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WO2024183127A1 - 氟碳相变微粒局部超声激励汽化和空化产生组织毁损的治疗用途 - Google Patents

氟碳相变微粒局部超声激励汽化和空化产生组织毁损的治疗用途 Download PDF

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WO2024183127A1
WO2024183127A1 PCT/CN2023/087820 CN2023087820W WO2024183127A1 WO 2024183127 A1 WO2024183127 A1 WO 2024183127A1 CN 2023087820 W CN2023087820 W CN 2023087820W WO 2024183127 A1 WO2024183127 A1 WO 2024183127A1
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cavitation
low
phase change
fluorocarbon
tissue
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French (fr)
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李辉
刘政
董小小
姚雷
何欢
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中国人民解放军陆军军医大学第二附属医院
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Publication of WO2024183127A1 publication Critical patent/WO2024183127A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0028—Disruption, e.g. by heat or ultrasounds, sonophysical or sonochemical activation, e.g. thermosensitive or heat-sensitive liposomes, disruption of calculi with a medicinal preparation and ultrasounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0028—Disruption, e.g. by heat or ultrasounds, sonophysical or sonochemical activation, e.g. thermosensitive or heat-sensitive liposomes, disruption of calculi with a medicinal preparation and ultrasounds
    • A61K41/0033—Sonodynamic cancer therapy with sonochemically active agents or sonosensitizers, having their cytotoxic effects enhanced through application of ultrasounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/08—Drugs for disorders of the urinary system of the prostate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention relates to the technical field of diseased tissue destruction by fluorocarbon phase change particles under low-frequency focused ultrasound excitation, and can be applied to the destruction treatment of diseased tissues such as malignant solid tumors and prostate hyperplasia, belonging to the technical field of non-invasive ultrasound treatment.
  • Histotripsy and boiling histotripsy are two methods of non-thermal mechanical tissue ablation using ultrasound. Both use extremely high sound pressure pulsed focused ultrasound (FUS) to induce the formation of the body's own microbubble cloud and cavitation to destroy the microbubbles, thereby non-invasively and homogenously destroying the target tissue in the body.
  • FUS extremely high sound pressure pulsed focused ultrasound
  • both methods require a large array of focused transducers, sometimes up to 20 cm in diameter, and a very high peak negative pressure (PNP) of about 15-30 MPa. This may bring great difficulties to clinical translation applications because these treatments require a wide ultrasound sound window, which rarely exists in the human body. As we know, the human body itself lacks a good sound window.
  • the abdominal sound window is not only narrow, but also changes dynamically due to breathing and intestinal gas movement.
  • the sound window generally only occupies 20-30% of the abdominal surface.
  • Tissue destruction is a precise mechanical ablation method, and precise focusing and sufficiently strong PNP amplitude are absolutely necessary to ensure cavitation destruction of target tissue. Therefore, any insufficient PNP or focus deviation may lead to incomplete ablation or damage to important structures such as surrounding large blood vessels. In this case, image-guided focusing and motion correction are necessary, which increases the complexity of the instrument and operation.
  • Some other mechanical destruction methods such as the use of pulsed high-intensity focused ultrasound (pHIFU) to try to destroy the dense stroma of pancreatic cancer, not only require image guidance, but also take up to 30 minutes to cover all spots even for small mouse tumors.
  • pHIFU pulsed high-intensity focused ultrasound
  • the existing tissue ablation and boiling tissue ablation using high-voltage pulsed focused ultrasound have the following problems: due to equipment limitations, it is necessary to find a wider ultrasound sound-transmitting window in the human body, but except for some surface organs, it is difficult to find parts of the human body that meet the requirements of the sound-transmitting window, which affects the efficiency and accuracy of treatment. Therefore, it is very necessary to develop a new type of ultrasound tissue ablation technology to overcome the above difficulties.
  • the present invention proposes a novel minimally invasive bubble histotripsy technique (Bubble histotripsy) that combines local injection of fluorocarbon phase change particles with ultrasonically stimulated vaporization and cavitation to produce diseased tissue damage.
  • the present invention mainly relates to the therapeutic effect and new use of fluorocarbon phase change particles under low-frequency, low-intensity focused ultrasound excitation to produce vaporization and cavitation effects, resulting in non-thermal mechanical damage to diseased tissue.
  • Diseased tissues here include: various malignant solid tumors, such as hepatocellular carcinoma, breast cancer, etc., as well as various benign diseased tissues such as prostate hyperplasia.
  • Fluorocarbon phase change particles refer to emulsified particles of fluorocarbon compounds (such as perfluoropentane/perfluoropentane, PFP) dispersed in liquid and having acoustic phase change characteristics.
  • fluorocarbon compounds such as perfluoropentane/perfluoropentane, PFP
  • the fluorocarbon phase change particles in the diseased tissue will undergo phase change vaporization and high-intensity inertial cavitation effect (Inertial cavitation), causing significant mechanical damage to the diseased tissue, which may then result in tumor antigen exposure, enhance the body's anti-tumor immune response, or ablate prostate hyperplasia tissue and other therapeutic effects.
  • the ultrasonic energy of the present invention refers to a pulsed or intermittent pulsed ultrasonic pulse energy form with relatively low sound pressure and low emission duty cycle (Figure 1).
  • the ultrasonic emission frequency range is 100-800kHz
  • the negative pressure range of the peak negative pressure is 0.5-5.0 MPa
  • the average sound intensity (I SPTA ) is less than 3.0 W/cm 2
  • the working duty cycle is less than 10%.
  • the acoustic parameters used in this study are ultrasonic emission frequency 596KHz, peak negative pressure 2.0MPa, pulse width 160 cycles, pulse repetition frequency 20Hz, intermittent pulsed ultrasonic emission (emission 1 second/interval 1 second), actual duty cycle 0.27%, and average sound intensity (I SPTA ) of only 20.3 mW/cm 2.
  • the experimental results show that this method produces significant cavitation mechanical damage to the target rabbit liver (Figure 2), and no temperature rise was found in temperature monitoring during treatment.
  • the present invention adopts the following technical scheme:
  • a new use of fluorocarbon phase change particles combined with low-frequency focused ultrasound in the preparation of tissue damage and/or ablation preparations The fluorocarbon phase change particles located in the diseased tissue undergo gas-liquid phase change/vaporization under the stimulation of pulsed, low-frequency, low-intensity focused ultrasound.
  • the resulting microbubble cloud/group undergoes microbubble cavitation resonance under the action of continuous low-frequency, low-intensity focused ultrasound, resulting in cavitation mechanical damage to the local tissue, thereby producing an ablation therapeutic effect on the diseased tissue.
  • fluorocarbon phase-change particles droplets
  • gas-liquid phase change i.e., vaporization
  • ultrasonic cavitation resonance of vaporized microbubbles occur, which then produce controllable cavitation and mechanical damage of local tissues, thereby producing ablative therapeutic effects on diseased tissues, named bubble histotripsy.
  • fluorocarbon phase-change droplets injected into the human body through local interventional puncture undergo phase change vaporization and expansion to form a large number of microbubble groups (i.e., so-called microbubble clouds) under the stimulation of pulsed low-frequency (frequency less than 800kHz) focused ultrasound; then, under the continuous ultrasonic resonance, the microbubble clouds produce cavitation and mechanical effects, causing the diseased tissues in the injection target area to be damaged by cavitation and mechanical effects.
  • the vaporized fluorocarbon phase-change microbubbles become ideal exogenous cavitation nuclei, which can significantly reduce the cavitation threshold and induce enhanced inertial cavitation effects.
  • This method has the characteristics of short treatment time, low required ultrasound sound pressure intensity, minimal tissue damage outside the target area, and easy operation, which improves the targeting and safety of the treatment.
  • this treatment method requires image guidance, it does not require very precise focusing, which reduces the difficulty of operation and has application prospects in multiple clinical treatment fields such as prostate hyperplasia and tumor immunotherapy.
  • the present technical solution proposes a bubble tissue destruction method that combines low-frequency focused ultrasound and local injection of fluorocarbon phase change microparticles.
  • the present solution uses perfluoropentane (PFP) droplets.
  • PFP perfluoropentane
  • Perfluoropentane droplets are a phase change emulsion.
  • ADV acoustic droplet vaporization
  • the present method uses low-frequency, low-intensity and low-sound pressure FUS to vaporize liquid fluorocarbon droplets, and stimulates the vaporized bubble cloud to produce cavitation effect, and mechanically destroys the target tissue through ultrasonic cavitation and mechanical effects.
  • FUS low-frequency, low-intensity and low-sound pressure
  • the existing non-thermal and mechanical tissue destruction method uses extremely high sound pressure pulsed focused ultrasound to induce the body's own microbubbles and cavitation to destroy the microbubbles to achieve tissue damage, but the above method may be limited by the narrow human acoustic window and is difficult to fully function. In addition, higher sound pressure intensity may also cause mechanical damage to normal tissues outside the target area.
  • This scheme can overcome the defects of the prior art. As a specific example, this scheme uses low-frequency, low-sound pressure, and low-intensity focused ultrasound to excite perfluoropentane droplets, and the droplets vaporize to form cavitation nuclei, thereby reducing the sound pressure threshold of focused ultrasound.
  • perfluoropentane droplets can be stimulated to produce cavitation.
  • the microbubble cloud undergoes cavitation expansion and compression or even collapses, releasing mechanical effects such as shock waves, microjets, and pulling and tearing, thereby causing mechanical damage to the tissue.
  • this approach can achieve low-frequency, low-sound-pressure, and low-intensity non-thermal tissue ablation by minimally invasive local injection of liquid fluorocarbon particles and focused ultrasound to stimulate vaporization and cavitation resonance.
  • This method can avoid many of the shortcomings of traditional tissue destruction techniques, thereby achieving safer tissue ablation.
  • the fluorocarbon phase-change particles are brought into contact with the diseased tissue through a puncture needle with an injection function
  • the puncture needle includes a multi-side hole alcohol injection needle and a coaxial puncture needle.
  • the fluorocarbon phase change particles are prepared by the following method: a liquid fluorocarbon compound is wrapped with a membrane material to form a suspension, and then dispersed into an emulsion state through mechanical or ultrasonic vibration; the membrane material includes lipids, human albumin and sugars; the liquid fluorocarbon compound includes perfluoropentane, Nonafluoro(trifluoromethyl)cyclopentane, Perfluorohexane, Perfluoroheptane, 2H,3H-decafluoropentane, Undecafluoro(trifluoromethyl)cyclohexane, Perfluorooctane, perfluorodichlorooctane, etc.
  • the pulsed, low-frequency, low-intensity ultrasound is generated by a focused ultrasonic transducer;
  • the focused ultrasonic transducer includes a concave transducer chip, or includes a planar transducer chip and an acoustic lens;
  • the ultrasonic parameter characteristics of the focused ultrasonic transducer are: an emission frequency range of 100-800kHz, a peak negative pressure range of 0.5-5.0 MPa, an average sound intensity lower than 20.3 W/ cm2 , and a working duty cycle lower than 10%.
  • the diameter of the fluorocarbon phase change particles is ⁇ 0.20 mm; the fluorocarbon phase change particles in an emulsion state are used to be dispersed in a lysozyme solution, and the components of the lysozyme solution include at least one of glucose, 1,2-propylene glycol, glycerol and mannitol.
  • the fluorocarbon phase change particles are prepared by the following method: lipids are dispersed in ultrapure water, and then a lysozyme solution is added after freeze-drying to obtain a lipid solvent; liquid fluorocarbon and lipid solvent are mixed, and shaken in an ice bath to obtain a fluorocarbon phase change particle suspension.
  • the usage ratio of liquid fluorocarbon and lipid solvent is 0.05-0.1:2.
  • the lysing solution consists of glucose solution, 1,2-propylene glycol and glycerol.
  • the fluorocarbon phase change microparticles are administered by direct injection into the target tissue; the amount of the fluorocarbon phase change microparticle suspension is 0.1 mL.
  • the above amount of fluorocarbon phase change microparticle suspension can generate a large amount of bubble clouds for destroying tissues under the action of focused ultrasound.
  • the average diameter of the fluorocarbon phase change particles is 0.1038 ⁇ 0.06 mm, and 99% of the perfluoropentane droplet microbubbles have a diameter of ⁇ 0.2 mm.
  • the frequency of the focused ultrasound is 596 kHz
  • the amplitude peak negative pressure is 2.05 MPa
  • the sound intensity is 20.3 mW/cm 2
  • the duty cycle is 0.27%
  • the pulse length is 160-cycle
  • the pulse repetition frequency is 20 Hz.
  • the focused ultrasound is provided by a small focused ultrasound device, which is connected to a transducer with an outer diameter of 35 mm.
  • the small focused ultrasound device of this solution is connected to a transducer, which includes an annular flat disk with an outer diameter of 35 mm and an inner diameter of 28 mm, and an aluminum focusing lens with a radius of curvature of 100 mm.
  • the above-mentioned device is small and easy to carry, especially the transducer is small in size, simple and convenient to operate, and can accurately target and damage the target tissue without a large acoustic window.
  • the distance from the transducer to the target tissue is 5 cm.
  • the peak negative pressure at 4.5 cm (focus) and 5 cm using the device of this solution is 2.10 MPa and 2.05 MPa respectively. Due to the 1 second on and 1 second off treatment scheme, the actual duty cycle is 0.27%, and the corresponding sound intensity ( ISPTA ) is 20.3 mW/ cm2 at a treatment distance of 5 cm.
  • the perfluoropentane droplet microbubbles are first injected into the target tissue, and then the part of the target tissue injected with the perfluoropentane droplet microbubbles is irradiated with low-frequency focused ultrasound for 10 minutes; the irradiation procedure is: irradiation for 3 minutes, pause for 1 minute, irradiation for 3 minutes, pause for 1 minute, and finally irradiation for 4 minutes.
  • the total treatment time is 10 minutes, which is divided into two 3-minute and one 4-minute sessions, with a pause of 1 minute in between. The above operation time can achieve effective ablation of liver tissue.
  • fluorocarbon phase change microparticles excited by specific ultrasonic pulses have the characteristics of high peak negative pressure, can induce high-intensity acoustic cavitation effect in tumor treatment, and have anti-angiogenic effect.
  • the characteristic of this application is that by exposing fluorocarbon phase change microparticles to pulsed ultrasound, they produce strong mechanical damage to the diseased tissue at an ultrasonic frequency of 100-800kHz, a peak negative pressure of 0.5-5.0MPa and a pulse length of 20-2000.
  • the biological effects of this high-intensity cavitation include rupture of blood vessels in the diseased tissue, cell fragmentation, and tissue homogenization.
  • the above treatment process can lead to complete blockage of tumor tissue microcirculation, inhibition of tumor growth and reduction of metastasis, and is a new and promising method for physical treatment of tumors or proliferative lesions.
  • the bubble tissue ablation (FUS+PFP) of the present technical solution utilizes low-frequency, low-sound-pressure weakly focused ultrasound irradiation to stimulate perfluoropentane (PFP) droplets injected into the target tissue to undergo acoustic droplet vaporization (ADV) in the target area.
  • PFP perfluoropentane
  • ADV acoustic droplet vaporization
  • a dense cloud of small bubbles is generated in the target area.
  • the bubble cloud expands and collapses thousands of times per second in the target area, exerting enormous pressure on the target tissue cells, thereby causing mechanical damage to the target tissue cells, and the target tissue can be completely destroyed.
  • the beneficial effects of this technical solution are: it can accurately target and damage the target tissue without a large acoustic window; the low-frequency, low-sound-pressure weakly focused ultrasound energy is extremely low (frequency is 596 kHz, peak amplitude negative pressure is 2 MPa, intensity is 20.3 mW/ cm2 ) and will not cause damage to surrounding important tissues and blood vessels; the focused transducer is small (outer diameter is 35 mm) and easy to operate; the treatment time is short (10 min) and the effect is significant; it mechanically destroys the diseased tissue cells, which is conducive to tumor tissue necrosis and tumor antigen exposure.
  • FIG. 1 shows the CK960 focused ultrasound device of Example 2 and the distribution of the peak negative pressure (PNP) of the amplitude.
  • PNP peak negative pressure
  • FIG. 2 is the animal experiment process of Example 2.
  • FIG3 is the ultrasound images of each group in Example 2 before and after treatment (2-D image, black arrows indicate bubble clouds).
  • FIG. 4 is an image of a liver stained with hematoxylin and eosin (HE) and a gross liver specimen of Experimental Example 2.
  • HE hematoxylin and eosin
  • the perfluoropentane droplet microbubbles in this scheme are prepared on the basis of Zhifuxian.
  • Zhifuxian is a lipid microbubble with a perfluoropropane gas core. Its preparation process can be found in the previous paper (“Liu P, Effects of a novel ultrasound contrast agent with long persistence on right ventricular pressure: Comparison with SonoVue. Ultrasonics. 2011;51(2):210-214.”).
  • the preparation process of perfluoropentane droplet microbubbles (a specific fluorocarbon phase change particle) is briefly described as follows:
  • Polyethylene glycol 4000 PEG-4000
  • DPPG 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol
  • DSPE 1,2-distearoyl-sn-glycero-3-phosphoethanolamine
  • the freeze-dried powder was then added to 1 ml of a lysozyme solution (the lysozyme solution was composed of a glucose solution, 1,2-propylene glycol and glycerol in a volume ratio of 8:1:1), and the volume ratio of the freeze-dried powder to the lysozyme solution was 1:1 to prepare a lipid solvent.
  • a lysozyme solution was composed of a glucose solution, 1,2-propylene glycol and glycerol in a volume ratio of 8:1:1
  • the volume ratio of the freeze-dried powder to the lysozyme solution was 1:1 to prepare a lipid solvent.
  • Example 2 Treatment process of perfluoropentane droplet microbubbles combined with low frequency focused ultrasound
  • a small focused ultrasound device (CK960, Mianyang Sonic Electronics Co., Ltd., China) was used in this study. As shown in Fig. 1a, b, the small focused ultrasound device was connected to a transducer, which consisted of a ring-shaped flat disk (PZT-82, Yuhai Electronic Ceramics Co., Ltd., China) with an outer diameter of 35 mm and an inner diameter of 28 mm, and an aluminum focusing lens with a radius of curvature of 100 mm. The transducer was operated at a frequency of 596 kHz, with a pulse length of 160-cycle and a pulse repetition frequency of 20 Hz.
  • a transducer which consisted of a ring-shaped flat disk (PZT-82, Yuhai Electronic Ceramics Co., Ltd., China) with an outer diameter of 35 mm and an inner diameter of 28 mm, and an aluminum focusing lens with a radius of curvature of 100 mm.
  • the transducer was operated at a frequency of 596 k
  • a needle pressure-sensitive detector HNA-0400, Onda Corporation adjusted by a precision three-dimensional positioning stage (Newport Electronics Co., Ltd.) was installed in a distilled water tank.
  • the results showed that the peak negative pressures at 4.5 cm (focus) and 5 cm were 2.10 MPa and 2.05 MPa, respectively (Fig. 1c, d).
  • the actual duty cycle was 0.27% and the corresponding acoustic intensity ( ISPTA ) at 5 cm (the treatment distance in this study) was 20.3 mW/ cm2 .
  • a total of 15 New Zealand white rabbits weighing 2-2.5 kg were randomly divided into three groups: PFP+FUS group, PFP group and FUS group, with five animals in each group.
  • 1% sodium pentobarbital 0.3 mL/kg was injected into the proximal ear vein for anesthesia, the upper abdominal hair was removed, the abdomen of the animals was surgically opened, the middle lobe of the liver was exposed in situ, and covered with saline-soaked gauze.
  • a 21G injection needle (Hakko, Japan) was used to inject 0.1 mL of the previously prepared PFP droplet into the center of the surgically exposed middle lobe, and the liver injection site was immediately treated with FUS (isolated with a 5-cm thick coupling pad).
  • FUS isolated with a 5-cm thick coupling pad.
  • This protocol only requires acoustic droplet vaporization.
  • the treatment transducer is handheld and can be aimed at the injection point during treatment. The total treatment time was 10 minutes, divided into two 3-minute and one 4-minute sessions, with a 1-minute pause in between ( Figure 2).
  • a handheld infrared thermal imager (UTi165A, Uni-trend Technology Co., Ltd., Ltd.) was used to measure the target area temperature before and immediately after FUS treatment (without gel pad).
  • liver damage is graded according to the number of vacuoles and the size of the necrotic area, and is divided into the following four grades:
  • Grade 3 More than 25 vacuoles or areas of tissue necrosis > 0.2 cm2 were observed.
  • a, d, e are grade 0: normal liver); b is grade 1: the liver has 1-9 vacuoles (black arrows) or ⁇ 0.1 cm2 of tissue necrosis (blue arrows); c, f are grade 3: the liver has more than 25 vacuoles or >0.2 cm2 of tissue necrosis.
  • Red arrows indicate inflammatory cell infiltration.
  • Yellow arrows indicate irregular necrotic areas with small surrounding patchy necrosis (h, I).
  • the temperature of the liver target area was measured before and immediately after all FUS treatments, and no significant differences were found before and after treatment (Fig. 6a, b).
  • the FUS device is a low-frequency, low-intensity focused ultrasound instrument with a transmission frequency of 596kHz, an amplitude peak negative pressure of 2.05 MPa, an acoustic intensity of 20.3mW/ cm2 , a duty cycle of 0.27%, a pulse length of 160-cycle, and a pulse repetition frequency of 20Hz.
  • the PFP droplet emulsion is prepared based on lipid microbubbles already available in the prior art. PFP droplets are encapsulated in a lipid membrane-like lipid solvent and are chemically stable dispersions.
  • PFP droplets are administered by local intervention injection rather than intravenous injection, so their particle size distribution has little effect on the technical effect.
  • the pressure threshold can even be lower than 1MPa. Therefore, our focused 2.05MPa pulse can effectively evaporate PFP droplets and cavitate bubbles.
  • the formation of bubble clouds after FUS treatment verifies the vaporization of PFP and the generation of cavitation effects (Figure 3).
  • the bubble cloud was dense, while only a small, slightly bright area was seen in the PFP group, and no bubble formation was seen in the FUS group.
  • the vaporized bubble cloud (cavitation nucleus) becomes an ideal target for low-intensity, low-pressure tissue destruction. Since PFP bubbles are insoluble in water, they can persist for 72 hours ( Figure 3), even if the FUS treatment time is only 10 minutes, which provides a wide range of treatment time options for FUS.

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Abstract

一种氟碳相变微粒作为机械毁损或消融病变组织的治疗消融剂的用途。氟碳相变微粒与病变组织接触后，在低频聚焦超声的激励下，发生气液相变（即汽化）和汽化微泡的超声空化谐振，继而产生局部组织的可控强度的空化和机械性毁损，从而产生病变组织的消融治疗作用。汽化的氟碳相变微泡成为理想的外源性空化核，能够显著降低空化阈值并诱导增强惯性空化效应。该方法具有治疗时间短、所需的超声声压和声强低、靶区以外组织损伤微小、操作方便等特点，提高了治疗的靶向性和安全性。该治疗方法虽然需要图像引导，但不需要非常精确的聚焦，降低了操作难度，在前列腺增生和肿瘤免疫治疗等多个临床治疗领域具有应用前景。

Description

氟碳相变微粒局部超声激励汽化和空化产生组织毁损的治疗用途 技术领域
本发明涉及氟碳相变微粒在低频聚焦超声激励下的病变组织毁损技术领域，可以应用于恶性实体肿瘤、前列腺增生等病变组织的毁损治疗，属于非创伤性超声治疗的技术领域。
 背景技术
组织毁损术（Histotripsy）和沸腾组织毁损术（Boiling histotripsy）是利用超声进行非热机械组织消融的两种方法。均使用极高声压的脉冲式聚焦超声（Focused ultrasound, FUS）诱导人体自身微泡云形成和 空化摧毁微气泡，从而对体内目标组织进行无创均匀化毁损。但这两种方法都需要较大阵列的聚焦换能器，有时直径可达20 cm，振幅峰值负压（Peak negative pressure, PNP）非常高，约为15-30 MPa。这可能会给临床转化应用带来很大的困难，因为这些治疗需要较宽广的超声透声窗，而这种透声窗在人体很少存在。正如我们所知，人体自身是缺乏良好的透声窗，除了像甲状腺、乳房这样的浅表器官，腹部声窗不仅狭窄，而且由于呼吸和肠道气体的运动而发生动态变化，透声窗一般只能占20-30%的腹部表面。组织毁损术是一种精确的机械消融方法，精确聚焦和足够强的PNP振幅对于保证靶区组织的空化毁损是绝对必要条件。因此，任何PNP不足或焦点偏移都可能导致不完全消融或周围大血管等重要结构的损伤。在这种情况下，图像引导聚焦和运动校正是必要的，这增加了仪器和操作的复杂性。一些其他的机械破坏方法，例如使用脉冲高强度聚焦超声(pHIFU)试图破坏胰腺癌密集的间质，这样的治疗方法不仅需要图像引导，即使是小的老鼠肿瘤，也需要长达30分钟的时间来覆盖所有的斑点。
综上，现有的利用高压脉冲聚焦超声的组织毁损术和沸腾组织毁损术存在以下问题：由于设备局限需要在人体寻找较宽的超声透声窗，但是人体除一些表层器官外，很难找到符合透声窗要求的部位，影响治疗效率和精确程度。因此，研发一种新型的超声组织毁损技术，克服以上困难，显得十分必要。
发明内容
本发明提出一种新型微创性的氟碳相变微粒局部注射结合超声激励汽化和空化产生病变组织毁损的泡泡毁损术（Bubble histotripsy）。本发明主要涉及氟碳相变微粒在低频、低声强的聚焦超声激励下，产生汽化和空化效应，导致病变组织非热机械性毁损的治疗作用和新用途。病变组织在这里包括：各种恶性实体肿瘤，例如肝细胞癌、乳腺癌等，以及前列腺增生等各种良性病变组织。氟碳相变微粒是指分散于液体中的、具有声致相变特性的氟碳化合物（如：全氟戊烷/perfluoropentane, PFP）乳化微粒。在本发明设计的低频超声作用下，病变组织中的氟碳相变微粒会发生相变汽化和强度较高的惯性空化效应（Inertial cavitation），造成显著的病变组织机械性毁损，进而可能产生肿瘤抗原暴露，提高机体抗肿瘤免疫反应，或者前列腺增生组织消融等治疗作用。本发明的超声能量是指一种脉冲式或者间歇脉冲式、相对较低声压、较低的发射占空比的超声脉冲能量形式（图1）。超声发射频率范围为100-800kHz、峰值负压的负压范围在0.5-5.0 MPa、平均声强（I SPTA）低于3.0 W/cm 2，工作占空比低于10%。例如：本研究使用的声学参数为超声发射频率596KHz，峰值负压2.0MPa，脉冲宽度160个周期，脉冲重复频率20Hz，间歇脉冲式超声发射（发射1秒/间歇1秒），实际占空比0.27%，平均声强仅（I SPTA）20.3 mW/cm 2。实验结果显示，该方法对靶区兔肝脏产生了显著的空化机械毁损（图2），而在治疗中温度监测未发现有温升现象。
为达到上述目的，本发明采用如下技术方案：
氟碳相变微粒联合低频聚焦超声在制备组织毁损和/或消融制剂中的新用途，位于病变组织中的氟碳相变微粒，在脉冲式、低频、低声强聚焦超声的激励下发生气液相变/汽化，继而产生的微泡云/群在持续的低频、低声强聚焦超声作用下发生微泡空化谐振，导致局部组织的空化机械性毁损，从而产生病变组织的消融治疗作用。
本技术方案的原理以及有益效果在于：
在本方案中，氟碳相变微粒（液滴）经局部介入穿刺方式注射进入人体的氟碳相变微粒，在低频聚焦超声的激励下发生气液相变（即汽化）和汽化微泡的超声空化谐振，继而产生局部组织的可控空化和机械性毁损，从而产生病变组织的消融治疗作用，命名为泡泡毁损术（Bubble histotripsy）。该氟碳相变液滴的治疗新用途特征在于：经局部介入穿刺注射进入人体的氟碳相变液滴微粒，在脉冲式低频（频率小于800kHz）聚焦超声的激励作用下，发生相变汽化膨胀形成大量微泡群（即所谓微泡云）；然后，在持续的超声谐振作用下，微泡云产生空化和机械效应，导致注射靶区的病变组织被空化效应和机械效应毁损破坏。汽化的氟碳相变微泡成为理想的外源性空化核，能够显著降低空化阈值并诱导增强惯性空化效应。本方法具有治疗时间短、所需的超声声压声强低、靶区以外组织损伤微小、操作方便等特点，提高了治疗的靶向性和安全性。本治疗方法虽然需要图像引导，但不需要非常精确的聚焦，降低了操作难度，在前列腺增生和肿瘤免疫治疗等多个临床治疗领域具有应用前景。
更具体地，本技术方案提出了一种结合低频聚焦超声和局部注射氟碳相变微粒的泡泡组织毁损术方法。作为具体示例，本方案使用了全氟戊烷（perfluoronpentane, PFP）液滴。全氟戊烷液滴是一种相变乳剂，在文献中声致液滴相变汽化（Acoustic droplet vaporization，ADV）方法被广泛应用于超声医学的基础研究中，是一种常用的液态惰性氟碳化合物。本方法采用低频、低强度和低声压的FUS对液态氟碳液滴进行汽化，并激励汽化的气泡云发生空化效应，通过超声空化和机械效应，机械性破坏靶区组织。显然，汽化的液态氟碳气泡成为了非常有效的空化核，能够显著诱导惯性空化并降低空化阈值。
现有的非热、机械性毁损组织方法是利用极高声压的脉冲式聚焦超声来诱导人体自身微泡发生并空化摧毁微泡实现对组织损毁，但上述方法可能受到人体声窗狭小的限制难以充分发挥作用。除此之外，较高的声压强度也可能对靶区以外的正常组织造成机械损伤损伤。本方案可以克服现有技术的缺陷。作为具体示例，本方案采用低频、低声压、低强度的聚焦超声来激励全氟戊烷液滴，液滴汽化形成空化核，进而降低聚焦超声的声压阈值。采用低频、低声压、低强度的聚焦超声，即可以激励全氟戊烷液滴产生空化现象。在低频超声激励下，微泡云发生空化膨胀和压缩甚至崩溃，释放冲击波、微射流、牵拉撕裂等机械效应，进而造成组织的机械性破坏。
综上，本方案通过微创局部注射液态氟碳微粒和聚焦超声以刺激汽化和空化谐振，可以实现低频、低声压和低强度的非热组织消融。这种方法可避免传统组织毁损术的诸多缺点，从而实现更安全的组织消融。
进一步，所述氟碳相变微粒通过带有注射功能的穿刺针与病变组织接触，所述穿刺针包括多侧孔酒精注射针和同轴穿刺针。
进一步，所述氟碳相变微粒由如下方法制备：用膜材料包裹液态氟碳化合物形成混悬液，然后通过机械振荡或超声振荡后分散呈乳化液状态；所述膜材料包括脂质、人血白蛋白和糖类；所述液态氟碳化合物包括全氟戊烷/perfluoropentane、全氟甲基环戊烷/Nonafluoro(trifluoromethyl)cyclopentane、全氟己烷/Perfluorohexane、全氟庚烷/Perfluoroheptane、2H,3H-十氟戊烷/2H,3H-Decafluoropentane、全氟甲基环己烷/Undecafluoro(trifluoromethyl)cyclohexane、十八氟辛烷/Perfluorooctane、perfluorodichlorooctane等。
进一步，脉冲式、低频、低声强超声由聚焦超声换能器产生；所述聚焦超声换能器包括凹面换能器晶片，或者包括平面换能器晶片和声透镜；所述聚焦超声换能器的超声参数特征为：发射频率范围为100-800kHz、峰值负压范围为0.5-5.0 MPa、平均声强低于20.3 W/cm 2，工作占空比低于10%。
进一步，所述氟碳相变微粒的直径≤0.20mm；乳化液状态的氟碳相变微粒用于分散于溶酶液中，所述溶酶液的成分包括葡萄糖、1,2-丙二醇、丙三醇和甘露醇中的至少一种。
进一步，作为一种优选，氟碳相变微粒（例如，全氟戊烷液滴微泡）由如下方法制备获得：将脂质分散于超纯水中，再经冻干处理之后加入溶酶液获得脂质溶剂；将液态氟碳和脂质溶剂混合，在冰浴中震荡后获得氟碳相变微粒悬液。
进一步，作为一种优选，液态氟碳和脂质溶剂的用量比为0.05-0.1：2。
进一步，作为一种优选，所述溶酶液由葡萄糖溶液、1,2-丙二醇和甘油组成。
进一步，作为一种优选，氟碳相变微粒的给药方式为直接注射至靶组织；氟碳相变微粒悬液的用量为0.1mL。上述用量的氟碳相变微粒悬液可在聚焦超声的作用下，产生大量的用于破坏组织的气泡云。
进一步，作为一种优选，氟碳相变微粒的平均直径为0.1038±0.06mm，99%的全氟戊烷液滴微泡直径＜0.2mm。
进一步，作为一种优选，聚焦超声的频率为596kHz，振幅峰值负压为2.05 MPa，声强为20.3mW/cm 2，占空比为0.27%，脉冲长度为160-cycle，脉冲重复频率为20Hz。
进一步，作为一种优选，所述聚焦超声由小型聚焦超声设备提供，小型聚焦超声设备连接有外径为35mm的换能器。本方案的小型聚焦超声设备连接换能器，换能器包括一个外径为35mm、内径为28mm的环形平面圆盘，以及一个半径曲率为100mm的铝聚焦透镜。上述设备小巧，便于携带，特别是换能器体积小，操作简单方便，无需较大声学窗口，就能精准靶向损毁目标组织。
进一步，作为一种优选，所述换能器到靶组织的距离为5 cm。采用本方案的设备，4.5 cm（焦点）和5 cm处的峰值负压分别为2.10 MPa和2.05 MPa。由于1秒打开和1秒关闭的治疗方案，实际占空比为0.27%，在5cm的治疗距离下，相应的声强度（I SPTA）为20.3mW/cm 2。
进一步，作为一种优选，所述全氟戊烷液滴微泡先注入靶组织，然后使用低频聚焦超声照射靶组织注入有全氟戊烷液滴微泡的部位10min；照射程序为：照射3min，暂停1min，再照射3min，暂停1min，最后照射4min。在本方案中，总治疗时间为10min，分为两次3min和一次4min进行，中间停顿1min，上述操作时间即可实现肝脏组织的有效消融。
综上所述，特定超声脉冲激发的氟碳相变微粒具有峰值负压高的特点，可在肿瘤治疗中诱导高强度声空化效应，并具备抗血管生成作用。本应用的特点在于通过将氟碳相变微粒暴露在脉冲超声波下，使其在100-800kHz的超声频率、0.5-5.0MPa的峰值负压和20-2000的脉冲长度下，对病变组织产生强烈的机械破坏。这种高强度空化的生物效应包括病变组织血管破裂、细胞破碎、组织匀浆化。上述治疗过程可导致肿瘤组织微循环完全阻断、肿瘤生长抑制和转移减少，是一种新的有前途的物理治疗肿瘤或增生性病变的方法。
更具体地，本技术方案的泡泡组织毁损术（FUS+PFP），其利用低频低声压弱聚焦超声照射激励注射到靶区组织的全氟戊烷（perfluoropentane, PFP）液滴在靶区发生声致液滴汽化（ADV），在低频低声压弱聚焦超声持续照射作用下在靶区产生密集小气泡云，气泡云在超声作用下在靶区每秒发生膨胀和崩溃数千次实现对靶区组织细胞施加巨大压力从而对靶区组织细胞产生机械性损毁，目标组织可被完全破坏。相对于现有技术，本技术方案的有益效果在于：无需较大声学窗口，就能精准靶向损毁目标组织；低频低声压弱聚焦超声能量极低（频率为596 kHz，振振幅峰值负压为2 MPa，强度为20.3 mW/cm 2）不会对周边重要组织和血管产生损伤；聚焦换能器小（外径为35 mm），操作简单方便；治疗时间短（10 min）、效果显著；机械性破坏病变组织细胞，利于肿瘤组织坏死和肿瘤抗原暴露。
附图说明
图1为实施例2的CK960聚焦超声设备以及振幅峰值负压(PNP)分布情况。
图2为实施例2的动物实验流程。
图3为实施例2的各组治疗前后的超声影像（2-D图像，黑色箭头表示气泡云）。
图4为实验例2的苏木精-伊红（HE）染色肝脏和肝脏大体标本图像。
图5为实验例2的治疗后肝脏的空泡数和坏死面积统计（mean±SD，**p＜0.01,***p＜0.001, n=5）。
图6为实验例2的温度变化检测结果（mean±SD，n=5）。
 实施方式
下面结合实施例对本发明做进一步详细的说明，但本发明的实施方式不限于此。若未特别指明，下述实施例以及实验例所用的技术手段为本领域技术人员所熟知的常规手段，且所用的材料、试剂等，均可从商业途径得到。
实施例1：全氟戊烷液滴微泡（perfluoropentane droplet）的制备
本方案的全氟戊烷液滴微泡在Zhifuxian的基础上制备。Zhifuxian为一种全氟丙烷气芯的脂质微泡，其制备过程可以参见在先论文（“Liu P, Effects of a novel ultrasound contrast agent with long persistence on right ventricular pressure: Comparison with SonoVue. Ultrasonics. 2011;51(2):210-214.”）。全氟戊烷液滴微泡（一种具体的氟碳相变微粒）制备过程简述如下： 
将质量比为394：3：3的聚乙二醇4000（PEG-4000）、1，2-二棕榈酰-sn-甘油-3-磷酸甘油（DPPG）和1，2-双硬脂酰-sn-甘油-3-磷酸乙醇胺（DSPE）溶于超纯水中，获得脂质悬液，然后将悬液分装入小瓶，再进行冻干处理。然后将冻干的粉末加入1ml溶酶液中（溶酶液由体积比为8：1：1的葡萄糖溶液、1,2-丙二醇和甘油组成）中，冻干的粉末和溶酶液的体积比为1：1，制成脂质溶剂。
将100 μL液态全氟戊烷（PFP，CAS 678-26-2，Strem Chemical，Incorporated，Newburyport，MA）与上述2 mL脂质溶剂混合，并将小瓶置于冰中至少5min，然后在冰浴中震荡15秒两次，间隔15秒。使用奥林巴斯立式显微镜（×100）拍照，并使用ImageJ软件（美国国立卫生研究院；http://www.imagej.softonic.de)分析，全氟戊烷液滴微泡（PFP液滴）的平均直径为0.1038±0.06mm，99%液滴小于0.2mm。
实施例2：全氟戊烷液滴微泡联合低频聚焦超声（low frequency focused ultrasound）的治疗过程
（1）聚焦超声（FUS）设备情况
本研究使用小型聚焦超声设备（CK960，绵阳索尼克电子有限公司，中国）。如图1a、b所示，该小型聚焦超声设备连接换能器，换能器包括一个外径为35mm、内径为28mm的环形平面圆盘（PZT-82，中国有限公司裕海电子陶瓷有限公司），以及一个半径曲率为100mm的铝聚焦透镜。换能器在596kHz的频率下工作，脉冲长度为160-cycle，脉冲重复频率为20Hz。为了测量换能器在尖端外45-50mm的范围内的峰值负压，在蒸馏水箱中安装了由精密三维定位平台（纽波特电子股份有限公司）调整的针式压敏检波器（HNA-0400，Onda Corporation）。结果表明，4.5 cm（焦点）和5 cm处的峰值负压分别为2.10 MPa和2.05 MPa（图1c，d）。由于1秒打开和1秒关闭的治疗方案，实际占空比为0.27%，在5cm（本研究中的治疗距离）的相应的声强（I SPTA）为20.3mW/cm 2。
（2）实验动物以及实验流程
共15只体重为2-2.5 kg的新西兰白兔随机分为三组：PFP+FUS组、PFP组和FUS组，每组五只动物。耳近端静脉注射1%戊巴比妥钠（0.3mL/kg）麻醉，去除上腹部毛发，手术打开动物腹部，原位暴露肝中叶，并用盐水浸泡的纱布覆盖。
本方案的实验流程参见图2，具体如下：在处理前，处理后即刻以及处理后72小时，使用带有X4-12L线性阵列换能器的VINNO 70超声诊断系统（VINNO Technology Co.Ltd，Suzhou，China），对所有的实验动物的肝脏进行超声影像观察研究。
对于PFP+FUS组，在超声影像学指导下，使用21G注射针（日本Hakko公司）将0.1mL 前文制备的PFP液滴注入手术暴露的中叶中心，并立即使用FUS处理肝脏注射部位（用5cm厚耦合垫隔离）。与现有技术中组织损毁术以及其他类型的FUS治疗不同，本方法不需要精确聚焦和成像指导，本方案只需要声致液滴汽化（acoustic droplet vaporization）。治疗用换能器是手持的，在治疗过程中瞄准注射点即可。总治疗时间为10分钟，分为两次3分钟和一次4分钟进行，中间停顿1分钟（图2）。使用手持式红外热成像仪（UTi165A，Uni-trend Technology Co.，Ltd.，Ltd.）在FUS治疗前和治疗后立即（无凝胶垫）测量靶区温度。
对于仅PFP组，PFP液滴在没有FUS暴露的情况下注射。对于US组，只进行FUS暴露，不注射PFP液滴。治疗后，关闭实验用兔腹部，动物保留72小时。然后，在实验结束时（72小时后），所有动物在静脉注射1%戊巴比妥钠溶液麻醉后处死，处死后立即采集肝中叶进行大体检查。
（3）实验结果
（3.1）影像学研究结果
经超声影像观察, 在2-D图像上，FUS组未发现显著变化(图3a、b)。在PFP组和FUS+PFP组中，刚完成治疗后，在目标区域可以看到PFP液滴蒸发的密集高回声气泡云（图3c，e）。72小时后，PFP组中的气泡云明显减少，仅在针尖周围看到一个小的明亮区域（图3d），但FUS+PFP组的影像比PFP组更亮（图3f）。
（3.2）病理学研究结果
组织学分级标准如下：根据空泡形成的数量和坏死区域的大小对肝脏损伤进行分级，分为以下四个等级：
0级：肝脏样品无空泡和坏死斑；
1级：肝组织可见1-9个空泡或≤0.1cm 2的组织坏死面积；
2级：肝组织可见10-25个空泡或0.1-0.2cm 2的组织坏死区；
3级：观察到超过25个空泡或＞0.2cm 2的组织坏死区域。
一般病理结果显示，FUS组和PFP组治疗后72小时的肝靶区未发现坏死区。而在PFP+FUS组中，肝脏靶区可见明显的片状米色空腔坏死，注射损伤周围可见弥漫性坏死（图4g，h，i）。HE染色显示FUS和PFP组的肝组织结构完整，基质均匀（图4a，b，d，e）。
a、d、e为0级：正常肝脏）；b为1级：肝脏有1-9个空泡（黑色箭头）或≤0.1cm 2组织坏死面积（蓝色箭头）；c、f为3级：肝脏有超过25个空泡或＞0.2 cm 2的组织坏死区域。红色箭头表示炎性细胞浸润。黄色箭头表示不规则的坏死区域，伴有小的周围斑片状坏死（h，I）。
特别是，FUS组未观察到坏死、炎性细胞浸润和米白色空泡。PFP组观察到一个坏死区域（0.005±0.008 cm 2）、炎性细胞浸润和白色空泡（7.00±12.88），但与FUS组相比没有显著差异（分别为p=0.22和p=0.259）（图5）。然而，在PFP+FUS组的肝组织中观察到大量不同大小的白色空泡（35.50±23.31），约为PFP组的5倍（图5a）。液泡形成的空间分布与ADV的空间格局有关，ADV主要分布在PFP液滴注射部位。与FUS和PFP相比，ADV发生后，PFP+FUS组存在明显的组织坏死（0.99±0.29 cm 2，p=0.001）和炎性细胞浸润区域，是PFP组的198倍（图5b）。
（3.3）肝脏目标区域温度变化检测
在所有FUS治疗前和治疗后立即测量肝脏靶区的温度，治疗前后没有显著差异（图6a，b）。
（4）总结
通过结合2.05MPa PNP、低强度FUS和局部注射相变PFP液滴，兔肝脏组织被显著破坏。FUS装置是一种低频、低声强聚焦超声仪器，发射频率为596kHz、振幅峰值负压为2.05 MPa，声强为20.3mW/cm 2，占空比为0.27%，脉冲长度为160-cycle，脉冲重复频率为20Hz。PFP液滴乳液是基于现有技术中已有的脂质微泡制备而成。PFP液滴被包裹在类脂膜状脂质溶剂中，是化学稳定的分散体。PFP液滴通过局部介入注射而不是静脉注射给药，所以其粒径分布情况对技术效果影响不大。在596kHz水平下，压力阈值甚至可以低于1MPa。因此，我们的聚焦2.05MPa脉冲可以有效地蒸发PFP液滴并空化气泡。通过FUS处理后气泡云的形成验证了PFP的汽化并产生空化效应（图3）。在FUS+PFP组中气泡云密集，而在PFP组中只看到一个小的微亮区域，在FUS组中没有看到气泡形成。汽化的气泡云（空化核）成为低强度、低压组织毁损术的理想目标。由于PFP气泡不溶于水，因此其可持续存在72小时（图3），即使FUS处理时间只有10min，这为FUS提供了广泛的治疗时间选择。
组织学检查中发现，PFP+FUS组注射部位周围可见外周斑片状坏死，进行大体检查，显微镜下还观察到大量不同大小的米白色空泡，这与HIFU热效应引起的凝固性坏死完全不同。温度测量表明，治疗前后没有显著差异，任何两组之间也没有任何差异（图6a，b），这与非热效应一致。通过本研究确认，PFP+FUS组合可导致兔肝脏组织损伤。首先，联合治疗具有良好的靶向性，这只会导致注射部位周围的坏死。其次，与凝固性坏死相比，这种空化效应具有更广阔的应用前景，这将是未来治疗的新途径。
通过微创局部注射PFP液滴和FUS以激励汽化和空化共振，可以实现低频、低声压和低强度的非热组织消融。这种方法有可能避免传统组织毁损术的诸多缺点，从而实现更安全的组织消融。
以上所述的仅是本发明的实施例，方案中公知的具体技术方案和/或特性等常识在此未作过多描述。应当指出，对于本领域的技术人员来说，在不脱离本发明技术方案的前提下，还可以作出若干变形和改进，这些也应该视为本发明的保护范围，这些都不会影响本发明实施的效果和专利的实用性。本申请要求的保护范围应当以其权利要求的内容为准，说明书中的具体实施方式等记载可以用于解释权利要求的内容。

Claims (5)

  1. 氟碳相变微粒联合低频聚焦超声在制备组织毁损和/或消融制剂中的新用途，其特征在于，位于病变组织中的氟碳相变微粒，在脉冲式、低频、低声强聚焦超声的激励下发生气液相变/汽化，继而产生的微泡云/群在持续的低频、低声强聚焦超声作用下发生微泡空化谐振，导致局部组织的空化机械性毁损，从而产生对病变组织的消融治疗作用。
  2. 根据权利要求1所述的新用途，其特征在于：所述氟碳相变微粒通过带有注射功能的穿刺针注射进入病变组织，所述穿刺针包括多侧孔酒精注射针和同轴穿刺针。
  3.  根据权利要求1所述的新用途，其特征在于：所述氟碳相变微粒的制备方法是，用膜材料包裹液态氟碳化合物形成混悬液，然后通过机械振荡或超声振荡后分散呈乳化液状态；所述膜材料包括脂质、人血白蛋白和糖类；
    所述液态氟碳化合物包括全氟戊烷/perfluoropentane、全氟甲基环戊烷/Nonafluoro(trifluoromethyl)cyclopentane、全氟己烷/Perfluorohexane、全氟庚烷/Perfluoroheptane、2H,3H-十氟戊烷/2H,3H-Decafluoropentane、全氟甲基环己烷/Undecafluoro(trifluoromethyl)cyclohexane、十八氟辛烷/Perfluorooctane、perfluorodichlorooctane等。
  4.  根据权利要求1所述的聚焦超声，其特征在于：脉冲式、低频、低声强聚焦超声由聚焦超声换能器产生；所述聚焦超声换能器包括凹面换能器晶片，或者包括平面换能器晶片和声透镜；所述聚焦超声换能器的超声参数特征为：发射频率范围为100-800kHz、峰值负压负压范围为0.5-5.0 MPa、平均声强低于3.0 W/cm 2，工作占空比低于10%。
  5.  根据权利要求3所述的氟碳相变微粒混悬液，其特征在于：所述氟碳相变微粒的直径≤0.20mm；乳化液状态的氟碳相变微粒分散于溶酶液中，所述溶酶液的成分包括葡萄糖、1,2-丙二醇、丙三醇和甘露醇中的至少一种。
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