A magnetic microbubble for microthrombus clearance and an interventional microthrombus clearance device

Abstract

The present invention provides a magnetic microbubble for microthrombus clearance and an interventional microthrombus clearance device. The magnetic microbubble for microthrombus clearance is composed of a core, a shell layer, and a ligand; wherein, the core contains magnetic particles; the ligand is a microthrombus-targeting ligand, and the ligand is connected to the outer side of the shell layer; the magnetic microbubble does not contain drugs. The present invention also provides an interventional microthrombus clearance device based on the above magnetic microbubble. For diffuse microthrombi, the magnetic microbubble with microthrombus targeting provided by the present invention can achieve specific binding with microthrombi and respond to a magnetic field, so as to achieve specific targeting and clearance of microthrombi without using thrombolytic drugs, and change the disadvantages of poor clearance effect or large side effects of the traditional method for diffuse microthrombi.

Description

Technical Field

[0001] The present invention relates to a magnetic microbubble for microthrombus clearance and an interventional microthrombus clearance device, belonging to the technical field of thrombus clearance. Background Art

[0002] Thrombosis is the main pathogenesis of acute ischemic cardiovascular diseases, seriously endangering life and health. At present, the main treatment methods for thrombus lesions are drug thrombolysis, surgical thrombectomy, and ultrasound thrombolysis.

[0003] Drug thrombolysis refers to injecting thrombolytic drugs through intravenous injection or catheter to directly or indirectly dissolve thrombi, thereby dredging the blocked blood vessels. These traditional thrombolytic drugs act on the whole body and are prone to increase the risk of systemic bleeding. Surgical thrombectomy is an effective means to remove large thrombi, but traditional surgical methods will cause greater trauma and longer recovery time. Ultrasound thrombolysis, especially ultrasound contrast agent microbubbles, shows its advantages in thrombus dissolution due to its precise local action and ultrasound-controlled drug release. However, the ultrasound power will not only affect the microbubble imaging effect but also may cause local endothelial damage and trigger adverse reactions.

[0004] Microthrombus refers to a tiny thrombus (with a diameter of 10 - 100 μm) occurring in the microvessels of the microcirculation, mainly composed of fibrin and its monomers, containing different numbers of platelets, white blood cells, or a small amount of red blood cells. Clinically, the formation of microthrombi is an important cause leading to insufficient perfusion, ischemia, or necrosis of important organs and may induce more serious thrombus problems. At the same time, microthrombi are also an important pathological transformation caused by novel coronavirus infection and an important cause of death and disability in novel coronavirus infection. Therefore, timely detection and clearance of microthrombi are crucial for shortening the treatment cycle and improving the prevention and treatment efficiency.

[0005] CN109045285A provides a drug-loaded magnetic microbubble, which can achieve a rapid response to an external magnetic field both in vitro and in vivo, thereby aggregating in the lesion area. Under the induction of an ultrasonic probe, the microbubble vibrates to controllably release the drug loaded thereon, so as to achieve the purpose of local thrombolysis. However, the release rate of the thrombolytic drug is related to the ultrasonic vibration frequency, and the thrombolytic drug will be dispersed during the release process, increasing the bleeding risk in non-lesion areas. At the same time, the ultrasonic action used to trigger the vibration and drug release of the microbubble may cause damage to endothelial cells, thereby triggering adverse reactions such as coagulation.

[0006] CN112587677A provides an iRGD magnetic targeting microbubble contrast agent. This contrast agent utilizes the dual targeting effects of magnetism and iRGD, improves the targeting of microbubbles to the angiogenesis of local tissues in the endometrium, uses magnetic particles for magnetic resonance imaging, and uses the internal gas components for ultrasonic imaging, achieving ultrasonic / magnetic resonance dual-modal imaging, which is beneficial for observing the process of changes in endometrial blood vessels. This technology mainly uses dual targeting to achieve dual imaging, but is only used for diagnosis and has no impact on treatment.

[0007] Due to the diffusivity of microthrombi, the above traditional thrombolysis methods are not applicable to microthrombi. Not only is it easy to cause an increase in the risk of systemic bleeding, but also the clearance effect is limited. As a contrast agent, microbubbles are increasingly used in thrombus diagnosis and clearance due to their non-invasive, low-cost, and easy-to-operate characteristics. In addition, magnetic microbubbles have gradually attracted attention due to their response effects on magnetic resonance and ultrasound and are applied to the diagnosis and treatment of diseases such as cancer. However, there are currently no domestic or foreign studies on the related reports of using magnetic microbubbles to target and clear microthrombi. Summary of the Invention

[0008] To solve the above technical problems, the purpose of the present invention is to provide a magnetic microbubble that can target and bind to microthrombi and can achieve magnetic force manipulation and aggregation with the help of an interventional magnetic wire.

[0009] To achieve the above purpose, the present invention provides a magnetic microbubble for microthrombus clearance, which is composed of a core, a shell layer, and a ligand;

[0010] Among them, the core contains magnetic particles;

[0011] The ligand is a microthrombus targeting ligand, and the ligand is connected to the outer side of the shell layer;

[0012] This magnetic microbubble does not contain drugs.

[0013] In the above magnetic microbubble, preferably, the magnetic particles are iron oxide nanoparticles; more preferably, the particle size of the iron oxide nanoparticles is less than or equal to 30nm. Fe3O4 nanoparticles belong to superparamagnetic nanoparticles, which have extremely strong magnetism itself. Due to their excellent size, non-toxicity, and excellent biocompatibility, they have good prospects. In addition, magnetic particles can significantly shorten the T2 relaxation time and reduce the magnetic resonance signal, which can provide an additional imaging function for the magnetic microbubbles of the present invention.

[0014] In the above magnetic wire microbubble, preferably, the core contains gas. This is related to the medium used in freeze-drying during the microbubble preparation process. The gas in the core can reduce the density of the overall microbubble, enabling it to not settle or deposit in the blood and having better hemodynamic characteristics.

[0015] In the above magnetic microbubbles, preferably, the material of the shell layer is poly(lactic-co-glycolic acid) (PLGA). PLGA is a biodegradable polymer synthesized by polycondensation of two monomers, lactic acid (LA) and glycolic acid (GA), in different proportions, and can be used in tissue engineering, medical materials, and drug carriers. PLGA has good biocompatibility, biodegradability, simple synthesis, high stability, adjustable degradation rate, and good plasticity. The PLGA microbubbles prepared using PLGA as the film-forming material have good stability, providing a good basis for the controllable operation of the targeted binding of the magnetic microbubbles and microthrombi in the present invention.

[0016] In the above magnetic microbubbles, preferably, the ligand includes the polypeptide arginine-glycine-aspartic acid (Ary-Gly-Asp, RGD) polypeptide that specifically binds to the platelet surface membrane glycoprotein IIb / IIIa receptor, or the glutamic acid-tryptophan-valine-aspartic acid-valine (Glu-Trp-Val-Asp-Val, EWVDV) polypeptide that binds to platelet P-selectin, or cysteine-arginine-glutamic acid-lysine-alanine (Cys-Arg-Glu-Lys-Ala, CREKA) that specifically binds to fibrin.

[0017] In the above magnetic microbubbles, preferably, the mass ratio of PLGA, the microthrombus-targeting ligand, and the iron oxide nanoparticles is 50:0.05 - 0.15:1 - 2.

[0018] In the above magnetic microbubbles, preferably, the particle size of the magnetic microbubbles is 1 - 10 microns.

[0019] The present invention also provides a preparation method of the above magnetic microbubbles for microthrombus clearance, wherein the preparation method includes the following steps:

[0020] S1. Disperse the magnetic particles in double-distilled water to form a suspension, and perform ultrasonic emulsification to disperse the magnetic particles to obtain a magnetic particle storage solution;

[0021] S2. Dissolve the material of the shell layer in an organic solvent, add the microthrombus-targeting polypeptide ligand after mixing and dissolving, and stir well to obtain a shell layer material solution;

[0022] S3. Mix the magnetic particle storage solution and the shell layer material solution, and perform primary emulsification under ultrasonic action to obtain a mixture;

[0023] S4. Slowly add the mixture prepared in step S3 to a 5% polyvinyl alcohol solution;

[0024] S5. Add the mixture obtained in step S4 into an isopropanol solution with a concentration of 2%, and stir at room temperature to solidify the surface of the microbubbles.

[0025] S6. Centrifuge the mixture obtained in step S5 to obtain microcapsules.

[0026] S7. Freeze-dry the microcapsules to prepare the magnetic microbubbles for microthrombus clearance.

[0027] In the above preparation method, preferably, in step S1, the concentration of the suspension is 1.6 mg / mL.

[0028] In the above preparation method, preferably, in step S2, the shell material is poly (lactic-co-glycolic acid). Among them, the concentration of the poly (lactic-co-glycolic acid) in the shell material solution is 0.625 - 10 mg / mL; the molar ratio of lactic acid to glycolic acid used for synthesizing the poly (lactic-co-glycolic acid) can be 25:75 or 50:50.

[0029] In the above preparation method, preferably, in step S2, the organic solvent includes ethyl acetate or chloroform.

[0030] In the above preparation method, in step S4, polyvinyl alcohol is used as a surfactant, which can reduce the surface tension and make the mixed emulsion form small liquid beads. Preferably, after adding the mixture into the polyvinyl alcohol solution, stir at a certain rotation speed. The stirring rotation speed can be controlled at 9500 rpm, and the stirring time can be controlled at 5 minutes.

[0031] In the above preparation method, in step S5, stirring at room temperature can be carried out at a rotation speed of 400 rpm for 1 h or at a rotation speed of 200 rpm for 4 - 6 h. Through stirring, the organic solvent can be completely volatilized.

[0032] In the above preparation method, in step S6, after obtaining the microcapsules, hexane can be added for three centrifugal washings.

[0033] The present invention also provides an interventional microthrombus clearance device, which includes: the above magnetic microbubbles for microthrombus clearance and an interventional magnetic wire;

[0034] The interventional magnetic wire includes a magnetic column and a housing;

[0035] The material of the magnetic column is neodymium iron boron, and the central magnetic induction intensity of the magnetic column is 0.1 - 0.6 T, preferably 0.4 T.

[0036] According to the specific implementation scheme of the present invention, preferably, the diameter of the magnetic column is 0.5 - 2, more preferably 1 mm.

[0037] According to a specific embodiment of the present invention, the magnetic column increases the radial magnetic field gradient through alternating magnetic poles, thereby enhancing the magnetic force on magnetic micro-elements; preferably, the magnetic poles of the magnetic column are assembled and arranged in an alternating pattern of N and S poles, for example Figure 3 in the form shown.

[0038] According to a specific embodiment of the present invention, preferably, the outer shell of the intrusive magnetic wire is a medical plastic for maintaining the position of the magnetic column. By presetting an interference fit, the protection of the magnetic column and the maintenance of the overall shape can be achieved.

[0039] The intrusive micro-thrombus removal device of the present invention uses precision-machined permanent magnet magnetic columns. Through the outer medical PDVF ultra-thin medical tube, the relative position control of the magnetic columns and the arrangement pattern of alternating magnetic poles of the magnetic columns are realized, enabling a magnetic field with a local high magnetic induction intensity and a high magnetic field gradient, and enabling the manipulation, aggregation, and capture of micro-thrombi in blood vessels.

[0040] The magnetic microbubbles provided by the present invention are a magnetically controlled microbubble system capable of targeting and collecting free micro-thrombi in blood and clearing them, and are microbubbles with both magnetism and thrombus targeting. Current domestic and international research has focused on the role of the acoustic and magnetic resonance signals of magnetic microbubbles in diagnostic imaging, and there are no reports on using magnetic microbubbles to target and clear micro-thrombi.

[0041] For diffuse micro-thrombi, the magnetic microbubbles with micro-thrombus targeting provided by the present invention can achieve specific binding with micro-thrombi and respond to the magnetic field, thereby realizing specific targeting and clearance of micro-thrombi without using thrombolytic drugs, and changing the drawbacks of poor clearance effect or large side effects of traditional methods for diffuse micro-thrombi. Description of the Drawings

[0042] Figure 1 Schematic diagram of the preparation process of magnetic microbubbles for micro-thrombus removal in Example 1.

[0043] Figure 2 Basic characterization results of magnetic microbubbles for micro-thrombus removal in Example 1.

[0044] Figure 3 Schematic diagram of the intrusive magnetic wire in Example 1.

[0045] Figure 4 Simulated magnetic field distribution diagram of the intrusive magnetic wire.

[0046] Figure 5 Measured magnetic field distribution diagram of the intrusive magnetic wire.

[0047] Figure 6 Magnetic field characterization results of magnetic microbubbles.

[0048] Figure 7 Verification experimental results of the microthrombus targeting property of the magnetic microbubbles in Example 1.

[0049] Figure 8 Schematic structural diagram of the microchannel used in the verification experiment of magnetic targeting property.

[0050] Figure 9 Verification experimental results of the magnetic targeting property of the magnetic microbubbles in Example 1. Detailed implementation manners

[0051] For a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention will be described in detail below, but it should not be construed as a limitation on the implementable scope of the present invention.

[0052] Example 1

[0053] This example provides a magnetic microbubble for microthrombus clearance, which is composed of three parts, specifically including: a microbubble shell made of PLGA, a microbubble core made of iron oxide nanoparticles and gas, and a microbubble exterior made of a microthrombus targeting ligand.

[0054] The preparation method of the magnetic microbubble for microthrombus clearance includes the following steps (as Figure 1 shown):

[0055] S1. Disperse the iron oxide nanoparticles in double-distilled water to form a suspension with a concentration of 1.6 mg / mL, and perform ultrasonic emulsification to disperse the nanoparticles to obtain a uniform storage solution of iron oxide nanoparticles.

[0056] S2. Dissolve the poly(lactic-co-glycolic acid) copolymer in an organic solvent (ethyl acetate or chloroform), add the microthrombus targeting polypeptide ligand (RGDS) after mixing and dissolving, and stir well to obtain a PLGA solution. Among them, the ratio of lactic acid to glycolic acid used in the preparation of PLGA is 50:50, and the concentration of the PLGA solution is 25 mg / mL.

[0057] S3. Mix the storage solution of iron oxide nanoparticles prepared in step S1 and the PLGA solution prepared in step S2, and perform primary emulsification by ultrasonic action to obtain a mixed solution.

[0058] S4. Slowly add the mixed solution prepared in step S3 to a 5% polyvinyl alcohol (PVA) solution, and stir at a speed of 9500 rpm for 5 minutes.

[0059] S5. Add the mixture obtained in step S4 into an isopropanol solution with a concentration of 2%, and stir at a speed of 400 rpm for 1 h (or stir at a speed of 200 rpm for 4 - 6 h) at room temperature to solidify the surface of the microbubbles and completely volatilize the organic solvent.

[0060] S6. Centrifuge the mixture obtained in step S5 to obtain microcapsules, and add hexane to centrifuge and wash three times.

[0061] S7. Freeze - dry the microcapsules obtained in step S6 to prepare magnetic microbubbles for micro - thrombus clearance.

[0062] In the above preparation process, the mass ratio of PLGA, ligand, and iron oxide nanoparticles is 50:0.1:1.6.

[0063] This embodiment also provides an interventional micro - thrombus clearance device, which includes: the above - mentioned magnetic microbubbles for micro - thrombus clearance and an interventional magnetic wire;

[0064] The interventional magnetic wire includes a magnetic column and a housing; the material of the magnetic column is neodymium iron boron, the central magnetic induction intensity of the magnetic column is 0.4 T, and the diameter is 1 mm; this interventional magnetic wire is as Figure 3 shown, and its simulated magnetic field distribution diagram and the actually measured magnetic field distribution diagram are respectively as Figure 4 and Figure 5 shown. The magnetic force received by the micro - thrombus is only related to the number of magnetic microbubbles adhering to its surface and the magnetic field distribution. The magnitude of the magnetic force received by the micro - thrombus adsorbed by n magnetic microbubbles is:

[0065]

[0066] V m is the effective magnetic response volume, μ0 is the spatial magnetic permeability, and χ is the magnetic susceptibility of the ferromagnetic material = permeability - 1. Therefore, increasing the magnetic induction intensity B and the magnetic induction intensity gradient can significantly increase the magnetic force, thereby improving the operation efficiency for micro - thrombus.

[0067] From Figure 4 and Figure 5 it can be seen that: compared with the arrangement of magnetic columns in the same direction, the arrangement method of the interventional magnetic wire provided by the present invention can make the radial induction intensity higher and the spatial change of the magnetic induction intensity larger, that is, the magnetic induction gradient is larger, thereby increasing the magnetic force received by the thrombus.

[0068] The material of the housing is medical plastic.

[0069] Comparative Example 1

[0070] The difference between Comparative Example 1 and Example 1 lies in step S1. Instead of adding Fe₃O₄ nanoparticles, 30 nm silica particles are used, and the other parameters and preparation methods are the same as those in Example 1.

[0071] Experimental Example 1. Basic Characterization of the Magnetic Microbubbles for Microthrombus Removal in Example 1

[0072] By macroscopically observing and comparing the appearance morphologies of the magnetic microbubbles in Example 1 and Comparative Example 1, as shown in Figure 2 Figure A and Figure B respectively, the appearance differences of the magnetic microbubbles with and without Fe₃O₄ nanoparticles are obvious.

[0073] By observing under the bright field of a ×40 eyepiece microscope, as shown in Figure 2 Figure C, the bilayer structure of the microbubbles can be observed.

[0074] The magnetic microbubbles prepared in Example 1 were observed and measured by scanning electron microscopy, and the results are as shown in Figure 2 Figure D.

[0075] Through a full-automatic image method particle size analyzer, an image of the mixed microbubble solution was obtained as shown in Figure 2 Figure E, which is an intuitive diagram of the particle size distribution of the microbubble group.

[0076] The average particle size distribution of the microbubbles was obtained through image analysis as shown in Figure 2 Figure F, and the average particle size is 1.805 μm.

[0077] The magnetic field characterization results of the magnetic microbubbles are as shown in Figure 6 , where 100 nm, 500 nm, and 1 μm respectively refer to the diameter of the microbubbles. According to Figure 6 it can be seen that: the larger the diameter, the better the magnetic field responsiveness. On the premise of not affecting the normal flow of blood, it is preferably to use microbubbles of about 1 μm for subsequent experiments.

[0078] Experimental Example 2. Verification Experiment on the Microthrombus Targeting of the Magnetic Microbubbles for Microthrombus Removal in Example 1

[0079] Fluorescent staining: Take 500 μl of blood, add 1.5 μL of a platelet staining agent labeled with DiOC6, and incubate at room temperature for 10 minutes; use rhodamine to perform fluorescent staining on the microbubbles prepared in Example 1.

[0080] Preparation of platelet-rich thrombus: Take the stained blood and centrifuge it at room temperature (1500 revolutions per minute, 10 minutes). After centrifugation, the blood is divided into two layers. The upper layer is platelet-rich plasma, and the lower layer consists of a large amount of red blood cells and a small amount of white blood cells. Extract the platelet-rich plasma and red blood cell suspension (volume ratio 9:1), mix them evenly, and then add a recalcification buffer with a volume ratio of 1:10 to restore the coagulation ability. Mix thoroughly and quickly. After 5 - 10 minutes, it can coagulate to obtain a platelet-rich thrombus.

[0081] Preparation of platelet-rich microthrombus: After the platelet-rich thrombus is prepared, place it in a normal temperature environment for about 3 hours and in a 4°C refrigerator for about 24 hours respectively to allow the thrombus to contract fully. Place the thrombus in liquid nitrogen for about 5 minutes to harden it, and then mechanically grind it to obtain microthrombus particles. First, filter it through a filter with a pore size of 100 microns, and collect the microthrombus particles that can pass through the filter. Then, filter it through a filter with a pore size of 10 microns, and collect the microthrombus particles that cannot pass through the filter, so as to obtain microthrombi with a size of 10 - 100 microns.

[0082] Dilute it with PBS solution to obtain a microthrombus buffer solution with a concentration of 10 5 cells / ml.

[0083] Add the stained magnetic microbubbles of Example 1 to the microthrombus buffer solution. Microscopically observe the fluorescence signals around the microthrombus before and after adding the microbubbles and find that: before adding the microbubbles, only the green fluorescence signal can be observed, while after adding the microbubbles, the red fluorescence signal can be clearly observed. The results show that the microbubbles can target and adhere to the surface of the microthrombus, as Figure 7 shown.

[0084] Test Example III: Verification experiment on the magnetic targeting of the magnetic microbubbles of Example 1 for microthrombus clearance

[0085] Adopt the microfluidic channel shown in Figure A of Figure 8 . Inject the magnetic microbubbles prepared as in Example 1 into the flow channel, inject and mix back and forth left and right to make the microbubble concentrations on both the left and right sides consistent. Apply a 200 mT magnetic field in the T direction shown in the figure. After acting for 10 minutes, observe the concentrations of the magnetic microbubbles in areas I and II, as shown in Figures B and C of Figure 8 . The experiment proves that under the action of the magnetic field, the microbubble concentration in area II is significantly greater than that in area I, that is, the magnetic microbubbles move from area I to area II, indicating that the magnetic microbubbles of Example 1 have good magnetic targeting and can be aggregated by using an intrusive magnetic wire. Figure 9 What is shown is the situation where the microthrombus migrates towards the wall surface under the traction of the microbubbles under magnetic field manipulation.

Claims

1. An interventional microthrombus removal device, comprising: Magnetic microbubbles and interventional magnetic wires for microthrombus clearance; The interventional magnetic wire includes a magnetic column and a housing; The material of the magnetic column is neodymium iron boron, the central magnetic induction intensity of the magnetic column is 0.1 - 0.6T, and the magnetic poles of the magnetic column are assembled and arranged in an alternating NS level manner; Among them, the magnetic microbubbles for microthrombus clearance are composed of a core, a shell layer, and a ligand; Among them, the core contains magnetic particles and gas, the magnetic particles are iron oxide nanoparticles, and the material of the shell layer is PLGA; The ligand is a microthrombus targeting ligand, and the ligand is connected to the outside of the shell layer. The mass ratio of PLGA, the microthrombus targeting ligand, and the iron oxide nanoparticles is 50:0.05 - 0.15:1 - 2; This magnetic microbubble does not contain drugs.

2. The interventional microthrombus removal device according to claim 1, wherein, The diameter of the magnetic column is 0.5 - 2mm.

3. The interventional micro-thrombus removal device according to claim 1, wherein, The particle size of the iron oxide nanoparticles is less than or equal to 30nm.

4. The interventional microthrombus removal device according to claim 1 or 3, wherein, The ligand includes a polypeptide arginine-glycine-aspartic acid polypeptide that specifically binds to the platelet surface membrane glycoprotein II b / III a receptor, or a glutamic acid-tryptophan-valine-aspartic acid-valine polypeptide that binds to platelet P-selectin, or a cysteine-arginine-glutamic acid-lysine-alanine that specifically binds to fibrin.

5. The interventional microthrombectomy device according to claim 1, wherein, The particle size of the magnetic microbubble is 1 - 10 microns.

6. The interventional microthrombus removal device according to claim 1, wherein, The preparation method of the magnetic microbubble includes the following steps: S1. Disperse the magnetic particles in double-distilled water to form a suspension, and perform ultrasonic emulsification to disperse the magnetic particles to obtain a magnetic particle storage solution; S2. Dissolve the material of the shell layer in an organic solvent, add the microthrombus targeting polypeptide ligand after mixing and dissolving, and stir evenly to obtain a shell layer material solution; S3. Mix the magnetic particle storage solution and the shell layer material solution, and perform primary emulsification under ultrasonic action to obtain a mixed solution; S4. Slowly add the mixed solution prepared in step S3 to a 5% polyvinyl alcohol solution; S5. Add the mixed solution obtained in step S4 to a 2% isopropyl alcohol solution, and stir at room temperature to cure the surface of the microbubbles; S6. Centrifuge the mixed solution obtained in step S5 to obtain microcapsules; S7. Freeze-dry the microcapsules to obtain the magnetic microbubbles for microthrombus clearance.

7. The interventional microthrombus removal device according to claim 6, wherein, In step S2, the material of the shell layer is poly(lactic acid - glycolic acid) copolymer.

8. The interventional microthrombus removal device according to claim 7, wherein, The concentration of the poly(lactic acid - glycolic acid) copolymer in the shell layer material solution is 0.625 - 10mg / mL.

9. The intrusive microthrombus removal device according to claim 7, wherein, The molar ratio of lactic acid to glycolic acid used in the synthesis of the poly(lactic acid - glycolic acid) copolymer is 25:75 or 50:50.

Images