In other examples, other ratios of 150-300 μg / mL were used, resulting in a more sensitive coupling reaction between the PSA-targeting antibody and the gas vesicles.

[0041] At the same time, this embodiment takes the example of first preparing gas vesicles and then activating antibodies targeting prostate-specific membrane antigen to introduce the acquisition methods and preparation steps of the two main raw materials of ultrasound contrast agents. In other embodiments, other methods that can achieve the effects provided by the present invention, such as first activating antibodies targeting prostate-specific membrane antigen and then preparing gas vesicles, or performing simultaneous operations, can also be used. This should not limit the scope of protection of the present invention.

[0042] (3) Mixing and purification.

[0043] The gas vesicles prepared in step (1) were added to the mixed solution containing the activated prostate-specific membrane antigen-targeting antibody obtained in step (2) at a mixing ratio of 1:1, and the mixture was incubated overnight on a shaker at 4°C to obtain a mixture. The mixture was then centrifuged at 250g for 2-3 hours to remove free EDC, NHS, and PSMA. This was repeated 3-4 times to obtain the ultrasound contrast agent PSMA-GVs, which were then stored in a refrigerator at 4°C.

[0044] In this example, a microplate reader was used to measure the absorbance of the gas vesicles at OD500. Gas vesicles with an OD500 of 3.0 were selected for preparation of ultrasound contrast agents, which improved the signal strength of the ultrasound contrast agent. In other examples, other gas vesicles with an OD500 of 2.5-3.0 and strong ultrasound signals could also be selected. Furthermore, in this example, the volume ratio of the mixed solution containing activated prostate-specific membrane antigen to the gas vesicles was 1:1. In other examples, other mixing ratios within the range of 1:1-2:3 could be used. In these cases, the coupling reaction between the prostate-specific membrane antigen-targeting antibody and the gas vesicles can proceed smoothly.

[0045] Example 2: Stability test

[0046] The ultrasound contrast agent PSMA-GVs obtained in Example 1 were observed using a transmission electron microscope. 1 mL of the ultrasound contrast agent was placed in a laser particle size analyzer cuvette and its particle size, distribution, and zeta potential were measured using a Zetasizer. As a control, the ultrasound contrast agent CTR (Control)-GVs were subjected to stability testing under the same conditions.

[0047] Results: The prepared ultrasound contrast agent exhibited a monodisperse, rugby-shaped structure with a regular morphology and uniform size. Specifically, Figure 2A shows the particle size distribution of the ultrasound contrast agent, showing a relatively uniform particle size distribution of approximately 213.60 ± 2.76 nm for the PSMA-GVs, which is relatively small. Figure 2B shows the potential distribution of the ultrasound contrast agent, showing a zeta potential of approximately -50.40 ± 2.46 mV, indicating that the particles have a negative surface charge. Due to the mutual repulsion of surface charges, they are less likely to settle and aggregate, effectively maintaining a dispersed state and exhibiting good stability.

[0048] Example 3: In vitro ultrasound imaging test

[0049] The ultrasound contrast agent PSMA-GVs obtained in Example 1 were placed in a 1% agarose phantom well at concentrations of OD500 = 0.5, 1.0, 1.5, 2.0, and 2.5. A Mindray Reson 7 linear array probe was then placed on one side of the agarose phantom, and imaging tests were performed in ultrasound contrast imaging mode at frequencies of 3-11 MHz. As a control, in vitro ultrasound imaging tests were performed using different concentrations of the non-targeted ultrasound contrast agent CTR-GVs under the same conditions.

[0050] Results: Figure 3 shows the results of in vitro ultrasound imaging tests. The vertical axis represents the test object, and the horizontal axis represents the concentration, increasing from left to right. It can be seen that as the ultrasound contrast agent concentration increases, the ultrasound signal becomes stronger. Furthermore, compared with the ultrasound contrast agent CTR-GVs, the ultrasound contrast agent PSMA-GVs produces a more powerful and stable contrast ultrasound signal, demonstrating relatively superior imaging properties.

[0051] Example 4: In vitro targeting ability test

[0052] 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PEN) were added to 1640 culture medium, and prostate cancer (LNCaP) cell line was inoculated therein and cultured at 37°C and 5% CO2.

[0053] LNCaP cells in the logarithmic growth phase were taken and 1×10 5 Cells were seeded at a density of 100 cells / well in a 24-well cell culture plate and incubated overnight. Afterwards, the cells were washed three times with PBS buffer and fixed with 4% paraformaldehyde for 15 minutes. After washing again with PBS buffer, the cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI).

[0054] After washing the LNCaP cells again with PBS, the fluorescein isothiocyanate (FITC)-labeled ultrasound contrast agent PSMA-GVs prepared in Example 1 were added. After incubation at room temperature for 5 minutes, the cells were observed using an inverted fluorescence microscope. As a control, the non-targeted ultrasound contrast agent CTR-GVs was tested under the same conditions.

[0055] Results: Figure 4 shows the results of in vitro targeting ability testing. The vertical axis represents the test object, and the horizontal axis represents DAPI, FITC cell staining, and a merged image of the two superimposed. It can be seen that the ultrasound contrast agent CTR-GVs group only showed DAPI fluorescence labeling, while the ultrasound contrast agent PSMA-GVs group showed cells labeled with both DAPI and FITC fluorescence, indicating that the ultrasound contrast agent PSMA-GVs can effectively target prostate cancer cell lines in vitro.

[0056] Example 5: In vivo ultrasound imaging test

[0057] 1×10 6 LNcap cells, wait until the tumor grows to 100cm 3 Afterwards, 100 μL of the ultrasound contrast agent PSMA-GVs prepared in Example 1 with an OD500 of 3.0 was injected into the mouse tail vein, and the results were video captured until the ultrasound contrast signal disappeared. As a control, half an hour later, the non-targeted ultrasound contrast agent CTR-GVs was tested under the same conditions. Subsequently, quantitative analysis was performed using the built-in analysis software of the Mindray Reson 7 device, and parameters including peak time, peak intensity, metabolic time, and area under the curve were statistically analyzed to compare the accumulation of ultrasound contrast agents PSMA-GVs and CTR-GVs in tumors.

[0058] Results: Figure 5A shows the in vivo ultrasound imaging test results. The vertical axis represents the object of examination, and the horizontal axis represents time, increasing from left to right. It can be seen that after 1 minute, the signal intensity of both the ultrasound contrast agents PSMA-GVs and CTR-GVs increased rapidly, and then showed a continuous downward trend. During this period, the ultrasound signal intensity of the ultrasound contrast agent PSMA-GVs was consistently higher than that of the ultrasound contrast agent CTR-GVs. In addition, compared with the ultrasound contrast agent CTR-GVs, PSMA-GVs have a longer circulation time in the tumor and a relatively slower decay curve, which facilitates more comprehensive imaging of deep tissues.

[0059] Figure 5B records the ultrasound signal intensity within 0-8 minutes after injection of the two ultrasound contrast agents, and Figure 5C compares the ultrasound signal intensity of the two ultrasound contrast agents at 30, 180, and 300 seconds. It can be seen that at 30 seconds after injection of the ultrasound contrast agent, the ultrasound signal intensity of the two groups did not show a difference, but a difference appeared at 1 minute. At 5 minutes after injection, the signal intensities of the ultrasound contrast agents PSMA-GVs and CTR-GVs were 51.89±8.63au and 18.64±10.14au, respectively, indicating a significant difference in ultrasound signal intensity between the two groups, and the signal intensity of the ultrasound contrast agent PSMA-GVs was significantly higher than that of CTR-GVs, indicating that the ultrasound contrast agent PSMA-GVs can effectively target tumors and has good ultrasound molecular imaging capabilities.

[0060] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements can be made without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. A preparation method of an ultrasound contrast agent, characterized in that Comprising: S1. Preparing gas vesicles; S2. Activating an antibody targeting prostate-specific membrane antigen; S3. Mixing the gas vesicles and the activated antibody targeting prostate-specific membrane antigen to obtain a mixture; and S4. Purifying the mixture to obtain the ultrasound contrast agent.

2. The preparation method of the ultrasound contrast agent according to claim 1, characterized in that, The step S1 includes: Adding a lysis solution to Halobacterium NRC-1 and centrifuging to obtain gas vesicles.

3. The preparation method of the ultrasonic contrast agent according to claim 2, characterized in that, In the step S1, the lysis solution is N-trimethyl chitosan.

4. The preparation method of the ultrasound contrast agent according to claim 3, wherein The step S2 includes: Dissolving the antibody targeting prostate-specific membrane antigen in a buffer solution and then activating it with a coupling activator.

5. The preparation method of the ultrasonic contrast agent according to claim 4, wherein, In the step S2, the dissolution concentration of the antibody targeting prostate-specific membrane antigen is 150 - 300 micrograms per milliliter.

6. The preparation method of the ultrasonic contrast agent according to claim 5, wherein, In the step S2, the coupling activator is 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide.

7. The preparation method of the ultrasound contrast agent according to any one of claims 1-6, characterized in that, In the step S3, gas vesicles with an OD500 of 2.5 - 3.0 are selected for mixing.

8. The preparation method of the ultrasound contrast agent according to claim 7, characterized in that, In the step S3, the volume ratio of the gas vesicles to the activated antibody targeting prostate-specific membrane antigen is 1∶1 - 2∶3.

9. An ultrasound contrast agent prepared by the preparation method of the ultrasound contrast agent according to any one of claims 1 - 8.

10. Use of the ultrasound contrast agent according to claim 9 in the preparation of a tumor diagnostic agent, a drug delivery agent, and / or an anti-tumor drug.

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