Therapeutic use of fluorocarbon phase-change micro-particle local ultrasonic excitation vaporization and cavitation to produce tissue destruction

Abstract

The present application relates to a new use of fluorocarbon phase transition microparticles as a therapeutic ablation agent for mechanically damaging or ablating diseased tissue. When fluorocarbon phase transition microparticles are in contact with diseased tissue, under the excitation of low-frequency focused ultrasound, gas-liquid phase transition (i.e. vaporization) and ultrasonic cavitation resonance of vaporized microbubbles occur, followed by controlled intensity cavitation and mechanical damage of local tissue, thereby producing an ablation therapeutic effect on diseased tissue. Vaporized fluorocarbon phase transition 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 ultrasonic pressure and sound intensity, minimal damage to tissues outside the target area, easy operation, etc., improving the targeting and safety of treatment. Although this treatment method requires image guidance, it does not require very precise focusing, reducing the difficulty of operation, and has application prospects in multiple clinical treatment fields such as prostate hyperplasia and tumor immunotherapy.

Description

Technical Field

[0001] This invention relates to the field of lesion tissue ablation technology using fluorocarbon phase change microparticles under low-frequency focused ultrasound excitation. It can be applied to the ablation treatment of lesions such as malignant solid tumors and benign prostatic hyperplasia, and belongs to the field of non-invasive ultrasound therapy. Background Technology

[0002] Histotripsy and boiling histotripsy are two methods of non-thermal mechanical tissue ablation using ultrasound. Both utilize highly focused ultrasound (FUS) with extremely high sound pressure levels to induce the formation and cavitation destruction of microbubbles within the body, thereby non-invasively and homogenously destroying the target tissue. However, both methods require large arrays of focused transducers, sometimes up to 20 cm in diameter, with very high peak negative pressure (PNP), approximately 15-30 MPa. This can pose significant challenges to clinical translation, as these treatments require a wide acoustic window, which is rare in the human body. As we know, the human body lacks a good acoustic window. Except for superficial organs like the thyroid and breast, the abdominal acoustic window is not only narrow but also dynamically changes due to respiration and intestinal gas movement; the acoustic window typically covers only 20-30% of the abdominal surface. Termination ablation is a precise mechanical ablation method, where accurate focusing and sufficiently strong PNP amplitude are absolutely necessary to ensure cavitation destruction of the target tissue. Therefore, any insufficient PNP or focus deviation can lead to incomplete ablation or damage to important structures such as surrounding large blood vessels. In such cases, image-guided focusing and motion correction are necessary, which increases the complexity of the instrument and the procedure. Some other mechanical ablation methods, such as using pulsed high-intensity focused ultrasound (pHIFU) to attempt to destroy the dense stroma of pancreatic cancer, require not only image guidance but also up to 30 minutes to cover all spots, even with small mouse tumors.

[0003] In summary, existing tissue ablation techniques using high-voltage pulse focused ultrasound and boiling tissue ablation have the following problems: due to equipment limitations, a wide ultrasound transmission window needs to be found in the human body, but apart from some superficial organs, it is difficult to find a site that meets the requirements for an ultrasound transmission window, affecting treatment efficiency and accuracy. Therefore, it is essential to develop a new ultrasound tissue ablation technique to overcome these difficulties. Summary of the Invention

[0004] This invention proposes a novel minimally invasive bubble histotripsy technique that combines local injection of fluorocarbon phase change microparticles with ultrasonic-induced vaporization and cavitation to destroy diseased tissue. The invention primarily relates to the therapeutic effects and new applications of fluorocarbon phase change microparticles under low-frequency, low-intensity focused ultrasound excitation, which induces vaporization and cavitation effects, leading to non-thermal mechanical destruction of diseased tissue. Diseased tissues include various malignant solid tumors, such as hepatocellular carcinoma and breast cancer, as well as various benign lesions such as benign prostatic hyperplasia. Fluorocarbon phase change microparticles refer to emulsion microparticles of fluorocarbon compounds (such as perfluoropentane, PFP) dispersed in a liquid and possessing acoustic phase change properties. Under the low-frequency ultrasound designed in this invention, the fluorocarbon phase change microparticles in the diseased tissue undergo phase change vaporization and a high-intensity inertial cavitation effect, resulting in significant mechanical destruction of the diseased tissue. This may lead to tumor antigen exposure, enhancing the body's anti-tumor immune response, or therapeutic effects such as ablation of benign prostatic hyperplasia tissue. The ultrasonic energy of this invention refers to a pulsed or intermittent pulsed ultrasonic pulse energy form with relatively low sound pressure and a low emission duty cycle. Figure 1 The ultrasonic emission frequency range is 100-800kHz, the peak negative pressure range is 0.5-5.0MPa, and the average sound intensity (I) is... SPTA Below 3.0 W / cm 2 The duty cycle is less than 10%. For example, the acoustic parameters used in this study are: ultrasonic emission frequency 596 kHz, peak negative pressure 2.0 MPa, pulse width 160 cycles, pulse repetition frequency 20 Hz, intermittent pulsed ultrasonic emission (1 second emission / 1 second interval), actual duty cycle 0.27%, and average sound intensity only (I SPTA 20.3mW / cm 2 Experimental results showed that this method caused significant cavitation mechanical damage to the target area of ​​the rabbit liver. Figure 2 However, no temperature rise was detected during treatment.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A novel application of fluorocarbon phase change microparticles combined with low-frequency focused ultrasound in the preparation of tissue ablation and / or necrolysis agents: Fluorocarbon phase change microparticles located in diseased tissue undergo gas-liquid phase change / vaporization under the excitation of pulsed, low-frequency, low-intensity focused ultrasound. The resulting microbubble clouds / clusters then undergo microbubble cavitation resonance under the continuous action of low-frequency, low-intensity focused ultrasound, leading to cavitation mechanical destruction of local tissues, thereby producing an ablation therapeutic effect on the diseased tissues.

[0007] The principle and beneficial effects of this technical solution are as follows:

[0008] In this protocol, fluorocarbon phase change microparticles (droplets) are injected into the human body via local interventional puncture. Under the excitation of low-frequency focused ultrasound, these microparticles undergo gas-liquid phase change (i.e., vaporization) and ultrasonic cavitation resonance of vaporized microbubbles, resulting in controllable cavitation and mechanical destruction of local tissues, thereby producing an ablation therapeutic effect on the diseased tissue. This protocol is named Bubble histotripsy. The novel therapeutic application of this fluorocarbon phase change droplet is characterized by the following: the fluorocarbon phase change droplet microparticles injected into the human body via local interventional puncture undergo phase change vaporization and expansion under the excitation of pulsed low-frequency (frequency less than 800kHz) focused ultrasound, forming a large number of microbubble clusters (i.e., so-called microbubble clouds). Then, under continuous ultrasonic resonance, the microbubble clouds generate cavitation and mechanical effects, leading to the destruction of the diseased tissue in the injection target area by cavitation and mechanical effects. The vaporized fluorocarbon phase change microbubbles become ideal exogenous cavitation nuclei, significantly reducing the cavitation threshold and inducing enhanced inertial cavitation effects. This method features short treatment time, low required ultrasound pressure, minimal damage to tissues outside the target area, and ease of operation, thus improving the targeting and safety of the treatment. Although this treatment method requires image guidance, it does not require highly precise focusing, reducing the difficulty of operation and showing promise for application in multiple clinical treatment areas such as benign prostatic hyperplasia and tumor immunotherapy.

[0009] More specifically, this technical solution proposes a bubble tissue ablation method combining low-frequency focused ultrasound (FUS) and local injection of fluorocarbon phase change microparticles. As a specific example, this method uses perfluoronpentane (PFP) droplets. Perfluoronpentane droplets are a phase change emulsion, and acoustic droplet vaporization (ADV) is a commonly used liquid inert fluorocarbon compound, widely applied in basic research in ultrasound medicine. This method uses low-frequency, low-intensity, and low-sound-pressure FUS to vaporize the liquid fluorocarbon droplets, exciting the vaporized bubble cloud to undergo cavitation. Through ultrasonic cavitation and mechanical effects, the target tissue is mechanically destroyed. Clearly, the vaporized liquid fluorocarbon bubbles become highly effective cavitation nuclei, significantly inducing inertial cavitation and lowering the cavitation threshold.

[0010] Existing non-thermal, mechanical tissue destruction methods utilize pulsed focused ultrasound with extremely high sound pressure levels to induce the formation and cavitation destruction of microbubbles within the human body, thereby achieving tissue damage. However, these methods may be limited by the small acoustic window of the human body, hindering their effectiveness. Furthermore, high sound pressure levels can also cause mechanical damage to normal tissue outside the target area. This proposed solution overcomes the shortcomings of existing technologies. As a specific example, this solution uses low-frequency, low-sound-pressure, and low-intensity focused ultrasound to excite perfluoropentane droplets. The droplets vaporize to form cavitation nuclei, thereby lowering the sound pressure threshold of the focused ultrasound. Using low-frequency, low-sound-pressure, and low-intensity focused ultrasound can effectively induce cavitation in perfluoropentane droplets. Under low-frequency ultrasound excitation, the microbubble cloud undergoes cavitation expansion and compression, and even collapses, releasing shock waves, microjets, and traction tearing mechanical effects, ultimately causing mechanical damage to the tissue.

[0011] In summary, this approach utilizes minimally invasive local injection of liquid fluorocarbon microparticles and focused ultrasound to stimulate vaporization and cavitation resonance, achieving low-frequency, low-sound-pressure, and low-intensity non-thermal tissue ablation. This method avoids many drawbacks of traditional tissue lesioning techniques, thus achieving safer tissue ablation.

[0012] Furthermore, the fluorocarbon phase change microparticles come into contact with the diseased tissue through a puncture needle with an injection function, the puncture needle including a multi-hole alcohol injection needle and a coaxial puncture needle.

[0013] Furthermore, the fluorocarbon phase change microparticles are prepared by the following method: encapsulating liquid fluorocarbon compounds with a membrane material to form a suspension, and then dispersing it into an emulsion state by mechanical or ultrasonic oscillation; the membrane material includes lipids, human serum albumin, and carbohydrates; the liquid fluorocarbon compounds include perfluoropentane, perfluoromethylcyclopentane, perfluorohexane, perfluoroheptane, 2H,3H-decafluoropentane, perfluoromethylcyclohexane, perfluorooctane, perfluorodichlorooctane, etc.

[0014] Furthermore, the pulsed, low-frequency, low-intensity ultrasound is generated by a focused ultrasound transducer; the focused ultrasound transducer includes a concave transducer wafer, or includes a planar transducer wafer and an acoustic lens; the ultrasonic parameters of the focused ultrasound transducer are characterized by: a transmission frequency range of 100-800kHz, a peak negative pressure range of 0.5-5.0MPa, and an average sound intensity below 20.3W / cm². 2 The work occupancy rate is less than 10%.

[0015] Furthermore, the diameter of the fluorocarbon phase change particles is ≤0.20 mm; the fluorocarbon phase change particles in emulsion state are used to disperse in a lysozyme solution, the components of which include at least one of glucose, 1,2-propanediol, glycerol and mannitol.

[0016] Furthermore, as a preferred method, fluorocarbon phase change microparticles (e.g., perfluoropentane droplet microbubbles) are prepared by dispersing lipids in ultrapure water, followed by freeze-drying and then adding lysozyme solution to obtain a lipid solvent; mixing liquid fluorocarbons and lipid solvents, and shaking in an ice bath to obtain a fluorocarbon phase change microparticle suspension.

[0017] Furthermore, as a preferred option, the ratio of liquid fluorocarbon to lipid solvent is 0.05-0.1:2.

[0018] Furthermore, as a preferred embodiment, the lysozyme solution is composed of glucose solution, 1,2-propanediol and glycerol.

[0019] Furthermore, as a preferred method of administration, the fluorocarbon phase change microparticles are administered via direct injection into the target tissue; the volume of the fluorocarbon phase change microparticle suspension is 0.1 mL. This volume of fluorocarbon phase change microparticle suspension, under the action of focused ultrasound, can generate a large number of bubble clouds that disrupt the tissue.

[0020] Furthermore, as a preferred embodiment, the average diameter of the fluorocarbon phase change microparticles is 0.1038±0.06 mm, and 99% of the perfluoropentane droplet microbubbles have a diameter of <0.2 mm.

[0021] Furthermore, as a preferred embodiment, the focused ultrasound frequency is 596 kHz, the peak amplitude negative pressure is 2.05 MPa, and the sound intensity is 20.3 mW / cm². 2 The duty cycle is 0.27%, the pulse length is 160 cycles, and the pulse repetition frequency is 20 Hz.

[0022] Furthermore, as a preferred embodiment, the focused ultrasound is provided by a miniature focused ultrasound device connected to a transducer with an outer diameter of 35 mm. The miniature focused ultrasound device in this solution is connected to a transducer comprising an annular planar 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. This device is compact and portable, and in particular, the transducer is small in size, making operation simple and convenient. It can precisely target and destroy target tissue without requiring a large acoustic window.

[0023] Furthermore, as a preferred embodiment, the distance from the transducer to the target tissue is 5 cm. Using this method, the peak negative pressures at 4.5 cm (focal point) and 5 cm are 2.10 MPa and 2.05 MPa, respectively. Due to the 1-second on / off treatment scheme, the actual duty cycle is 0.27%, and at a treatment distance of 5 cm, the corresponding sound intensity (IL) is... SPTA The value is 20.3 mW / cm. 2 .

[0024] Furthermore, as a preferred embodiment, the perfluoropentane droplet microbubbles are first injected into the target tissue, and then the area containing the perfluoropentane droplet microbubbles is irradiated with low-frequency focused ultrasound for 10 minutes. The irradiation procedure is as follows: irradiate for 3 minutes, pause for 1 minute, irradiate for another 3 minutes, pause for 1 minute, and finally irradiate for 4 minutes. In this protocol, the total treatment time is 10 minutes, divided into two 3-minute sessions and one 4-minute session, with a 1-minute pause in between. The above operation time is sufficient to achieve effective ablation of liver tissue.

[0025] In summary, fluorocarbon phase change microparticles excited by specific ultrasound pulses possess high peak negative pressure, which can induce high-intensity acoustic cavitation effects in tumor treatment and exhibit anti-angiogenic effects. The key feature of this application lies in exposing fluorocarbon phase change microparticles to pulsed ultrasound, causing them to exert strong mechanical damage on diseased tissue at ultrasound frequencies of 100-800 kHz, peak negative pressures of 0.5-5.0 MPa, and pulse lengths of 20-2000 Hz. The biological effects of this high-intensity cavitation include rupture of blood vessels in the diseased tissue, cell fragmentation, and tissue homogenization. This treatment process can lead to complete blockage of tumor microcirculation, inhibition of tumor growth, and reduction of metastasis, representing a novel and promising method for physical therapy of tumors or proliferative lesions.

[0026] More specifically, this technique, FUS+PFP (Follicular Unit Extraction and Pulsed Physiolysis), utilizes low-frequency, low-sound-pressure, weakly focused ultrasound to excite perfluoropentane (PFP) droplets injected into the target tissue, causing acoustic vaporization (ADV) in the target area. Under continuous irradiation by the low-frequency, low-sound-pressure, weakly focused ultrasound, a dense cloud of small bubbles is generated in the target area. This bubble cloud expands and collapses thousands of times per second under the influence of ultrasound, applying immense pressure to the target tissue cells and thus mechanically destroying them, completely destroying the target tissue. Compared to existing technologies, the advantages of this technique are: precise targeting and destruction of the target tissue can be achieved without a large acoustic window; the low-frequency, low-sound-pressure, weakly focused ultrasound energy is extremely low (frequency 596 kHz, peak amplitude negative pressure 2 MPa, intensity 20.3 mW / cm²). 2 It will not damage surrounding important tissues and blood vessels; the focused transducer is small (outer diameter of 35mm), and is simple and convenient to operate; the treatment time is short (10min) and the effect is significant; it mechanically destroys diseased tissue cells, which is conducive to tumor tissue necrosis and tumor antigen exposure. Attached Figure Description

[0027] Figure 1 This shows the CK960 focused ultrasound device in Example 2 and the distribution of peak amplitude negative pressure (PNP).

[0028] Figure 2 This is the animal experiment procedure for Example 2.

[0029] Figure 3 The images shown are ultrasound images (2-D images, with black arrows indicating bubble clouds) of each group before and after treatment in Example 2.

[0030] Figure 4 Images of the liver and gross liver specimen stained with hematoxylin and eosin (HE) in Experiment Example 2.

[0031] Figure 5 The number of vacuoles and the area of ​​necrosis in the liver after treatment in Experiment Example 2 are statistically analyzed (mean±SD, **p<0.01,***p<0.001,n=5).

[0032] Figure 6 The temperature change detection results for Experiment Example 2 are shown (mean ± SD, n = 5). Detailed Implementation

[0033] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials and reagents used are all commercially available.

[0034] Example 1: Preparation of perfluoropentane droplet microbubbles

[0035] The perfluoropentane droplet microbubbles in this scheme are prepared based on Zhifuxian. Zhifuxian is a lipid microbubble with a perfluoropropane gas core, and its preparation process can be found in the previous paper (“Liu P, Effects of a novel ultrasonic contrast agent with long persistence on right ventricular pressure: Comparison with SonoVue. Ultrasonics. 2011; 51(2):210-214.”). The preparation process of the perfluoropentane droplet microbubbles (a specific fluorocarbon phase change microparticle) is briefly described as follows:

[0036] A lipid suspension was prepared by dissolving polyethylene glycol 4000 (PEG-4000), 1,2-dipalmitoyl-sn-glycerol-3-phosphate (DPPG), and 1,2-distearate-sn-glycerol-3-phosphate ethanolamine (DSPE) in ultrapure water at a mass ratio of 394:3:3. The suspension was then aliquoted into vials and lyophilized. The lyophilized powder was then added to 1 ml of lysozyme solution (composed of glucose solution, 1,2-propanediol, and glycerol at a volume ratio of 8:1:1), with a volume ratio of 1:1, to prepare the lipid solvent.

[0037] 100 μL of liquid perfluoropentane (PFP, CAS 678-26-2, Strem Chemical, Incorporated, Newburyport, MA) was mixed with 2 mL of the above-mentioned lipid solvent, and the vial was placed on ice for at least 5 min. The vial was then shaken twice in an ice bath for 15 seconds, with a 15-second interval. Images were taken using an Olympus vertical microscope (×100), and analysis was performed using ImageJ software (National Institutes of Health; http: / / www.imagej.softonic.de). The average diameter of the perfluoropentane droplet microbubbles (PFP ​​droplets) was 0.1038 ± 0.06 mm, and 99% of the droplets were less than 0.2 mm.

[0038] Example 2: Treatment process of perfluoropentane droplet microbubbles combined with low-frequency focused ultrasound

[0039] (1) Focused Ultrasound (FUS) Equipment Status

[0040] This study used a small focused ultrasound device (CK960, Mianyang Sonic Electronics Co., Ltd., China). Figure 1 As shown in a and b, the small focused ultrasound device is connected to a transducer, which includes a ring-shaped planar 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 operates at a frequency of 596 kHz, with a pulse length of 160 cycles and a pulse repetition frequency of 20 Hz. To measure the peak negative pressure of the transducer within a range of 45-50 mm from the tip, a needle-type pressure-sensitive detector (HNA-0400, Onda Corporation) adjusted by a precision three-dimensional positioning platform (Newport Electronics Co., Ltd.) was installed in a distilled water tank. The results show that the peak negative pressures at 4.5 cm (focal point) and 5 cm are 2.10 MPa and 2.05 MPa, respectively. Figure 1 c, d). Due to the 1-second on and 1-second off treatment protocol, the actual duty cycle is 0.27%, and the corresponding sound intensity (I) at 5cm (the treatment distance in this study) is... SPTA The value is 20.3 mW / cm. 2 .

[0041] (2) Laboratory animals and experimental procedures

[0042] Fifteen 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. The animals were anesthetized by intravenous injection of 1% sodium pentobarbital (0.3 mL / kg) near the ear, the hair on the upper abdomen was removed, the abdomen was surgically opened, the middle lobe of the liver was exposed in situ, and covered with gauze soaked in saline.

[0043] The experimental procedure for this scheme can be found in [link to relevant documentation]. Figure 2 Specifically, the livers of all experimental animals were observed using ultrasound imaging with a VINNO 70 ultrasound diagnostic system (VINNO Technology Co. Ltd., Suzhou, China) equipped with an X4-12L linear array transducer before treatment, immediately after treatment, and 72 hours after treatment.

[0044] For the PFP+FUS group, under ultrasound imaging guidance, 0.1 mL of the previously prepared PFP droplet was injected into the surgically exposed median lobe using a 21G injection needle (Hakko Corporation, Japan), and the liver injection site was immediately treated with FUS (isolated with a 5 cm thick coupling pad). Unlike existing tissue ablation techniques and other types of FUS treatments, this method does not require precise focusing and imaging guidance; this protocol only requires acoustic droplet vaporization. The transducer used for treatment is handheld and can be aimed at the injection point during treatment. The total treatment time is 10 minutes, divided into two 3-minute sessions and one 4-minute session, with a 1-minute pause in between. Figure 2 The target area temperature was measured before and immediately after FUS treatment (without gel pad) using a handheld infrared thermal imager (UTi165A, Uni-trend Technology Co., Ltd.).

[0045] For the PFP-only group, PFP drops were injected without FUS exposure. For the US group, FUS exposure was performed only, without PFP drop injection. After treatment, the experimental rabbit abdomens were closed, and the animals were left in place for 72 hours. Then, at the end of the experiment (72 hours later), all animals were euthanized after intravenous anesthesia with 1% sodium pentobarbital solution, and the middle lobe of the liver was immediately harvested for gross examination.

[0046] (3) Experimental Results

[0047] (3.1) Results of imaging studies

[0048] Ultrasound imaging revealed no significant changes in the FUS group on 2D images. Figure 3 a, b). In both the PFP and FUS+PFP groups, immediately after treatment, dense, hyperechoic bubble clouds of PFP droplets evaporating could be observed in the target area. Figure 3 c, e). After 72 hours, the bubble cloud in the PFP group significantly decreased, with only a small bright area visible around the needle tip. Figure 3 d), but the image of the FUS+PFP group is brighter than that of the PFP group. Figure 3 f).

[0049] (3.2) Pathological study results

[0050] The histological grading criteria are as follows: Liver injury is graded according to the number of vacuoles and the size of the necrotic area, and is divided into the following four grades:

[0051] Grade 0: Liver samples showed no vacuoles or necrotic spots;

[0052] Grade 1: 1-9 vacuoles or ≤0.1cm are visible in the liver tissue.2 The area of ​​tissue necrosis;

[0053] Grade 2: 10-25 vacuoles or 0.1-0.2 cm vacuoles are visible in the liver tissue. 2 Necrotic tissue areas;

[0054] Level 3: More than 25 vacuoles observed or >0.2cm 2 The necrotic area of ​​the tissue.

[0055] General pathological results showed no necrotic areas in the liver target area 72 hours after treatment in the FUS and PFP groups. However, in the PFP+FUS group, obvious patchy beige cavitary necrosis was observed in the liver target area, and diffuse necrosis was visible around the injection site. Figure 4 g, h, i). HE staining showed that the liver tissue structure of the FUS and PFP groups was intact and the matrix was homogeneous. Figure 4 a, b, d, e).

[0056] a, d, and e represent grade 0: normal liver; b represents grade 1: liver with 1-9 vacuoles (black arrows) or ≤0.1cm. 2 Area of ​​tissue necrosis (blue arrow); c and f are grade 3: the liver has more than 25 vacuoles or >0.2 cm. 2 The areas of tissue necrosis are shown. Red arrows indicate inflammatory cell infiltration. Yellow arrows indicate irregular areas of necrosis with small, surrounding patchy necrosis (h, I).

[0057] In particular, no necrosis, inflammatory cell infiltration, or off-white vacuoles were observed in the FUS group. One necrotic area (0.005 ± 0.008 cm) was observed in the PFP group. 2 Inflammatory cell infiltration and white vacuoles (7.00±12.88) were observed, but there were no significant differences compared with the FUS group (p=0.22 and p=0.259, respectively). Figure 5 However, a large number of white vacuoles of varying sizes (35.50±23.31) were observed in the liver tissue of the PFP+FUS group, approximately five times that of the PFP group. Figure 5 a) The spatial distribution of vacuolar formation is related to the spatial pattern of ADV, with ADV mainly distributed at the PFP droplet injection site. Compared with FUS and PFP, the PFP+FUS group showed significant tissue necrosis (0.99±0.29 cm) after ADV formation. 2 (p=0.001) and the area of ​​inflammatory cell infiltration was 198 times that of the PFP group (p=0.001). Figure 5 b).

[0058] (3.3) Detection of temperature changes in the target area of ​​the liver

[0059] The temperature of the liver target area was measured before and immediately after all FUS treatments, and there was no significant difference before and after treatment. Figure 6 a, b).

[0060] (4) Summary

[0061] By combining 2.05 MPa PNP, low-intensity FUS, and local injection of phase-change PFP droplets, rabbit liver tissue was significantly disrupted. The FUS device is a low-frequency, low-intensity focused ultrasound instrument with a transmission frequency of 596 kHz, a peak amplitude negative pressure of 2.05 MPa, and an acoustic intensity of 20.3 mW / cm². 2 The duty cycle was 0.27%, the pulse length was 160 cycles, and the pulse repetition frequency was 20 Hz. PFP droplet emulsions were prepared based on existing lipid microbubbles. The PFP droplets were encapsulated in a lipid-like film solvent, forming a chemically stable dispersion. Since the PFP droplets were administered via local injection rather than intravenous injection, their particle size distribution had little impact on the efficacy. At the 596 kHz level, the pressure threshold could even be below 1 MPa. Therefore, our focused 2.05 MPa pulse effectively evaporated the PFP droplets and cavitated the bubbles. The formation of a bubble cloud after FUS treatment validated the vaporization of PFP and the cavitation effect. Figure 3 In the FUS+PFP group, the bubble cloud was dense, while only a small, faintly lit area was observed in the PFP group, and no bubble formation was observed in the FUS group. Vaporized bubble clouds (cavitation nuclei) became ideal targets for low-intensity, low-pressure tissue ablation. Because PFP bubbles are insoluble in water, they can persist for up to 72 hours. Figure 3 Even though the FUS treatment time is only 10 minutes, this provides a wide range of treatment time options for FUS.

[0062] Histological examination revealed peripheral patchy necrosis around the injection site in the PFP+FUS group. Gross examination and microscopic examination also revealed numerous off-white vacuoles of varying sizes, which are completely different from the coagulative necrosis caused by the thermal effect of HIFU. Temperature measurements showed no significant difference before and after treatment, and no difference between any two groups. Figure 6 a) and b) are consistent with non-thermal effects. This study confirms that the PFP+FUS combination can cause liver tissue damage in rabbits. First, the combined treatment has good targeting, resulting only in necrosis around the injection site. Second, compared with coagulative necrosis, this cavitation effect has broader application prospects, which will be a new approach for future treatment.

[0063] By stimulating vaporization and cavitation resonance through minimally invasive local injection of PFP droplets and FUS, low-frequency, low-sound-pressure, and low-intensity non-thermal tissue ablation can be achieved. This method has the potential to avoid many of the drawbacks of traditional tissue lesioning techniques, thus enabling safer tissue ablation.

[0064] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. The use of fluorocarbon phase change microparticles combined with low-frequency, low-intensity focused ultrasound in the preparation of a system for tissue mechanical ablation, characterized in that, Fluorocarbon phase change particles located in the diseased tissue undergo gas-liquid phase change / vaporization under the excitation of pulsed, low-frequency, low-intensity focused ultrasound. The resulting microbubble clouds / clusters undergo microbubble cavitation resonance under the continuous action of low-frequency, low-intensity focused ultrasound, leading to cavitation mechanical damage to the local tissue, thereby producing an ablation therapeutic effect on the diseased tissue. The system includes a focused ultrasound transducer for generating pulsed, low-frequency, low-intensity focused ultrasound and fluorocarbon phase change microparticles. The fluorocarbon phase change microparticles are prepared by the following method: Polyethylene glycol 4000, 1,2-dipalmitoyl-sn-glycerol-3-phosphate, and 1,2-distearate-sn-glycerol-3-phosphate ethanolamine were dissolved in ultrapure water to obtain a lipid suspension, which was then lyophilized to obtain a lyophilized powder. The lyophilized powder was added to a lysozyme solution composed of glucose solution, 1,2-propanediol, and glycerol to prepare a lipid solvent. Perfluoropentane and the lipid solvent were mixed and shaken in an ice bath to obtain the fluorocarbon phase change microparticles. The ultrasonic parameters of the focused ultrasonic transducer are as follows: transmission frequency of 596 kHz, peak negative pressure of 2.05 MPa, and average sound intensity of 20.3 mW / cm². 2 The duty cycle is 0.27%.

2. The use according to claim 1, characterized in that: The fluorocarbon phase change microparticles are injected into the lesion tissue through a puncture needle with an injection function, the puncture needle including a multi-hole alcohol injection needle and a coaxial puncture needle.

3. The use according to claim 1, characterized in that: The focused ultrasound transducer includes a concave transducer wafer, or a planar transducer wafer and an acoustic lens.

4. The use according to claim 1, characterized in that: The diameter of the fluorocarbon phase change particles is ≤0.20 mm; the fluorocarbon phase change particles in emulsion state are dispersed in lysozyme solution.

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