Targeted CMVD ultrasonic contrast agent as well as preparation method and application thereof

Abstract

The invention relates to the technical field of ultrasonic medicine, in particular to a targeted CMVD ultrasonic contrast agent and a preparation method and application thereof. The targeted CMVD ultrasonic contrast agent comprises micron-sized lipid microbubbles, perfluorinated carbon gas existing in the lipid microbubbles and a targeted cyclic peptide fragment cRGD existing on the surfaces of lipid shells of the lipid microbubbles, and the ultrasonic contrast agent can adhere thrombus in a targeted mode and is stable in property and uniform in particle size distribution. In-vitro experiments fully prove that the ultrasonic contrast agent has good biological safety, imaging characteristic and thrombus targeting property.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of ultrasonic medical technology, and particularly relates to a targeted CMVD ultrasonic contrast agent and a preparation method and application thereof. BACKGROUND

[0002] The information disclosed in the background of the present application is merely intended to increase the understanding of the overall background of the present application and should not necessarily be regarded as acknowledging or implicitly suggesting that this information constitutes prior art known to those skilled in the art.

[0003] Coronary microvascular dysfunction (CMVD) refers to a pathological state of abnormal structure and / or function of the micro coronary artery (diameter < 500 microns) of the heart, resulting in reduced myocardial blood flow reserve. It is an important cause of chest pain (especially in female patients), myocardial ischemia, heart failure and even adverse cardiovascular events. Traditional coronary angiography can clearly show the lesions of epicardial large vessels, but it cannot directly image and effectively evaluate the structure and function of the microcirculation with small diameter and extensive distribution. The diagnosis of CMVD in clinical practice mainly relies on indirect functional tests, such as invasive measurements of coronary flow reserve (CFR) and microcirculation resistance index (IMR), or non-invasive tests such as drug loading echocardiography and cardiac magnetic resonance imaging (CMR) perfusion scanning. However, these methods are either invasive, complex to operate, and costly (such as CMR), or have limited resolution and low specificity, making it difficult to accurately identify the specific cause and location of microvascular dysfunction, such as whether there is in situ thrombus formation or a prothrombotic state in the microvessels.

[0004] Ultrasound contrast imaging technology uses gas-filled microbubbles as contrast agents to significantly enhance the acoustic contrast of tissues. Microbubbles, due to their size (usually 2-3 microns in diameter), are similar to red blood cells and can enter the systemic circulation through the pulmonary circulation and distribute in the coronary microcirculation with blood flow, making them an ideal medium for real-time evaluation of myocardial perfusion and microcirculatory hemodynamics. The second generation of phospholipid or high-molecular polymer-coated inert gas microbubbles has good stability and high safety, and has been widely used in clinical practice. However, traditional ultrasound microbubble contrast agents mainly reflect blood pool volume and blood flow velocity information, and are "passive" tracers. They can indirectly assess myocardial perfusion and microvascular obstruction (such as after myocardial infarction) through time-intensity curves, but lack the ability to target specific pathological markers of microvascular walls and cannot directly and visually diagnose specific pathological changes (such as microthrombosis, inflammatory infiltration, and endothelial dysfunction) that cause CMVD. "Active targeting" ultrasound microbubbles usually modify specific ligands (such as antibodies, peptide segments, small molecules, aptamers, etc.) on the surface of the microbubble shell, allowing them to specifically bind to specific molecular markers (such as cell adhesion molecules, coagulation factors, platelet receptors, and inflammatory factor receptors) on the vascular endothelium or thrombus. Research on thrombus-targeting microbubbles is particularly active, mainly used for the diagnosis and monitoring of thrombolytic therapy in thromboembolic diseases (such as deep vein thrombosis, peripheral arterial thrombosis, atrial fibrillation-related thrombus, and acute myocardial infarction coronary thrombus). These targeted microbubbles (e.g., targeting fibrin, activated platelet glycoprotein IIb / IIIa, P-selectin, etc.) have been shown to effectively locate and enhance the ultrasound signal of thrombus in experimental and preclinical studies.

[0005] Ultrasound microbubble technology targeting thrombus has made significant progress, but its application in the field of coronary microvessels, especially for the detection of in situ microthrombus (microthrombus) in the context of microvascular dysfunction (CMVD), has significant gaps and challenges: 1. Lack of specific diagnostic tools for microthrombus in the context of CMVD. There are almost no direct, non-invasive, and specific imaging tools for diffuse microthrombus in the myocardial microcirculation, which is microscopically small (much smaller than 1 mm 3 ), and may have different causes. 2. Challenges in adapting to the microvascular microenvironment: The microvascular environment is different from larger vessels, with narrower space and different blood flow shear force, and the target (thrombus-related molecules) may be more diffuse or in an early state; existing targeting strategies designed for larger thrombus (such as antibody types, affinity, microbubble size, and stiffness) may not effectively adapt to the microvascular environment, achieving sufficient binding efficiency and signal-to-noise ratio. 3. High diagnostic accuracy is required: The diagnosis of CMVD needs to accurately locate the pathological changes in the complex microcirculation; non-targeted microbubbles can only provide indirect blood flow information, while existing other targeted microbubbles (such as those targeting inflammatory markers) are related to the lesion, but cannot directly reflect the key event of local microthrombus formation that causes microvascular obstruction. SUMMARY

[0006] Therefore, the present application provides a targeted CMVD ultrasound contrast agent and a preparation method and application thereof. The microbubble ultrasound contrast agent of the present application is an ultrasound contrast agent specially designed and optimized for thrombus-related target points in coronary microvessels, which is used for non-invasive, real-time and high-specificity detection of microthrombi or prothrombotic states in the myocardial microcirculation of CMVD patients, and can reveal the role of microthrombi in the pathophysiology of CMVD, which has urgent clinical needs and important scientific value.

[0007] In order to achieve the above-mentioned purpose, the present application is realized by the following technical scheme: In a first aspect, the present application provides a targeted CMVD ultrasound contrast agent, which comprises a micro-sized lipid microbubble, a perfluorocarbon gas existing in the lipid microbubble, and a targeting cyclic peptide segment cRGD existing on the surface of the lipid shell of the lipid microbubble. The lipid shell material is distearoyl phosphatidylcholine (DSPC), distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-maleimide (DSPE-PEG2000-Mal). The perfluorocarbon gas is selected from perfluoropropane, perfluorobutane, and perfluorohexane; preferably, the fluorocarbon gas is selected from perfluoropropane. The thiol group of the targeting cyclic peptide segment cRGD (cyclic arginine-glycine-aspartic acid peptide) is connected to the surface of the lipid shell through a thiol coupling reaction with maleimide, and the cRGD is a targeting peptide segment of thrombus surface platelet glycoprotein IIb / IIIa (Glycoprotein IIb / IIIa, GPIIb / IIIa).

[0008] The targeted CMVD ultrasound contrast agent can target CMVD, and can be used for preparing an ultrasound contrast agent for detecting CMVD, and has excellent imaging effect in preclinical models; in animal experiments, it has a targeted adhesion effect on the thrombus of the pathological region of the coronary microcirculation disorder of the pig heart.

[0009] Preferably, the molar ratio of DSPC, DSPE-PEG2000, and DSPE-PEG2000-Mal is 17-19:1-2:1.

[0010] Preferably, in the targeted CMVD ultrasound contrast agent, the molar ratio of cRGD to DSPE-PEG2000-Mal is 20-60:1; more preferably, the molar ratio is 20-40:1.

[0011] Preferably, the average particle size of the CMVD-targeted ultrasound contrast agent is 2-3 microns.

[0012] In a second aspect, the present application provides a method for preparing the CMVD-targeted ultrasound contrast agent as described above, comprising the following steps: (1) Preparation of maleimide ultrasound microbubbles (MB-Mal) Using the thin film hydration method, DSPC, DSPE-PEG2000, and DSPE-PEG2000-Mal are dissolved in chloroform, and after evaporation of the organic solvent under a nitrogen stream, a thin film is formed. After hydration and oscillation, the air is replaced with a perfluorocarbon gas, and the lipid microbubbles are formed by oscillation. (2) Preparation of CMVD-targeted ultrasound contrast agent (MB-cRGD) Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) is dissolved in water, cRGD is added, followed by 1-5 x 10 9 The lower liquid is removed, and the upper microbubbles are retained as the CMVD-targeted ultrasound contrast agent.

[0013] Preferably, the perfluorocarbon gas is selected from perfluoropropane, perfluorobutane, and perfluorohexane; more preferably, the fluorocarbon gas is perfluoropropane. Preferably, in the preparation of the CMVD-targeted ultrasound contrast agent, the molar ratio of cRGD to DSPE-PEG2000-Mal is 20-60:1.

[0014] Preferably, in step (1), the molar ratio of DSPC, DSPE-PEG2000, and DSPE-PEG2000-Mal is 17-19:1-2:1.

[0015] Preferably, in step (2), the water is ultrapure water, and the concentration of TCEP is 40-50 mM.

[0016] Preferably, in step (2), the reaction is carried out in the dark.

[0017] In a third aspect, the present application provides the use of the CMVD-targeted ultrasound contrast agent of the first aspect in the preparation of an ultrasound contrast agent for detecting CMVD.

[0018] Preferably, the CMVD-targeted ultrasound contrast agent is a lipid-based contrast agent, and the CMVD-targeted ultrasound contrast agent can be administered by injection, preferably by intravenous injection.

[0019] Compared with the prior art, the present application has the following beneficial effects: (1) The present application uses a lipid shell to wrap fluorocarbon gas to prepare a new type of targeted CMVD ultrasound contrast agent. The ultrasound contrast agent can target and adhere to thrombus, is stable in nature, and has uniform particle size distribution. In vitro experiments fully prove that the targeted CMVD ultrasound contrast agent has good biological safety, imaging characteristics and thrombus targeting.

[0020] (2) The targeted CMVD ultrasound contrast agent prepared by the present application has excellent imaging effect in a preclinical model, and has a targeted adhesion effect on the thrombus of the pathological region of the coronary microcirculation disorder of the pig heart in animal experiments, and has good diagnostic effect. BRIEF DESCRIPTION OF DRAWINGS

[0021] The drawings accompanying the specification of the present application serve to provide further understanding of the present application, and the illustrative embodiments of the present application and the description thereof serve to explain the present application, and do not constitute an improper limitation on the present application.

[0022] Figure 1 Synthesis diagram and structure schematic diagram of the targeted CMVD ultrasound contrast agent of the present application; Figure 2 Morphology observation diagram of the targeted CMVD ultrasound contrast agent prepared in Example 1; Figure 3 Zeta potential and particle size distribution diagram of the targeted CMVD ultrasound contrast agent prepared in Example 1; Figure 4 Biological toxicity test diagram of the targeted CMVD ultrasound contrast agent prepared in Example 1; Figure 5 In vitro ultrasonic imaging diagram of the targeted CMVD ultrasound contrast agent prepared in Example 1; Figure 6 In vitro targeting verification diagram of the targeted CMVD ultrasound contrast agent prepared in Example 1; Figure 7 In vivo targeting verification diagram of the targeted CMVD ultrasound contrast agent prepared in Example 1. DETAILED DESCRIPTION

[0023] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art to which the present application belongs.

[0024] The technical solutions of the present application will be further described below in combination with specific embodiments.

[0025] Example 1 (1) Preparation of maleimide ultrasound microbubbles (MB-Mal): Weigh the following components and dissolve them in the organic solvent chloroform: 9 mg of distearoylphosphatidylcholine (DSPC); 0.5 mg of distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000); and 0.5 mg of distearoylphosphatidylethanolamine-polyethylene glycol 2000-maleimide (DSPE-PEG2000-mal). Ultrasonically dissolve the above lipid mixture in a 40-50°C water bath to form a 1 mg / mL uniform lipid solution. Then, inject the lipid solution into a glass flask of a rotary evaporator, rotate at a speed of 100 rpm under a 40-45°C water bath temperature, and simultaneously introduce a high-purity nitrogen gas flow. Evaporate the chloroform by negative pressure (≤0.1 MPa) for 30 minutes until a uniform lipid film is formed on the bottle wall. Continue to introduce the nitrogen gas flow for 5 minutes to completely remove the solvent residues. Then, add a phosphate buffer (PBS) with a pH of 7.4 (volume ratio of 1:1 with the initial lipid solution) to the flask, and maintain the water bath temperature at 40-45°C. Under a nitrogen atmosphere, mechanically shake at 300 rpm for 1 hour to hydrate the lipid film and form a multi-compartment liposome suspension. Then, continuously introduce a high-density inert gas perfluoropropane into the hydrated liposome suspension at a flow rate of 100 mL / min for 5 minutes to completely replace the air in the system. Next, place the sealed container in a shaker at a high speed of 4500 rpm for 60 seconds to form lipid microbubbles encapsulating C3F8 by gas shearing. Finally, let the microbubble suspension stand for 15 minutes, separate the layers, and then suck the middle layer of the homogeneous emulsion. Remove the unencapsulated lipid fragments by low-speed centrifugation (500g, 5 minutes), and collect the upper layer of microbubbles. Resuspend the microbubbles in PBS to the target concentration (5 x 10 9 bubbles / mL) for subsequent preparation of CMVD-targeted microbubbles.

[0026] (2) Preparation of CMVD-targeted ultrasound contrast agent (MB-cRGD): Weigh 3 mg of cyclic arginine-glycine-aspartic acid peptide (cRGD) and dissolve it in 10 mL of ultrapure water containing 50 mM tris(2-carboxyethyl)phosphine hydrochloride. Stir at room temperature for 15 minutes to activate and reduce the disulfide bond in the peptide chain. Then, add 10 9 microbubbles of maleimide lipid, gently mix, and then react in the dark for 3 hours to promote thiol coupling between the free thiol group (-SH) of cRGD and the maleimide group at the end of the biotin-PEG on the surface of the microbubbles. After the reaction is completed, remove the unbound peptide chains and byproducts in the lower layer of the supernatant by low-speed centrifugation at 400g for 5 minutes, and then wash twice with PBS (pH=7.4). Finally, the upper layer of the white microbubbles is obtained.

[0027] Example 2 The present application connects a specific targeting polypeptide cRGD to the surface of a maleimide ultrasonic microbubble to prepare a targeted CMVD ultrasonic contrast agent MB-cRGD (a synthesis diagram of which is shown in Figure 1 ).

[0028] Morphological observation: The prepared MB-cRGD was diluted 100 times with ultrapure water, and the morphology and distribution were observed under an environmental scanning microscope. Under the microscope, it was observed that the MB-cRGD was round and evenly distributed, with a particle size of 2-3 microns (as shown in Figure 2 ).

[0029] Particle size and potential detection: After diluting the MB-cRGD with ultrapure water, the surface potential was detected by adding it into a nanoparticle size and potential analyzer. The results showed that the average particle size was about 2 microns, and the potential was negative-25 mV (as shown in Figure 3 ).

[0030] Biological safety detection: The biological safety of the prepared targeted CMVD ultrasonic contrast agent was detected by MTT method. The results showed that the microbubble concentration in the range of 10 5 -10 9 / mL did not affect the activity and proliferation of endothelial cells (as shown in Figure 4 ).

[0031] Imaging effect verification: Different concentrations of microbubbles were imaged using a 24MHz linear array probe, and similar acoustic enhancement effects were obtained, and an acoustic intensity-targeted microbubble concentration curve was obtained. When the microbubble concentration was greater than 10 5 / mL, a clear acoustic enhancement effect was obtained (as shown in Figure 5 ).

[0032] In vitro targeting ability verification: The parallel plate flow chamber experiment was used to verify the targeting ability of MB-cRGD. The plate bottom of the parallel plate flow chamber was coated with platelet glycoprotein IIb / IIIa protein and BSA, and MB-cRGD and MB-Mal were incubated with lipid fluorescent dye Dil (green) to make them labeled with fluorescence. MB-cRGD and MB-Mal were made to flow evenly through the parallel plate flow chamber, and the adhesion of the microbubbles was observed under a microscope. The results showed that only MB-cRGD had obvious adhesion in the parallel plate flow chamber coated with platelet glycoprotein IIb / IIIa protein (as shown in Figure 6 ), indicating that MB-cRGD has specific targeting.

[0033] In vivo targeting ability verification: using coronary microcirculatory dysfunction of small pigs as experimental subjects, using commercially available Sonovue, MB-Mal, MB-cRGD in rest state, then using three kinds of microbubbles for myocardial acoustic contrast after adenosine loading (AD), finally injecting MB-cRGD-FITC microbubbles. The results confirm that MB-Mal and MB-cRGD have good acoustic contrast effect in normal left ventricular wall segments of small pig model in rest and adenosine loading state, which is better than that of commercially available Sonovue Figure 7 .A). Under adenosine loading, there is no significant difference in acoustic contrast signal of MB-Mal in the modeling area and the non-modeling area, and delayed contrast enhancement can be observed in the modeling area of MB-cRGD microbubbles, where the red arrow is the MB-cRGD signal Figure 7 .B). To verify the targeting ability of the ultrasound contrast agent, we connected the FITC group on the MB-cRGD, and observed that the targeted microbubbles were specifically attached to the myocardium in the modeling area after myocardial biopsy, and the fluorescence signal in the myocardial tissue close to the microvessel was more obvious Figure 7 .C), the above results show that MB-cRGD has good clinical effect for the diagnosis of CMVD.

[0034] The above only describes the preferred embodiments of the present application and is not used to limit the present application. For those skilled in the art, the present application can have various changes and variations. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A targeted CMVD ultrasound contrast agent, characterized in that, The targeting CMVD ultrasound contrast agent comprises micro-sized lipid microbubbles, perfluorocarbon gas existing in the lipid microbubbles, and a targeting cyclic peptide segment cRGD existing on the surface of the lipid shell of the lipid microbubbles. The lipid shell material is DSPC, DSPE-PEG2000 and DSPE-PEG2000-Mal.

2. The ultrasound contrast agent as set forth in claim 1, wherein, The perfluorocarbon gas is selected from perfluoropropane, perfluorobutane and perfluorohexane; preferably, the fluorocarbon gas is selected from perfluoropropane.

3. The ultrasound contrast agent as set forth in claim 1, wherein, The thiol group of the targeting cyclic peptide segment cRGD is connected to the surface of the lipid shell through a thiol coupling reaction with a maleimide, and the cRGD is a targeting peptide segment of thrombus surface platelet glycoprotein IIb / IIIa.

4. The ultrasound contrast agent of claim 1, wherein, The molar ratio of the DSPC, DSPE-PEG2000 and DSPE-PEG2000-Mal is 17-19:1-2:1; and / or, the molar ratio of the cRGD to the DSPE-PEG2000-Mal in the ultrasound contrast agent is 20-60:

1.

5. The ultrasound contrast agent of claim 1, wherein, The average particle size of the targeting CMVD ultrasound contrast agent is 2-3 microns.

6. The method of claim 1, wherein the CMVD-targeted ultrasound contrast agent is prepared by the steps of: (a) mixing a CMVD-targeted ultrasound contrast agent with a pharmaceutically acceptable carrier; and (b) sterilizing the mixture. The method comprises the following steps: (1) Preparation of maleimide ultrasound microbubbles: dissolve DSPC, DSPE-PEG2000 and DSPE-PEG2000-Mal in chloroform, form a thin film after evaporation of the organic solvent under a nitrogen stream, replace air with perfluorocarbon gas after hydration and oscillation, and then oscillate to obtain the maleimide ultrasound microbubbles; (2) Preparation of CMVD-targeted ultrasound contrast agent: TCEP was dissolved in water, cRGD was added, followed by 1-5 x 10 9 maleimide ultrasound microbubbles, and the reaction was carried out at room temperature for 2-5 hours. The lower liquid was removed, and the upper microbubbles were reserved to obtain the product.

7. The production method according to claim 6, wherein The molar ratio of the cRGD to the DSPE-PEG2000-Mal is 20-60:1; and / or, the molar ratio of the DSPC, DSPE-PEG2000 and DSPE-PEG2000-Mal is 17-19:1-2:

1.

8. The production method according to claim 6, wherein In the step (2), the reaction is carried out in the dark; and / or, the concentration of tris(2-carboxyethyl)phosphine hydrochloride is 40-50 mM.

9. Use of the targeting CMVD ultrasound contrast agent of claim 1 in the preparation of an ultrasound contrast agent for detecting CMVD.

10. Use according to claim 9, wherein the compound is ###00003### or a pharmaceutically acceptable salt thereof. The ultrasound contrast agent is a lipid-based contrast agent; preferably, the ultrasound contrast agent can be administered by injection; preferably, the ultrasound contrast agent is administered by intravenous injection. The ultrasound contrast agent is a lipid-based contrast agent; preferably, the ultrasound contrast agent can be administered by injection; preferably, the ultrasound contrast agent is administered by intravenous injection.

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