A low-temperature phase change nano-material for promoting ultrasonic-induced cavitation chain reaction and a system and method for intensifying tissue pulverization

Abstract

The application provides a low-temperature phase-change nanoparticle, which comprises: (a) a liquid core comprising a fluorocarbon compound or azeotropic composition thereof with a boiling point of -20-50 DEG C at normal temperature and pressure; and (b) a shell composed of a phospholipid material or a polymer material and optionally modified with a targeting molecule; wherein the morphology of the nanoparticle is regular or irregular spherical particles, or regular or irregular microflaky, or regular or irregular shaped micelles; the low temperature refers to a temperature of -20-50 DEG C; and the particle size of the nanoparticle is 100-5000 nm. The application also provides an ultrasonic treatment system for realizing chain reaction cavitation using the low-temperature phase-change nanoparticle and a method for realizing target tissue pulverization using the ultrasonic treatment system.

Description

Technical Field

[0001] This application belongs to the technical field of non-invasive ultrasound therapy, and relates to the non-invasive tissue fragmentation of low-temperature phase change nanoparticles at low ultrasound energy, which can be applied to tumor ablation and fragmentation, lymph node dissection, thrombolysis and other fields. Background Technology

[0002] Traditional high-intensity focused ultrasound (HIFU) technology relies on high energy (typically requiring a peak negative pressure of 20–30 MPa) to induce acoustic cavitation effects to mechanically destroy diseased tissue, showing potential applications in areas such as tumor ablation and thrombolysis. However, it suffers from drawbacks such as bulky equipment, high risk of thermal damage, limited penetration (affected by bone and gas interference), and insufficient treatment precision.

[0003] Therefore, this application aims to overcome these problems in the prior art and develop a novel non-invasive tissue pulverization system and technology that reduces the ultrasound energy requirement by orders of magnitude and significantly improves the safety and portability of treatment. Summary of the Invention

[0004] This application proposes a novel non-invasive tissue fragmentation system and technology based on a combination of low-temperature phase change nanoparticles and a chain reaction cavitation model. This system achieves an order-of-magnitude reduction in ultrasound energy requirements (peak negative pressure reduced to 0.5–15 MPa) and significantly improves treatment safety and portability. The core of this invention is the introduction of low-temperature phase change nanoparticles as cavitation nuclei, particularly fluorocarbon droplets with a boiling point of 35–45 °C. These fluorocarbon droplets can efficiently trigger chain reaction cavitation under low-energy ultrasound. The principle lies in the inventors' innovative selection of compounds within the aforementioned boiling point range, enabling significant acoustic evaporation and localized cavitation triggering through localized heating or single ultrasound irradiation. For a boiling point of 40°C to 45°C, high-energy ultrasound triggers evaporation and breakup effects, causing fluorocarbon droplets to vaporize into bubbles and then break into several smaller bubbles. These smaller bubbles rapidly diffuse locally. Simultaneously, because the local human tissue temperature is around 36°C (below the boiling point of 40-45°C), condensation quickly occurs, leading to the formation of multiple broken fluorocarbon flakes or irregular fluorocarbon droplet nuclei from a single droplet. These broken droplets, under ultrasound irradiation, undergo further acoustic evaporation and breakup. This triggers a chain cavitation reaction, causing a single fluorocarbon droplet to rapidly form an exponential number of broken droplets and acoustic evaporation effects. This results in a highly effective low-energy ultrasound-triggered cavitation chain reaction, achieving the clinical therapeutic goal of breaking up local tissue.

[0005] Specifically, to achieve the above objectives, this application adopts the following technical solution: 1. A low-temperature phase change nanoparticle, characterized in that the low-temperature phase change nanoparticle comprises: (a) A liquid core comprising a fluorocarbon compound or an azeotropic composition thereof having a boiling point of -20°C to 50°C at ambient temperature and pressure; and (b) A shell composed of a phospholipid or polymer material and optionally modified with a targeting molecule; in: The nanoparticles are in the form of regular or irregular spherical particles, regular or irregular microplates, or regular or irregular micromicelles. The low temperature refers to a temperature range of -20°C to 50°C; The nanoparticles have a particle size of 100-800 nm.

[0006] 2. The low-temperature phase change nanoparticles according to item 1, wherein, The outer shell is capable of expanding, contracting, or rupturing within the local microenvironment of the tissue. After the liquid core is injected into the body, it is used to heat the target area externally or drive it with ultrasound irradiation. This process achieves a cyclical chain of phase change cavitation within the tissue, involving micro-boiling, cavitation, fragmentation, condensation, and penetration. The mechanical force generated by the phase change then breaks down the cells and tissue structures at the target location.

[0007] 3. According to the low-temperature phase change nanoparticles described in item 1, wherein, after the liquid core is injected into the body, external heating of the intended treatment site refers to using techniques such as local hot compresses, infrared heating, and microwave heating to raise the temperature of the site where the low-temperature phase change nanoparticles are aggregated to 37-50°C.

[0008] 4. The low-temperature phase change nanoparticles according to any one of items 2 to 3, wherein the injection into the body refers to injection into the lesion site via blood vessels or local percutaneous or cavitary injection for related treatment, or injection into the lymphatic drainage area for chain cavitation treatment of lymphatic vessels and lymph nodes.

[0009] 5. The low-temperature phase change nanoparticles according to item 1, wherein the fluorocarbon compound of the liquid core has a boiling point of 35°C to 50°C at room temperature and pressure.

[0010] 6. The low-temperature phase change nanoparticles according to item 1, wherein the fluorocarbon compound of the liquid core has a boiling point of 40°C to 50°C at room temperature and pressure.

[0011] 7. The low-temperature phase change nanoparticles according to item 1, wherein the fluorocarbon compound of the liquid core is selected from: 1H-perfluoropentane, decafluorobutane, dodecafluoropentane, cis-1-chloro-3,3,3-trifluoropropene (HCFO-1233zd(Z)), 1,1,1,3,3-pentafluorobutane (HFC-365mfc), perfluorohexane, perfluorocyclopentane, perfluorodimethylcyclobutane, perfluoromethylcyclopentane, perfluorotert-butylamine, perfluorotrimethylcyclopropane, perfluoromethylcyclohexane, perfluorobutylethane, perfluoroheptane, perfluoro(3-methylhexane), perfluoro(2-methylpentane), perfluoropentane and its branched isomers, perfluorooctane, hydrofluoroalkanes, hydrofluoroolefins, perfluoro-1,2-dimethylcyclobutane, or combinations thereof.

[0012] 8. The low-temperature phase change nanoparticles according to item 1, wherein the fluorocarbon compound of the liquid core is selected from: decafluorobutane, cis-1-chloro-3,3,3-trifluoropropene (HCFO-1233zd(Z)), 1,1,1,3,3-pentafluorobutane (HFC-365mfc), 1H-perfluoropentane, perfluorocyclopentane, perfluorodimethylcyclobutane, perfluorotert-butylamine, perfluorotrimethylcyclopropane, perfluorobutylethane, perfluoro(3-methylhexane), perfluoro(2-methylpentane), hydrofluoroalkanes, hydrofluoroolefins, perfluoro-1,2-dimethylcyclobutane, or combinations thereof.

[0013] 9. The low-temperature phase change nanoparticles according to item 1, wherein the fluorocarbon compound of the liquid core is selected from: cis-1-chloro-3,3,3-trifluoropropene (HCFO-1233zd(Z)), 1,1,1,3,3-pentafluorobutane (HFC-365mfc), 1H-perfluoropentane, and combinations thereof.

[0014] 10. The low-temperature phase change nanoparticles according to any one of claims 1 to 9, wherein the shell material is selected from: 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC), cholesterol, 1,2-distearyl-sn-glycerol-3-phosphoethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), Tween, lecithin, dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylcholine-polyethylene glycol 2000 (DPPC-PEG2000), matrix metalloproteinase-2 (MMP-2), or matrix metalloproteinase-9 (MMP-2). MMP-9) cleavable peptide polymers, disulfide-containing polymers, polyhistidine, polyethylene glycol-polylactic acid (PEG-PLA), polylactic acid-glycolic acid copolymer (PLGA), poloxamer F127, poloxamer F68, chitosan, poly-N-isopropylacrylamide (PNIPAM) or copolymers thereof, cyclodextrin-based supramolecular assemblies, mesoporous silica, metal-organic frameworks (MOFs), silica nanoparticles, fluorocarbon-MOF composites, perfluorooctanoic acid-hydrazone-drug conjugates, perfluorooctane sulfonate diethylamine salt, other antibody conjugates or combinations thereof.

[0015] 11. The low-temperature phase change nanoparticles according to any one of claims 1 to 9, wherein the shell material is selected from: 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC), 1,2-distearyl-sn-glycerol-3-phosphoethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), lecithin, and combinations thereof.

[0016] 12. The low-temperature phase change nanoparticles according to any one of claims 1 to 11, wherein the shell of the low-temperature phase change nanoparticles is modified with a targeting molecule.

[0017] 13. The low-temperature phase transition nanoparticles according to item 12, wherein the targeting molecule is selected from: integrin αvβ3 receptor, folic acid receptor, prostate-specific membrane antigen, pancreatic stellate cells, phosphatidylinositol proteoglycan-3 (GPC-3), desialyl glycoprotein receptor (ASGPR), mesothelin (MSLN), carcinoembryonic antigen-associated cell adhesion molecule 5 (CEACAM5), carbonic anhydrase IX (CAIX), EGFR (epidermal growth factor receptor), c-Met, and combinations thereof.

[0018] 14. The low-temperature phase change nanoparticles according to any one of items 1 to 13, wherein the particle size of the nanoparticles is selected from the following ranges: 100-600 nm, 100-400 nm, 200-600 nm, 200-400 nm.

[0019] 15. An ultrasound therapy system for achieving chain reaction cavitation, comprising: A nanoparticle delivery module for delivering the low-temperature phase change nanoparticles as described in any one of items 1 to 14 to a target tissue region; An ultrasonic transducer array configured to emit ultrasonic pulses toward the target tissue region and receive echo signals for imaging; A controller, which is communicatively connected to the ultrasonic transducer array to control the ultrasonic transducer array.

[0020] 16. The ultrasound therapy system according to claim 15, wherein the controller comprises the following unit: a. A cavitation nucleus status monitoring unit that estimates the density or distribution of effective cavitation nuclei within a target area based on echo signals; b. An adaptive pulse sequence generation unit that dynamically generates and controls the ultrasonic pulse sequence emitted by the ultrasonic transducer array based on the estimated density or distribution of effective cavitation nuclei in the target area. The parameters in the adaptive pulse sequence generation unit are set to trigger and maintain a self-sustaining chain reaction cavitation process. c. Safety monitoring unit, which monitors safety parameters in real time, including mechanical index (MI) and spatial peak time average intensity (ISPTA), and adjusts pulse parameters to keep them within preset safety thresholds.

[0021] 17. The ultrasound therapy system according to claim 16, characterized in that the adaptive pulse sequence generation unit is configured to generate a double-pulse sequence or a single-pulse sequence, the double-pulse sequence or double-pulse sequence comprising: Leader pulse: Its peak negative pressure is based on the acoustic evaporation (ADV) threshold pressure of the nanoparticles. This setting is used to trigger droplet vaporization; Main pulse: Its peak negative pressure is less than or equal to that of the pilot pulse. It is used to drive the generated cavitation bubbles to undergo inertial collapse and generate mechanical shear force.

[0022] 18. The ultrasound therapy system according to item 16 or 17, characterized in that the ultrasound pulse sequence adopts any of the following pulse sequences: Single-pulse sequence: center frequency 1.0-13 MHz, peak negative voltage 1-13 MPa, pulse length 1-50 cycles, pulse repetition frequency (PRF) 1-50 Hz, duty cycle less than 1%; Dual-pulse sequence: center frequency 1.0-13 MHz, leader pulse length 2-20 cycles, pulse repetition frequency (PRF) 1-50 Hz, peak negative voltage 1-13 MPa; main pulse length 1-30 cycles, pulse repetition frequency (PRF) 1-50 Hz, peak negative voltage 1-13 MPa; interval between two pulses 5-800 ms.

[0023] 19. The ultrasound therapy system according to item 16 or 17, characterized in that the ultrasound pulse sequence adopts any of the following pulse sequences: Single-pulse sequence: center frequency 1.5-5 MHz, peak negative voltage 1-5 MPa, pulse length 1-30 cycles, pulse repetition frequency (PRF) 4-20 Hz, duty cycle less than 1%; Dual-pulse sequence: center frequency is 1.0-5 MHz, leader pulse length is 5-10 cycles, pulse repetition frequency (PRF) is 10-15 Hz, peak negative voltage is 1-5 MPa; main pulse length is 5-25 cycles, pulse repetition frequency (PRF) is 1-50 Hz, peak negative voltage is 1-3 MPa; the interval between the two pulses is 5-800 ms.

[0024] 20. The ultrasound therapy system according to any one of items 15 to 19, characterized in that the controller selects the optimal particle size range and adjusts the pulse sequence parameters based on the ultrasound center frequency, wherein: (1) When the center frequency is 2-5MHz, the pulse repetition frequency (PRF) is set to 10-100 Hz, the peak negative pressure is 1.0-10 MPa, and the particle size of the nanoparticles is 200-2000 nm. (2) When the center frequency is 10-13MHz, the pulse repetition frequency (PRF) is set to 5-500 Hz, the peak negative pressure is 2.0-13 MPa, and the particle size of the nanoparticles is 200-600 nm.

[0025] 21. The ultrasound therapy system according to any one of items 15 to 20, characterized in that the ultrasound transducer array is a handheld probe, which adopts a concentric array or interlaced array structure, wherein the central high-frequency array element is used for ultrasound imaging, and the peripheral mid- and low-frequency array elements are used for emitting therapeutic ultrasound pulses.

[0026] 22. The use of the ultrasonic therapy system described in any one of items 15 to 21 for achieving the purpose of fragmenting target tissue, characterized in that the achievement of said purpose includes the following steps: (I) Introducing the low-temperature phase change nanoparticles described in any one of items 1 to 14 into the target tissue; (II) Monitoring the cavitation nucleus state of the target area using ultrasonic imaging; (III) Based on monitoring results, adaptively emit ultrasonic pulse sequences to trigger chain reaction cavitation and optionally maintain chain reaction cavitation; and (IV) Monitor safety parameters in real time and adjust ultrasound energy output to ensure treatment safety.

[0027] This application achieves the following core innovations: (1) Breakthrough in materials science: Fluorocarbon compounds with boiling points between -20℃ and 50℃ (such as perfluoropentane and HCFO-1233zd(Z)) were screened as phase change working fluids, and stable low-temperature phase change nanoparticles were constructed by combining them with biocompatible shell encapsulation technology. In particular, fluorocarbon compounds and their combinations with boiling points between 35-45℃ were adopted.

[0028] (2) Energy efficiency leap: By establishing an acoustic-phase transition-mechanical coupling mechanism, the required ultrasound energy is reduced to the diagnostic level (MI<2.5, ISPTA<500 mW / cm). 2 ), supporting the development of handheld devices.

[0029] (3) Innovative strategies and methods to enhance chain reactions: Direct injection of fluorocarbon nanodroplets into the target tissue or peripheral intravenous injection of targeted nanoparticles integrating tumor targets, so that they are evenly distributed in the local interstitial tissue. The formation of exogenous cavitation nuclei is beneficial to significantly reduce the cavitation effect and strong shear force of the ultrasound energy. This ability of fluorocarbon droplets to form a powerful energy-reducing and efficiency-enhancing cavitation has been proven by research more than ten years ago, but it has not been widely used in actual clinical work. One of the main reasons is that exogenous low-boiling-point fluorocarbon droplets are easily depleted under ultrasound irradiation. The newly launched high-energy ultrasound (HIFU) tissue fragmentation technology (energy ≥20MPa) continuously and repeatedly cavitates the focused area with high energy. Under the optimization of high-energy, low-repetition-frequency pulse sequences, it achieves the purpose of fragmenting tissue without significant temperature rise. Whether it is traditional HIFU or this tissue fragmentation technology, it is necessary to focus on a certain point on the organ tissue for continuous irradiation. Its deeper potential meaning is that the tissue cells in the focused area are always present, and there is no problem of cavitation material depletion.

[0030] When injecting fluorocarbon particles with boiling points below body temperature to introduce exogenous cavitation nuclei, theoretically, strong shear and mechanical forces can be generated with very low energy. However, the broken and collapsed bubbles quickly disperse and disappear, thus being rapidly depleted. In reality, what truly matters is the tiny three-dimensional space within the focused area of ​​the ultrasound. Within this tiny space, the number of locally injected carbon droplets is limited and more easily consumed. To address this, the inventors creatively proposed a comprehensive strategy based on a unique ultrasound irradiation sequence to induce a chain reaction, significantly reducing both the energy required for tissue fragmentation and the amount of fluorocarbon droplets used. The inventors preferentially selected fluorocarbon compounds with boiling points greater than 37°C and less than 50°C. These fluorocarbon compounds have a relatively high boiling point (above body temperature) that is not too high to cause damage to normal tissue. Furthermore, because their boiling point is above body temperature, they can rapidly condense after acoustic vaporization. This droplet-cavitation-condensation process easily forms a cycle when ultrasound is applied locally, creating a chain-like reaction. This greatly reduces the rapid consumption of exogenous fluorocarbon droplets, providing a continuous supply of cavitation nuclei for acoustically fragmented tissue. The inventors conducted in-depth theoretical analysis and in vitro experiments, achieving a significant breakthrough.

[0031] (4) Precision control system: integrates real-time ultrasound imaging feedback and model prediction control algorithm, dynamically adjusts pulse parameters, realizes visualization and closed-loop control of cavitation process, and improves treatment selectivity and repeatability.

[0032] In summary, this application not only solves the problem of excessive energy in traditional HIFU, but also promotes the transformation of non-invasive surgery from "experience-based operation" to "quantitative engineering" through a design paradigm guided by mathematical modeling. Attached Figure Description

[0033] Figure 1 This is an electron microscope image of nanoparticle 1.

[0034] Figure 2 The particle size distribution of nanoparticle 1 is shown, wherein the particle size is in the range of 300-500 nm, the average particle size is about 400 nm, and the PDI is <0.2.

[0035] Figure 3 The image shows that nanoparticle 1, captured by a high-speed camera, instantly produces bubbles after the first pulse is activated.

[0036] Figure 4 The image shows that after multiple injections of nanoparticles 1 into the liver, ultrasonic irradiation created a honeycomb-like fragmented area in the local area.

[0037] Figure 5 The results show that the ultrasonic energy required to achieve a significant tissue fragmentation effect is significantly reduced when nanoparticles 1 are used.

[0038] Figure 6This shows the local tissue dissolution area formed on an isolated porcine liver by local injection of 3 ml of nanoparticles and low-energy ultrasound irradiation.

[0039] Figure 7 This is an HE staining image of the excised liver tissue of specimen 6 after it has been pulverized and dissolved. In this image, a large amount of light red staining of dissolved necrotic material can be seen in the central part.

[0040] Figure 8 The image shows fragmented porcine liver tissue formed by the sequential injection of 1H-perfluoropentane fluorocarbon particles of different sizes in layers. Detailed Implementation

[0041] The following description provides exemplary embodiments of this application, including various details to aid understanding, and should be considered merely exemplary. Therefore, those skilled in the art will recognize that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of this application. Similarly, for clarity and brevity, descriptions of well-known functions and structures are omitted in the following description.

[0042] Unless otherwise stated, the terms used in this application have the meanings commonly known in the art.

[0043] In the first aspect, this application establishes a novel algorithm capable of theoretical calculations and design for technologies based on a combination of low-temperature phase change nanoparticles and a chain reaction cavitation model. Based on this novel algorithm, this application can also preliminarily screen suitable raw materials and ultrasonic parameters for preparing nanoparticles that can initiate chain reactions at low ultrasonic energies, thereby facilitating the design of nanoparticles and ultrasonic therapy systems.

[0044] In a second aspect, this application provides a low-temperature phase change nanoparticle comprising: (a) a liquid core containing a fluorocarbon compound or an azeotropic composition thereof having a boiling point of -20°C to 50°C at room temperature and pressure; and (b) a shell composed of a phospholipid material or a polymer material and optionally modified with targeting molecules; wherein: the morphology of the nanoparticle is a regular or irregular spherical particle, or a regular or irregular microplate, or a regular or irregular micromicelle; the low temperature refers to a temperature of -20°C to 50°C; and the particle size of the nanoparticle is 100-800 nm.

[0045] The term "particle size" as used in this application refers to the diameter of a particle. Since most particles are not perfectly spherical but irregular, the equivalent particle size is often used to describe their size. The equivalent particle size is the diameter of an irregular particle converted into an equivalent sphere using an equivalent method, used to describe its size. Therefore, in this application, "particle size" and "equivalent particle size" are used interchangeably. In this application, the particle size of the nanoparticles is measured using scanning electron microscopy (SEM). Specifically, by selecting a number of particles (e.g., 10, 50, 100, etc.) from the SEM image, adding the diameters of these particles, and then dividing by the total number of particles, the linear average diameter of the particles is obtained. That is, both "particle size" and "equivalent particle size" mentioned in this application refer to the aforementioned average diameter.

[0046] In one embodiment, the particle size of the nanoparticles of this application ranges from a lower limit of 100 nm, or 150 nm, or 200 nm, or 250 nm, or 300 nm, or 350 nm, or 400 nm, or 450 nm, or 500 nm, or 550 nm, or 600 nm, or 650 nm, or 750 nm to an upper limit of 150 nm, or 200 nm, or 250 nm, or 300 nm, or 350 nm, or 400 nm, or 450 nm, or 500 nm, or 550 nm, or 600 nm, or 650 nm, or 750 nm, or 800 nm. For example, the particle size of the nanoparticles of this application can be selected from the following ranges: 100-800 nm, 100-600 nm, 100-400 nm, 200-600 nm, 200-400 nm.

[0047] In one embodiment, the outer shell is capable of expanding, contracting, or rupturing within the local microenvironment of the tissue; the liquid core, driven by external temperature or ultrasonic irradiation, achieves a cyclical chain-like phase change cavitation process of micro-boiling-cavitation-breakup-condensation-penetration within the tissue, utilizing the mechanical force generated by the phase change to lyse the cells and tissue structures at the location. In another embodiment, after the liquid core is injected into the body, external heating of the intended treatment site refers to using techniques such as local hot compresses, infrared heating, or microwave heating to raise the temperature of the area containing the low-temperature phase change nanoparticles to 37-50°C. In this application, "injection into the body" refers to intravascular injection, local percutaneous or cavity injection into the lesion site for related treatment, or injection into the lymphatic drainage area to perform chain-like cavitation treatment on lymphatic vessels and lymph nodes.

[0048] In one embodiment, the fluorocarbon compound contained in the low-temperature phase change nanoparticles of this application has a boiling point of 35°C to 50°C, particularly 36°C to 50°C, particularly 37°C to 50°C, particularly 38°C to 50°C, particularly 39°C to 50°C, and preferably 40°C to 50°C, at room temperature and pressure. The boiling point is measured using a boiling point meter.

[0049] In one embodiment, the fluorocarbon compound is selected from: 1H-perfluoropentane, decafluorobutane, dodecafluoropentane, cis-1-chloro-3,3,3-trifluoropropene (HCFO-1233zd(Z)), 1,1,1,3,3-pentafluorobutane (HFC-365mfc), perfluorohexane, perfluorocyclopentane, perfluorodimethylcyclobutane, perfluoromethylcyclopentane, perfluorotert-butylamine, perfluorotrimethylcyclopropane, perfluoromethylcyclohexane, and so on. Fluorobutylethane, perfluoroheptane, perfluoro(3-methylhexane), perfluoro(2-methylpentane), perfluoropentane and its branched isomers, perfluorooctane, hydrofluoroalkanes, hydrofluoroolefins, perfluoro-1,2-dimethylcyclobutane, or combinations thereof; more preferably cis-1-chloro-3,3,3-trifluoropropene (HCFO-1233zd(Z)), 1,1,1,3,3-pentafluorobutane (HFC-365mfc), or 1H-perfluoropentane.

[0050] In one embodiment, the shell material of the low-temperature phase change nanoparticles of this application is selected from: 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC), cholesterol, 1,2-distearyl-sn-glycerol-3-phosphoethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), Tween, lecithin, dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylcholine-polyethylene glycol 2000 (DPPC-PEG2000), matrix metalloproteinase-2 (MMP-2) or matrix metalloproteinase-9 (MMP-9) cleavable peptide polymers, disulfide-containing polymers, polyhistidine, polyethylene glycol-polylactic acid (PEG-P) LA), polylactic acid-glycolic acid copolymer (PLGA), poloxamer F127, poloxamer F68, chitosan, poly-N-isopropylacrylamide (PNIPAM) or copolymers thereof, cyclodextrin-based supramolecular assemblies, mesoporous silica, metal-organic frameworks (MOFs), silica nanoparticles, fluorocarbon-MOFs composites, perfluorooctanoic acid-hydrazone-drug conjugates, perfluorooctane sulfonate diethylamine salt, other antibody conjugates or combinations thereof; more preferably 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC), 1,2-distearyl-sn-glycerol-3-phosphoethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), lecithin.

[0051] In one embodiment, the shell of the nanoparticles of this application is modified with a targeting molecule. The targeting molecule is selected from: integrin αvβ3 receptor, folic acid receptor, prostate-specific membrane antigen, pancreatic stellate cells, phosphatidylinositol proteoglycan-3 (GPC-3), desialyl glycoprotein receptor (ASGPR), mesothelin (MSLN), carcinoembryonic antigen-associated cell adhesion molecule 5 (CEACAM5), carbonic anhydrase IX (CAIX), EGFR (epidermal growth factor receptor), c-Met, and combinations thereof.

[0052] In one embodiment, the acoustic droplet evaporation (ADV) threshold pressure of the nanoparticles of this application. P th The range is defined by the following relation:

[0053] in Where is the particle radius, The pressure is standard atmosphere, and σ is surface tension. This is the vapor pressure.

[0054] In one embodiment, the nanoparticles of this application are prepared using a sequential injection strategy, wherein the particle size of the first injected nanoparticles is 200-600 nm, and the particle size of the subsequently injected nanoparticles is 1000-6000 nm or 1000-5000 nm; the volume ratio of the first injection to the subsequent injections is 0.1:1 to 10:1, or 1:1 to 10:1, or 0.1:1 to 1:1. In the sequential injection strategy, for nanoparticles with a particle size of 200-600 nm, the mass concentration of the nanoparticles is greater than or equal to 0.5 mg / mL, and the particle number concentration is 10. 7 Up to 10 10 For nanoparticles with a diameter of 1000-6000 nm, the mass concentration of nanoparticles is greater than or equal to 0.5 mg / mL, and the number concentration of nanoparticles is 10. 5 Up to 10 8 per mL.

[0055] In one embodiment, the reliquefaction time constant of the bubble fragments generated by the nanoparticles of this application after ultrasonically triggered phase transition. The time ranges from 50 milliseconds to 1000 milliseconds.

[0056] In one embodiment, the particle size of the nanoparticles of this application is selected based on the center frequency of the ultrasound therapy system to satisfy the resonance matching condition: (1) When the center frequency is 2 to 5 MHz, the particle size of the nanoparticles is 200-2000 nm; (2) When the center frequency is 10 to 13 MHz, the particle size of the nanoparticles is 200-600 nm.

[0057] In a third aspect, this application provides an ultrasound therapy system for achieving chain reaction cavitation, comprising: A nanoparticle delivery module for delivering the low-temperature phase change nanoparticles described in this application to a target tissue region; An ultrasonic transducer array configured to emit ultrasonic pulses toward the target tissue region and receive echo signals for imaging; A controller, which is communicatively connected to the ultrasonic transducer array to control the ultrasonic transducer array.

[0058] In one embodiment, the controller of the ultrasound therapy system includes the following units: a. A cavitation nucleus status monitoring unit, which estimates the density or distribution of effective cavitation nuclei within the target area based on echo signals; a. A cavitation nucleus status monitoring unit that estimates the density or distribution of effective cavitation nuclei within a target area based on echo signals; b. An adaptive pulse sequence generation unit that dynamically generates and controls the ultrasonic pulse sequence emitted by the ultrasonic transducer array based on the estimated density or distribution of effective cavitation nuclei in the target area. The parameters in the adaptive pulse sequence generation unit are set to trigger and maintain a self-sustaining chain reaction cavitation process. c. Safety monitoring unit, which monitors safety parameters in real time, including mechanical index (MI) and spatial peak time average intensity (ISPTA), and adjusts the pulse parameters to keep them within preset safety thresholds.

[0059] In one embodiment, the adaptive pulse sequence generation unit of the ultrasound therapy system is configured to generate a dual-pulse sequence or a single-pulse sequence, the dual-pulse sequence or dual-pulse sequence comprising: Leader pulse: Its peak negative pressure is based on the acoustic evaporation (ADV) threshold pressure of the nanoparticles. P th This setting is used to trigger droplet vaporization; Main pulse: Its peak negative pressure is less than or equal to that of the pilot pulse. It is used to drive the generated cavitation bubbles to undergo inertial collapse and generate mechanical shear force.

[0060] In one embodiment, the ultrasound pulse sequence employs any of the following pulse sequences: The single-pulse sequence has a center frequency of 1.0-13 MHz, preferably 1.0-5 MHz, more preferably 1.5-5 MHz, a peak negative voltage of 1-13 MPa, preferably 1-10 MPa, more preferably 1-5 MPa, a pulse length of 1-50 cycles, preferably 1-30 cycles, a pulse repetition frequency (PRF) of 1-50 Hz, preferably 10-20 Hz, and a duty cycle of less than 1%. The dual-pulse sequence has the following characteristics: a center frequency of 1.0-13 MHz, preferably 1.0-10 MHz, more preferably 1.0-5 MHz; a leader pulse length of 2-20 cycles, preferably 5-10 cycles; a pulse repetition frequency (PRF) of 1-50 Hz, preferably 10-15 Hz; and a peak negative voltage of 1-13 MPa, preferably 1-10 MHz, more preferably 1-5 MPa. The main pulse length is 1-30 cycles, preferably 5-25 cycles; the pulse repetition frequency (PRF) is 1-50 Hz; and the peak negative voltage is 1-13 MPa, preferably 1-5 MPa, more preferably 1-3 MPa. The interval between the two pulses is 5-800 ms.

[0061] In one embodiment, the ultrasonic transducer array is a handheld probe that employs a concentric or interlaced array structure, with the central high-frequency array element used for ultrasonic imaging and the peripheral mid-to-low-frequency array elements used for emitting therapeutic ultrasonic pulses.

[0062] In one implementation, the cavitation nucleus state monitoring unit estimates the cavitation nucleus density using an iterative algorithm. N The algorithm is based on a chain reaction kinetic model, the general formula of which is: ,in k λ is the fragmentation proliferation factor, λ is the tissue loss rate, and Δ is the tissue loss rate. t The pulse interval.

[0063] In one embodiment, the ultrasound therapy system is configured to optimize mechanical force density. To set treatment parameters, the density and cavitation core density are... N Bubble collapse pressure Pulse frequency and tissue-specific correction coefficient and efficiency coefficient η Positive correlation.

[0064] In one implementation, the peak negative pressure of the pilot pulse is obtained by querying a nanoparticle parameter database pre-stored in the controller, the database containing at least the ADV threshold pressure corresponding to nanoparticles of different sizes and compositions. P th The measured or calculated value.

[0065] In one embodiment, in the ultrasound therapy system, the controller selects the optimal particle size range and adjusts the pulse sequence parameters based on the ultrasound center frequency, wherein: (1) When the center frequency is 2-5 MHz, the pulse repetition frequency (PRF) is set to 10-100 Hz, the peak negative pressure is 1.0-10.0 MPa, and the particle size of the nanoparticles is 200-2000 nm; (2) When the center frequency is 10-13 MHz, the pulse repetition frequency (PRF) is set to 5-500 Hz, the peak negative pressure is 2.0-13 MPa, and the particle size of the nanoparticles is 200-600 nm.

[0066] In a fourth aspect, this application provides the use of the ultrasonic therapy system of this application for achieving the fragmentation of target tissue, characterized in that the achievement of said use includes the following steps: (I) Introducing the low-temperature phase change nanoparticles described in this application into the target tissue; (II) Monitoring the cavitation nucleus state of the target area using ultrasonic imaging; (III) Based on monitoring results, adaptively emit ultrasonic pulse sequences to trigger chain reaction cavitation and optionally maintain chain reaction cavitation; and (IV) Monitor safety parameters in real time and adjust ultrasound energy output to ensure treatment safety.

[0067] In one embodiment, step (III) of maintaining chain reaction cavitation includes: controlling the pulse repetition frequency (PRF) such that the pulse interval is greater than the reliquefaction time constant of the nanoparticle bubble fragments in the target tissue environment. .

[0068] In one implementation, the cavitation nucleus state monitoring unit estimates the cavitation nucleus density using an iterative algorithm. N The algorithm is based on a chain reaction kinetic model, the general formula of which is: ,in k λ is the fragmentation proliferation factor, λ is the tissue loss rate, and Δ is the tissue loss rate. t The pulse interval.

[0069] In one embodiment, the ultrasound therapy system is configured to optimize mechanical force density. To set treatment parameters, the density and cavitation core density are... N Bubble collapse pressure Pulse frequency and tissue-specific correction coefficient and efficiency coefficient η Positive correlation.

[0070] In one embodiment, step (I) of introducing nanoparticles employs the following layered injection strategy: first, nanoparticles with a particle size of 200-600 nm are injected, allowing them to diffuse into the deep interstitial tissue; after an interval of 2-5 minutes, nanoparticles with a particle size of 1000-6000 nm are injected to form a cavitation-enhancing core in the superficial to middle tissue layers; the strategy improves the efficiency of the chain reaction and the treatment coverage through a synergistic effect.

[0071] In one embodiment, in step (III), the step does not include maintaining chain reaction cavitation, but instead directly triggers the cavitation effect through an ultrasonic pulse sequence, wherein the pulse repetition frequency (PRF) is set to 10-1000 Hz, the peak negative pressure is set to 0.5-15 MPa, the center frequency is set to 1.5-20 MHz, and the duty cycle is less than 0.1%; the fluorocarbon compound of the liquid core has a boiling point of -10℃ to 50℃, so as to achieve effective tissue fragmentation by optimizing the cavitation threshold and mechanical force output, without relying on the chain reaction mechanism; wherein, fluorocarbon compounds with a boiling point below 37℃ reduce the ADV threshold through their high vapor pressure characteristics, and fluorocarbon compounds with a boiling point above 37℃ enhance the cavitation controllability through phase change stability.

[0072] Example Example 1: System Modeling and Design Example 1-1: Mathematical Modeling and Calculation of the Core Theoretical Model System This embodiment will provide a detailed explanation of the derivation process of the entire algorithm for the model.

[0073] 1. Calculation model for the threshold of acoustically induced droplet evaporation (ADV) ADV threshold pressure This is a key parameter triggering the phase transition of fluorocarbon droplets, derived based on classical nucleation theory. A shape correction coefficient is introduced. The corrected formula is as follows:

[0074] in: ● (Environmental hydrostatic pressure) ● (Surface tension at the liquid-gas interface, taking perfluorohexane as an example). ● (equivalent radius, (for shape correction factor) ● The saturated vapor pressure at the operating temperature (e.g., perfluorohexane at 37°C). ), ● (Correction factor, compensating for tissue heterogeneity and nucleation barrier).

[0075] ● Mathematical modeling and examples of shape correction factor Shape correction factor Used to quantify the effect of non-spherical fragments (such as sheet-like or irregular shapes) on cavitation dynamics. Defined as follows: ●Mathematical definition: ,in This represents the actual surface area of ​​the fragment. This is the surface area of ​​an equivalent sphere of the same volume. For spherical fragments, For non-spherical fragments, (Typical value 1.2-2.0).

[0076] ●Physical meaning: At that time, the surface area of ​​the fragments increases, affecting the reliquefaction time, proliferation factor, and loss rate.

[0077] ●Example calculation: Assuming the spherical fragment has a diameter d = 1 μm and a surface area of... If the actual fragments are in the form of flakes, ,but This results in a shorter reliquefaction time (due to the larger surface area and faster condensation), but a lower proliferation factor (poorer fragment stability). Shape correction coefficients will be integrated into subsequent models to enhance realism.

[0078] This model reveals the relationship between particle size and energy demand: smaller particles, due to higher Laplace pressure, require stronger ultrasonic negative pressure to trigger a phase transition. After shape correction, When the equivalent radius decreases, the Laplace pressure term increases, and the ADV threshold rises.

[0079] Numerical examples: ●Nanoparticles (radius) N , ):

[0080]

[0081] ● Micron-sized particles (radius) , ):

[0082] Treatment sound pressure settings: To ensure reliable cavitation, the peak ultrasonic pressure is negative. Set to 5-10 times the ADV threshold: ●Nanoparticles: (rounded to the nearest integer) ), ●Micron-sized particles: (rounded to the nearest integer) ).

[0083] 2. The principle and analysis of chain reaction-enhanced low-energy tissue fragmentation 2.1 Bubble Breaking and Fragment Generation Bubbles grow during ultrasonic negative pressure cycles and collapse during positive pressure cycles, producing fragments. The number of fragments depends on the bubble size and sound pressure. Collapse generates a high-speed jet and fragments. The number of fragments depends on the bubble size and sound pressure. Simplified according to the Rayleigh-Plesset equations, the maximum bubble radius η is related to the sound pressure:

[0084] in The initial droplet radius. Collapse time. for:

[0085] in (Tissue density) .

[0086] After the collapse, each bubble produces Number of fragments Typically 2-10 (based on experimental data). Fragment particle size is usually smaller than the original droplet, making it easier to disperse. However, droplets with a boiling point of 42°C are prone to condensation and reliquefaction at body temperature, thus reducing dispersion depletion.

[0087] After introducing shape correction, the fragment proliferation factor is corrected to:

[0088] in (Spherical fragment proliferation factor), α ≈ 0.5 (empirical index). When k' decreases.

[0089] After the bubble bursts, the fragments lose the coating of the fluorocarbon droplet film, the Laplace pressure term disappears, and the ADV threshold decreases.

[0090] Numerical examples:

[0091] This value is lower than the initial ADV threshold, indicating that the fragments are more prone to cavitation, which is conducive to chain reactions.

[0092] 2.2 Chain Reaction Kinetic Model The chain reaction involves a cycle of cavitation, breakup, condensation, and recavitation. Droplet density updates are based on a discrete-time model, with shape corrections introduced:

[0093] in: ● The droplet density after the nth pulse (unit: droplets / mm) 3 ), ● (Modified proliferation factor) ● (Corrected loss rate, β ≈ 0.5, λ = 0.5 s) -1 ), ● (Pulse interval, unit: s).

[0094] Chain reaction constraint model The chain reaction requires that the cavitation debris can be liquefied again to serve as new cavitation nuclei, which affects the choice of particle size. ●Reliquefaction time: (Based on a diffusion model), therefore, larger particle sizes require longer reliquefaction times.

[0095] ● Proliferation factor: The number of fragments k may vary with particle size, but the document assumes k = 5 (a constant). After shape correction... (α ≈ 0.5).

[0096] ●Loss rate: After shape correction (β ≈ 0.5, λ = 0.5 s) -1 ).

[0097] Chain reaction sustainability requires pulse intervals satisfy Therefore, larger particle size allows for lower PRF.

[0098] Initial conditions: droplets / mm 3 (Typical injection concentration).

[0099] Saturation condition: droplets / mm 3 (Tissue porosity limitation).

[0100] Saturation pulse number satisfy ,

[0101] Saturation time .

[0102] Numerical examples: ● Micron-sized particles: PRF = 50 Hz ( ), λ' = 0.5 × 1.5^{0.5} ≈ 0.61 s -1 , k'= 5 × 1.5^{-0.5} ≈ 4.08:

[0103] ● Nanoparticles: PRF = 10 Hz ( ), λ' = 0.61 s -1 k' = 4.08

[0104] 2.3 Consideration of condensation time: Condensation time Depending on the fragment size and undercooling, after introducing shape correction:

[0105] Where m ≈ 0.5. Typical value: ●Nano fragments (200-600 nm): , ●Micron-sized fragments (1-5 μm): The PRF must satisfy... To ensure condensation is complete: ●Nanoparticles: PRF < 5-10 Hz ( At that time, τ_cond' ≈ 122-244 ms, PRF < 4.1-8.2 Hz. Micron-sized particles: PRF < 10-100 Hz ( At that time, τ_cond' ≈ 12.2-122 ms, PRF < 8.2-82 Hz.

[0106] 2.4 Spatial Correction Coefficient and Efficiency Correction Coefficient Since cavitation occurs in the extracellular space, a correction factor needs to be introduced: ●Spatial correction factor: Definition: Based on tissue cell volume fraction (Typical soft tissue), ECM volume fraction .therefore, (Cavitation is limited to ECM).

[0107] (ECM volume fraction) ●Efficiency Correction Factor: Definition: The efficiency of intracellular cavitation directly pulverizing cells. Extracellular cavitation requires shear force to be generated through the cell membrane, which is inefficient. (Based on experimental estimation).

[0108] Therefore, for fluorocarbon droplets, (Extracellular cavitation efficiency).

[0109] ● Tissue complexity correction: The loss rate λ may vary depending on the tissue type (e.g., high-flow tissue λ ≈ 1-2 s). -1 Fibrotic tissue λ≈0.3 s -1 ). Saturation density It may also change (such as fibrotic tissue) ).

[0110] 2.5 Calculation of Mechanical Force Density Mechanical force density (Unit: MPa / s) Quantitative mechanical force output per unit volume per unit time:

[0111] in: ●Based on the simplification of the Rayleigh-Plesset equation (bubble collapse pressure,) ), ● (Pulse frequency).

[0112] Numerical comparison: ●Conventional HIFU (CFC-free droplets): ○ (Natural nucleus) ○ , ○ (Typical PRF) ○ (Cavitation is everywhere) ○ (Intracellular cavitation) , , , ,

[0113] ●Fluorocarbon droplets (involved in chain reactions): ○ (Saturation density) ○ , ○ (Nanoparticle PRF) ○ , ○ , droplets / mm3 , , , , , ,

[0114] ○ Mechanical force ratio:

[0115] The mechanical strength of fluorocarbon droplet-enhanced cavitation is approximately 4.7 times that of conventional HIFU.

[0116] 2.6 Differences between nanometer and micrometer particles and modulation strategies Summary of differences: ● Nanoparticles (200-600 nm): High ADV threshold (0.3-0.5 MPa), requiring high... (3-5 MPa), low PRF (5-10 Hz), k' decreases after shape correction.

[0117] ● Micron-sized particles (1-5 μm): Low ADV threshold (0.1-0.2 MPa), requiring low... (1-2 MPa), high PRF (10-20 Hz), relatively high k'.

[0118] Modulation strategy: ●Nanoparticles: Preferred for initial injection (3-5 μL, concentration) (droplets / mL) to achieve deep distribution, PRF set to 5-10 Hz. .

[0119] ● Micron-sized particles: used to enhance shear force, PRF set to 20-50 Hz. It can be injected in combination with nanoparticles.

[0120] 2.7 Boiling Point Selection Droplets with boiling points >37°C (such as perfluorohexane) are preferred to allow chain reactions; chain reactions are not feasible for droplets with boiling points <37°C (such as perfluorobutane) and are not recommended. Shape modification does not affect boiling point selection, but it does affect fragment kinetics.

[0121] 2.8 Safety Parameter Calculation: MI and ISPTA ● Machinery Index (MI): , where f is the center frequency (in MHz). For example, f = 2MHz It is recommended to keep it below 2.5.

[0122] ● Spatial Peak Temporal Average Intensity (ISPTA): (Unit: mW / cm) 2 ).For example, PRF = 10 Hz, f = 2 MHz It is far below the safety limit (100 mW / cm). 2 ).

[0123] 2.9 Actual chain reaction duration versus treatment time Chain reaction saturation time (0.1 s for micron-sized particles, 0.5 s for nanoparticles). Treatment time is typically 10-30 seconds to ensure complete tissue fragmentation. Influencing factors: The tissue loss rate λ may prolong the saturation time (e.g., λ = 1 s). -1 At that time, t_sat increases by about 20%.

[0124] 3. Safety Parameter Control Model To avoid damage to non-target tissues, safety indicators must be strictly controlled: • Machinery Index (MI): It is recommended to keep it below 2.5 (1.5–2.0 is commonly used for treatment). • Spatial Peak Time Mean Intensity (ISPTA): It is recommended not to exceed 100 mW / cm 2 The above models are all embedded in the controller algorithm to achieve real-time safety monitoring.

[0125] 4. Boiling Point Optimization Model To determine the optimal boiling point of the fluorocarbon compound for enhancing the cavitation chain reaction, a multi-objective optimization model was established, with the following core trade-offs: ●Energy optimization objective: Minimize the ADV threshold. ●Chain reaction efficiency target: Maximize the sustainability of cavitation cloud proliferation.

[0126] ●Key parameters: ● Boiling point of fluorocarbons (in °C). ● (Human body temperature) ● (Supercooling).

[0127] 4.1 Model of the Influence of Boiling Point on Energy Demand Based on the acoustically induced droplet evaporation (ADV) threshold formula, the boiling point affects energy demand through the vapor pressure term:

[0128] Vapor pressure-boiling point relationship (simplified Antoine equation):

[0129] in For latent heat of vaporization, is the gas constant.

[0130] Analysis conclusion: ●When (Right now ), ADV threshold Minimize, with the lowest energy requirement.

[0131] ●But too small This can cause droplets to nearly spontaneously evaporate at body temperature, resulting in poor stability.

[0132] 4.2 Model of the Influence of Boiling Point on the Duration of Chain Reactions The chain reaction requires that bubble fragments be reliquefied within the pulse interval to serve as nuclei for subsequent cavitation. The reliquefaction time constant... With supercooling Related:

[0133] Pulse Repetition Frequency (PRF) Constraint (to ensure complete reliquefaction):

[0134] Analysis conclusion: ● The larger the value (the higher the boiling point), the faster the reliquefaction, the higher the permissible PRF, and the faster the chain reaction proliferation rate.

[0135] ● But too large This will lead to an increase in the ADV threshold and an increase in energy demand.

[0136] 4.3 Multi-objective optimization model and optimal point solution Objective function:

[0137] in As a weighting factor, it reflects the relative importance of energy efficiency and chain reaction efficiency.

[0138] Constraints: 1. (To ensure the stability of the liquid at body temperature) 2. (Maximum negative pressure that the equipment can provide) 3. (Can be reliquefied within the pulse interval) Numerical solution and parameter fitting: ● Fluorocarbons with a boiling point of 56℃ exhibit weak chain reactions (PRF limited). ● Perfluoropentane (boiling point 29°C) case: low energy requirement but poor stability. ● Intermediate boiling point compounds (such as HCFO-1233zd(Z), boiling point 40℃) exhibit balanced performance Optimization results: ●Optimal boiling point range: (Right now ) ●Theoretical basis: ○ The ADV threshold for this interval is moderate. It can be triggered by low-energy ultrasound. ○ Reliquefaction time PRF is allowed at 10-100 Hz to meet the requirements of chain reaction. ○ The droplets are well stable at body temperature (they do not easily evaporate spontaneously). 4.4 Candidate Material Screening Preferred fluorocarbon compounds include: perfluorohexane (boiling point 56℃), HCFO-1233zd(Z) (boiling point 40℃), and perfluoropentane (boiling point 29℃). Based on the boiling point optimization model, compounds with boiling points of 40-50℃ are recommended.

[0139] 4.5 Preferred boiling point

[0140] Through mathematical modeling and theoretical analysis, the following key conclusions can be drawn: 1. Optimal boiling point range: It is the optimal range for balancing energy efficiency and chain reaction performance. 2. Physical Mechanism: This range provides sufficient supercooling (3-8℃) to ensure the reliquefaction rate while maintaining a low ADV threshold. 3. Parameter Coordination: It is recommended that the ultrasound parameters be set to PRF = 10-50 Hz. To match the optimal boiling point characteristics This optimization model provides a theoretical basis for screening fluorocarbons, and further optimization of the weighting factors is needed through experimental verification. The value of .

[0141] 5. Optimal particle size analysis of fluorocarbon droplets The optimal particle size r needs to be minimized. And maximize the chain reaction efficiency. Solve for r to make Maximize, with the following constraints: (The maximum negative pressure of the equipment is typically 15 MPa) and (Maximum pulse interval corresponds to minimum PRF).

[0142] 5.1 Comprehensive Evaluation System for Chain Reaction Efficiency Establish a three-dimensional evaluation model for the influence of particle size:

[0143] The key sub-performance indicators are: 1) Activation efficacy (negatively correlated with ADV threshold)

[0144] 2) Proliferation efficiency (positively correlated with fragment quantity and regeneration capacity)

[0145] in It is a particle size-related fragment proliferation factor. This is the reliquefaction efficiency function.

[0146] 3) Mechanical efficiency (positively correlated with bubble collapse energy)

[0147] 5.2 Particle Size Dependence Key Parameter Model 1. ADV threshold particle size effect:

[0148] The function increases sharply when r < 100 nm and tends to level off when r > 2 μm.

[0149] 2. Correlation between fragmentation proliferation factor and particle size: Experimental data fitting showed that smaller particle sizes produced more fragments, but the fragment stability was poor. After shape correction... .

[0150] 3. Reliquefaction timescale:

[0151] in , ΔT represents the degree of subcooling.

[0152] 5.3 Multi-objective optimization and Pareto front analysis The overall performance function was obtained through weight optimization (α=0.4, β=0.4, γ=0.2). Numerical results show that the optimal solution appears in the range of 750-1200 nm.

[0153] Numerical solution and recommended range Numerical calculations, based on file parameters: ● σ = 0.02 N / m (Typical value).

[0154] ● ,in , .

[0155] ● .

[0156] calculate For curves with r ranging from 100 nm to 5 μm, the peak appears near r ≈ 1.0 μm. Considering practical constraints (injection feasibility, tissue penetration), the recommended particle size range is: ● Nanoparticles: 200 - 600 nm (for high-permeability applications). ● Micron-sized particles: 1.0 - 3.0 μm (for low-energy triggering).

[0157] Example calculation: ● For r = 300 nm (nanoparticles). , :

[0158] ●For r=1.5 μm (micron particles) , :

[0159] micron-sized particles Lower values ​​make cavitation more likely to be triggered.

[0160] 6. Frequency and Particle Size Selection Analysis Based on the physical principles of acoustic cavitation and chain reaction kinetics, the optimal particle size selection at center frequencies of 2 MHz, 3.5 MHz, and 10 MHz is analyzed.

[0161] 6.1 Establishment of a General Mathematical Model 1. ADV threshold optimization model: The optimal particle size is based on minimizing P_th and maximizing the chain reaction efficiency.

[0162] 2. Multi-objective optimization weight function: Define a comprehensive scoring function S(r) to balance resonance efficiency, penetration ability and cavitation facilitation.

[0163] 6.2 Three-Frequency Modeling and Analysis ●Frequency 1: 2 MHz analysis (deep treatment): Penetration depth (5-8 cm), recommended particle size 1.0-3.0 μm (micrometer level), low P_th (0.1-0.2 MPa), PRF=5-10 Hz.

[0164] ●Frequency 2: 3.5 MHz analysis (medium depth): moderate penetration (3-5 cm), recommended particle size 0.5-1.0 μm (submicron), medium P_th (0.2-0.3 MPa), PRF=10-15 Hz.

[0165] ●Frequency 3: 10 MHz analysis (superficial treatment): shallow penetration (1-2 cm), recommended particle size 200-500 nm (nanometer level), high P_th (0.3-0.5 MPa), PRF=10-20 Hz.

[0166] After shape correction, particle size adjustment is based on However, the recommended scope remains unchanged.

[0167] Mathematical correlation: Frequency itself does not directly determine particle size, but it can be influenced by adjusting ultrasonic parameters (such as PRF and...). To adapt to the particle size. For example: ● For high frequencies of 10 MHz, a higher PRF (10-20 Hz) and higher [unclear] are typically required. (3-5 MPa) to trigger small particle cavitation.

[0168] ● For low frequencies of 2 MHz, a lower PRF (5-10 Hz) and a lower (1-2 MPa) is used for large particle size.

[0169] Recommended examples: ● 2 MHz: Particle size 1.0-3.0 μm, PRF=5-10 Hz .

[0170] ● 3.5 MHz: Particle size 0.5-1.0 μm, PRF=10-15 Hz .

[0171] ● 10 MHz: Particle size 200-500 nm, PRF = 10-20 Hz .

[0172] ● Integration and impact of shape correction Shape correction factor Effective in all models: ● ADV threshold: Increase Therefore, larger particle size compensation is required.

[0173] ● Chain reaction: Reduce proliferation factors and increase loss rate However, it shortens the reliquefaction time. .

[0174] ●Optimization results: At this time, the optimal particle size range shifts slightly towards larger particle sizes (for example, nanoparticles are adjusted from 200-600 nm to 250-700 nm, and microparticles are adjusted from 1.0-3.0 μm to 1.2-3.5 μm), but considering practical constraints, the recommended range remains unchanged.

[0175] Example calculation: When r = 300 nm, (10% taller than a sphere), therefore it needs to be even higher. However, by adjusting the PRF, the chain reaction can still continue.

[0176] ●Complete example calculation Hypothetical treatment scenario: deep liver tumor, using 2 MHz ultrasound, preferably micron-sized particles.

[0177] ●Parameters: , , σ = 0.02 N / m.

[0178] ● ADV threshold: calculated .

[0179] ●Therapeutic ultrasound settings: (Rounded to 1.0 MPa), PRF = 10 Hz (based on τ_cond ≈ 100 ms for micron particles).

[0180] ●Chain reaction simulation: N0 = 1000 droplets / mm 3 k' = 4.08, λ' = 0.61 s -1 Δt = 0.1s:

[0181] Saturation time t_sat ≈ 0.5 s (reaching N_max = 10) 6 droplets / mm 3 ).

[0182] This setting ensures security (MI= ) and effective.

[0183] Example 1-2: System Design 1. Engineering design of nanoparticles Monodisperse and stable phase change nanoparticles were prepared using high-pressure homogenization, thin-film hydration-extrusion, or microfluidic technology.

[0184] • Core materials: Fluorocarbon compounds with boiling points close to body temperature are preferred (such as perfluoropentane with a boiling point of 29°C and HCFO-1233zd(Z) with a boiling point of 40°C) to facilitate low-energy triggering; • Shell structure: Commonly used phospholipid mixtures (such as DSPC:cholesterol:DSPE-PEG2000 = 70:20:10 molar ratio) provide good biocompatibility and targeted modification sites; • Size control: Narrowly distributed particles (PDI < 0.2) can be obtained through membrane extrusion (pore size 100 nm–1 μm) or microfluidic adjustment. 200–600 nm particles facilitate deep penetration, while 1–5 μm particles enhance local shear forces.

[0185] 2. Intelligent Ultrasonic Control System The system consists of an ultrasonic transducer array, a controller, and an imaging module, and adopts a model predictive control (MPC) architecture.

[0186] • Transducer design: Handheld probe with concentric array – central high-frequency element (5–10 MHz) for B-mode imaging, and peripheral mid-to-low frequency elements (1–3 MHz) for therapeutic emission; • Control algorithm flow: • State estimator: Cavitation kernel density retrieved from echo signals ; • Adaptive pulse generator: dynamically adjusts single / double pulse sequence parameters; • Security monitoring module: Real-time calculation of MI and ISPTA, and automatic amplitude limiting.

[0187] Pulse sequence strategy:

[0188] 3. Treatment parameter optimization matrix Model-guided personalized treatment plans:

[0189] Injection strategy: Sequential injection method is adopted—first inject nanoparticles (200–600 nm, concentration 10) 9 To achieve widespread distribution, particles per mL are injected, followed by the injection of micron-sized particles (1–5 μm, concentration 10). 7 To enhance the shear effect, the total volume ratio was controlled between 1:1 and 10:1 (particles / mL).

[0190] Example 2: Preparation and Characterization of Nanoparticles In this embodiment, phospholipid-encapsulated fluorocarbon nanoparticles were prepared using phospholipids and a fluorocarbon compound, and the nanoparticles were characterized. The materials and equipment used are shown below.

[0191] Materials and Equipment

[0192]

[0193] Example 2-1: Preparation of nanoparticles 1 Step 1: Formula Calculation This example uses the preparation of a 10 ml nanoparticle suspension as an example, with a target concentration of 1.8 mg / ml.

[0194] The total mass of the nanoparticles = 1.8 mg / ml × 10 ml = 18 mg. To ensure stability, it is assumed that the phospholipid shell mass fraction is 10%, the phospholipid mass = 1.8 mg, and the fluorocarbon mass = 16.2 mg. In actual preparation, to compensate for losses, excess phospholipid (e.g., 2 mg) and fluorocarbon (e.g., 16 mg) are used, for a total mass of 18 mg. Solvent volume: 1 ml chloroform (to dissolve the phospholipid), 9 ml aqueous phase (PBS).

[0195] Step 2: Preparation of organic phase Weigh 2 mg of DSPC and dissolve it in 1 ml of chloroform, vortexing over approximately 5 minutes until completely dissolved. Add 16 mg (approximately 10.2 μl) of 1H-perfluoropentane (density approximately 1.573 g / ml) and gently vortex, avoiding vigorous stirring to prevent evaporation of the 1H-perfluoropentane.

[0196] Step 3: Prepare the aqueous phase Measure 9 ml of PBS and place it in an open container.

[0197] Step 4: Preliminary emulsification The organic phase prepared above was slowly injected into the aqueous phase prepared above, while the mixture was stirred at 10,000 rpm for 2 minutes using an Ultra-Turrax high-speed shear mixer to form a crude emulsion.

[0198] Step 5: High-pressure homogenization at low temperature Transfer the crude emulsion obtained in step 4 to the sample cell of a high-pressure homogenizer and cycle it 10 times at a temperature of 3-4°C. Check the temperature after each cycle to ensure stability. If the particle size is too large, increase the pressure to 2000 bar or the number of cycles to 15; if the particle size is too small, decrease the pressure to 1000 bar.

[0199] Step 6: Remove solvent Chloroform was evaporated under reduced pressure at 40°C using a rotary evaporator for approximately 30 minutes.

[0200] Alternatively, place the emulsion into a dialysis bag and dialyze it against the PBS for 24 hours. Change the solution every 8 hours.

[0201] Step 7: Adjust the concentration Measure the final volume; if less than 10 ml, make up the difference with PBS. Take a small amount of sample and calculate the concentration using a gravimetric method (dry weighing) or a phospholipid assay (such as Bartlett's phosphate assay). Adjust the concentration to 1.8 mg / ml.

[0202] Step 8: Storage Store the nanoparticle suspension at 4°C away from light and avoid freezing.

[0203] Step 9: Quality Control Verify the concentration using gravimetric or UV spectrophotometric methods. Measure particle size and concentration periodically (on days 1, 7, and 14) to check for precipitation or aggregation.

[0204] Step 10: Characterization Particle size and polydispersity index (PDI) were measured immediately after each preparation using DLS. Particle morphology (spherical, core-shell structure) was observed by transmission electron microscopy (TEM).

[0205] result: The prepared nanoparticles 1 all have the following parameters: - Particle size: At a pressure of 1500 bar, the average particle size is approximately 100-400 nm, and the PDI is <0.2.

[0206] - Concentration: Through optimization, the concentration is 1.8 mg / ml ± 0.2 mg / ml.

[0207] - Encapsulation efficiency: >90% (fluorocarbon compounds are effectively encapsulated by phospholipids).

[0208] - Stability: Stable at 4°C for at least 4 weeks with particle size change <10%.

[0209] Figure 1 Electron micrographs of the prepared nanoparticles 1 are shown. Figure 2 The particle size distribution of nanoparticle 1 is shown.

[0210] Example 2-2: Preparation of nanoparticles 2 Step 1: Formula Calculation This example uses the preparation of a 10 ml nanoparticle suspension as an example, with a target concentration of 1.8 mg / ml.

[0211] The total mass of the nanoparticles = 1.8 mg / ml × 10 ml = 18 mg. To ensure stability, it is assumed that the phospholipid shell mass fraction is 10%, the phospholipid mass = 1.8 mg, and the fluorocarbon mass = 16.2 mg. In actual preparation, to compensate for losses, excess phospholipid (e.g., 2 mg) and fluorocarbon (e.g., 16 mg) are used, for a total mass of 18 mg. Solvent volume: 1 ml chloroform (to dissolve the phospholipid), 9 ml aqueous phase (PBS).

[0212] Step 2: Preparation of organic phase Weigh 2 mg of DSPE and dissolve it in 1 ml of chloroform, vortexing over approximately 5 minutes until completely dissolved. Add 16 mg (approximately 10.2 μl) of 1H-perfluoropentane (density approximately 1.573 g / ml) and gently vortex, avoiding vigorous stirring to prevent evaporation of the 1H-perfluoropentane.

[0213] Step 3: Prepare the aqueous phase Measure 9 ml of PBS and place it in an open container.

[0214] Step 4: Preliminary emulsification The organic phase prepared above was slowly injected into the aqueous phase prepared above, while the mixture was stirred at 10,000 rpm for 2 minutes using an Ultra-Turrax high-speed shear mixer to form a crude emulsion.

[0215] Step 5: High-pressure homogenization at low temperature Transfer the crude emulsion obtained in step 4 to the sample cell of a high-pressure homogenizer and cycle it 10 times at a temperature of 3-4°C. Check the temperature after each cycle to ensure stability. If the particle size is too large, increase the pressure to 2000 bar or the number of cycles to 15; if the particle size is too small, decrease the pressure to 1000 bar.

[0216] Step 6: Remove solvent Chloroform was evaporated under reduced pressure at 40°C using a rotary evaporator for approximately 30 minutes.

[0217] Alternatively, place the emulsion into a dialysis bag and dialyze it against the PBS for 24 hours. Change the solution every 8 hours.

[0218] Step 7: Adjust the concentration Measure the final volume; if less than 10 ml, make up the difference with PBS. Take a small amount of sample and calculate the concentration using a gravimetric method (dry weighing) or a phospholipid assay (such as Bartlett's phosphate assay). Adjust the concentration to 1.8 mg / ml.

[0219] Step 8: Storage Store the nanoparticle suspension at 4°C away from light and avoid freezing.

[0220] Step 9: Quality Control Verify the concentration using gravimetric or UV spectrophotometric methods. Measure particle size and concentration periodically (on days 1, 7, and 14) to check for precipitation or aggregation.

[0221] Step 10: Characterization Particle size and polydispersity index (PDI) were measured immediately after each preparation using DLS. Particle morphology (spherical, core-shell structure) was observed by transmission electron microscopy (TEM).

[0222] result: The prepared nanoparticles 2 all have the following parameters: - Particle size: At a pressure of 1500 bar, the average particle size is approximately 100-400 nm, and the PDI is <0.2.

[0223] - Concentration: Through optimization, the concentration is 1.8 mg / ml ± 0.2 mg / ml.

[0224] - Encapsulation efficiency: >90% (fluorocarbon compounds are effectively encapsulated by phospholipids).

[0225] - Stability: Stable at 4°C for at least 4 weeks with particle size change <10%.

[0226] The morphology and particle size distribution of nanoparticle 2 are similar to those of nanoparticle 1.

[0227] Examples 2-3: Preparation of nanoparticles 3 Step 1: Formula Calculation This example uses the preparation of a 10 ml nanoparticle suspension as an example, with a target concentration of 1.8 mg / ml.

[0228] The total mass of the nanoparticles = 1.8 mg / ml × 10 ml = 18 mg. To ensure stability, it is assumed that the phospholipid shell mass fraction is 10%, the phospholipid mass = 1.8 mg, and the fluorocarbon mass = 16.2 mg. In actual preparation, to compensate for losses, excess phospholipid (e.g., 2 mg) and fluorocarbon (e.g., 16 mg) are used, for a total mass of 18 mg. Solvent volume: 1 ml chloroform (to dissolve the phospholipid), 9 ml aqueous phase (PBS).

[0229] Step 2: Preparation of organic phase Weigh 2 mg of DSPC and dissolve it in 1 ml of chloroform, vortexing over approximately 5 minutes until completely dissolved. Add 16 mg (approximately 12.6 μl) of HFC-365mfc (density approximately 1.27 g / ml) and gently vortex to mix, avoiding vigorous stirring which could cause HFC-365mfc to evaporate.

[0230] Step 3: Prepare the aqueous phase Measure 9 ml of PBS and place it in an open container.

[0231] Step 4: Preliminary emulsification The organic phase prepared above was slowly injected into the aqueous phase prepared above, while the mixture was stirred at 10,000 rpm for 2 minutes using an Ultra-Turrax high-speed shear mixer to form a crude emulsion.

[0232] Step 5: Perform the procedure at low temperature using a liposome extruder. The crude emulsion from step 4 was extruded through a 0.4 μm membrane 20 times.

[0233] Step 6: Remove solvent Chloroform was evaporated under reduced pressure at 40°C using a rotary evaporator for approximately 30 minutes.

[0234] Alternatively, place the emulsion into a dialysis bag and dialyze it against the PBS for 24 hours. Change the solution every 8 hours.

[0235] Step 7: Adjust the concentration Measure the final volume; if less than 10 ml, make up the difference with PBS. Take a small amount of sample and calculate the concentration using a gravimetric method (dry weighing) or a phospholipid assay (such as Bartlett's phosphate assay). Adjust the concentration to 1.8 mg / ml.

[0236] Step 8: Storage Store the nanoparticle suspension at 4°C away from light and avoid freezing.

[0237] Step 9: Quality Control Verify the concentration using gravimetric or UV spectrophotometric methods. Measure particle size and concentration periodically (on days 1, 7, and 14) to check for precipitation or aggregation.

[0238] Step 10: Characterization Particle size and polydispersity index (PDI) were measured immediately after each preparation using DLS. Particle morphology (spherical, core-shell structure) was observed by transmission electron microscopy (TEM).

[0239] result: The prepared nanoparticles 3 all have the following parameters: - Particle size: Average particle size is approximately 100-400 nm, PDI < 0.2.

[0240] - Concentration: Through optimization, the concentration is 1.8 mg / ml ± 0.2 mg / ml.

[0241] - Encapsulation efficiency: >90% (fluorocarbon compounds are effectively encapsulated by phospholipids).

[0242] - Stability: Stable at 4°C for at least 4 weeks with particle size change <10%.

[0243] The morphology and particle size distribution of nanoparticle 3 are similar to those of nanoparticle 1.

[0244] Examples 2-4: Preparation of Nanoparticles 4 Step 1: Formula Calculation This example uses the preparation of a 10 ml nanoparticle suspension as an example, with a target concentration of 1.8 mg / ml.

[0245] The total mass of the nanoparticles = 1.8 mg / ml × 10 ml = 18 mg. To ensure stability, it is assumed that the phospholipid shell mass fraction is 10%, the phospholipid mass = 1.8 mg, and the fluorocarbon mass = 16.2 mg. In actual preparation, to compensate for losses, excess phospholipid (e.g., 2 mg) and fluorocarbon (e.g., 16 mg) are used, for a total mass of 18 mg. Solvent volume: 1 ml chloroform (to dissolve the phospholipid), 9 ml aqueous phase (PBS).

[0246] Step 2: Preparation of organic phase Weigh 2 mg of DSPE and dissolve it in 1 ml of chloroform, vortexing over approximately 5 minutes until completely dissolved. Add 16 mg (approximately 12.6 μl) of HFC-365mfc (density approximately 1.27 g / ml) and gently vortex to mix, avoiding vigorous stirring which could cause HFC-365mfc to evaporate.

[0247] Step 3: Prepare the aqueous phase Measure 9 ml of PBS and place it in an open container.

[0248] Step 4: Preliminary emulsification The organic phase prepared above was slowly injected into the aqueous phase prepared above, while the mixture was stirred at 10,000 rpm for 2 minutes using an Ultra-Turrax high-speed shear mixer to form a crude emulsion.

[0249] Step 5: High-pressure homogenization at low temperature Transfer the crude emulsion obtained in step 4 to the sample cell of a high-pressure homogenizer and cycle it 10 times at a temperature of 3-4°C. Check the temperature after each cycle to ensure stability. If the particle size is too large, increase the pressure to 2000 bar or the number of cycles to 15; if the particle size is too small, decrease the pressure to 1000 bar.

[0250] Step 6: Remove solvent Chloroform was evaporated under reduced pressure at 40°C using a rotary evaporator for approximately 30 minutes.

[0251] Alternatively, place the emulsion into a dialysis bag and dialyze it against the PBS for 24 hours. Change the solution every 8 hours.

[0252] Step 7: Adjust the concentration Measure the final volume; if less than 10 ml, make up the difference with PBS. Take a small amount of sample and calculate the concentration using a gravimetric method (dry weighing) or a phospholipid assay (such as Bartlett's phosphate assay). Adjust the concentration to 1.8 mg / ml.

[0253] Step 8: Storage Store the nanoparticle suspension at 4°C away from light and avoid freezing.

[0254] Step 9: Quality Control Verify the concentration using gravimetric or UV spectrophotometric methods. Measure particle size and concentration periodically (on days 1, 7, and 14) to check for precipitation or aggregation.

[0255] Step 10: Characterization Particle size and polydispersity index (PDI) were measured immediately after each preparation using DLS. Particle morphology (spherical, core-shell structure) was observed by transmission electron microscopy (TEM).

[0256] result: The prepared nanoparticles 4 all have the following parameters: - Particle size: At a pressure of 1500 bar, the average particle size is approximately 100-400 nm, and the PDI is <0.2.

[0257] - Concentration: Through optimization, the concentration is 1.8 mg / ml ± 0.2 mg / ml.

[0258] - Encapsulation efficiency: >90% (fluorocarbon compounds are effectively encapsulated by phospholipids).

[0259] - Stability: Stable at 4°C for at least 4 weeks with particle size change <10%.

[0260] The morphology and particle size distribution of nanoparticle 4 are similar to those of nanoparticle 1.

[0261] Examples 2-5: Preparation of nanoparticles 5 Step 1: Formula Calculation This example uses the preparation of a 10 ml nanoparticle suspension as an example, with a target concentration of 1.8 mg / ml.

[0262] The total mass of the nanoparticles = 1.8 mg / ml × 10 ml = 18 mg. To ensure stability, it is assumed that the phospholipid shell mass fraction is 10%, the phospholipid mass = 1.8 mg, and the fluorocarbon mass = 16.2 mg. In actual preparation, to compensate for losses, excess phospholipid (e.g., 2 mg) and fluorocarbon (e.g., 16 mg) are used, for a total mass of 18 mg. Solvent volume: 1 ml chloroform (to dissolve the phospholipid), 9 ml aqueous phase (PBS).

[0263] Step 2: Preparation of organic phase Weigh 2 mg of DSPC and dissolve it in 1 ml of chloroform, vortexing over approximately 5 minutes until completely dissolved. Add 16 mg (approximately 12.2 μl) of HCFO-1233zd(Z) (density approximately 1.312 g / ml) and gently vortex to mix, avoiding vigorous stirring which could cause HCFO-1233zd(Z) to evaporate.

[0264] Step 3: Prepare the aqueous phase Measure 9 ml of PBS and place it in an open container.

[0265] Step 4: Preliminary emulsification The organic phase prepared above was slowly injected into the aqueous phase prepared above, while the mixture was stirred at 10,000 rpm for 2 minutes using an Ultra-Turrax high-speed shear mixer to form a crude emulsion.

[0266] Step 5: High-pressure homogenization at low temperature Transfer the crude emulsion obtained in step 4 to the sample cell of a high-pressure homogenizer and cycle it 10 times at a temperature of 3-4°C. Check the temperature after each cycle to ensure stability. If the particle size is too large, increase the pressure to 2000 bar or the number of cycles to 15; if the particle size is too small, decrease the pressure to 1000 bar.

[0267] Step 6: Remove solvent Chloroform was evaporated under reduced pressure at 40°C using a rotary evaporator for approximately 30 minutes.

[0268] Alternatively, place the emulsion into a dialysis bag and dialyze it against the PBS for 24 hours. Change the solution every 8 hours.

[0269] Step 7: Adjust the concentration Measure the final volume; if less than 10 ml, make up the difference with PBS. Take a small amount of sample and calculate the concentration using a gravimetric method (dry weighing) or a phospholipid assay (such as Bartlett's phosphate assay). Adjust the concentration to 1.8 mg / ml.

[0270] Step 8: Storage Store the nanoparticle suspension at 4°C away from light and avoid freezing.

[0271] Step 9: Quality Control Verify the concentration using gravimetric or UV spectrophotometric methods. Measure particle size and concentration periodically (on days 1, 7, and 14) to check for precipitation or aggregation.

[0272] Step 10: Characterization Particle size and polydispersity index (PDI) were measured immediately after each preparation using DLS. Particle morphology (spherical, core-shell structure) was observed by transmission electron microscopy (TEM).

[0273] result: The prepared nanoparticles 5 all have the following parameters: - Particle size: At a pressure of 1500 bar, the average particle size is approximately 100-400 nm, and the PDI is <0.2.

[0274] - Concentration: Through optimization, the concentration is 1.8 mg / ml ± 0.2 mg / ml.

[0275] - Encapsulation efficiency: >90% (fluorocarbon compounds are effectively encapsulated by phospholipids).

[0276] - Stability: Stable at 4°C for at least 4 weeks with particle size change <10%.

[0277] The morphology and particle size distribution of nanoparticle 5 are similar to those of nanoparticle 1.

[0278] Examples 2-6: Preparation of Nanoparticles 6 Step 1: Formula Calculation This example uses the preparation of a 10 ml nanoparticle suspension as an example, with a target concentration of 1.8 mg / ml.

[0279] The total mass of the nanoparticles = 1.8 mg / ml × 10 ml = 18 mg. To ensure stability, it is assumed that the phospholipid shell mass fraction is 10%, the phospholipid mass = 1.8 mg, and the fluorocarbon mass = 16.2 mg. In actual preparation, to compensate for losses, excess phospholipid (e.g., 2 mg) and fluorocarbon (e.g., 16 mg) are used, for a total mass of 18 mg. Solvent volume: 1 ml chloroform (to dissolve the phospholipid), 9 ml aqueous phase (PBS).

[0280] Step 2: Preparation of organic phase Weigh 2 mg of DSPE and dissolve it in 1 ml of chloroform, vortexing over approximately 5 minutes until completely dissolved. Add 16 mg (approximately 12.2 μl) of HCFO-1233zd(Z) (density approximately 1.312 g / ml) and gently vortex to mix, avoiding vigorous stirring which could cause HCFO-1233zd(Z) to evaporate.

[0281] Step 3: Prepare the aqueous phase Measure 9 ml of PBS and place it in an open container.

[0282] Step 4: Preliminary emulsification The organic phase prepared above was slowly injected into the aqueous phase prepared above, while the mixture was stirred at 10,000 rpm for 2 minutes using an Ultra-Turrax high-speed shear mixer to form a crude emulsion.

[0283] Step 5: High-pressure homogenization at low temperature Transfer the crude emulsion obtained in step 4 to the sample cell of a high-pressure homogenizer and cycle it 10 times at a temperature of 3-4°C. Check the temperature after each cycle to ensure stability. If the particle size is too large, increase the pressure to 2000 bar or the number of cycles to 15; if the particle size is too small, decrease the pressure to 1000 bar.

[0284] Step 6: Remove solvent Chloroform was evaporated under reduced pressure at 40°C using a rotary evaporator for approximately 30 minutes.

[0285] Alternatively, place the emulsion into a dialysis bag and dialyze it against the PBS for 24 hours. Change the solution every 8 hours.

[0286] Step 7: Adjust the concentration Measure the final volume; if less than 10 ml, make up the difference with PBS. Take a small amount of sample and calculate the concentration using a gravimetric method (dry weighing) or a phospholipid assay (such as Bartlett's phosphate assay). Adjust the concentration to 1.8 mg / ml.

[0287] Step 8: Storage Store the nanoparticle suspension at 4°C away from light and avoid freezing.

[0288] Step 9: Quality Control Verify the concentration using gravimetric or UV spectrophotometric methods. Measure particle size and concentration periodically (on days 1, 7, and 14) to check for precipitation or aggregation.

[0289] Step 10: Characterization Particle size and polydispersity index (PDI) were measured immediately after each preparation using DLS. Particle morphology (spherical, core-shell structure) was observed by transmission electron microscopy (TEM).

[0290] result: The prepared nanoparticles 6 all have the following parameters: - Particle size: At a pressure of 1500 bar, the average particle size is approximately 100-400 nm, and the PDI is <0.2.

[0291] - Concentration: Through optimization, the concentration is 1.8 mg / ml ± 0.2 mg / ml.

[0292] - Encapsulation efficiency: >90% (fluorocarbon compounds are effectively encapsulated by phospholipids).

[0293] - Stability: Stable at 4°C for at least 4 weeks with particle size change <10%.

[0294] The morphology and particle size distribution of nanoparticle 6 are similar to those of nanoparticle 1.

[0295] Examples 2-7: Preparation of Nanoparticles 7 Step 1: Formula Calculation This example uses the preparation of a 10 ml nanoparticle suspension as an example, with a target concentration of 1.8 mg / ml.

[0296] The total mass of the nanoparticles = 1.8 mg / ml × 10 ml = 18 mg. To ensure stability, it is assumed that the phospholipid shell mass fraction is 10%, the phospholipid mass = 1.8 mg, and the fluorocarbon mass = 16.2 mg. In actual preparation, to compensate for losses, excess phospholipid (e.g., 2 mg) and fluorocarbon (e.g., 16 mg) are used, for a total mass of 18 mg. Solvent volume: 1 ml chloroform (to dissolve the phospholipid), 9 ml aqueous phase (PBS).

[0297] Step 2: Preparation of organic phase Weigh 2 mg of DSPC and dissolve it in 1 ml of chloroform, vortexing until completely dissolved over approximately 5 minutes. Add 16 mg (approximately 10.0 μl) of perfluoropentane (density approximately 1.60 g / ml), and gently vortex in an ice bath, keeping the temperature below 10°C to avoid vigorous stirring that could cause the perfluoropentane to volatilize.

[0298] Step 3: Prepare the aqueous phase Measure 9 ml of PBS and place it in an open container.

[0299] Step 4: Preliminary emulsification The organic phase prepared above was slowly injected into the aqueous phase prepared above, while the mixture was stirred at 10,000 rpm for 2 minutes using an Ultra-Turrax high-speed shear mixer to form a crude emulsion.

[0300] Step 5: High-pressure homogenization at low temperature Transfer the crude emulsion obtained in step 4 to the sample cell of a high-pressure homogenizer and cycle it 10 times at a temperature of 3-4°C. Check the temperature after each cycle to ensure stability. If the particle size is too large, increase the pressure to 2000 bar or the number of cycles to 15; if the particle size is too small, decrease the pressure to 1000 bar.

[0301] Step 6: Remove solvent Chloroform was evaporated under reduced pressure at 40°C using a rotary evaporator for approximately 30 minutes.

[0302] Alternatively, place the emulsion into a dialysis bag and dialyze it against the PBS for 24 hours. Change the solution every 8 hours.

[0303] Step 7: Adjust the concentration Measure the final volume; if less than 10 ml, make up the difference with PBS. Take a small amount of sample and calculate the concentration using a gravimetric method (dry weighing) or a phospholipid assay (such as Bartlett's phosphate assay). Adjust the concentration to 1.8 mg / ml.

[0304] Step 8: Storage Store the nanoparticle suspension at 4°C away from light and avoid freezing.

[0305] Step 9: Quality Control Verify the concentration using gravimetric or UV spectrophotometric methods. Measure particle size and concentration periodically (on days 1, 7, and 14) to check for precipitation or aggregation.

[0306] Step 10: Characterization Particle size and polydispersity index (PDI) were measured immediately after each preparation using DLS. Particle morphology (spherical, core-shell structure) was observed by transmission electron microscopy (TEM).

[0307] result: The prepared nanoparticles 7 all have the following parameters: - Particle size: At a pressure of 1500 bar, the average particle size is approximately 100-400 nm, and the PDI is <0.2.

[0308] - Concentration: Through optimization, the concentration is 1.8 mg / ml ± 0.2 mg / ml.

[0309] - Encapsulation efficiency: >90% (fluorocarbon compounds are effectively encapsulated by phospholipids).

[0310] - Stability: Stable at 4°C for at least 4 weeks with particle size change <10%.

[0311] The morphology and particle size distribution of nanoparticle 7 are similar to those of nanoparticle 1.

[0312] Examples 2-8: Preparation of Nanoparticles 8 Step 1: Formula Calculation This example uses the preparation of a 10 ml nanoparticle suspension as an example, with a target concentration of 1.8 mg / ml.

[0313] The total mass of the nanoparticles = 1.8 mg / ml × 10 ml = 18 mg. To ensure stability, it is assumed that the phospholipid shell mass fraction is 10%, the phospholipid mass = 1.8 mg, and the fluorocarbon mass = 16.2 mg. In actual preparation, to compensate for losses, excess phospholipid (e.g., 2 mg) and fluorocarbon (e.g., 16 mg) are used, for a total mass of 18 mg. Solvent volume: 1 ml chloroform (to dissolve the phospholipid), 9 ml aqueous phase (PBS).

[0314] Step 2: Preparation of organic phase Weigh 2 mg of DSPE and dissolve it in 1 ml of chloroform, vortexing over approximately 5 minutes until completely dissolved. Add 16 mg (approximately 10.0 μl) of perfluoropentane (density approximately 1.60 g / ml), and gently vortex in an ice bath, keeping the temperature below 10°C to avoid vigorous stirring that could cause the perfluoropentane to volatilize.

[0315] Step 3: Prepare the aqueous phase Measure 9 ml of PBS and place it in an open container.

[0316] Step 4: Preliminary emulsification The organic phase prepared above was slowly injected into the aqueous phase prepared above, while the mixture was stirred at 10,000 rpm for 2 minutes using an Ultra-Turrax high-speed shear mixer to form a crude emulsion.

[0317] Step 5: High-pressure homogenization at low temperature Transfer the crude emulsion obtained in step 4 to the sample cell of a high-pressure homogenizer and cycle it 10 times at a temperature of 3-4°C. Check the temperature after each cycle to ensure stability. If the particle size is too large, increase the pressure to 2000 bar or the number of cycles to 15; if the particle size is too small, decrease the pressure to 1000 bar.

[0318] Step 6: Remove solvent Chloroform was evaporated under reduced pressure at 40°C using a rotary evaporator for approximately 30 minutes.

[0319] Alternatively, place the emulsion into a dialysis bag and dialyze it against the PBS for 24 hours. Change the solution every 8 hours.

[0320] Step 7: Adjust the concentration Measure the final volume; if less than 10 ml, make up the difference with PBS. Take a small amount of sample and calculate the concentration using a gravimetric method (dry weighing) or a phospholipid assay (such as Bartlett's phosphate assay). Adjust the concentration to 1.8 mg / ml.

[0321] Step 8: Storage Store the nanoparticle suspension at 4°C away from light and avoid freezing.

[0322] Step 9: Quality Control Verify the concentration using gravimetric or UV spectrophotometric methods. Measure particle size and concentration periodically (on days 1, 7, and 14) to check for precipitation or aggregation.

[0323] Step 10: Characterization Particle size and polydispersity index (PDI) were measured immediately after each preparation using DLS. Particle morphology (spherical, core-shell structure) was observed by transmission electron microscopy (TEM).

[0324] result: The prepared nanoparticles 8 all have the following parameters: - Particle size: At a pressure of 1500 bar, the average particle size is approximately 100-400 nm, and the PDI is <0.2.

[0325] - Concentration: Through optimization, the concentration is 1.8 mg / ml ± 0.2 mg / ml.

[0326] - Encapsulation efficiency: >90% (fluorocarbon compounds are effectively encapsulated by phospholipids).

[0327] - Stability: Stable at 4°C for at least 4 weeks with particle size change <10%.

[0328] The morphology and particle size distribution of nanoparticle 8 are similar to those of nanoparticle 1.

[0329] Examples 2-9: Preparation of nanoparticles 9 Step 1: Formula Calculation This example uses the preparation of a 10 ml nanoparticle suspension as an example, with a target concentration of 1.8 mg / ml.

[0330] The total mass of the nanoparticles = 1.8 mg / ml × 10 ml = 18 mg. To ensure stability, it is assumed that the phospholipid shell mass fraction is 10%, the phospholipid mass = 1.8 mg, and the fluorocarbon mass = 16.2 mg. In actual preparation, to compensate for losses, excess phospholipid (e.g., 2 mg) and fluorocarbon (e.g., 16 mg) are used, for a total mass of 18 mg. Solvent volume: 1 ml chloroform (to dissolve the phospholipid), 9 ml aqueous phase (PBS).

[0331] Step 2: Preparation of organic phase Weigh 2 mg of DSPC and dissolve it in 1 ml of chloroform, vortexing over approximately 5 minutes until completely dissolved. Add 16 mg (approximately 9.7 μl) of perfluorohexane (density approximately 1.656 g / ml) and gently vortex, avoiding vigorous stirring to prevent the perfluorohexane from evaporating.

[0332] Step 3: Prepare the aqueous phase Measure 9 ml of PBS and place it in an open container.

[0333] Step 4: Preliminary emulsification The organic phase prepared above was slowly injected into the aqueous phase prepared above, while the mixture was stirred at 10,000 rpm for 2 minutes using an Ultra-Turrax high-speed shear mixer to form a crude emulsion.

[0334] Step 5: High-pressure homogenization at low temperature Transfer the crude emulsion obtained in step 4 to the sample cell of a high-pressure homogenizer and cycle it 10 times at a temperature of 3-4°C. Check the temperature after each cycle to ensure stability. If the particle size is too large, increase the pressure to 2000 bar or the number of cycles to 15; if the particle size is too small, decrease the pressure to 1000 bar.

[0335] Step 6: Remove solvent Chloroform was evaporated under reduced pressure at 40°C using a rotary evaporator for approximately 30 minutes.

[0336] Alternatively, place the emulsion into a dialysis bag and dialyze it against the PBS for 24 hours. Change the solution every 8 hours.

[0337] Step 7: Adjust the concentration Measure the final volume; if less than 10 ml, make up the difference with PBS. Take a small amount of sample and calculate the concentration using a gravimetric method (dry weighing) or a phospholipid assay (such as Bartlett's phosphate assay). Adjust the concentration to 1.8 mg / ml.

[0338] Step 8: Storage Store the nanoparticle suspension at 4°C away from light and avoid freezing.

[0339] Step 9: Quality Control Verify the concentration using gravimetric or UV spectrophotometric methods. Measure particle size and concentration periodically (on days 1, 7, and 14) to check for precipitation or aggregation.

[0340] Step 10: Characterization Particle size and polydispersity index (PDI) were measured immediately after each preparation using DLS. Particle morphology (spherical, core-shell structure) was observed by transmission electron microscopy (TEM).

[0341] result: The prepared nanoparticles 9 all have the following parameters: - Particle size: At a pressure of 1500 bar, the average particle size is approximately 100-400 nm, and the PDI is <0.2.

[0342] - Concentration: Through optimization, the concentration is 1.8 mg / ml ± 0.2 mg / ml.

[0343] - Encapsulation efficiency: >90% (fluorocarbon compounds are effectively encapsulated by phospholipids).

[0344] - Stability: Stable at 4°C for at least 4 weeks with particle size change <10%.

[0345] The morphology and particle size distribution of nanoparticle 9 are similar to those of nanoparticle 1.

[0346] Examples 2-10: Preparation of nanoparticles 10 Step 1: Formula Calculation This example uses the preparation of a 10 ml nanoparticle suspension as an example, with a target concentration of 1.8 mg / ml.

[0347] The total mass of the nanoparticles = 1.8 mg / ml × 10 ml = 18 mg. To ensure stability, it is assumed that the phospholipid shell mass fraction is 10%, the phospholipid mass = 1.8 mg, and the fluorocarbon mass = 16.2 mg. In actual preparation, to compensate for losses, excess phospholipid (e.g., 2 mg) and fluorocarbon (e.g., 16 mg) are used, for a total mass of 18 mg. Solvent volume: 1 ml chloroform (to dissolve the phospholipid), 9 ml aqueous phase (PBS).

[0348] Step 2: Preparation of organic phase Weigh 2 mg of DSPE and dissolve it in 1 ml of chloroform, vortexing over approximately 5 minutes until completely dissolved. Add 16 mg (approximately 9.7 μl) of perfluorohexane (density approximately 1.656 g / ml) and gently vortex, avoiding vigorous stirring to prevent the perfluorohexane from evaporating.

[0349] Step 3: Prepare the aqueous phase Measure 9 ml of PBS and place it in an open container.

[0350] Step 4: Preliminary emulsification The organic phase prepared above was slowly injected into the aqueous phase prepared above, while the mixture was stirred at 10,000 rpm for 2 minutes using an Ultra-Turrax high-speed shear mixer to form a crude emulsion.

[0351] Step 5: High-pressure homogenization at low temperature Transfer the crude emulsion obtained in step 4 to the sample cell of a high-pressure homogenizer and cycle it 10 times at a temperature of 3-4°C. Check the temperature after each cycle to ensure stability. If the particle size is too large, increase the pressure to 2000 bar or the number of cycles to 15; if the particle size is too small, decrease the pressure to 1000 bar.

[0352] Step 6: Remove solvent Chloroform was evaporated under reduced pressure at 40°C using a rotary evaporator for approximately 30 minutes.

[0353] Alternatively, place the emulsion into a dialysis bag and dialyze it against the PBS for 24 hours. Change the solution every 8 hours.

[0354] Step 7: Adjust the concentration Measure the final volume; if less than 10 ml, make up the difference with PBS. Take a small amount of sample and calculate the concentration using a gravimetric method (dry weighing) or a phospholipid assay (such as Bartlett's phosphate assay). Adjust the concentration to 1.8 mg / ml.

[0355] Step 8: Storage Store the nanoparticle suspension at 4°C away from light and avoid freezing.

[0356] Step 9: Quality Control Verify the concentration using gravimetric or UV spectrophotometric methods. Measure particle size and concentration periodically (on days 1, 7, and 14) to check for precipitation or aggregation.

[0357] Step 10: Characterization Particle size and polydispersity index (PDI) were measured immediately after each preparation using DLS. Particle morphology (spherical, core-shell structure) was observed by transmission electron microscopy (TEM).

[0358] result: The prepared nanoparticles 10 all have the following parameters: - Particle size: At a pressure of 1500 bar, the average particle size is approximately 100-400 nm, and the PDI is <0.2.

[0359] - Concentration: Through optimization, the concentration is 1.8 mg / ml ± 0.2 mg / ml.

[0360] - Encapsulation efficiency: >90% (fluorocarbon compounds are effectively encapsulated by phospholipids).

[0361] - Stability: Stable at 4°C for at least 4 weeks with particle size change <10%.

[0362] The morphology and particle size distribution of nanoparticle 10 are similar to those of nanoparticle 1.

[0363] Table 1 below summarizes the composition and parameters of the ten nanoparticles prepared in Example 2.

[0364] Table 1

[0365] Example 3: Verification of chain reaction kinetics under a two-pulse scheme This embodiment verifies the effectiveness of inducing a chain reaction in isolated porcine liver using ten nanoparticles under a dual-pulse scheme. Specifically, nanoparticles were injected into isolated porcine liver, followed by ultrasonic irradiation (PRF = 10 Hz, P_ = 1.5 MPa), and the droplet density change was monitored.

[0366] Example 3-1: Dual-pulse scheme 1 Ten types of nanoparticles were respectively loaded at a density of 1×10⁻⁶. 9 A concentration of nanoparticles / mL was injected into different parts of an isolated pig liver to a depth of 3-4 cm. The pig liver was then immersed in de-aerated water, and an ultrasound probe was submerged in the water and brought into contact with the injected nanoparticle sites on the liver surface. Each injection point was irradiated for 10 seconds. After irradiation, the corresponding area was excised, photographed, and the degree and extent of tissue fragmentation were determined.

[0367] The corresponding parameters used in dual-pulse scheme 1 are as follows: • First pulse (trigger pulse): A short pulse with high PNP, quickly overcoming the Laplace pressure (ΔP = 2γ / r) and triggering droplet vaporization: Peak negative pressure (PNP): PNP = 3.2–3.5 MPa. PNP needs to exceed the threshold by 10–20% to ensure vaporization; PNP is used to mechanically overcome ΔP.

[0368] • Pulse length: 5–10 cycles (20–50 μs @ 2 MHz).

[0369] • Center frequency: 1.0 MHz (balanced penetration and threshold).

[0370] Waveform: Sine wave • Second pulse (driving cavitation): A lower PNP longer pulse drives the oscillation and collapse of cavitation bubbles, generating mechanical forces (micro-jet, shock wave) for tissue disruption pauses: Allows partial reliquefaction (especially for fluorocarbons with boiling points close to body temperature), reduces energy accumulation and thermal risks, enhances the mechanical effect of cavitation bubbles, and requires continuous energy to drive oscillation and collapse.

[0371] • Peak negative pressure (PNP): PNP = 2.0–2.5 MPa • Pulse length: 20–25 cycles (100–125 μs @ 2 MHz). Longer pulses enhance energy deposition and drive bubble dynamics; increasing pulse length can lower the effective threshold.

[0372] • Center frequency: Same as the first pulse (1.0 MHz).

[0373] • Waveform: Sine wave.

[0374] Sequence Design: PRF, Duty Cycle, and Total Duration The dual-pulse sequence needs to integrate the first and second pulses and control the overall energy.

[0375] Pulse repetition frequency (PRF): 10–15 Hz (overall PRF, with an interval of 67–100 ms between each pair of pulses).

[0376] Basis: Low PRF (10–20 Hz) allows for reliquefaction during downtime (especially for fluorocarbons with a boiling point of 42°C, where the body temperature of 37°C is close to the boiling point, making reliquefaction easier); bubbles can partially condense within 100 ms.

[0377] Pulse timing: There is no interval between the first and second pulses (continuous transmission), but there is a pause after each pair of pulses in the overall sequence. For PRF=10 Hz, a pair of pulses is transmitted every 100 ms (first pulse 5 μs + second pulse 10 μs), followed by an 85 μs pause (100 ms - 15 μs).

[0378] Duty cycle: Total pulse length = 5 μs (first) + 10 μs (second) = 15 μs; PRF = 10 Hz; Duty cycle = 15 μs × 10 Hz = 0.00015 = 0.015%.

[0379] Safety: Far below 0.1%, thermal effect is negligible.

[0380] Total duration: 10 s Example 3-2: Dual-pulse scheme 2 Ten types of nanoparticles were respectively loaded at a density of 1×10⁻⁶. 9 A concentration of nanoparticles / mL was injected into different parts of an isolated pig liver to a depth of 3-4 cm. The pig liver was then immersed in de-aerated water, and an ultrasound probe was submerged in the water and brought into contact with the injected nanoparticle sites on the liver surface. Each injection point was irradiated for 10 seconds. After irradiation, the corresponding area was excised, photographed, and the degree and extent of tissue fragmentation were determined.

[0381] The corresponding parameters used in dual-pulse scheme 2 are shown below: • First pulse (trigger pulse): A short pulse with high PNP, quickly overcoming the Laplace pressure (ΔP = 2γ / r) and triggering droplet vaporization: Peak negative pressure (PNP): PNP = 7.5–10 MPa. PNP needs to exceed the threshold by 10–20% to ensure vaporization; PNP is used to mechanically overcome ΔP.

[0382] • Pulse length: 1–2 cycles (50–100 μs @ 2 MHz).

[0383] • Center frequency: 1.0 MHz (balanced penetration and threshold).

[0384] Waveform: Sine wave • Second pulse (driving cavitation): A lower PNP longer pulse drives the oscillation and collapse of cavitation bubbles, generating mechanical forces (micro-jet, shock wave) for tissue disruption pauses: Allows partial reliquefaction (especially for fluorocarbons with boiling points close to body temperature), reduces energy accumulation and thermal risks, enhances the mechanical effect of cavitation bubbles, and requires continuous energy to drive oscillation and collapse.

[0385] • Peak negative pressure (PNP): PNP = 5–8 MPa • Pulse length: 32–35 cycles (150–250 μs @ 2 MHz). Longer pulses enhance energy deposition and drive bubble dynamics; increasing pulse length can lower the effective threshold.

[0386] • Center frequency: Same as the first pulse (1.0 MHz).

[0387] • Waveform: Sine wave.

[0388] Sequence Design: PRF, Duty Cycle, and Total Duration The dual-pulse sequence needs to integrate the first and second pulses and control the overall energy.

[0389] Pulse repetition frequency (PRF): 10–15 Hz (overall PRF, with an interval of 67–100 ms between each pair of pulses).

[0390] Basis: Low PRF (10–20 Hz) allows for reliquefaction during downtime (especially for fluorocarbons with a boiling point of 42°C, where the body temperature of 37°C is close to the boiling point, making reliquefaction easier); bubbles can partially condense within 100 ms.

[0391] Pulse timing: There is no interval between the first and second pulses (continuous transmission), but there is a pause after each pair of pulses in the overall sequence. For PRF=10 Hz, a pair of pulses is transmitted every 100 ms (first pulse 5 μs + second pulse 10 μs), followed by an 85 μs pause (100 ms - 15 μs).

[0392] Duty cycle: Total pulse length = 5 μs (first) + 10 μs (second) = 15 μs; PRF = 10 Hz; Duty cycle = 15 μs × 10 Hz = 0.00015 = 0.015%.

[0393] Safety: Far below 0.1%, thermal effect is negligible.

[0394] Total duration: 10 s.

[0395] Experimental results: In Example 3-1, nanoparticles 1, 3, and 5 instantly produced bubbles after the first pulse of the high-speed camera was activated, such as... Figure 3 The results for nanoparticle 1 are shown, while nanoparticles 3 and 5 show similar results. It is evident that the key to the first pulse is high-speed triggering, which avoids unnecessary energy deposition. In other words, high-speed camera observations confirm that short pulses can instantaneously vaporize droplets. In Examples 3-2, although nanoparticles 1, 3, and 5 can also trigger a chain reaction under high-energy ultrasound, the energy used is clearly too high, posing a risk of tissue damage when used clinically for in vivo tissue ablation. Nanoparticles 2, 4, 6, and 7-10, even under the high-energy ultrasound of Examples 3-2, cannot trigger a chain reaction.

[0396] Nanoparticles 1, 3, and 5, under low-energy ultrasonic irradiation in Example 3-1 or high-energy ultrasonic irradiation in Example 3-2, can all locally form honeycomb-like pulverized regions, such as... Figure 4 The results for nanoparticle 1 are shown, while nanoparticles 3 and 5 show similar results. It is evident that significant tissue fragmentation and voids are generated at all injection sites under ultrasonic irradiation. However, as mentioned above, the ultrasonic energy used in Examples 3-2 is relatively high, posing a risk of tissue damage when used clinically for in vivo tissue ablation. Nanoparticles 2, 4, 6, and 7-10, even under the high-energy ultrasonic irradiation of Examples 3-2, failed to trigger a chain reaction and thus did not generate tissue fragmentation and voids.

[0397] In summary, only nanoparticles formed from specific materials can create significant tissue fragmentation and voids under low-energy dual-pulse ultrasound irradiation.

[0398] Example 4: Verification of chain reaction kinetics under single-pulse sequence This embodiment verifies the effectiveness of inducing a chain reaction in isolated porcine liver using ten nanoparticles under a single-pulse protocol. Specifically, nanoparticles were injected into isolated porcine liver, followed by ultrasound irradiation. The chain reaction results were then evaluated using imaging, temperature monitoring, and histological analysis.

[0399] The ultrasound equipment used in this embodiment is a conventional therapeutic focused ultrasound device, which is equipped with a transducer (spherical or planar focused transducer, frequency range 0.8-3 MHz) and a drive system (independent radio frequency power amplifier, supporting pulse waveform control, i.e., pulse number, PRF, and duty cycle are adjustable), and the equipment parameters are as follows: (1) Sound power: - Range: 100-200 W (target peak negative pressure 5-10 MPa).

[0400] (2) Pulse parameters: - Cycle count / pulse: 5,000–10,000 cycles / pulse (millisecond pulses).

[0401] - PRF: 0.5–1Hz.

[0402] - Pulse length: 5-10ms - Duty cycle: 0.5-1% - Total time: 60-300 seconds.

[0403] (3) Frequency selection: - Clock speed: 1-2 MHz.

[0404] Example 4-1: Single Pulse Scheme 1 Step 1: Prepare nanoparticles The ten nanoparticles 1-10 prepared in Example 2 were used at a concentration of 0.1-2 mg / mL.

[0405] Step 2: Sample Preparation Prepare isolated pig livers and locally inject ten different types of nanoparticles. Then, degas the livers in a vacuum chamber for 30 minutes to prevent stray cavitation.

[0406] Step 3: Ultrasonic irradiation The following ultrasonic irradiation scheme was adopted. Pulse sequence: Each pulse contains 5-10 ms, pulse length is 1-30 cycles, PRF=10-20 Hz, center frequency is 1.5-5 MHz, peak negative pressure is 1-5 MPa, and an allowable interval (1-2 seconds) is used for gas dispersion and nanoparticle reliquefaction (to prevent excessive bubble dispersion).

[0407] Pulse-on: 5 ms emission promotes nanoparticle vaporization and cavitation.

[0408] Pulse-off: 995 ms interval (for PRF=10 Hz), allowing for cooling and tissue response.

[0409] Ultrasonic imaging after ultrasonic irradiation: B-mode and contrast-enhanced ultrasound (CEUS) monitoring of cavitation cloud formation (microbubble echo enhancement after nanoparticle activation).

[0410] Sound field mapping was performed by measuring the focal sound pressure using a hydrophone and ensuring that the peak negative pressure was ≥5 MPa (nanoparticle activation threshold), thereby calibrating the equipment.

[0411] Step 4: Temperature Monitoring For the exposed surface of ex vivo tissue, temperature is continuously recorded using thermocouples or captured by an infrared camera. Temperature calibration is performed by embedding thermocouples in the focal area and verifying the accuracy of the infrared thermal imaging (error <1℃).

[0412] Step 5: Histological analysis H&E staining was used to assess the degree of cell damage (0-5 point scale): Where 0 = complete, 5 = completely liquefied And focus on observing the boundary between mechanical fracture and thermal damage.

[0413] Example 4-2: Single Pulse Scheme 2 Step 1: Prepare nanoparticles The ten nanoparticles 1-10 prepared in Example 2 were used at a concentration of 0.1-2 mg / mL.

[0414] Step 2: Sample Preparation Prepare isolated pig livers and locally inject ten different types of nanoparticles. Then, degas the livers in a vacuum chamber for 30 minutes to prevent stray cavitation.

[0415] Step 3: Ultrasonic irradiation The following ultrasonic irradiation scheme was adopted. Pulse sequence: Each pulse contains 10-20 ms, the pulse length is 55 cycles, PRF=10-20 Hz, the center frequency is 1.5-5 MHz, the peak negative pressure is 7.5-10 MPa, and an allowable interval (1-2 seconds) is provided for gas dispersion and nanoparticle reliquefaction (to prevent excessive bubble dispersion).

[0416] Pulse-on: 5 ms emission promotes nanoparticle vaporization and cavitation.

[0417] Pulse-off: 995 ms interval (for PRF=10 Hz), allowing for cooling and tissue response.

[0418] Ultrasonic imaging after ultrasonic irradiation: B-mode and contrast-enhanced ultrasound (CEUS) monitoring of cavitation cloud formation (microbubble echo enhancement after nanoparticle activation).

[0419] Sound field mapping was performed by measuring the focal sound pressure using a hydrophone and ensuring that the peak negative pressure was ≥5 MPa (nanoparticle activation threshold), thereby calibrating the equipment.

[0420] Step 4: Temperature Monitoring For the exposed surface of ex vivo tissue, temperature is continuously recorded using thermocouples or captured by an infrared camera. Temperature calibration is performed by embedding thermocouples in the focal area and verifying the accuracy of the infrared thermal imaging (error <1℃).

[0421] Step 5: Histological analysis H&E staining was used to assess the degree of cell damage (0-5 point scale): Where 0 = complete, 5 = completely liquefied And focus on observing the boundary between mechanical fracture and thermal damage.

[0422] Experimental results: Table 2 below shows some of the results measured in this embodiment.

[0423] Table 2

[0424] Figure 5 The ultrasonic energy was significantly reduced, demonstrating a substantial tissue-disrupting effect in nanoparticle 1. Nanoparticles 3 and 5 showed similar results.

[0425] Figure 6 The image shows a localized tissue dissolution area formed on an isolated porcine liver by local injection of 3 ml of nanoparticle 1. Nanoparticles 3 and 5 show similar results.

[0426] Figure 7 yes Figure 6 HE staining image of excised liver tissue after pulverization and dissolution. The central part of the image shows a large amount of dissolved necrotic material with light red staining. Nanoparticles 3 and 5 show similar results.

[0427] According to Table 2 and Figure 5-7 The results show that nanoparticles 1, 3, and 5 can form significant tissue dissolution areas at low ultrasonic energy. Although they can also form significant tissue dissolution areas at high ultrasonic energy, the ultrasonic energy used is obviously higher, which leads to a certain increase in temperature and poses a risk of tissue damage.

[0428] Nanoparticles 2, 4, 6, and 7-10 cannot trigger a chain reaction under any ultrasonic energy, thus failing to ablate tissue.

[0429] Example 5: Layered sequential injection of nanoparticles and microparticles can enhance tissue disruption. Step 1: Preparation of nanoparticles and microparticles (1) Preparation of nanoparticles Nanoparticles were prepared using a lipid encapsulation emulsification method to ensure monodispersity and stability. The target size of the nanoparticles was 300-500 nanometers.

[0430] A lipid mixture was prepared by mixing DPPC (dispalmitoylphosphatidylcholine), LPC (lysophosphatidylcholine), and DPPE-PEG2000 in a ratio of 70:20:10. This lipid mixture was dissolved in chloroform at a total lipid concentration of 20 mg / mL, and the mixture was rotary evaporated in a round-bottom flask at 40°C under negative pressure to form a thin film. The film was then vacuum dried overnight to remove residual solvent.

[0431] The lipid film formed above was hydrated in a -20°C environment using pre-cooled 4°C HEPES buffer (pH 7.4) to achieve a final concentration of 10 mg / mL. The solution was then sonicated in an ice bath using a probe sonicator for preliminary emulsification. The sonication conditions were: 20 kHz, 50% amplitude, 30-second pulses, 30-second intervals, and a total duration of 5 minutes.

[0432] The condensed liquid 1H-perfluoropentane obtained by cooling the gas with dry ice was added to the hydration solution at a volume ratio of 1H-perfluoropentane:lipid solution of 1:10. The mixture was homogenized at 10,000 rpm for 5 minutes at -20°C, and then extruded at -20°C using a cryogenic extruder (Avestin LiposoFast) with a 100 nm pore size polycarbonate membrane. This process was repeated 20 times to reduce particle size and ensure monodispersity. After extrusion, the emulsion was centrifuged at 3000 rpm for 15 minutes at 4°C to remove uncoated 1H-perfluoropentane, precipitating it and resuspending it in HEPES buffer.

[0433] The concentration of the obtained nanoparticles was verified by dynamic light scattering (DLS) (target size 150-300 nm), and they were stored in sealed vials at 4°C to avoid temperature fluctuations. Stability tests showed that the obtained nanoparticles remained without significant aggregation for 7 days at 4°C.

[0434] (2) Preparation of micron-sized particles Micron-sized particles were prepared using a mechanical emulsification method and stabilized with a lipid or polymer shell. The target size of the micron-sized particles was 1–5 μm.

[0435] A lipid mixture was prepared by mixing DPPC (dispalmitoylphosphatidylcholine), LPC (lysophosphatidylcholine), and DPPE-PEG2000 in a ratio of 70:20:10. This lipid mixture was dissolved in chloroform at a total lipid concentration of 20 mg / mL, and the mixture was rotary evaporated in a round-bottom flask at 40°C under negative pressure to form a thin film. The film was then vacuum dried overnight to remove residual solvent.

[0436] The lipid film formed above was hydrated with PBS buffer (pH 7.4) at room temperature to a final concentration of 10 mg / mL. 1H-perfluoropentane was added to a volume ratio of 1H-perfluoropentane to lipid solution of 1:5, followed by initial vortex mixing.

[0437] The emulsion was coarsely emulsified at 15,000 rpm for 10 minutes using a high-speed homogenizer at low temperature, and then extruded at room temperature through a 1 μm pore size polycarbonate membrane. This process was repeated 10 times to control the emulsion droplet size within the range of 1 to 5 micrometers.

[0438] The emulsion was allowed to stand at low temperature for 1 hour to stabilize it, and then centrifuged at 1000 rpm for 10 minutes to remove large particles. The supernatant contained micron-sized particles of 1-3 μm. The successful preparation of micron-sized particles was verified by microscopy or DLS. The micron-sized particles were stored in a light-protected container at room temperature and remained stable for up to 14 days.

[0439] (3) Sequential injection Sequential injection aims to sequentially inject nanoparticles and microparticles to evaluate synergistic effects.

[0440] The concentration of the nanoparticles prepared above was adjusted to 1×10⁻⁶. 9 The concentration of the micron-sized particles prepared above was adjusted to 1 × 10⁻⁶ particles / mL. 7 Particles / mL. The nanoparticle suspension and the microparticle suspension were respectively connected to a dual-syringe pump.

[0441] Fresh, isolated porcine liver samples were collected and kept in physiological saline at 37°C to simulate in vivo conditions. The injection needle (27G) of a dual-syringe pump was inserted into the blood vessels or parenchyma of the porcine liver.

[0442] Then, two types of particles were sequentially injected. First, nanoparticles were injected: 0.1 mL volume, 0.5 mL / min flow rate, allowing for extravasation or distribution into the interstitial space. After a 5-minute interval, micron-sized particles were injected: 0.1 mL volume, 0.5 mL / min flow rate, to enhance cavitation nuclei density. After injection, the mixture was allowed to stand for 2 minutes to allow for uniform particle distribution, followed by ultrasonic irradiation.

[0443] (4) Ultrasonic irradiation Ultrasonic irradiation aims to vaporize particles and induce cavitation. The ultrasonic equipment used, equipped with an immersion point-focusing probe, was purchased from Eindico Technologies (Shanghai) Co., Ltd., and has the following parameters: center frequency 1-2.25MHz, crystal size 13mm, and focusing range 20-48mm.

[0444] The pulse sequence and focusing adjustments are based on the following: nanoparticles have low boiling points and require lower ultrasonic energy; micron-sized particles have high boiling points and require higher energy. For larger micron-sized particles, the focal spot can be slightly larger; for smaller nanoparticles, the focal spot needs to be tighter to increase energy density, thereby ensuring that the sound field covers the target area.

[0445] Under real-time ultrasound monitoring, sequential irradiation was performed in the following order: - First, low-energy ultrasound (1.5 MHz, MI 1.0) is applied to activate the nanoparticles and generate cavitation nuclei.

[0446] - Subsequently, high-energy ultrasound (3 MHz, MI 1.8) was applied to target micron-sized particles to enhance the cavitation effect.

[0447] - Total ultrasound irradiation time is 15 minutes, including intervals, to ensure synergistic effects.

[0448] - Temperature control: The pig liver samples were maintained at 37°C, and the temperature was monitored (<50°C) during ultrasound irradiation to avoid thermal damage.

[0449] Experimental results: Figure 8 Images of fragmented porcine liver tissue formed by stratified sequential injection of 1H-perfluoropentane particles of different sizes are shown. It is evident that the strategy of sequentially injecting nanoparticles and microparticles, along with sequential irradiation injection of both types of particles, can enhance tissue fragmentation.

[0450] Example 6: Targeted enrichment ability of nanoparticles with surface-modified targeting molecules after intravenous injection In this embodiment, several nanoparticles with surface-modified targeting molecules were prepared. After intravenous injection, the molecular targets that could satisfy the chain reaction enrichment, tumor universality, and nanoscale adaptability were observed.

[0451] The following four target molecules were selected in this embodiment.

[0452] Integrin αvβ3 receptor: It is widely and highly expressed in various malignant tumors (such as breast, prostate, and glioma) and tumor neovascular endothelial cells, but its expression is extremely low in normal tissues.

[0453] Folic acid receptor: It is overexpressed in a variety of epithelial malignancies (such as ovarian, lung and breast tumors) and is a recognized broad-spectrum tumor target.

[0454] Prostate-specific membrane antigen (PSMA): In addition to being highly expressed in prostate cancer, PSMA is also widely present in the neovascular endothelium of various solid tumors (such as renal cell carcinoma and thyroid cancer).

[0455] Pancreatic stellate cells (PSCs) and related matrix targets: Targeting targets that activate pancreatic stellate cells (PSCs) (such as integrins or collagen) is valuable for tumors with dense matrix, such as pancreatic cancer. Although these targets are not directly expressed by tumor cells, they are ubiquitous in the malignant tumor microenvironment and are important for improving the intratumoral distribution of nanoparticles.

[0456] Step 1: Preparation of nanoparticles with surface-modified targeting molecules The above-mentioned nanoparticles 1, integrin αvβ3 receptor and RGD cyclic peptide were placed in a container filled with DMSO at a molar ratio of 1:100:100 and stirred at room temperature for 24 hours to obtain nanoparticles 11 (with a particle size of about 250-300 nm) with surface modified integrin αvβ3 receptor.

[0457] Nanoparticles 12 (approximately 380 nm in diameter) with folic acid receptors, nanoparticles 13 (approximately 250 nm in diameter) with prostate-specific membrane antigens, and nanoparticles 14 (approximately 300 nm in diameter) with pancreatic stellate cells were prepared in a similar manner.

[0458] Step 2: Intravenous injection of modified nanoparticles 11-14 The modified nanoparticles 11-14 were intravenously injected into liver cancer H_(22) tumor-bearing mice (purchased from Wuhan Shangen Biotechnology Co., Ltd.).

[0459] Step 3: Ultrasonic irradiation Following intravenous injection, the tumor site in the human patient was irradiated with ultrasound using the single-pulse scheme 1 in Example 4-1.

[0460] Experimental results: After intravenous injection, nanoparticle 11 can achieve efficient enrichment at the tumor site (i.e., the enrichment amount can reach more than 10% of the injected dose within 12 hours) and successfully trigger ultrasonic phase transition and cavitation effect. It has high targeting efficiency and good compatibility with nanoparticles with a diameter of 200-400 nm.

[0461] Nanoparticle 12 can penetrate tumor blood vessels through an active targeting mechanism and accumulate in the tumor stroma. After intravenous injection, its enrichment is sufficient to enhance photoacoustic / ultrasound dual-modal imaging signals and support subsequent treatment procedures, demonstrating good compatibility with nanoparticle size.

[0462] Nanoparticle 13 can reach peak enrichment in the tumor area within 12 hours after intravenous injection, providing sufficient focal concentration for chain reaction cavitation.

[0463] Nanoparticle 14 can be partially retained at the tumor site after intravenous injection (the matrix barrier may affect the penetration of nanoparticles), thereby enabling subsequent chain reaction cavitation.

[0464] In summary, integrin αvβ3 and folate receptors are promising targets due to their widespread high expression in various malignant tumors, good compatibility with 200-400 nm nanoparticle sizes, and significant tumor enrichment capacity demonstrated after intravenous injection. PSMA is an ideal choice for specific cancer types.

[0465] In summary, this application not only innovatively develops a novel algorithm capable of theoretical calculation and design for technologies combining low-temperature phase change nanoparticles with a chain reaction cavitation model, but also unexpectedly discovers through experiments that only nanoparticles made with specific fluorocarbon compounds (within a specific boiling point range) as the core and specific phospholipids as the shell can initiate a cavitation chain reaction under low-energy dual-pulse or single-pulse schemes, thereby significantly fragmenting and ablating the target tissue. Furthermore, the strategy of sequentially injecting nanoparticles and microparticles, as well as sequentially irradiating both types of particles, can enhance tissue fragmentation. Additionally, by modifying the surface of the nanoparticles in this application with specific targeting molecules, the resulting modified nanoparticles exhibit significant tumor enrichment capacity and tumor universality.

[0466] Although the embodiments of this application have been described above in conjunction with the accompanying drawings, this application is not limited to the specific embodiments and application fields described above. The specific embodiments described above are merely illustrative and instructive, not restrictive. Those skilled in the art can make many other forms based on the guidance of this specification and without departing from the scope of protection of the claims of this application, and these are all within the scope of protection of this application.

Claims

1. A low-temperature phase change nanoparticle, characterized in that, The low-temperature phase change nanoparticles include: (a) A liquid core comprising a fluorocarbon compound or an azeotropic composition thereof having a boiling point of -20°C to 50°C at ambient temperature and pressure; and (b) A shell composed of a phospholipid or polymer material and optionally modified with a targeting molecule; in: The nanoparticles are in the form of regular or irregular spherical particles, regular or irregular microplates, or regular or irregular micromicelles. The low temperature refers to a temperature range of -20°C to 50°C; The nanoparticles have a particle size of 100-5000 nm.

2. The low-temperature phase change nanoparticles according to claim 1, wherein, After the liquid core is injected into the body, external heating of the target area refers to using techniques such as local hot compresses, infrared heating, microwave heating, or combined ultrasonic irradiation to raise the temperature of the area containing low-temperature phase change nanoparticles to 37-50°C.

3. The low-temperature phase change nanoparticles according to claim 1, wherein the fluorocarbon compound in the liquid core has a boiling point of 35°C to 45°C at room temperature and pressure.

4. The low-temperature phase change nanoparticles according to claim 1, wherein the fluorocarbon compound of the liquid core is selected from: 1H-perfluoropentane, decafluorobutane, dodecafluoropentane, cis-1-chloro-3,3,3-trifluoropropene (HCFO-1233zd(Z)), 1,1,1,3,3-pentafluorobutane (HFC-365mfc), perfluorohexane, perfluorocyclopentane, perfluorodimethylcyclobutane, perfluoromethylcyclopentane, perfluorotert-butylamine, perfluorotrimethylcyclopropane, perfluoromethylcyclohexane, perfluorobutylethane, perfluoroheptane, perfluoro(3-methylhexane), perfluoro(2-methylpentane), perfluoropentane and its branched isomers, perfluorooctane, hydrofluoroalkanes, hydrofluoroolefins, perfluoro-1,2-dimethylcyclobutane, or combinations thereof.

5. The low-temperature phase change nanoparticles according to any one of claims 1 to 4, wherein the shell material is selected from: 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC), cholesterol, 1,2-distearyl-sn-glycerol-3-phosphoethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), Tween, lecithin, dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylcholine-polyethylene glycol 2000 (DPPC-PEG2000), matrix metalloproteinase-2 (MMP-2), or matrix metalloproteinase-9. (MMP-9) includes cleavable peptide polymers, disulfide-containing polymers, polyhistidine, polyethylene glycol-polylactic acid (PEG-PLA), polylactic acid-glycolic acid copolymer (PLGA), poloxamer F127, poloxamer F68, chitosan, poly-N-isopropylacrylamide (PNIPAM) or copolymers thereof, cyclodextrin-based supramolecular assemblies, mesoporous silica, metal-organic frameworks (MOFs), silica nanoparticles, fluorocarbon-MOF composites, perfluorooctanoic acid-hydrazone-drug conjugates, perfluorooctane sulfonate diethylamine salt, other antibody conjugates or combinations thereof.

6. The low-temperature phase change nanoparticles according to any one of claims 1 to 5, wherein the shell of the low-temperature phase change nanoparticles is modified with a targeting molecule.

7. The low-temperature phase transition nanoparticles according to claim 6, wherein the targeting molecule is selected from: integrin αvβ3 receptor, folic acid receptor, prostate-specific membrane antigen, pancreatic stellate cells, phosphatidylinositol proteoglycan-3 (GPC-3), desialyl glycoprotein receptor (ASGPR), mesothelin (MSLN), carcinoembryonic antigen-associated cell adhesion molecule 5 (CEACAM5), carbonic anhydrase IX (CAIX), EGFR (epidermal growth factor receptor), c-Met, and combinations thereof.

8. The low-temperature phase change nanoparticles according to any one of claims 1 to 7, wherein the particle size of the nanoparticles is selected from the following ranges: 100-600 nm, 100-400 nm, 200-600 nm, 200-400 nm, 200-5000 nm.

9. An ultrasound therapy system for achieving chain reaction cavitation, comprising: A nanoparticle delivery module for delivering the low-temperature phase change nanoparticles as described in any one of claims 1 to 8 to a target tissue region; An ultrasonic transducer array configured to emit ultrasonic pulses toward the target tissue region and receive echo signals for imaging; A controller, which is communicatively connected to the ultrasonic transducer array to control the ultrasonic transducer array.

10. The use of the ultrasonic therapy system of claim 9 for achieving target tissue fragmentation, characterized in that, The implementation of the stated purpose includes the following steps: (I) Introducing the low-temperature phase change nanoparticles of any one of claims 1 to 8 into the target tissue; (II) Monitoring the cavitation nucleus state of the target area using ultrasonic imaging; (III) Based on monitoring results, adaptively emit ultrasonic pulse sequences to trigger chain reaction cavitation and optionally maintain chain reaction cavitation; and (IV) Monitor safety parameters in real time and adjust ultrasound energy output to ensure treatment safety.

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