High-stability magnetic steering microbubble capable of enhancing ultrasound contrast and preparation method and application of high-stability magnetic steering microbubble

Abstract

The invention discloses a high-stability magnetic steering microbubble for enhancing ultrasound contrast, the high-stability magnetic steering microbubble is composed of a shell layer and an inner core, the shell layer comprises magnetic particles and a polylactic acid-glycolic acid copolymer, the inner core comprises pure water, the magnetic particles are oleic acid modified Fe3O4 nano particles, and the magnetic particles are magnetic particles and polylactic acid-glycolic acid copolymer. The mass ratio of the magnetic particles to the polylactic acid-glycolic acid copolymer is 1: (100-250). The invention further discloses a preparation method and application of the high-stability magnetic steering microbubble, the internal water phase of the microbubble is set to be water, so that the microbubble can generate a strong echo signal under the action of ultrasonic waves, the contrast ratio between tissues and blood vessels is remarkably improved, ultrasonic imaging is more facilitated, and the resolution ratio is improved; by regulating and controlling the mass ratio of the magnetic particles to the polylactic acid-glycolic acid copolymer (PLGA), the concentration of the emulsifier polyvinyl alcohol and the stirring speed, the microbubbles with small particle sizes are prepared, and the microbubbles have stability.

Description

Technical Field

[0001] The invention belongs to the technical field of medical imaging, and in particular relates to a high-stability magnetically guided microbubble for enhanced ultrasound contrast imaging, and a preparation method and application thereof. Background Art

[0002] Ultrasound imaging is one of the most widely used non-invasive examination methods in clinical practice. It forms images by emitting ultrasound waves and receiving signals reflected by biological tissues, thereby helping doctors diagnose diseases. However, due to the limitations of the acoustic properties of tissues, traditional ultrasound imaging technology is difficult to provide sufficient contrast and resolution in some cases, limiting its application in the detection of complex lesions.

[0003] In order to overcome the above limitations, ultrasound contrast imaging technology came into being. Ultrasound contrast agents are usually composed of gas-filled microbubbles, which can produce strong echo signals under the action of ultrasound, thereby significantly improving the contrast between tissues and blood vessels. However, the core of the microbubbles currently used on the market is generally fluorocarbon gas, which has certain toxicity; and this type of microbubble reaches various tissues through blood circulation, and does not have the ability to focus on specific tissues and conduct long-term imaging.

[0004] In order to enhance nuclear magnetic resonance imaging, the prior art loads magnetic particles on the outer shell of microbubbles, but this causes the particle size of the microbubbles to increase, causing damage to tissues and is not conducive to maintaining the stability of the microbubbles. Summary of the invention

[0005] In order to overcome the deficiencies of the above prior art, the object of the present invention is to provide a high-stability magnetically guided microbubble for enhancing ultrasound contrast imaging, which not only improves the visibility of the microbubble on CT images but also has high stability.

[0006] Another object of the present invention is to provide a method for preparing high-stability magnetically guided microbubbles for enhancing ultrasound contrast imaging.

[0007] Another object of the present invention is to provide an application of high-stability magnetically guided microbubbles for enhancing ultrasound contrast imaging.

[0008] The purpose of the present invention is achieved through the following technical solutions: A high-stability magnetically guided microbubble for enhanced ultrasound contrast imaging, consisting of a shell layer and an inner core, wherein the shell layer comprises magnetic particles and a polylactic acid-glycolic acid copolymer, the inner core is pure water, the magnetic particles are Fe3O4 nanoparticles modified with oleic acid, and the mass ratio of the magnetic particles to the polylactic acid-glycolic acid copolymer is 1:100-250.

[0009] Preferably, the particle size of the magnetically guided microbubbles is 1-5 μm.

[0010] Preferably, the particle size of the Fe3O4 nanoparticles is 5-40 nm.

[0011] Preferably, the polylactic acid-glycolic acid copolymer is polymerized from lactic acid and glycolic acid, and the molar ratio of the lactic acid to the glycolic acid is 2-3:1-2.

[0012] A method for preparing high-stability magnetically guided microbubbles for enhanced ultrasound contrast imaging comprises the following steps: (1) Dissolve the oleic acid-modified ferroferric oxide nanoparticles, lactic acid and glycolic acid respectively, mix the two solutions and disperse them in an organic solvent, and ultrasonicate the mixture in an ice-water bath for 1-15 minutes to obtain a uniformly dispersed magnetic oil phase solution A; (2) In an ice-water bath, pure water is used as the aqueous phase solution B, the aqueous phase solution B is mixed with the magnetic oil phase solution A, an ultrasonic oscillator is used in an ice-water bath to obtain a W / O colostrum, a surfactant C is added to the W / O colostrum, and a W / O / W emulsion is obtained after homogenization; (3) Add the W / O / W emulsion to a 2-5% by mass isopropanol solution, stir at room temperature until the surface of the microbubbles solidifies, wash and centrifuge the obtained product several times, and finally freeze-dry to obtain high-stability magnetically guided microbubbles for enhanced ultrasound contrast imaging.

[0013] Preferably, the organic solvent in step (1) is dichloromethane.

[0014] Preferably, in step (2), the surfactant C is polyvinyl alcohol, and its mass fraction is 2-7wt%.

[0015] Preferably, the volume ratio of the three solutions in step (2) is magnetic oil phase solution A: aqueous phase solution B: surfactant C = 2: (0.3-0.8): 10.

[0016] Preferably, the product obtained in step (3) is washed and centrifuged 2-3 times.

[0017] Preferably, the stirring speed in step (3) is 200-1000 r / min.

[0018] The invention discloses an application of high-stability magnetically guided microbubbles for enhancing ultrasound contrast, which are applied to ultrasound imaging as ultrasound contrast agents, and the concentration of the injected microbubbles is 10-80 mg / mL.

[0019] The present invention has the following advantages and beneficial effects compared with the prior art: (1) The present invention sets the inner aqueous phase of the microbubbles to water. Compared with water-soluble substances as the inner aqueous phase, water can volatilize into a purer cavity through freeze drying, making the microbubbles purer and more stable. The ultrasound contrast microbubbles filled with air can generate a strong echo signal under the action of ultrasound, thereby significantly improving the contrast between tissues and blood vessels, which is more conducive to ultrasound imaging and improves resolution.

[0020] (2) The present invention prepares microbubbles with smaller particle sizes by regulating the mass ratio of magnetic particles to polylactic acid-glycolic acid copolymer (PLGA), the concentration of emulsifier polyvinyl alcohol and the stirring speed. The higher the emulsifier concentration and the faster the stirring speed, the smaller the particle size of the prepared microbubbles and the higher the stability. However, when a certain threshold is reached, the particle size of the microbubbles hardly changes, thereby avoiding waste of resources. The present invention sets the mass fraction of the emulsifier to 2-7wt% and the stirring speed to 200-1000r / min. By setting the mass ratio of magnetic particles to PLGA to 1:100~250, Fe3O4, as a material with a higher atomic number, has a natural X-ray attenuation ability, making it an effective contrast medium, thereby enhancing the visibility of microbubbles on CT images. As the Fe3O4 content increases, the number of particles that can absorb X-rays increases, resulting in a proportional enhancement of the signal on the CT image, which provides the possibility of accurately controlling the contrast of clinical images. However, too much Fe3O4 will cause the contrast enhancement index to show a downward trend. Therefore, the mass ratio of magnetic particles to PLGA is set to 1:100~250 in the present invention.

[0021] (3) The present invention uses oleic acid to modify the ferroferric oxide nanoparticles. This treatment makes the surface of the Fe3O4 particles lipophilic, thereby improving their dispersion ability in organic media and promoting effective bonding with high molecular polymers, so that they are more evenly distributed on the shell surface of the composite material. At the same time, these modified Fe3O4 nanoparticles also have excellent biocompatibility. In addition, the Fe3O4 nanoparticles exhibit superparamagnetic characteristics, that is, they only show magnetism under the action of an external magnetic field, and return to a non-magnetic state after the magnetic field is removed. This characteristic enables them to produce a significant contrast effect in magnetic resonance imaging (MRI), which helps to improve image quality and provide more accurate diagnostic information. In addition, these nanoparticles have good biocompatibility and rarely cause immune responses or toxicity problems.

[0022] (4) The present invention discloses a simple and directional method for preparing magnetic ultrasound contrast microbubbles. The materials used have been approved, ensuring good biosafety. The material is a degradable functional polymer compound that can be made into a variety of forms according to needs, such as thin films, microspheres, nanoparticles and porous scaffolds, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the effect of Fe3O4 / PLGA mass ratio on microbubble particle size.

[0024] Figure 2 This is the effect of emulsifier concentration on microbubble particle size.

[0025] Figure 3 This is the effect of stirring speed on microbubble particle size.

[0026] Figure 4 Schematic diagram of extracorporeal ultrasound imaging.

[0027] Figure 5 CT images of the microbubbles prepared in Examples 1-4 and the grayscale values ​​of the microbubble images.

[0028] Figure 6 The figure shows the effect of different contents of magnetic particles on ultrasound imaging and the ultrasound contrast enhancement index of the corresponding microbubbles.

[0029] Figure 7 The effects of different concentrations of microbubbles on ultrasound imaging and their corresponding microbubble ultrasound contrast enhancement indexes.

[0030] Figure 8 These are H&E stained sections of rabbit organs on day 7.

[0031] Fig. 9 To perform real-time ultrasound imaging of rabbit blood vessels.

[0032] Fig.10 B-mode ultrasound imaging of the thrombus location. DETAILED DESCRIPTION

[0033] The purpose of the present invention is further described in detail below with reference to the accompanying drawings and specific examples. The examples cannot be described one by one here, but the implementation methods of the present invention are not therefore limited to the following examples.

[0034] Example 1 A method for preparing high-stability magnetically guided microbubbles for enhanced ultrasound contrast imaging comprises the following steps: (1) PLGA (polylactic acid-glycolic acid copolymer) was dissolved in dichloromethane, and then oleic acid-modified nano-iron tetroxide particles (20 nm) were added, and then ultrasonicated in an ice-water bath for 10 min to obtain a uniformly dispersed magnetic oil phase solution A (O). Among them, the ratio of lactic acid to glycolic acid used in PLGA was 3:1, the molecular weight was 19000, the PLGA concentration was 50 mg / mL, the magnetic particle concentration was 0.5 mg / mL, and the mass ratio of magnetic particles to PLGA was 1:100.

[0035] (2) Place pure water in an ice water bath to prepare the aqueous phase solution B (W).

[0036] (3) Take 300 μL of solution B and add it to 2 mL of solution A. Then use an ultrasonic oscillator (100w) to stir and shake for 50 seconds to obtain colostrum (W / O).

[0037] (4) Colostrum was added to 10 mL of 4 wt % polyvinyl alcohol solution and then homogenized for 5 min using a homogenizer (700 rpm / min) to obtain a double emulsion (W / O / W).

[0038] (5) Add the emulsion into 10 mL of 2% isopropanol solution and stir at room temperature (700 rpm / min) for 1 hour to solidify the microbubble surface and completely evaporate the organic solvent.

[0039] (6) The mixed solution obtained in step (5) is added with pure water and centrifuged for three times, and then freeze-dried to obtain high-stability magnetically guided microbubbles. The particle size of the prepared microbubbles is 1-3 μm.

[0040] In vivo toxicity test of small animals: In order to determine the toxicity of the material in vivo, high-stability magnetically guided microbubbles with a concentration of 10 mg / mL were prepared with normal saline and injected intravenously at a mass of 1 mL / kg of body weight. The control group was injected with an equal amount of normal saline. The experimental rabbits were raised for 7 days and then killed. The heart, liver, spleen, lung, and kidney tissues were taken for histological analysis. The results of the in vivo toxicity test are shown in Figure 2. Figure 6 shown.

[0041] Example 2 Except for the following technical features, the other technical features of this embodiment are the same as those of Embodiment 1: The mass ratio of magnetic particles to PLGA was set to 1:500.

[0042] Example 3 Except for the following technical features, the other technical features of this embodiment are the same as those of Embodiment 1: The mass ratio of magnetic particles to PLGA was set to 1:250.

[0043] Example 4 Except for the following technical features, the other technical features of this embodiment are the same as those of Embodiment 1: The mass ratio of magnetic particles to PLGA was set to 1:50.

[0044] Example 5 Except for the following technical features, the other technical features of this embodiment are the same as those of Embodiment 1: The mass ratio of magnetic particles to PLGA was set to 1:25.

[0045] Example 6 Except for the following technical features, the other technical features of this embodiment are the same as those of Embodiment 1: The mass fraction of polyvinyl alcohol is 1 wt%.

[0046] Example 7 Except for the following technical features, the other technical features of this embodiment are the same as those of Embodiment 1: The mass fraction of polyvinyl alcohol is 2 wt%.

[0047] Example 8 Except for the following technical features, the other technical features of this embodiment are the same as those of Embodiment 1: The mass fraction of polyvinyl alcohol is 4 wt%.

[0048] Example 9 Except for the following technical features, the other technical features of this embodiment are the same as those of Embodiment 1: The mass fraction of polyvinyl alcohol is 7 wt%.

[0049] Example 10 Except for the following technical features, the other technical features of this embodiment are the same as those of Embodiment 1: The mass fraction of polyvinyl alcohol is 10 wt%.

[0050] Embodiment 11 Except for the following technical features, the other technical features of this embodiment are the same as those of Embodiment 1: The stirring speed is 200 r / min.

[0051] Example 12 Except for the following technical features, the other technical features of this embodiment are the same as those of Embodiment 1: The stirring speed is 300 r / min.

[0052] Embodiment 13 Except for the following technical features, the other technical features of this embodiment are the same as those of Embodiment 1: The stirring speed is 500 r / min.

[0053] Embodiment 14 Except for the following technical features, the other technical features of this embodiment are the same as those of Embodiment 1: The stirring speed is 1000 r / min.

[0054] Embodiment 15 In vivo ultrasound imaging of small animals: Adult New Zealand white rabbits were used as animal models to demonstrate the steps of in vivo ultrasound angiography. Before the experiment, 3% sodium pentobarbital was used for intravenous general anesthesia. After the anesthesia took effect, the hair of the rabbit's hind limbs was cleaned with depilatory cream, and then a high-resolution ultrasound imaging system was used to perform ultrasound imaging of the rabbit's hind limb veins at baseline, and the relevant images and data were recorded for comparison with the data after subsequent treatment. Afterwards, samples containing 0, 10, 20, 30, 60, and 80 mg / mL microbubbles (1 mL / kg) were slowly injected through the rabbit's ear vein. After the injection of the microbubbles, continuous real-time ultrasound imaging was performed immediately to observe the distribution and movement of the microbubbles in the blood vessels, as well as their enhancement effect on the intravascular signals. All operations must strictly comply with animal ethics and welfare guidelines to ensure the health and safety of the animals. The results of in vivo ultrasound imaging are as follows: Figure 7 shown.

[0055] Figure 1-3 The effect of Fe3O4 / PLGA mass ratio, emulsifier concentration and stirring speed on microbubble particle size is shown in Figure 2. Figure 1-3 It can be seen that under the condition of reasonable utilization of resources, the optimal preparation conditions for preparing microbubbles with smaller particle size are as follows: the mass ratio of Fe3O4 to PLGA is between 1 / 250-100, the emulsifier concentration is between 2 wt%-7 wt%, and the reasonable stirring speed range is 500-700r / min.

[0056] Figure 4 Schematic diagram of extracorporeal ultrasound imaging. Figure 4 It can be seen that ultrasonic imaging forms an image by emitting ultrasonic waves and receiving signals reflected by biological tissues. In the present invention, the inner aqueous phase of the microbubbles is set to water, and the aqueous phase in the inner core is volatilized into a cavity by freeze-drying. The ultrasound contrast microbubbles filled with air can generate strong echo signals under the action of ultrasonic waves, thereby significantly improving the contrast between tissues and blood vessels.

[0057] Figure 5 The CT images of the microbubbles prepared in Examples 1-4 and the grayscale values ​​of the microbubble images, wherein (a) air; (b) Fe3O4 / PLGA mass ratios are 0 / 500 (Example 2), 1 / 250 (Example 3), 1 / 100 (Example 1), and 1 / 50 (Example 4), respectively. Figure 5 It can be seen that the CT imaging of microbubbles is affected by the content of magnetic particles. As a material with a high atomic number, Fe3O4's natural X-ray attenuation ability makes it an effective contrast medium, which enhances the visibility of microbubbles on CT images. As the Fe3O4 content increases, the number of particles that can absorb X-rays increases, resulting in a proportional increase in the signal on the CT image, providing the possibility of precise control of contrast for clinical images.

[0058] Figure 6 The effect of different contents of magnetic particles on ultrasound imaging and the corresponding ultrasound contrast enhancement index of microbubbles are shown in Figure 1. Figure 6 It can be seen that with the increase of Fe3O4 content, its contrast enhancement index shows a downward trend. This may be because Fe3O4 nanoparticles are easy to agglomerate at high concentrations, which may disrupt the uniform distribution within the microbubbles and affect their ultrasonic reflection ability.

[0059] Figure 7 The effect of different concentrations of microbubbles on ultrasound imaging and the corresponding microbubble ultrasound contrast enhancement index diagram are shown in Figure 1. Figure 7 It can be seen that with the increase of microbubble concentration, the contrast enhancement index shows an upward trend. The increase in the number of microbubbles increases the reflection interface of ultrasound. Each microbubble acts as an independent reflection point and can scatter ultrasound. Multiple such scattering points mean that more signals are reflected back to the ultrasound transducer, resulting in enhanced imaging contrast.

[0060] Figure 8 The H&E stained sections of rabbit organs on the 7th day (AE: heart, liver, spleen, lung, and kidney in the control group; ae: heart, liver, spleen, lung, and kidney in the experimental group) are shown in Figure 1. Figure 8 It can be seen that no obvious changes occurred in various organs, and no phenomena such as cell swelling, inflammatory response, cell death (apoptosis or necrosis) and fibrosis occurred. Therefore, the microbubbles did not show obvious tissue toxicity and had good biocompatibility.

[0061] Fig. 9 To perform real-time ultrasound imaging of rabbit blood vessels, the images in B mode are (a) before microbubble injection and (b) after microbubble injection. Before contrast agent injection, the blood vessel lumen is relatively dark with almost no echo signal; after microbubble samples are injected intravenously into the rabbit, the blood vessel signal becomes brighter with the microbubbles brought in by the blood flow. This dynamic process fully demonstrates that the microbubble has a good effect of enhancing ultrasound imaging in vivo.

[0062] Fig.10 In order to use B-mode ultrasound to develop the location of thrombus, by comparing the B-mode echo intensity of the obstruction segment in the blank control group and the microbubble group, it can be seen that the microbubble group has a higher contrast than the blank group, the blood circulation area is more obviously gray, and the gray value of the obstruction is low; while in the blank group, the gray values ​​of both the circulation area and the obstruction area are low, and it is difficult to distinguish the obstruction area. This result shows that the microbubble can enhance the ultrasound development effect in vivo, and can perform angiography of local tissues according to personal needs, and has a wide range of application prospects.

[0063] The above specific implementation modes are preferred embodiments of the present invention and cannot be used to limit the present invention. Any other changes or other equivalent replacement methods that do not deviate from the technical solution of the present invention are included in the protection scope of the present invention.

Claims

1. A high-stability magnetically guided microbubble for enhanced ultrasound contrast imaging, characterized in that: It consists of a shell layer and an inner core, wherein the shell layer includes magnetic particles and polylactic acid-glycolic acid copolymer, the inner core is pure water, the magnetic particles are Fe3O4 nanoparticles modified by oleic acid, and the mass ratio of the magnetic particles to the polylactic acid-glycolic acid copolymer is 1:100-250.

2. The high-stability magnetically guided microbubble for enhanced ultrasound contrast imaging according to claim 1, characterized in that: The particle size of the magnetically guided microbubbles is 1-5 μm.

3. The high-stability magnetically guided microbubble for enhanced ultrasound contrast imaging according to claim 1, characterized in that: The particle size of the Fe3O4 nanoparticles is 5-40nm.

4. The high-stability magnetically guided microbubble for enhanced ultrasound contrast imaging according to claim 1, characterized in that: The polylactic acid-glycolic acid copolymer is polymerized by lactic acid and glycolic acid, and the molar ratio of the lactic acid to the glycolic acid is 2-3:1-2.

5. The method for preparing high-stability magnetically guided microbubbles for enhanced ultrasound contrast imaging according to any one of claims 1 to 4, characterized in that: The following steps are involved: (1) Dissolve the oleic acid-modified ferroferric oxide nanoparticles, lactic acid and glycolic acid respectively, mix the two solutions and disperse them in an organic solvent, and ultrasonicate the mixture in an ice-water bath for 1-15 minutes to obtain a uniformly dispersed magnetic oil phase solution A; (2) In an ice-water bath, pure water is used as the aqueous phase solution B, the aqueous phase solution B is mixed with the magnetic oil phase solution A, an ultrasonic oscillator is used in an ice-water bath to obtain a W / O colostrum, a surfactant C is added to the W / O colostrum, and a W / O / W emulsion is obtained after homogenization; (3) Add the W / O / W emulsion to a 2-5% by mass isopropanol solution, stir at room temperature until the surface of the microbubbles solidifies, wash and centrifuge the obtained product several times, and finally freeze-dry to obtain high-stability magnetically guided microbubbles for enhanced ultrasound contrast imaging.

6. The method for preparing high-stability magnetically guided microbubbles for enhanced ultrasound contrast according to claim 5, characterized in that: In step (2), the surfactant C is polyvinyl alcohol, and its mass fraction is 2-7wt%.

7. The method for preparing high-stability magnetically guided microbubbles for enhanced ultrasound contrast according to claim 5, characterized in that: The stirring speed in step (3) is 200-1000 r / min.

8. The use of high-stability magnetically guided microbubbles for enhanced ultrasound contrast imaging according to any one of claims 1 to 4, characterized in that: Applied to ultrasound imaging as ultrasound contrast agent, the concentration of injected microbubbles is 10-80 mg / mL.

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