WO2020199788A1 - 一种血小板膜自组装纳米气泡及其制备方法和应用 - Google Patents

一种血小板膜自组装纳米气泡及其制备方法和应用 Download PDF

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WO2020199788A1
WO2020199788A1 PCT/CN2020/076443 CN2020076443W WO2020199788A1 WO 2020199788 A1 WO2020199788 A1 WO 2020199788A1 CN 2020076443 W CN2020076443 W CN 2020076443W WO 2020199788 A1 WO2020199788 A1 WO 2020199788A1
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platelet membrane
gas
self
nanobubbles
platelet
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French (fr)
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杨芳
李明熹
顾宁
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东南大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
    • A61K49/221—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by the targeting agent or modifying agent linked to the acoustically-active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
    • A61K49/222—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by a special physical form, e.g. emulsions, liposomes
    • A61K49/223—Microbubbles, hollow microspheres, free gas bubbles, gas microspheres

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  • the invention belongs to the technical field of biomedicine, and specifically relates to a platelet membrane self-assembled nano bubble and a preparation method and application thereof.
  • Cardiovascular and cerebrovascular diseases are still the main cause of death in the world. On the whole, the prevalence and mortality of cardiovascular diseases in China are still on the rise. With the acceleration of social aging and urbanization, unhealthy lifestyles are prevalent in my country. Residents' cardiovascular disease (CVD) risk factors are generally exposed, showing a trend of rapid growth and individual aggregation among younger age and low-income groups.
  • CVD cardiovascular disease
  • the estimated number of cardiovascular diseases is 290 million, of which 13 million are stroke, 11 million are coronary heart disease, 5 million are cor pulmonale, 4.5 million are heart failure, 2.5 million are rheumatic heart disease, 2 million are congenital heart disease, and hypertension 270 million. Cardiovascular disease deaths account for more than 40% of residents’ disease deaths.
  • CT computerized tomography
  • MRI magnetic resonance imaging
  • MRI digital subtraction angiography
  • transcranial Doppler CT angiography
  • MRI magnetic resonance imaging
  • MRI magnetic resonance imaging
  • MRI digital subtraction angiography
  • transcranial Doppler CT angiography
  • MRI magnetic resonance imaging
  • the advantage of ultrasound imaging is that it is simple and fast to operate, and it can observe the dynamic development of vascular injury lesions in real time, which is economical and affordable, and reduces the economic pressure on patients.
  • the disadvantage is that the resolution of ultrasound imaging is low, and a contrast agent is needed to enhance the contrast of ultrasound imaging and improve the resolution.
  • Traditional ultrasound contrast agent probes are microbubbles wrapped in lipids, albumin, and polymers. Because the membrane material of the microbubbles uses foreign substances such as lipids or polymers, the problem of biological safety is increased. The instability of the microbubbles results in a shorter cycle time of the contrast agent. At the same time, the larger size of the microbubbles also limits its application and development in molecular imaging.
  • Platelet is one of the important components in human blood. It is a small biologically active cytoplasm that is lysed and shed from the mature megakaryocyte cytoplasm of bone marrow. It is used in the physiological process of identifying and repairing blood vessel damage and maintaining the integrity of blood vessel wall. Play an important role, and platelets are also one of the culprits of cardiovascular and cerebrovascular diseases such as thrombosis and atherosclerosis.
  • the present invention provides a platelet membrane self-assembled nanobubble and a preparation method thereof.
  • the present invention can obtain nanobubbles with nanometer size and retain the natural properties of the platelet membrane, and has a high biological phase. Capacitive and targeted for vascular injury, it can be used for ultrasound imaging diagnosis of vascular injury sites, and solve the problem of difficult targeted ultrasound imaging diagnosis of small and micro lesions in the early stage of cardiovascular and cerebrovascular diseases.
  • the platelet membrane nanobubbles prepared by the invention have natural thrombus targeting ability because the membrane shell completely retains the protein and lipid components of the platelet membrane, and can quickly target the lesion in the detection and diagnosis of vascular injury.
  • the present invention also provides the application of platelet membrane self-assembled nanobubbles.
  • the platelet membrane nanobubbles prepared by the present invention can be used to prepare cell membrane bionic nanobubble contrast agents that provide ultrasound image enhancement for vascular damage, and can protect the damaged blood vessels at the same time , To prevent the further development of thrombus.
  • a method for preparing platelet membrane self-assembled nanobubbles according to the present invention includes the following steps:
  • the platelets are subjected to repeated freezing and thawing, and then washed to obtain a purified platelet membrane vesicle suspension; homogenization is carried out by ultrasonic action in a water bath;
  • the platelet membrane fragments are self-assembled and reorganized at the gas-liquid interface to construct the platelet membrane-coated nanobubbles .
  • the self-assembly recombination using the gas-liquid interface includes ultrasonic cavitation of the homogenized platelet membrane vesicle suspension, while gas is introduced to form nano gas nuclei under the action of ultrasonic cavitation, and then applied
  • the milder ultrasonic cavitation effect promotes the self-assembly and reorganization of platelet membrane fragments on the surface of the air core; or by repeatedly compressing the gas into the homogenized platelet membrane vesicle suspension and then returning to atmospheric pressure, the platelet membrane fragments are repeatedly squeezed The surface of free nanobubbles formed in the self-assembly.
  • step (1) specifically includes: (a) centrifugal separation of fresh platelets, and washing to remove plasma; (b) resuspending the pure platelet components obtained in step (a) and then freezing; (c) performing step (b) The frozen platelets are thawed at room temperature, centrifuged at a high speed, and then resuspended and washed to separate the platelet cell membrane vesicles from the organelles; (d) repeat step (c) to obtain platelet membrane vesicles; (e) perform step (d) The obtained platelet membrane suspension was crushed and homogenized in a water bath ultrasound.
  • the self-assembly recombination using the gas-liquid interface described in step (2) includes ultrasonic cavitation of the homogenized platelet membrane vesicle suspension, and at the same time gas is introduced to form nano gas nuclei under the action of ultrasonic cavitation, and then Gentle ultrasonic cavitation is applied to promote the self-assembly and reorganization of platelet membrane fragments on the surface of the air core, specifically: (a) The homogenized platelet membrane vesicle suspension is broken by ultrasound at a higher power, in this process (B) Reduce the power of ultrasonic cavitation, so that the platelet membrane fragments broken by ultrasonic cavitation will be adsorbed on the nano gas-liquid interface formed by cavitation, and recombined to form platelet membrane-coated nano Bubble suspension; (c) centrifugal separation of the prepared platelet nanobubble suspension to obtain platelet membrane nanobubbles.
  • step (2) by repeatedly compressing the gas into the homogenized platelet membrane vesicle suspension and then returning to atmospheric pressure, the platelet membrane fragments are self-assembled on the surface of the free nanobubbles formed during the repeated extrusion process, specifically: (a) The platelet membrane vesicle suspension is contained in a container such as a vial, and the upper side of the container, the communication tube and the variable volume squeezing device such as a syringe are filled with gas to form a closed system; (b) the compression device is forced The plunger presses part of the gas into the liquid through the connecting tube, increasing the pressure of the closed system to one to five times the original; (c) Remove the external force, the pressure in the closed system returns to normal pressure, free bubbles are formed, and the platelet membrane is in the air core Self-assembly of the air-liquid interface; (d) Repeated pressure to return to normal pressure, so that the platelet membrane is fully assembled and fused to form a platelet membrane-
  • the pressure of the system is changed by adjusting the volume of the squeezing device to generate a pressure difference, and then return to normal pressure to generate free nanobubbles.
  • the platelet membrane fragments are adsorbed on the gas-liquid interface of the nanobubbles and fully fused and assembled.
  • the container used is preferably a vial containing a platelet membrane nanobubble suspension containing a specific gas (such as sulfur hexafluoride) in a closed state, and also serves as a nanobubble storage device.
  • the variable volume extruding device can change the pressure of the system by adjusting the volume of the extruding device to generate a pressure difference.
  • the variable volume device can drive part of the liquid and gas through the connecting pipe by changing the system pressure, and the shear generated The effect makes the gas and liquid phases mix thoroughly.
  • step (a) the crushing is performed at a power of 400-1000W for 10-40s; in step (b), the ultrasonic cavitation power is reduced to crushing at a power of 80-200W for 60- In 90s, the platelet membrane fragments broken by ultrasonic cavitation are adsorbed on the gas-liquid interface of nanobubbles formed under cavitation, and recombined to form a nanobubble suspension covered by platelet membranes.
  • crushing under higher power is 30s under 500W power; reducing ultrasonic cavitation power is 90s under 100W power
  • the repeated pressurization-return to normal pressure process is repeated pressurization 50-200 times of pressurization-return to normal pressure, and the pressure of the system after each pressurization is 0.1-0.5 MPa.
  • each pressurization makes the pressure of the system 0.3 MPa, and pressurization is to increase the pressure of the closed system to three times the original.
  • the air pressure in the closed system before compression defaults to one atmosphere, that is, 0.1 MPa.
  • the gas is one or two of air, oxygen, nitrogen, hydrogen, nitric oxide, helium, and sulfur hexafluoride. Most preferably, sulfur hexafluoride is used.
  • the platelet membrane nanobubbles are prepared by the method for preparing platelet membrane self-assembled nanobubbles of the present invention.
  • the platelet membrane nanobubbles prepared by the invention have a smaller particle size, ranging from 100 to 250 nm.
  • platelets participate in various physiological and pathological processes of the human body, and play an important role in hemostasis, thrombosis, atherosclerosis and other diseases, and use biological autologous platelet membrane to prepare nano-sized bubble imaging It has high biocompatibility and safety, and can quickly target the lesion site to cause adhesion and aggregation in the early stage of vascular disease, improve the detection sensitivity of ultrasound images, realize early real-time dynamic diagnosis of vascular damage, and monitor blood vessels
  • the occurrence and development of injury diseases provide a basis for the early diagnosis and intervention treatment of clinical vascular injury diseases.
  • the present invention has the following advantages:
  • the preparation method of platelet membrane nanobubbles of the present invention is simple, can be batched, and has good repeatability;
  • the platelet membrane nanobubbles preparation material of the present invention is derived from the organism itself, has good biological safety, can escape the screening of the immune system, and prolong the circulation time of nanobubbles in the body;
  • the platelet membrane nanobubbles prepared by the present invention have a size of 100-250 nm, which can pass through a variety of biological barriers to achieve contrast enhancement of extravascular tissues;
  • the platelet membrane nanobubbles prepared by the present invention have a natural thrombus targeting property because the membrane shell completely retains the protein and lipid components of the platelet membrane, and can quickly target the lesion in the detection and diagnosis of vascular injury;
  • the gas contained in the platelet membrane nanobubbles prepared by the present invention is sulfur hexafluoride, helium and other gases, which is safe for clinical use, and the membrane shell elasticity can generate echo signals in response to ultrasound energy, which improves the spatial resolution of ultrasound images To achieve accurate diagnosis of the occurrence and development process of vascular injury through ultrasound image enhancement, and to provide a basis for the early diagnosis and treatment of clinical vascular injury diseases.
  • Figure 1 is a transmission electron microscopic structure characterization diagram of platelet membrane nano-sulfur hexafluoride bubbles prepared by ultrasonic cavitation method in Example 1 of the present invention
  • Example 2 is a structural characterization diagram of the platelet membrane helium nanobubbles prepared by the gas-liquid mixing repeated extrusion method in Example 2 of the present invention
  • Fig. 3 is an ultrasonic imaging experiment result of platelet membrane nanobubbles in Example 1 of the present invention, which characterizes its ultrasonic imaging function.
  • the fresh platelets with a concentration of 1 ⁇ 10 9 /ml were separated by centrifugation at 500 g for 10 min, and resuspended in normal saline to remove the plasma. After that, the platelets were frozen in a refrigerator at -80 °C. The frozen platelets were thawed at room temperature and used at 4000 g Centrifuge for 5 min to separate, and resuspend and wash three times with physiological saline solution to separate the platelet cell membrane vesicles from the organelles to obtain a purified platelet membrane vesicle suspension.
  • the obtained platelet vesicle suspension was homogenized by 100W, 42KHz water bath ultrasound for 5 minutes, it was broken by ultrasonic cavitation at 500W power for 30s, and sulfur hexafluoride gas was introduced in the process; the ultrasonic cavitation was reduced Power to 100W for 90s to make the platelet membrane fragments broken by ultrasonic cavitation adsorb to the nano gas-liquid interface formed by the cavitation, and recombine to form a platelet membrane-coated nanobubble suspension; the prepared The platelet nanobubble suspension was centrifuged at 600g for 5 minutes to obtain a platelet nanobubble suspension with a smaller particle size.
  • a transmission electron microscope was used to characterize the microstructure of the prepared platelet membrane nanobubbles. As shown in Figure 1, the average particle size of the nanobubbles is 100 ⁇ 50nm, which has a good bubble structure.
  • the method for extracting platelet membrane in this embodiment is the same as that in embodiment 1.
  • the device for preparing nanobubbles the fixed volume of the vial is 3mL; the volume of the syringe of the variable-volume squeezing device can be changed in the range of 0-5mL; the volume of the connecting tube is 0.5mL, one end is connected to the variable volume device, and the other end is inserted into the generating container cilin
  • the water-purified platelet membrane vesicle suspension contained in the bottle is 3mL; the volume of the syringe of the variable-volume squeezing device can be changed in the range of 0-5mL; the volume of the connecting tube is 0.5mL, one end is connected to the variable volume device, and the other end is inserted into the generating container cilin
  • the water-purified platelet membrane vesicle suspension contained in the bottle is the fixed volume of the vial is 3mL; the volume of the syringe of the variable-volume
  • part of the platelet membrane vesicle suspension and SF 6 enter the variable-volume extrusion device through the connecting tube, and the shearing action further makes the SF 6 and the platelet membrane vesicle suspension fully mixed; repeat the addition
  • a transmission electron microscope was used to characterize the microstructure of the prepared platelet membrane nanobubbles. As shown in Fig. 2, the average particle size of the platelet membrane nanobubbles is 250 ⁇ 50nm, which has a good bubble structure. The assembled structure of the platelet membrane on the surface of the nanobubbles can be clearly observed.
  • the platelet membrane extraction method in this example is the same as that of Example 1; the preparation method of platelet membrane-coated nanobubbles is the same as that of Example 2, except that the sulfur hexafluoride gas used in Example 1 is replaced with helium.
  • a transmission electron microscope was used to characterize the microstructure of the prepared platelet membrane nanobubbles, which had a good bubble structure, and the assembled structure of the platelet membrane on the surface of the nanobubbles could be clearly observed.
  • Example 4 is the same as Example 1, except that: after homogenization, ultrasonic cavitation is used for crushing at 1000W power for 10s, and nitrogen is introduced during this process; the ultrasonic cavitation power is reduced to 200W for 60s.
  • Example 4 is the same as Example 1, except that: after homogenization, ultrasonic cavitation is used for 40 s at 400 W, and sulfur hexafluoride is introduced during this process; the ultrasonic cavitation power is reduced to 80 W for 90 s.
  • Example 6 is the same as Example 2, but the difference lies in that the process of pressurizing to 0.5Mpa and returning to normal pressure is repeated 50 times.
  • Example 7 is the same as Example 2, except that: the process of increasing the pressure to 0.1Mpa and returning to normal pressure is repeated 200 times.
  • mice Nine stroke model mice were selected as the observation objects of 18MHz ultrasound imaging, and 3 mice injected with 10 ⁇ l/g PBS were randomly selected as the blank group, and 3 mice injected with 10 ⁇ l/g frozen homogenized
  • 3 mice with platelet membrane vesicle suspensions were injected with platelet membrane nanobubbles prepared by the ultrasonic cavitation method provided in Example 1 of the present invention.
  • Contrast-enhanced ultrasound was used as the experimental group. Collected before injection, 0, 5, 10, 15, 20, 25, 30, 40, 50, 60 minutes, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, after injection. Ultrasound contrast signal of mouse head at 24, 48, 72, and 168 hours. The results are shown in Figure 3.
  • the contrast-enhanced ultrasound signal of the head of the mice in the blank group did not change significantly during the acquisition period, within the range of 800-1000a.u.; in the control group, there was no gas in the platelet membrane vesicles During the signal collection period, the enhanced ultrasound signal of the mouse head was similar to that of the blank group, and the signal intensity was in the range of 800-1000a.u.; while the experimental group was injected with platelet membrane nanobubbles.
  • the increased signal of contrast-enhanced ultrasound was observed to increase at 40 minutes, and reached a peak at 24 hours, at about 1800a.u., indicating that platelet membrane nanobubbles can be targeted to the lesions of cerebral stroke in mice to achieve enhanced ultrasound imaging signal ;
  • mice Nine stroke model mice were selected as the observation objects of the small animal in vivo near-infrared fluorescence imaging system, and three mice injected with 10 ⁇ l/g PBS were randomly selected as the blank group, and three mice were randomly selected to be injected with 10 ⁇ l/g
  • the frozen homogenized platelet membrane vesicle suspension of the mouse head with near-infrared fluorescence signals was used as a control group, and 3 mice were randomly selected and injected with 10 ⁇ l/g platelet membrane nanobubbles prepared by the ultrasonic cavitation method provided in Example 1 of the present invention
  • the near-infrared fluorescence signal of mouse head was used as the experimental group.
  • the near-infrared fluorescence signals of the mouse head were collected before injection and 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 24 hours after injection.
  • the intensity of the ultrasound near-infrared fluorescence signal on the head of the mice in the blank group did not change significantly during the collection period, within the range of 5 ⁇ 10 6 -7.5 ⁇ 10 6 (p/s/cm 2 /sr); in the control group, Because the size of platelet membrane vesicles is 1-2 ⁇ m, it is not easy to accumulate in the focal site of stroke.
  • the intensity of the near-infrared fluorescence signal on the head of the mouse is similar to that of the blank group without significant changes, and the signal intensity is 5 ⁇ 10 6- Within the range of 10 ⁇ 10 6 (p/s/cm 2 /sr); in the experimental group, due to the injection of platelet membrane nanobubbles, the intensity of the near-infrared fluorescence signal of the mouse head was observed to rise rapidly at 0.5 h, and the fluorescence at 12 h The intensity is high, ranging from 15 ⁇ 10 6 -25 ⁇ 10 6 (p/s/cm 2 /sr), indicating that platelet membrane nanobubbles can quickly target the vascular injury lesions in the head of mice to achieve accumulation and near-infrared fluorescence The signal strength is enhanced. In addition, the results of mice injected with helium nanobubbles on platelet membranes prepared by gas-liquid mixing and repeated extrusion were similar to those of Test Examples 1 and 2.

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Abstract

一种血小板膜自组装纳米气泡及其制备方法和应用，制备过程包括如下步骤：(1)将血小板经过反复冻融，经过洗涤得到纯化的血小板膜囊泡悬液；并通过水浴超声作用进行匀质化；(2)将匀质化的血小板膜囊泡悬液经过超声空化破碎或气液混合反复挤压后，实现血小板膜碎片在气液界面自组装重组，构建形成血小板膜包覆的纳米气泡。血小板膜纳米气泡的制备方法简单，具有纳米尺寸、保留血小板膜天然性质的纳米气泡，具有很高的生物相容性以及血管损伤靶向性，可用于血管损伤部位的超声影像诊断，解决心脑血管疾病早期小微病灶靶向超声影像诊断困难的问题。

Description

一种血小板膜自组装纳米气泡及其制备方法和应用 技术领域
本发明属于生物医药技术领域，具体涉及一种血小板膜自组装纳米气泡及其制备方法和应用。
背景技术
心脑血管疾病仍然是全世界死亡的主要原因，总体上看，中国心血管病患病率及死亡率仍处于上升阶段，随着社会老龄化和城市化进程加快，居民不健康生活方式流行，我国居民心血管病(CVD)危险因素普遍暴露，呈现在低龄化、低收入群体中快速增长及个体聚集趋势。推算心血管病现患人数2.9亿，其中脑卒中1300万，冠心病1100万，肺原性心脏病500万，心力衰竭450万，风湿性心脏病250万，先天性心脏病200万，高血压2.7亿。心血管病死亡占居民疾病死亡构成40％以上，在心脑血管发生病变损伤时，往往由于在发病早期对病灶部位不能进行准确的定位，而导致病情进一步发展。目前临床对血管疾病的诊断方法主要是电子计算机断层扫描成像(CT)、磁共振成像(MRI)，以及能够早期发现血管狭窄的数字减影血管造影，经颅多普勒，CT血管造影以及核磁共振血管造影等。
超声成像的优势在于其操作简单快速，并且可实时观察血管损伤病灶的动态发展，经济实惠，减轻了患者的经济压力。其不足之处在于超声成像分辨率较低，需要造影剂增强超声成像的对比度，提高分辨率。传统的超声造影剂探针是以脂质、白蛋白以及聚合物包裹的微气泡，由于微泡的膜材使用脂质或者聚合物等外来物质，增大了其生物安全性方面的问题，同时微泡的不稳定性导致造影剂在体循环时间较短，同时由于微泡的尺寸较大也限制了其在分子影像方面的应用与发展。
血小板是人类血液中的重要组成成分之一，是从骨髓成熟的巨核细胞胞质裂解脱落下来的具有生物活性的小块胞质，在识别、修复血管损伤、维护血管壁完整性等生理过程中发挥重要作用，同时血小板也是导致血栓形成，动脉粥样硬化等心脑血管疾病发生的元凶之一。
发明内容
发明目的：针对现有技术存在的问题，本发明提供一种血小板膜自组装纳米气泡及其制备方法，本发明能够获得具有纳米尺寸、保留血小板膜天然性质的纳米气泡，具有很高的生物相容性以及血管损伤靶向性，可用于血管损伤部位的超声影像诊断，解决心脑血管疾病早期小微病灶靶向超声影像诊断困难的问题。
本发明制备得到的血小板膜纳米气泡由于其膜壳完整的保留了血小板膜的蛋白和脂质成分，具有天然的血栓靶向性，在血管损伤的检测诊断中可以快速靶向病灶。
本发明还提供血小板膜自组装纳米气泡的应用，本发明制备得到的血小板膜纳米气泡可以用于制备为血管损伤提供超声影像增强的细胞膜仿生纳米气泡造影剂，同时可以对损伤的血管产生保护作用，阻止血栓进一步发展。
技术方案：为了实现上述目的，如本发明的所述的一种血小板膜自组装纳米气泡的制备方法，包括如下步骤：
(1)将血小板经过反复冻融，经过洗涤得到纯化的血小板膜囊泡悬液；并水浴超声作用进行匀质化；
(2)将匀质化的血小板膜囊泡悬液经过超声空化破碎或气液混合反复挤压后，再实现血小板膜碎片在气液界面自组装重组，构建形成血小板膜包覆的纳米气泡。
其中，所述利用气液界面自组装重组包括通将匀质化的血小板膜囊泡悬液进行超声空化，同时通入气体，在超声空化的作用下形成纳米气核，然后再施以较为温和的超声空化作用，促进血小板膜碎片在气核表面自组装重组；或者通过反复压缩气体至匀质化血小板膜囊泡悬液中再恢复到大气压，使血小板膜碎片在反复挤压过程中形成的自由纳米气泡表面自组装。
其中，步骤(1)具体为：(a)将新鲜血小板离心分离，并清洗去除血浆；(b)将步骤(a)所得纯血小板成分重悬后进行冷冻；(c)将步骤(b)中冷冻的血小板在室温下解冻，并高速离心分离后重悬洗涤，使血小板细胞膜囊泡与细胞器分离；(d)重复步骤(c)，得到血小板膜囊泡；(e)将步骤(d)中获得的血小板膜悬液置于水浴超声中破碎匀质化。
其中，步骤(2)所述利用气液界面自组装重组包括将匀质化血小板膜囊泡悬液进行超声空化，同时通入气体，在超声空化的作用下形成纳米气核，然后再施以温和的超声空化作用，促进血小板膜碎片在气核表面自组装重组，具体为：(a)将匀质化血小板膜囊泡悬液通过超声在较高功率下进行破碎，在此过程中通入特定气体；(b)降低超声空化功率，使被超声空化作用破碎的血小板膜碎片吸附在空化作用下形成的纳米气核气液界面，并重组形成血小板膜包覆的纳米气泡悬浊液；(c)将制备得到的血小板纳米气泡悬浊液进行离心分离，得到血小板膜纳米气泡。
其中，步骤(2)所述通过反复压缩气体至匀质化血小板膜囊泡悬液中再恢复到大气压，使血小板膜碎片在反复挤压过程中形成的自由纳米气泡表面自组 装，具体为：(a)将血小板膜囊泡悬液收容于容器如西林瓶，容器上侧、连通管和体积可变挤压装置如注射器中填充气体，构成密闭系统；(b)施力压缩挤压装置的柱塞，将部分气体通过连通管压入液体中，增加密闭系统压力至原来的一到五倍；(c)除去外力，密闭系统中的压力恢复常压，自由气泡形成，血小板膜在气核的气液界面自组装；(d)重复加压恢复成常压过程，使血小板膜充分组装融合，形成血小板膜包覆的纳米气泡悬浊液；(e)将制备得到的血小板纳米气泡悬浊液进行离心分离，得到血小板纳米气泡。通过调节挤压装置的体积改变系统的压力，产生压力差，再恢复成常压，产生自由纳米气泡，在这个过程中使血小板膜碎片吸附在纳米气泡气液界面上，并充分融合组装。
其中，所用容器优选为西林瓶以密闭状态收容生成的内含特定气体(如六氟化硫)的血小板膜纳米气泡的混悬液，兼做纳米气泡储存装置。所述的体积可变挤压装置，通过调节挤压装置的体积改变系统的压力，产生压力差，体积可变装置，通过改变系统压力，可驱动部分液体和气体通过连通管，产生的剪切作用使气液两相充分混合。
作为优选，步骤(a)所述在较高功率下进行破碎为在400-1000W功率下破碎10-40s；步骤(b)所述在降低超声空化功率为在80-200W功率下破碎60-90s，使被超声空化作用破碎的血小板膜碎片吸附在空化作用下形成的纳米气泡的气液界面，重组形成血小板膜包覆的纳米气泡悬浊液。优先，较高功率下进行破碎为在500W功率下破碎30s；降低超声空化功率为在100W功率下破碎90s
进一步地，所述重复加压-恢复成常压过程为重复加压50-200次加压-恢复成常压，每次加压后系统的压力为0.1-0.5MPa。优选，每次加压使系统的压力为0.3MPa，加压为增加密闭系统压力至原来的三倍，压缩之前密闭系统内气压默认为一个大气压即0.1MPa。
作为优选，所述气体为空气、氧气、氮气、氢气、一氧化氮、氦气、六氟化硫中的一种或两种。最优选采用六氟化硫。
本发明所述的血小板膜自组装纳米气泡的制备方法所制备的血小板膜纳米气泡。本发明制备的血小板膜纳米气泡粒径尺寸较小，在100-250nm。
本发明所述的血小板膜自组装纳米气泡的制备方法所制备的血小板膜纳米气泡在制备用于血管损伤部位的超声影像诊断造影剂中的应用。
本发明使用血小板膜作为纳米气泡膜材的优势在于：血小板参与人体多种生理病理过程，在止血，血栓，动脉粥样硬化等疾病中起重要作用，利用生物自体血小板膜制备纳米尺寸的气泡造影剂，具有较高的生物相容性和安全性，并且可以在血管疾病发生早期快速靶向到病灶部位产生粘附和聚集，提高超声影像的检 测灵敏度，实现血管损伤早期实时动态诊断，监测血管损伤疾病的发生与发展过程，为临床血管损伤疾病的早期诊断和干预治疗提供判断依据。
有益效果：与现有技术相比，本发明具有如下优点：
1、本发明血小板膜纳米气泡的制备方法简单、可批量化，重复性良好；
2、本发明的血小板膜纳米气泡制备材料来源于生物自体，具有良好的生物安全性，能够逃逸免疫系统的筛查，延长纳米气泡在体内的循环时间；
3、本发明制备得到的血小板膜纳米气泡尺寸在100-250nm，可通过多种生物屏障，实现血管外组织的超声造影增强；
4、本发明制备得到的血小板膜纳米气泡由于其膜壳完整的保留了血小板膜的蛋白和脂质成分，具有天然的血栓靶向性，在血管损伤的检测诊断中可以快速靶向病灶；
5、本发明制备得到的血小板膜纳米气泡内部所包裹的气体为六氟化硫、氦气等气体，临床使用安全，且膜壳弹性可以响应超声能量产生回波信号，提高超声影像的空间分辨率，实现通过超声影像增强准确诊断血管损伤的发生、发展过程，为临床血管损伤疾病的早期诊断与治疗干预提供判断依据。
附图说明
图1是本发明中实施例1超声空化法制备血小板膜纳米六氟化硫气泡的透射电子显微结构表征图；
图2是本发明中实施例2气液混合反复挤压法制备的血小板膜氦气纳米气泡透射电镜结构表征图；
图3是本发明中实施例1血小板膜纳米气泡的超声成像实验结果，表征其超声显影功能。
具体实施方式
以下结合实施例和附图对本发明作进一步说明。
实施例1
将浓度为1×10 9/毫升的新鲜血小板由500g转速离心10min分离，并用生理盐水重悬清洗去除血浆，之后将血小板放置于-80℃冰箱内冷冻，冷冻的血小板在室温下解冻，并用4000g转速离心5min分离，并用生理盐水溶液重悬清洗三次，使血小板细胞膜囊泡与细胞器分离，得到纯化的血小板膜囊泡悬浊液。将所得血小板囊泡悬浊液通过100W，42KHz的水浴超声作用5min匀质化之后，通过超声空化在500W功率下进行破碎30s，在此过程中通入六氟化硫气体；降低超声空化功率至100W作用90s，使被超声空化作用破碎的血小板膜碎片吸附在空化作用下形成的纳米气核气液界面，并重组形成血小板膜包覆的纳米气泡悬 浊液；将制备得到的血小板纳米气泡悬浊液进行600g离心分离5min，得到较小粒径的血小板纳米气泡悬液。
采用透射电子显微镜表征制备所得血小板膜纳米气泡的微观结构，如图1所示，其平均粒径为100±50nm，具有良好的气泡结构。
实施例2
本实施例中血小板膜提取方法同实施例1。制备纳米气泡的装置，西林瓶的固定体积为3mL；体积可变挤压装置注射器的容积可在0-5mL范围改变；连通管体积为0.5mL，一端连接体积可变装置，一端插入生成容器西林瓶收容的水纯化的血小板膜囊泡悬浊液中。
将2mL的血小板膜囊泡悬液和4.5mL的99.99％的高纯六氟化硫(SF 6)收容于由纳米气泡装置中，构成密闭系统，此时体积可变挤压装置的平衡容积为3mL；施力压缩体积可变装置，将3mL SF 6通过连通管压入水中，此时SF 6总体积为1.5mL，系统压力为0.3MPa；去除外力，使柱筛回复至与大气压平衡位置，密闭系统压力降为常压，此时部分血小板膜囊泡悬液和SF 6通过连通管进入体积可变挤压装置，剪切作用进一步使SF 6和血小板膜囊泡悬液充分混合；重复加压到0.3Mpa和常压过程150次，利用血小板膜在SF 6纳米气泡表面自组装作用，制备获得血小板膜包覆的六氟化硫纳米气泡。
采用透射电子显微镜表征制备所得血小板膜纳米气泡的微观结构，如图2所示，其平均粒径为250±50nm，具有良好的气泡结构，可清晰观察血小板膜在纳米气泡表面的组装结构。
实施例3
本实施例中血小板膜提取方法同实施例1；血小板膜包覆的纳米气泡制备方法同实施例2，仅有的区别在于将实施例1中所使用的六氟化硫气体替换为氦气。采用透射电子显微镜表征制备所得血小板膜纳米气泡的微观结构，具有良好的气泡结构，可清晰观察血小板膜在纳米气泡表面的组装结构。
实施例4
实施例4同实施例1，不同之处在于：匀质化之后，通过超声空化在1000W功率下进行破碎10s，在此过程中通入氮气；降低超声空化功率至200W作用60s。
实施例5
实施例4同实施例1，不同之处在于：匀质化之后，通过超声空化在400W用40s，在此过程中通入六氟化硫；降低超声空化功率至80W作用90s。
实施例6
实施例6同实施例2，不同之处在于：重复加压到0.5Mpa和恢复常压过程 50次。
实施例7
实施例7同实施例2，不同之处在于：重复加压到0.1Mpa和恢复常压过程200次。
试验例1
选取由超声空化法制备的100±50nm的血小板纳米气泡悬液
选取9只脑卒中模型小鼠作为18MHz超声成像观测对象，随机选取3只注射10μl/g PBS的小鼠头部超声造影增强信号作为空白组，随机选取3只注射10μl/g冷冻后的匀质化血小板膜囊泡悬液的小鼠头部超声造影增强信号作为对照组，随机选取3只注射10μl/g本发明实施例1提供的超声空化法制备的血小板膜纳米气泡的小鼠头部超声造影增强信号作为实验组。分别采集注射前，注射后0，5，10，15，20，25，30，40，50，60分钟，2，3，4，5，6，7，8，9，10，11，12，24，48，72，168小时的小鼠头部超声contrast信号。结果如图3所示，空白组中的小鼠头部超声造影增强信号在采集时间段内没有明显变化，在800-1000a.u.范围内；对照组中，由于血小板膜囊泡中没有气体，在信号采集时间段中小鼠头部超声造影增强信号与空白组类似，没有明显变化，信号强度在在800-1000a.u.范围内；而实验组由于注射了血小板膜纳米气泡，小鼠头部超声造影增强信号在40分钟观察到上升趋势，并且在24小时时达到峰值，为1800a.u.左右，说明血小板膜纳米气泡可靶向小鼠头部卒中血管损伤病灶部位实现超声显影信号增强；用匀质化的血小板膜囊泡悬液作为对照组，由于单纯的血小板膜囊泡没有被制备成为血小板纳米气泡，则没有超声影像增强效果。
试验例2
选取由超声空化法制备的100±50nm的血小板纳米气泡悬液。
选取9只脑卒中模型小鼠作为小动物活体近红外荧光成像系统观测对象，随机选取3只注射10μl/g PBS的小鼠头部近红外荧光信号作为空白组，随机选取3只注射10μl/g冷冻后的匀质化血小板膜囊泡悬液的小鼠头部近红外荧光信号作为对照组，随机选取3只注射10μl/g本发明实施例1提供的超声空化法制备的血小板膜纳米气泡的小鼠头部近红外荧光信号作为实验组。分别采集注射前，注射后0.5，1，2，3，4，5，6，7，8，9，10，11，12，24小时的小鼠头部近红外荧光信号。空白组中的小鼠头部超声近红外荧光信号强度在采集时间段内没有明显变化，在5×10 6-7.5×10 6(p/s/cm 2/sr)范围内；对照组中，由于血小板膜囊泡尺寸在1-2μm，不易在脑卒中病灶部位聚集，在信号采集时间段中小鼠头部近红外荧光信号强度与空白组类似，没有明显变化，信号强度在5×10 6-10×10 6 (p/s/cm 2/sr)范围内；而实验组由于注射了血小板膜纳米气泡，小鼠头部近红外荧光信号强度在0.5h观察到快速上升，并在12小时荧光强度均较高，为15×10 6-25×10 6(p/s/cm 2/sr)，说明血小板膜纳米气泡可快速靶向小鼠头部卒中血管损伤病灶部位实现累积与近红外荧光信号强度增强。此外，注射气液混合反复挤压法制备的血小板膜氦气纳米气泡的的小鼠与试验例1和2结果类似。

Claims (10)

  1. 一种血小板膜自组装纳米气泡的制备方法，其特征在在于，包括如下步骤：
    (1)将血小板经过反复冻融，经过洗涤得到纯化的血小板膜囊泡悬液；并通过水浴超声作用进行匀质化；
    (2)将匀质化的血小板膜囊泡悬液经过超声空化破碎或气液混合反复挤压后，再实现血小板膜碎片在气液界面自组装重组，构建形成血小板膜包覆的纳米气泡。
  2. 根据权利要求1所述的血小板膜自组装纳米气泡的制备方法，其特征在在于，步骤(2)所述利用气液界面自组装重组包括将匀质化血小板膜囊泡悬液进行超声空化，同时通入气体，在超声空化的作用下形成纳米气核，然后再施以温和的超声空化作用，促进血小板膜碎片在气核表面自组装重组；或者通过反复压缩气体至匀质化血小板膜囊泡悬液中再恢复到大气压，使血小板膜碎片在反复挤压过程中形成的自由纳米气泡表面自组装。
  3. 根据权利要求1所述的血小板膜自组装纳米气泡的制备方法，其特征在在于，步骤(1)具体为：(a)将新鲜血小板离心分离，并清洗去除血浆；(b)将步骤(a)所得纯血小板成分重悬后进行冷冻；(c)将步骤(b)中冷冻的血小板在室温下解冻，并高速离心分离后重悬洗涤，使血小板细胞膜囊泡与细胞器分离；(d)重复步骤(c)，得到血小板膜囊泡；(e)将步骤(d)中获得的血小板膜悬液置于水浴超声中破碎匀质化。
  4. 根据权利要求2所述的血小板膜自组装纳米气泡的制备方法，其特征在在于，步骤(2)所述利用气液界面自组装重组包括将匀质化血小板膜囊泡悬液进行超声空化，同时通入气体，在超声空化的作用下形成纳米气核，然后再施以温和的超声空化作用，促进血小板膜碎片在气核表面自组装重组具体为：(a)将匀质化血小板膜囊泡悬液通过超声在较高功率下进行破碎，在此过程中通入气体；(b)降低超声空化功率，使被超声空化作用破碎的血小板膜碎片吸附在空化作用下形成的纳米气核气液界面，并重组形成血小板膜包覆的纳米气泡悬浊液；(c)将制备得到的血小板纳米气泡悬浊液进行离心分离，得到血小板膜纳米气泡。
  5. 根据权利要求2所述的血小板膜自组装纳米气泡的制备方法，其特征在于，步骤(2)所述通过反复压缩气体至匀质化血小板膜囊泡悬液中再恢复到大气压，使血小板膜碎片在反复挤压过程中形成的自由纳米气泡表面自组装具体为：(a)将匀质化的血小板膜囊泡悬液收容于容器，容器上侧、连通管和体积可变挤压装置，构成密闭系统；(b)施力压缩挤压装置的柱塞，将部分气体通过连 通管压入液体中，增加密闭系统压力至原来的一到五倍；(c)除去外力，密闭系统中的压力恢复常压，自由气泡形成，血小板膜碎片在气核气液界面自组装；(d)重复加压恢复成常压过程，使血小板膜碎片充分组装融合，形成血小板膜包覆的纳米气泡悬浊液；(e)将制备得到的血小板纳米气泡悬浊液进行离心分离，得到血小板纳米气泡。
  6. 根据权利要求4所述的血小板膜自组装纳米气泡的制备方法，其特征在在于，步骤(a)所述在较高功率下进行破碎为在400-1000W功率下破碎10-40s；步骤(b)所述在降低超声空化功率为在80-200W功率下破碎60-90s，使被超声空化作用破碎的血小板膜碎片吸附在空化作用下形成的纳米气泡的气液界面重组，形成血小板膜包覆的纳米气泡悬浊液。
  7. 根据权利要求5所述的血小板膜自组装纳米气泡的制备方法，其特征在在于，所述重复加压恢复成常压过程为重复加压50-200次加压-恢复成常压，每次加压后系统压力为0.1-0.5MPa。
  8. 根据权利要求4所述的血小板膜自组装纳米气泡的制备方法，其特征在在于，所述气体优选为空气、氧气、氮气、氢气、一氧化氮、氦气、六氟化硫中的一种或两种。
  9. 一种权利要求1所述的血小板膜自组装纳米气泡的制备方法所制备的血小板膜纳米气泡。
  10. 一种权利要求1所述的血小板膜自组装纳米气泡的制备方法所制备的血小板膜纳米气泡在制备用于血管损伤部位的超声影像诊断造影剂中的应用。
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