Nuclear magnetic resonance imaging contrast agent RM / HD22 (at) Gd based on thrombin aptamer as well as preparation method and application of nuclear magnetic resonance imaging contrast agent RM / HD22 (at) Gd

Abstract

The invention discloses a thrombin aptamer-based nuclear magnetic resonance imaging contrast agent RM / HD22 (at) Gd and a preparation method and application thereof.The construction method of the contrast agent comprises the steps that an extracted SD rat red cell membrane (RBCM) serves as a carrier matrix, the thrombin aptamer HD22 is anchored to the surface of the RBCM through DSPE-PEG, then gadolinium ions (Gd) are wrapped, and the contrast agent RM / HD22 (at) Gd is obtained; the nuclear magnetic resonance contrast agent for targeting thrombus imaging is formed. The contrast agent is used for realizing in-vivo visual detection of early targeting and sensitivity of thrombus by means of molecular imaging aiming at a microenvironment in which the content of local thrombin in early-stage lesion of thrombus is abnormally increased. By exploring the dynamic change of the thrombin content and activity, the thrombus formation risk, formed thrombus and physiological or pathological hypercoagulable state can be distinguished, the time node of thrombus visualization is advanced, the timeliness of thrombus diagnosis is improved, and a new molecular imaging tool is provided for accurate diagnosis and treatment of thrombotic vascular diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the multi-disciplinary field of material science, thrombotic vascular disease, medical imaging, and the like, and in particular to a thrombin aptamer-based magnetic resonance imaging contrast agent RM / HD22@Gd, as well as a preparation method and application thereof. BACKGROUND

[0002] Thrombosis is the cause of acute myocardial infarction, ischemic stroke, pulmonary embolism and other fatal diseases. However, the existing clinical thrombus imaging detection methods, including magnetic resonance imaging (MRI), computed tomography (CT) angiography and ultrasound, mainly show blood flow loss or stenosis, rather than directly imaging thrombus, and have limited ability to image early thrombus. Thrombin is a key enzyme involved in the coagulation cascade and plays a crucial role in thrombus formation, growth and stability, and is an ideal target for in vivo thrombus imaging. To date, conventional coagulation tests (e.g., activated partial thromboplastin time (APTT) and prothrombin time (PT)) are not necessarily able to quickly and accurately distinguish between patients at risk of bleeding or thrombosis and healthy controls, which is not accurate for predicting in situ thrombus formation events. The mechanisms of thrombin imaging such as fluorescence imaging, MRI and positron emission tomography (PET) are mostly not directly bound to thrombin, but rely on protease-cleavable peptides activated by thrombin protease, so once the protein is inactivated by cleavage, it is difficult to stably image thrombus.

[0003] A nucleic acid aptamer is a small piece of single-stranded oligomeric DNA or RNA selected in vitro, which can specifically and highly bind to target molecules. Among them, HD22 thrombin aptamer, also known as TBA29 or DNA60-18, is a thrombin aptamer that has been studied in depth in recent years. Numerous studies have shown that HD22 has high affinity for thrombin and only binds to thrombin, not to prothrombin or thrombin-antithrombin complex, and can be used as an effective anti-platelet, anti-coagulation and anti-thrombus imaging agent. Currently, most thrombus imaging techniques based on aptamer principles use fluorescence imaging methods, and there has been no research and development of MRI thrombus imaging using thrombin aptamer.

[0004] In view of this, the present application develops a thrombin aptamer-based MRI contrast agent RM / HD22@Gd that can solve the above technical problems, which can quickly and accurately diagnose thrombus formation sites and achieve early detection, early diagnosis and early treatment of thrombotic vascular disease. SUMMARY

[0005] The application aims to provide a thrombin aptamer-based MRI contrast agent RM / HD22@Gd and a preparation method and application thereof.

[0006] To achieve the above-mentioned purpose, the application adopts the following technical solutions. In a first aspect of the application, a nuclear magnetic resonance imaging contrast agent RM / HD22@Gd is provided, a structural schematic diagram of the contrast agent is shown in FIG. 1. Figure 1 As shown in FIG. 1. In a second aspect of the application, a preparation method of the nuclear magnetic resonance imaging contrast agent RM / HD22@Gd is provided, steps of the preparation method are shown in FIG. 2, and the preparation method specifically comprises the following steps. Figure 1 (1) collecting blood samples from anesthetized SD rats, obtaining red blood cell membranes after centrifugation, separation, washing, ultrasonic treatment and purification; (2) oscillating the red blood cell membranes in a HEPES buffer containing CaCl2, adding DSPE and membrane phospholipids to pre-synthesized HD22-PEG-DSPE, and obtaining red blood cell membranes modified with HD22 (RM / HD22) after oscillation, temperature rising and rapid temperature dropping; (3) placing the RM / HD22 in a citric acid and glucose solution, adding GdCl3, oscillating, adjusting pH, and obtaining the final product RM / HD22@Gd.

[0007] In step (1), the blood collection includes one or more of external jugular vein blood collection.

[0008] In step (1), the centrifugation and separation are preferably performed at a temperature of 4℃, a rotation speed of 1500xg, and a centrifugation time of 10 minutes.

[0009] In step (1), the washing solution used in the washing is preferably PBS, and the washing is performed for 3 times or more until the supernatant is clear and transparent.

[0010] In step (1), the ultrasonic treatment is preferably performed at a power of 100 W and a time of 3x30 s.

[0011] In step (1), the concentration of the sucrose gradient solution for purification is preferably 30%-60%, and the purified red blood cell membranes are preferably stored in PBS containing protease inhibitors at a storage temperature of 4℃. ​

[0012] In step (2), the pH of the HEPES buffer is 7.0, the concentration of CaCl2 is preferably 1.5 mM, the shaking time is preferably 15 min, and the temperature is preferably 37℃.

[0013] In step (2), the molar ratio of DSPE to membrane phospholipid is preferably 1:150, the shaking time is preferably 4 h, the temperature is preferably 25℃, and the shaking speed is preferably 200 rpm.

[0014] In step (2), the temperature is preferably raised to 42℃ after shaking, the holding time is preferably 5 min, and the temperature is preferably lowered to 4℃ rapidly, and the holding time is preferably 10 min. In step (3), the RM / HD22 is placed in a citric acid and glucose solution, wherein the concentration of citric acid is preferably 300 mM, the concentration of glucose is preferably 5 mM, and the pH of the solution is preferably pH 4.0.

[0015] In step (3), the final concentration of GdCl3 is preferably 40 mM, the shaking temperature is preferably 37℃, the shaking time is preferably 30 min, and the pH is preferably adjusted to pH 7.4 after shaking, and the adjusting agent is preferably 0.1 M NaOH.

[0016] In a third aspect of the present application, the use of the above-mentioned MRI contrast agent RM / HD22@Gd is provided, and the use includes at least one of the following uses: use in preparing an imaging tool for detecting acute thromboembolism, use in preparing an MRI contrast agent for detecting thrombus formation, use in preparing an MRI contrast agent for detecting thrombus formation related diseases, and use in preparing an imaging tool for predicting thrombus formation related risks.

[0017] The present application has the following technical effects or advantages: (1) The present application expands the method of thrombin imaging, and uses the characteristics of high stability, strong affinity and strong binding ability of aptamers to target molecules to achieve precise positioning of thrombus in vivo. (2) The MRI contrast agent based on thrombin aptamer of the present application can solve the problem that acute ischemic stroke is almost invisible in ordinary stroke MRI sequences, and further distinguish the formation of old thrombus and fresh thrombus. (3) The method of the present application is simple, low in production cost, and easy to scale up; (4) The required reaction conditions and reagents are conventional and easily available; The above is a summary of the technical scheme of the present application, and the present application will be further described in conjunction with the drawings and specific embodiments. BRIEF DESCRIPTION OF DRAWINGS Figure 1is the RM / HD22@Gd synthetic route map Figure 2 is the electron microscope picture after cell membrane loading contrast agent Figure 3 is the establishment of an arterial thrombus model. Among them, Figure A is the in situ thrombus model photo, Figure B is the vascular H&E staining imaging collected from the rabbit thrombus model, and Figure C is the TOF 3D MR imaging of the in situ arterial thrombus model. DETAILED DESCRIPTION

[0018] The advantages and various effects of the present application will be more clearly presented hereinafter in conjunction with specific embodiments and examples. Those skilled in the art should understand that these specific embodiments and examples are used to illustrate the present application, not to limit the present application.

[0019] Throughout the specification, unless otherwise specifically indicated, the terms used herein are to be understood as having the meanings commonly used in the art. Therefore, unless otherwise defined, 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. If there is a conflict, the present specification takes precedence.

[0020] Unless otherwise specifically stated, the various raw materials, reagents, instruments and equipment used in the present application can be purchased from the market or obtained by existing methods.

[0021] As an optional embodiment of the present application, the present application provides a preparation method of a nuclear magnetic resonance imaging contrast agent RM / HD22@Gd, as shown in the attached Figure 1 The preparation method comprises the following steps: (1) Collect about 8-10 mL of SD rat blood in a centrifuge tube containing an anticoagulant, and obtain red blood cell membranes after centrifugation, separation, washing, ultrasonic treatment and purification.

[0022] (2) Shake the obtained cell membranes in a HEPES buffer containing CaCl2, then add DSPE and membrane phospholipids, and obtain red blood cell membranes modified with HD22 through the process of shaking, warming and cooling.

[0023] (3) Place RM / HD22 in a solution containing citric acid and glucose, then add GdC3, and obtain the final product RM / HD22@Gd through shaking and pH adjustment.

[0024] As one of the preferred schemes of the specific embodiment, in step (1), the centrifugation conditions are temperature 4℃, rotation speed 1500xg, and time 10 minutes.

[0025] As one of the preferred schemes of the specific embodiment, in step (1), the ultrasonic treatment conditions are power 100W, and time 3x30s.

[0026] As one of the preferred embodiments of the specific embodiment, in step (2), the concentration of CaCl2 used is 1.5 mM, and the molar ratio of DSPE: membrane phospholipid is 1:150.

[0027] As one of the preferred embodiments of the specific embodiment, in step (2), after the addition of DSPE and membrane phospholipid, the solution is shaken at 25°C for 4 h, then warmed to 42°C for 5 min, and then rapidly cooled to 4°C for 10 min.

[0028] As one of the preferred embodiments of the specific embodiment, in step (3), the concentrations of citric acid and glucose in the solution are 300 mM and 5 mM, respectively.

[0029] As one of the preferred embodiments of the specific embodiment, in step (3), the final concentration of GdCl3 is 40 mM, and the reaction is carried out by shaking at 37°C for 30 min, followed by rapid adjustment of the solution pH to 7.4 using 0.1 M NaOH.

[0030] A nuclear magnetic resonance imaging contrast agent RM / HD22@Gd and a preparation method and application thereof will be described in detail below with reference to examples, comparative examples, and experimental data.

[0031] Example 1, a nuclear magnetic resonance imaging contrast agent based on a thrombin aptamer and a preparation method thereof 1. First, red blood cell membranes RM / HD22 modified with a thrombin aptamer HD22 are prepared, and the preparation process and related characterization results are as shown in Table 1. Figure 1 The preparation method of the RM / HD22 is as follows: (1) A centrifuge tube containing an anticoagulant (sodium heparin, 0.1 mM EDTA) is used to collect blood samples from SD rats, and after centrifugation at 4°C, red blood cells are collected and washed with PBS multiple times to obtain red blood cells. The red blood cells are then subjected to ultrasonic treatment (100 W, 3x30 s) to obtain red blood cell membrane vesicles, which are then purified by a sucrose gradient solution (30%-60% gradient) to obtain red blood cell membranes, which are suspended in PBS containing protease inhibitors and stored at 4°C.

[0032] (2) The obtained red blood cell membranes are placed in a HEPES buffer containing 1.5 mM CaCl2 and shaken at 37°C for 15 minutes, then HD22-PEG-DSPE is pre-synthesized by adding DSPE: membrane phospholipid at a molar ratio of 1:150, and then shaken at 25°C for 4 h, followed by warming to 42°C for 5 min, and then rapidly cooling to 4°C for 10 min to obtain red blood cell membranes modified with HD22 (RM / HD22).

[0033] 2. Add RM / HD22 into the solution containing 300 mM citric acid and 5 mM glucose (pH 4.0), then add GdCl3 (final concentration 40 mM) into the solution, shake at 37°C for 30 min, then adjust the pH of the solution to 7.4 with 0.1 M NaOH to obtain the contrast agent RM / HD22@Gd.

[0034] Example 1 The contrast agent RM / HD22@Gd obtained in Example 1 was observed under a transmission electron microscope. As shown in FIG. 1, the typical morphology of RM / HD22@Gd was a complete and clear spherical vesicle (128 ± 8 nm in diameter), and discrete convex nodules were visible on the surface, which were the anchoring sites of HD22-PEG-DSPE. In addition, scattered nodular electron-dense shadows were visible in the lumen of the RM / HD22@Gd membrane, which proved that Gd was successfully loaded. Based on the above electron microscope characterization, it can be proved that RM / HD22@Gd synchronously realizes the surface anchoring of the targeting unit and the intraluminal loading of the contrast agent on the basis of retaining the structure of the red blood cell membrane vesicle, and does not cause membrane fusion or deformation, which provides an ideal carrier morphological basis for thrombus-targeted MRI. Figure 2

[0035] Example 2 First, a mouse external carotid artery thrombosis model was established: the anesthetized mouse was fixed in a supine position, and a median neck incision was made after the neck was disinfected and fully exposed the right sternocleidomastoid muscle. The right common carotid artery (CCA) was bluntly separated, as shown in FIG. 2A, a piece of plastic wrap was placed under the CCA to isolate the surrounding tissue. A 2 × 2 mm2filter paper piece was immersed in a FeCl3solution of different concentrations for 1 min, and the excess liquid was scraped off and covered on the surface of the middle segment of the CCA, and stood for 10 min. After observing the local blood vessel texture hardening and color deepening (indications of vascular endothelial injury) during the operation, the filter paper was removed and the incision was sutured. Figure 3

[0036] Figure 3 B shows that the right external carotid artery vascular section shows the formation of a mixed thrombus of platelets and fibrin in the lumen; as shown in FIG. 2C, coronal / sagittal / axial images show local lumen stenosis and strip-shaped filling defects in the right external carotid artery, which confirms the successful establishment of the mouse external carotid artery thrombosis model. Figure 3

[0037] Therefore, the thrombus magnetic resonance imaging contrast agent based on the thrombin aptamer using the contrast agent has feasibility, and is expected to achieve imaging of early thrombus and provide a new effective molecular imaging tool for accurate diagnosis and treatment of thrombotic vascular diseases.

[0038] ​​​Finally, it should be noted that the terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but can also include other elements not expressly listed or inherent to such process, method, article, or apparatus.

[0039] While the preferred embodiments of the application have been described, additional variations and modifications can be made to the preferred embodiments by those of skill in the art once they have the benefit of the present disclosure without departing from the spirit and scope of the application. Accordingly, it is intended that the appended claims encompass all such variations and modifications as falling within the scope of the application.

[0040] Obviously, numerous modifications and variations of the present application are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims and their equivalents, the application can be practiced otherwise than as specifically described.

Claims

1. A magnetic resonance imaging contrast agent RM / HD22@Gd, characterized in that, The structural schematic of the contrast agent RM / HD22@Gd is shown in Figure 1.

2. A method for synthesizing the RM / HD22@Gd nuclear magnetic resonance imaging contrast agent based on thrombin aptamer HD22 as described in claim 1, characterized in that, The schematic diagram of the RM / HD22@Gd synthesis process described in the method is shown in Figure 1, and includes the following steps: S1: Extraction and purification of erythrocyte membranes from SD rats; S2: HD22-PEG-DSPE was anchored onto the rat erythrocyte membrane using the calcium-activated insertion method; S3: Using the biomimetic channel method to transfer Gd 3+ It is loaded across the membrane into the HD22-modified erythrocyte membrane.

3. The method for synthesizing a magnetic resonance imaging contrast agent (RM / HD22@Gd) based on thrombin aptamer HD22 according to claim 2, characterized in that, The method for obtaining red blood cell membranes in step S1 includes: S11: After anesthetizing SD rats, about 1-1.5 mL of blood was collected from each rat via the external jugular vein and placed into a centrifuge tube containing anticoagulants (sodium heparin, 0.1 mM EDTA); S12: Centrifuge the blood sample obtained in S11 at 1500×g for 10 minutes at 4℃, discard the upper plasma and white blood cell layer (pale yellow layer), collect the bottom red blood cells, add an equal volume of PBS to resuspend, and wash with PBS three times, centrifuging at 1500×g for 10 minutes each time, until the supernatant is clear. S13: The red blood cells obtained in S12 were sonicated (100 W, 3 × 30 s) to obtain red blood cell membrane vesicles; then the membrane fragments were further purified using a sucrose gradient solution (30%-60% gradient). The purified red blood cell membrane was then suspended in PBS containing protease inhibitors and stored at 4°C for later use to obtain the red blood cell membrane.

4. The method for synthesizing a magnetic resonance imaging contrast agent (RM / HD22@Gd) based on thrombin aptamer HD22 according to claim 2, characterized in that, The method for preparing RM / HD22 in step S2 includes: S21: The red blood cell membrane obtained in step S1 was shaken in HEPES buffer (pH 7.0) containing 1.5 mM CaCl2 for 15 min (37℃). S22: Add pre-synthesized HD22-PEG-DSPE at a molar ratio of DSPE:membrane phospholipid = 1:

150. After shaking at 25℃ for 4 h (200 rpm), first raise the temperature to 42℃ and maintain for 5 min, then quickly lower it to 4℃ and maintain for 10 min to obtain erythrocyte membrane modified with HD22 (RM / HD22).

5. The method for synthesizing MRI contrast agent (RM / HD22@Gd) based on thrombin aptamer HD22 according to claim 2, characterized in that, The method for preparing RM / HD22@Gd in step S3 includes: S31: Place the RM / HD22 obtained in step S2 into a solution containing 300 mM citric acid and 5 mM glucose (pH 4.0); S32: Add GdCl3 to make the final concentration 40 mM, shake at 37℃ for 30 min, and then quickly adjust the pH of the solution to 7.4 with 0.1 M NaOH to obtain the final product RM / HD22@Gd.

6. The method for synthesizing and extending the application of the thrombin aptamer-based MRI contrast agent according to claim 2, characterized in that: The principle of MRI thrombosis imaging using thrombin aptamers is that the thrombin aptamer constructed on the contrast agent can be of different types, including but not limited to thrombin aptamer HD22; at the same time, the cell membrane can also carry a series of substances such as fibrinolytic agents, photosensitizers, and targeted drugs according to different application scenarios.

7. A nuclear magnetic resonance contrast agent RM / HD22@Gd obtained by the preparation method according to any one of claims 1-6.

8. The extended application of the magnetic resonance imaging contrast agent RM / HD22@Gd according to claim 7 in thrombotic vascular diseases.

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