CN117982690A - Ultrasonic contrast agent and preparation method and application thereof - Google Patents
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Abstract
The invention relates to the field of biological medicine, in particular to an ultrasonic contrast agent and a preparation method and application thereof. The invention connects the antibody targeting the prostate specific membrane antigen to the surface of the bioprotein nanobubble for the first time, obtains the ultrasonic molecular probe targeting the prostate specific membrane antigen by taking the gas vesicle as a carrier, improves the imaging performance and tumor targeting of the ultrasonic contrast agent, simplifies the preparation process, and provides assistance for early drug development, accurate diagnosis and accurate treatment of tumors such as prostate cancer and the like.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to an ultrasonic contrast agent and a preparation method and application thereof.
Background
In recent years, as a powerful tool of an ultrasonic imaging technology, an ultrasonic contrast agent benefits from the remarkable advantages of no wound, no radiation, low cost and the like, and has been widely applied to diagnosis of clinical diseases such as thrombus, tumor, inflammation and the like. Besides imaging performance, part of ultrasonic contrast agents also have the capability of releasing drugs to lesion areas in a targeted manner, so that the aim of diagnosis and treatment integration is fulfilled, and new possibility is provided for improving disease treatment.
The traditional ultrasonic contrast agent is mainly prepared by wrapping gas with membrane materials such as phospholipid, albumin and the like, and the gas-containing microbubbles can effectively display the distribution and integrity of a blood system through the systemic circulation and the pulmonary circulation. However, the particle size of conventional ultrasound contrast agents is mainly on the order of microns, and cannot penetrate the endothelial gap of tumor vessels, so that the efficiency of ultrasound molecular imaging and drug delivery is affected. To overcome this challenge, the search for more penetrating ultrasound contrast agents has become an important research direction in improving the application of ultrasound technology in tumor diagnosis and treatment.
In many research directions, nanobubbles are a nano-scale ultrasound contrast agent which is of great interest, such as gas vesicles (GAS VESICLE, GVS) generated based on marine bacteria and archaeal cells, have the advantages of strong permeability and high stability, and can be used for purposefully transmitting drugs or genes to tumor parenchymal cells in an ultrasound-assisted manner, thereby bringing new possibilities for tumor treatment. However, in contrast to the small size of nanoscale ultrasound contrast agents, experiments have not shown that they can bring about a strong imaging contrast, and thus imaging performance remains to be optimized.
In addition to focusing on imaging performance, researchers have also focused on developing targeted ultrasound contrast agents that are capable of binding at the molecular level to target tissue-specific antigens or receptors to achieve tissue-specific visualization effects. For example, some studies have incorporated lipid phosphocerebroside into the outer membrane fraction of microbubbles, leading to the initial acquisition of ultrasound contrast agents that can be directed against tumor tissue. However, since the microvesicles do not specifically bind to tumor cells, the targeting of the microvesicles to tumor tissues is not high enough, and the sensitivity of the microvesicles in tumor detection is relatively low, which is a challenging task, namely, designing an ultrasound contrast agent which realizes high unique binding to target tissues.
In addition, the existing preparation technology of the ultrasonic contrast agent depends on various high-precision instruments and equipment, has high requirements on experimental operation and high preparation difficulty, and increases the manufacturing cost. For example, in order to extract microbubbles of high purity, it is necessary to continuously distill the volatile solvent under high temperature conditions using a reduced pressure rotary evaporator; for another example, in order to release solid particles or biological cell components in the liquid formulation, an operation link such as ultrasonic disruption is required. At the same time, the solvents and volatile substances involved also present certain safety hazards. Thus, process preparation optimization of ultrasound contrast agents remains an important aspect of future research.
In summary, the problems of poor imaging performance, low tumor targeting and high preparation difficulty of the ultrasound contrast agent in the prior art are needed to be solved.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, and the ultrasonic contrast agent for targeting prostate specific membrane antigen positive tumors is obtained by mixing nanobubbles and antibodies for targeting prostate specific membrane antigen, and a preparation method and application thereof.
Specifically, the invention provides the following technical scheme:
In a first aspect of the present invention, there is provided a method of preparing an ultrasound contrast agent comprising: s1, preparing gas vesicles; s2, activating an antibody targeting a prostate specific membrane antigen; s3, mixing the gas vesicle and the activated antibody of the targeted prostate specific membrane antigen to obtain a mixed solution; and S4, purifying the mixed solution to obtain the ultrasonic contrast agent.
In some embodiments, the step S1 includes: adding the lysate into NRC-1 bacillus and centrifuging to obtain gas vesicles.
In some embodiments, in the step S1, the lysate is N-trimethyl chitosan.
In some embodiments, the step S2 includes: the antibodies targeting the prostate specific membrane antigen are dissolved in a buffer and then activated with a coupling activator.
In some embodiments, in step S2, the dissolved concentration of the antibody targeting the prostate-specific membrane antigen is 150-300 μg/ml.
In some embodiments, in step S2, the coupling activator is 1-ethyl- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide.
In some embodiments, in the step S3, the gas vesicles with OD500 of 2.5-3.0 are selected for mixing.
In some embodiments, the volume ratio of the gas vesicle to the activated prostate-specific membrane antigen-targeted antibody is 1:1-2:3.
In a second aspect of the present invention, there is provided an ultrasound contrast agent produced by the above-described ultrasound contrast agent production method.
In a third aspect, the present invention provides an application of the above ultrasound contrast agent in preparing tumor diagnostic agent, drug delivery agent and/or anti-tumor drug.
Compared with the prior art, the invention has the beneficial effects that,
(1) The ultrasonic contrast agent provided by the invention has the components of the targeted prostate specific membrane antigen, can be aggregated in tumor cell areas such as prostate cancer and the like, specifically combines with tumor cells, has good tumor targeting function, improves the sensitivity of tumor diagnosis and detection, and can play an application value in drug delivery and drug preparation.
(2) The ultrasonic contrast agent provided by the invention has the antibody of the targeted prostate specific membrane antigen, has small antigenicity, can not generate immune response at normal tissues of patients, has relatively mild performance, and reduces the influence of sequelae.
(3) The ultrasonic contrast agent provided by the invention adopts the gas vesicle extracted from NRC-1 halophiles as a carrier, and shows higher ultrasonic signal intensity and slower attenuation curve, so that the imaging performance is good.
(4) The ultrasonic contrast agent provided by the invention has the advantages that the preparation process is simple, the adopted experimental equipment is low in price, the time and money cost are reduced, and the possibility of large-scale floor production is increased.
Drawings
FIG. 1 is a flow chart of ultrasound contrast agent preparation in one embodiment;
FIG. 2 is a graph of an ultrasonic contrast agent stability test in one embodiment, wherein FIG. 2A is a particle size distribution graph and FIG. 2B is a potential distribution graph;
FIG. 3 is an in vitro ultrasound imaging test chart in one embodiment;
FIG. 4 is an in vitro targeting ability test chart in one embodiment;
fig. 5 is an in vivo ultrasound imaging test chart in one embodiment, wherein fig. 5A is an ultrasound imaging chart, fig. 5B is an ultrasound signal intensity record chart within 0-8min of injection of ultrasound contrast agent, and fig. 5C is an ultrasound signal intensity statistical chart at 30, 180s, 300s of injection of ultrasound contrast agent.
Detailed Description
The technical solutions of the present patent will be described in further detail below with reference to specific embodiments, and it should be noted that the following detailed description is exemplary, and is intended to provide further explanation of the present application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
Example 1: preparation of ultrasound contrast agent PSMA-GVs
As shown in fig. 1, the method for preparing an ultrasound contrast agent provided in this embodiment includes: s1, preparing gas vesicles; s2, activating an antibody targeting a prostate specific membrane antigen; s3, mixing the gas vesicle and the activated antibody targeting the prostate specific membrane antigen to obtain a mixed solution; s4, purifying the mixed solution to obtain the ultrasonic contrast agent. The method comprises the following steps:
(1) Preparation of gas vesicles.
NRC-1 Salmonella (Halobacteria NRC-1, halo) was inoculated into ATCC medium and placed in a shaker and incubated at 37℃for 7-9 days at 220 rpm/min. After the culture is finished, transferring the culture medium into a separating funnel, standing, removing the culture solution at the lower layer after the strain floats on the surface of the liquid, and separating to obtain the Halo bacteria.
The isolated Halo bacteria were added to an equal volume of N-trimethylchitosan (N, N, N-TRIMETHYL CHITOSAN, TMC) lysate and centrifuged at 300g for 4h and repeated three times. The obtained gas vesicles were then washed with PBS buffer and centrifuged at 250g for 4h, after three repetitions, pure white gas vesicles were obtained and stored in a refrigerator at 4 ℃ for later use.
In this embodiment, the extraction of gas vesicles from NRC-1 bacillus as a raw material of an ultrasound contrast agent can improve the imaging capability of the ultrasound contrast agent, and in other embodiments, other microorganisms can be used.
(2) Activation of antibodies (PSMA/GCPII Polyclonal antibody, proteintech, 13163-1-AP) targeting prostate specific membrane antigen (Prostate Specific Membrane Antigen, PSMA).
200. Mu.g of the antibody targeting the prostate-specific membrane antigen was dissolved in 1mL of PBS buffer, then 3mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) and 3mg of N-hydroxysuccinimide (N-Hydroxysuccinimide, NHS) were added, and incubated on a horizontal shaker at room temperature for 2-3 hours to obtain a mixed solution containing the activated antibody targeting the prostate-specific membrane antigen.
In the embodiment, EDC and NHS are used as coupling activators to participate in the reaction of activating carboxyl to form amide bonds, and the two reagents have the advantages of no toxicity and good biocompatibility. In addition, in this embodiment, the placement ratio of the antibody targeting the prostate specific membrane antigen to the PBS buffer is 200. Mu.g/mL, and in other embodiments, other mixing ratios of 150-300. Mu.g/mL may be used, where the coupling reaction between the antibody targeting the prostate specific membrane antigen and the gas vesicle is more sensitive.
Meanwhile, in this embodiment, taking the method of preparing the gas vesicle first and then activating the antibody targeting the prostate-specific membrane antigen as an example, the method of obtaining two main raw materials of the ultrasound contrast agent and the preparation steps are described, and in other embodiments, other methods of preparing the gas vesicle first and then activating the antibody targeting the prostate-specific membrane antigen, or simultaneously operating and the like may be adopted, which should not limit the scope of protection of the present invention.
(3) Mixing and purifying.
Adding the gas vesicle prepared in the step (1) into the mixed solution containing the activated antibody of the targeted prostate specific membrane antigen obtained in the step (2) at a mixing ratio of 1:1, and incubating on a shaking table at 4 ℃ overnight to obtain a mixture. Thereafter, the mixture was centrifuged at 250g for 2-3 hours to remove free EDC, NHS and PSMA, and after repeating 3-4 times, an ultrasound contrast agent PSMA-GVs was obtained and stored in a refrigerator at 4 ℃.
In this embodiment, the absorbance of the gas vesicle at the OD500 is measured by using an enzyme-labeled instrument, and the gas vesicle with the OD500 of 3.0 is selected to prepare the ultrasonic contrast agent, so that the signal intensity of the ultrasonic contrast agent is improved, and in other embodiments, other gas vesicles with strong ultrasonic signals with the OD500 of 2.5-3.0 can be selected. In addition, in this embodiment, the mixing volume ratio of the mixed solution containing the activated prostate-specific membrane antigen and the gas vesicle is 1:1, and in other embodiments, other mixing methods in the range of 1:1-2:3 may be adopted, and at this time, the coupling reaction between the antibody targeting the prostate-specific membrane antigen and the gas vesicle may be smoothly performed.
Example 2: stability test
The ultrasonic contrast agent PSMA-GVs obtained in example 1 was observed by a transmission electron microscope, 1mL of the ultrasonic contrast agent was taken in a cuvette dedicated to a laser particle diameter instrument, and the particle diameter, distribution and zeta potential were measured by a Zetasizer particle diameter instrument. As a Control, stability test was performed under the same conditions using ultrasound contrast agent CTR (Control, control group) -GVs.
Results: the ultrasound contrast agent obtained was observed to exhibit a monodispersed rugby-shaped structure, regular morphology and uniform size. Specifically, fig. 2A shows the particle size distribution of the ultrasound contrast agent, and it can be seen that the particle size distribution of the ultrasound contrast agent PSMA-GVs is relatively uniform, about 213.60 ±2.76nm, and relatively small. Fig. 2B shows the potential distribution of the ultrasound contrast agent, and it can be seen that the zeta potential of the ultrasound contrast agent PSMA-GVs is about-50.40 ±2.46mV, which indicates that the surface of the particles has negative charges, and is not easy to deposit and agglomerate due to the mutual repulsion of the surface charges, so that the dispersion state can be effectively maintained, and the stability is better.
Example 3: in vitro ultrasound imaging test
The ultrasonic contrast agent PSMA-GVs obtained in example 1 was placed in 1% agarose model wells at concentration od500=0.5, 1.0, 1.5, 2.0, 2.5, and then a line probe of the clinical ultrasonic diagnostic apparatus Mindray Reson was placed on the agarose model side, and imaging test was performed in the ultrasonic contrast imaging mode at a frequency of 3 to 11 MHz. As a control, in vitro ultrasound imaging tests were performed under identical conditions using different concentrations of non-targeted ultrasound contrast agent CTR-GVs.
Results: figure 3 shows the results of an in vitro ultrasonic imaging test, the vertical axis representing the test object, the horizontal axis representing the concentration, and the horizontal axis increasing from left to right. It has been found that with increasing concentration of ultrasound contrast agent, the ultrasound signal is stronger and, at the same time, the ultrasound contrast agent PSMA-GVs can produce a more powerful and stable contrast ultrasound signal with a relatively good imageability compared to the ultrasound contrast agent CTR-GVs.
Example 4: in vitro targeting capability test
10% Fetal bovine serum (Fetal Bovine Serum, FBS) and 1% Penicillin-streptomycin (Penicillin-Streptomycin) were added to 1640 medium, and the prostate cancer (Lymph Node Carcinoma of the Prostate, LNCaP) cell line was inoculated therein and cultured at 37℃under 5% CO 2.
LNCaP cells in the logarithmic growth phase were seeded at a density of 1X 10 5 cells/well in 24-well cell culture plates and incubated overnight. After three washes with PBS buffer, cells were fixed with 4% paraformaldehyde for 15min, and after a subsequent wash with PBS buffer, nuclei were stained with 4',6-diamidino-2-phenylindole (4', 6-diamidino-2-phenylindole, DAPI).
After washing the LNCaP cell line again with PBS buffer, the ultrasound contrast agent PSMA-GVs prepared in example 1 labeled with fluorescein isothiocyanate (Fluorescein Isothiocyanate, FITC) was added thereto, and after incubation for 5min at room temperature, observation was carried out with an inverted fluorescence microscope. As a control, the same-condition test was performed using non-targeted ultrasound contrast agents CTR-GVs.
Results: fig. 4 shows the results of in vitro targeting ability test, the vertical axis represents the test object, and the horizontal axis sequentially refers to DAPI, FITC cell staining, and Merge images superimposed on the two. It was found that the ultrasound contrast agent CTR-GVs group showed only DAPI fluorescent label, while the ultrasound contrast agent PSMA-GVs group showed cells with both DAPI fluorescent label and FITC fluorescent label, indicating that the ultrasound contrast agent PSMA-GVs could effectively target prostate cancer cell lines in vitro.
Example 5: in vivo ultrasound imaging test
LNcap cells of 1X 10 6 were subcutaneously injected into the right thigh of C57BL/6 male mice, and after the tumor had grown to 100cm 3, 100. Mu.L of the ultrasound contrast agent PSMA-GVs prepared in example 1 having an OD500 of 3.0 was intravenously injected into the tail of the mice, and the results were video-captured until the ultrasound contrast signal disappeared. As a control, testing was performed under the same conditions after half an hour using non-targeted ultrasound contrast agents CTR-GVs. Subsequently, quantitative analysis is carried out by utilizing analysis software built in Mindray Reson equipment, parameters including peak reaching time, peak reaching intensity, metabolism time, area under a curve and the like are counted, so that the aggregation conditions of the ultrasonic contrast agents PSMA-GVs and CTR-GVs in tumors are compared.
Results: fig. 5A shows in vivo ultrasonic imaging test results, with the vertical axis representing the test object and the horizontal axis representing time, increasing from left to right. It was found that after 1min, the signal intensities of the ultrasound contrast agents PSMA-GVs and CTR-GVs both increased rapidly, and then also showed a decreasing trend, during which the ultrasound signal intensity of the ultrasound contrast agent PSMA-GVs was always higher than that of the ultrasound contrast agent CTR-GVs. In addition, PSMA-GVs have longer circulation times in tumors than do the ultrasound contrast agents CTR-GVs, and their decay curves are also relatively slow, which is advantageous for more comprehensive imaging of deep tissues.
Fig. 5B records the ultrasound signal intensity for the two ultrasound contrast agents injected within 0-8min, and fig. 5C compares the ultrasound signal intensity for the two ultrasound contrast agents at 30, 180s, 300 s. It can be seen that the ultrasound signal intensities of the two groups did not show a difference at 30s of injection of the ultrasound contrast agent, and a difference was shown at 1 min. At 5min after injection, the signal intensities of the ultrasonic contrast agents PSMA-GVs and CTR-GVs are 51.89+/-8.63 a.u and 18.64+/-10.14 a.u respectively, namely, the two groups of ultrasonic signal intensities show significant differences, and the signal intensity of the ultrasonic contrast agents PSMA-GVs is obviously higher than that of the CTR-GVs, which indicates that the ultrasonic contrast agents PSMA-GVs can effectively target tumors and have good ultrasonic molecular imaging capability.
The foregoing are merely some embodiments of the invention. It will be apparent to those skilled in the art that various modifications and improvements can be made without departing from the spirit of the invention.
Claims (10)
1. A method of preparing an ultrasound contrast agent, comprising:
s1, preparing gas vesicles;
s2, activating an antibody targeting a prostate specific membrane antigen;
S3, mixing the gas vesicle and the activated antibody of the targeted prostate specific membrane antigen to obtain a mixed solution; and
And S4, purifying the mixed solution to obtain the ultrasonic contrast agent.
2. The method for preparing an ultrasound contrast agent according to claim 1, wherein the step S1 comprises:
adding the lysate into NRC-1 bacillus and centrifuging to obtain gas vesicles.
3. The method for preparing an ultrasound contrast agent according to claim 2, wherein in the step S1, the lysate is N-trimethyl chitosan.
4. A method of preparing an ultrasound contrast agent according to claim 3, wherein step S2 comprises:
The antibodies targeting the prostate specific membrane antigen are dissolved in a buffer and then activated with a coupling activator.
5. The method of preparing an ultrasound contrast agent according to claim 4, wherein the dissolved concentration of the antibody targeting the prostate-specific membrane antigen in step S2 is 150 to 300 μg/ml.
6. The method for preparing an ultrasound 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 method of any one of claims 1-6, wherein in step S3, the gas vesicles having an OD500 of 2.5-3.0 are selected for mixing.
8. The method of preparing an ultrasound contrast agent according to claim 7, wherein in the step S3, the volume ratio of the gas vesicle and the activated antibody targeting a prostate-specific membrane antigen is 1:1-2:3.
9. An ultrasound contrast agent produced by the process for producing an ultrasound contrast agent according to any one of claims 1 to 8.
10. Use of an ultrasound contrast agent according to claim 9 for the preparation of a tumor diagnostic agent, a drug delivery agent and/or an anti-tumor drug.
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