Ultrasonic-temperature dual-triggered dual-liquid gas phase-change contrast agent and preparation method thereof

Abstract

The application discloses an ultrasonic-temperature double-triggered double-liquid gas phase change contrast agent and a preparation method thereof. The preparation steps comprise the following steps: adding an isotonic buffer solution into a prepared composite phospholipid film, oscillating after ultrasonic dispersion, obtaining a targeted phospholipid vesicle dispersion; then high-pressure double-liquid co-loading is carried out, and the phase change temperature and the ultrasonic response are calibrated; filtering through a filter membrane, collecting the precipitate after centrifugation, adding a protective solution after washing with a PBS buffer solution, and sealing and storing to obtain the double-liquid gas phase change contrast agent. After the contrast agent enters the body, under the ultrasonic double triggering of the physiological temperature and an ultrasonic imaging instrument, the double-liquid gas synchronously phase changes into a gaseous state, drives the contrast agent to rapidly expand from a nano-sized particle into a micron-sized microbubble, realizes ultrasonic enhanced imaging, and has a significant clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of ultrasound medical imaging technology, specifically relating to an ultrasound-temperature dual-triggered dual liquid-gas phase change contrast agent and its preparation method. Background Technology

[0002] Ultrasound contrast agents are core materials for improving the accuracy of ultrasound diagnosis, and their performance depends on trigger responsiveness, phase transition efficiency, and imaging stability. Traditional phase transition ultrasound contrast agents mostly rely on single temperature triggering, which has the following key technical bottlenecks: First, the phase transition triggering condition is singular, and the response speed is slow (usually >5s) when relying solely on temperature triggering, making it difficult to quickly achieve imaging switching; second, the temperature difference between some lesion areas and normal tissues is small, and single temperature triggering can easily lead to non-specific phase transitions, affecting imaging accuracy; third, when dual gases are co-carried, the gaseous and liquid states are mixed, resulting in low encapsulation efficiency and poor stability, and the lack of a design for ultrasound and temperature synergistic triggering limits the flexibility of clinical applications.

[0003] To overcome the shortcomings of dual-gas co-loading, researchers attempted to use perfluorobutane (phase change temperature -2℃~1℃) and perfluoropentane (phase change temperature 29℃~31℃) to achieve dual liquid gas co-loading during the preparation process through low-temperature control. However, existing technologies have not yet achieved effective integration of ultrasound-temperature dual triggering with dual liquid gas co-loading, mainly due to the following problems: First, the design of a dual triggering mechanism is lacking, and the relationship between ultrasound parameters and phase change efficiency is not clear; second, during dual liquid gas co-loading, ultrasound triggering may cause vesicle membrane rupture, affecting the stability of microbubbles after phase change; third, the quantitative correlation between the molar ratio of the two gases and the dual-trigger phase change temperature and response time is unclear, making precise control impossible.

[0004] Therefore, developing a nano- to micro-converted phospholipid ultrasound contrast agent based on dual liquid gas co-carrying, ultrasound-temperature dual triggering, and adjustable phase transition temperature has significant technological breakthrough and clinical value. Summary of the Invention

[0005] To address at least one of the aforementioned problems, the present invention provides an ultrasound-temperature dual-triggered dual liquid-gas phase change contrast agent and its preparation method.

[0006] To achieve the above objectives, the present invention employs the following technical means: This invention provides a method for preparing a dual-liquid-gas phase change contrast agent triggered by both ultrasound and temperature, comprising the following steps: S1. A composite phospholipid composition consisting of natural / synthetic phospholipids, targeted modified phospholipids, stabilizers, and membrane elasticity modifiers is dissolved in a mixed organic solvent, and the organic solvent is removed by rotary evaporation at 35-50 °C to form a uniform and dense composite phospholipid film. S2. Add an isotonic buffer solution to the composite phospholipid film, disperse it by ultrasonication, and then oscillate to obtain a targeted phospholipid vesicle dispersion. S3. Transfer the targeted phospholipid vesicle dispersion to a high-pressure reactor. First, introduce liquid high phase change temperature gas and gradually increase the pressure to 2.5 MPa at a rate of 0.3 MPa / 20 min, while controlling the temperature to not exceed 25 ℃. Keep it at this temperature and let it stand. Then, introduce liquid low phase change temperature gas, adjust the total pressure to 4 MPa, control the temperature to 1-3 ℃, and stir. S4. The product generated in step S3 is calibrated for phase transition temperature and ultrasonic response. S5. Filter the dispersion using a filter membrane, collect the precipitate after centrifugation, wash with PBS buffer solution, add protective solution, seal and store to obtain the dual liquid-gas phase change contrast agent.

[0007] In some embodiments of the present invention, the mass ratio of natural phospholipids / synthetic phospholipids, targeted modified phospholipids, stabilizers, and membrane elasticity modifiers in the composite phospholipid composition of step S1 is 1:(0.2-0.6):(0.01-0.04):(0.001-0.005).

[0008] In some embodiments of the present invention, in step S1, an antioxidant with a mass fraction of 0.05%-0.3% is added to the composite phospholipid composition, wherein the antioxidant is selected from one or more of ascorbyl palmitate and glutathione.

[0009] In some embodiments of the present invention, in step S1, the mixed organic solvent is a chloroform-methanol mixture with a volume ratio of (1-6):1 or an ethanol-dichloromethane mixture with a volume ratio of (2-8):1; the mass-volume ratio of the composite phospholipid composition to the mixed organic solvent is 1 g: (25-80) mL; the temperature of the low-temperature mixed organic solvent is maintained at ≤10°C by an ice bath or a low-temperature thermostat.

[0010] In some embodiments of the present invention, the natural phospholipid is one or more of soybean lecithin and egg yolk lecithin; the synthetic phospholipid is one or more of DPPA, DPPC, DSPA, DSPC, DOPC, POPC, DSPE-PEG, and DPPE-PEG.

[0011] In some embodiments of the present invention, the targeted modified phospholipid is one or more of folic acid modified phospholipid, RGD peptide modified phospholipid, galactose modified phospholipid, and transferrin modified phospholipid.

[0012] In some embodiments of the present invention, the stabilizer is one or more of cholesterol, polyethylene glycol, and vitamin E succinate; the membrane elasticity modifier is one or more of palmitic acid, stearic acid, and oleic acid.

[0013] In some embodiments of the present invention, in step S2, the isotonic buffer solution is one of phosphate buffer solution, physiological saline or HEPES buffer solution, with a pH value of 7.2-7.5, an osmotic concentration of 280-330 mOsm / kg, and a mass-to-volume ratio of composite phospholipid membrane to isotonic buffer solution of 1 g: (50-120) mL.

[0014] In some embodiments of the present invention, the phase change temperature and ultrasonic response calibration method in step S4 is as follows: the particle size at different temperatures is combined with the phase change response time detected by the ultrasonic triggering test system. If the phase change temperature deviates from the target value of 25℃~37℃ or the response time is >3s, the molar ratio of the two liquid gases is adjusted and the operation is repeated until both the phase change temperature and the response time reach the target value.

[0015] In some embodiments of the present invention, the phase transition temperature detection conditions are as follows: the contrast agent is placed in a latex finger sleeve and placed in a heating bath, the heating rate is 0.5-2℃ / min, the test range is 20℃~40℃, the sample amount is 8-15mg, and the sample temperature is maintained at ≤10℃ before testing; the detection conditions of the ultrasonic triggering test system are as follows: the ultrasonic contrast instrument probe is placed in the heating bath, the ultrasonic frequency is 1-15MHz, preferably 2-15MHz, the test environment temperature simulates the human physiological temperature: 25℃~37℃, the particle size change is monitored in real time by a dynamic light scattering instrument and a microscope, and the conversion time from nanoscale to microscale is recorded as the phase transition response time.

[0016] In some embodiments of the present invention, in step S5, the protective solution contains 3%-8% mannitol, 1%-2% trehalose, and 0.5%-1% proline by mass.

[0017] In some embodiments of the present invention, in step S5, the dispersion is centrifuged at ≤15°C and 4000-8000 r / min for 5-15 min, the precipitate is collected, and washed 2-4 times with an isotonic buffer solution pre-cooled to ≤10°C.

[0018] A second aspect of the present invention provides an ultrasound-temperature dual-triggered dual-liquid-gas phase change contrast agent, said dual-liquid-gas phase change contrast agent being prepared according to the method described in the first aspect.

[0019] In some embodiments of the present invention, the initial particle size of the dual liquid-gas phase change contrast agent is 100-800 nm, the molar ratio is (1:9)-(9:1), the phase change temperature is 25℃~37℃, and after the phase change, it expands into micron-sized microbubbles of 1-10 μm. Under dual triggering of ultrasonic frequency 1-15 MHz and temperature 25℃~37℃, the phase change response time is ≤3s.

[0020] In some embodiments of the present invention, the quantitative relationship between the molar ratio of the two liquid phase change gases and the phase change temperature during the preparation of the dual liquid-gas phase change contrast agent is as follows: When the ratio of low phase change temperature gas to high phase change temperature gas is 7:3, the phase change temperature is 27℃~29℃; When the ratio of low phase change temperature gas to high phase change temperature gas is 5:5, the phase change temperature is 30℃~32℃; When the ratio of low phase change temperature gas to high phase change temperature gas is 3:7, the phase change temperature is 34℃~37℃; When the ratio of low phase change temperature gas to high phase change temperature gas is 9:1, the phase change temperature is 25℃~26℃. When the ratio of low phase transition temperature gas to high phase transition temperature gas is 1:9, the phase transition temperature is 36℃~37℃.

[0021] In some embodiments of the present invention, the low phase change temperature gas is perfluorobutane (C4F10), with a phase change temperature of -1℃ to 1℃, and it is in a liquid state when the total pressure is adjusted to 4MPa and the temperature is 1-3℃ during the preparation process; the high phase change temperature gas is perfluoropentane (C5F12), with a phase change temperature of 29℃ to 31℃, and it is in a liquid state when the temperature is controlled to be ≤25℃ during the preparation process.

[0022] The present invention also provides the application of a dual-liquid-gas phase change contrast agent, as described in the second aspect, in ultrasound medical imaging diagnosis.

[0023] The applications include ultrasound-enhanced imaging and precise delineation of lesion boundaries for thyroid nodules, breast lesions, liver tumors, kidney space-occupying lesions, cardiovascular stenosis, cerebral vascular malformations, and skin tumors. The application method is intravenous injection. After the contrast agent enters the body, under the dual triggering of the local temperature of the lesion (25℃~37℃) and the ultrasound of the ultrasound contrast instrument (frequency 1-15MHz), the two liquid gases synchronously change to gaseous state, completing the nano-micro phase transition transformation, and achieving targeted and efficient imaging.

[0024] In this application, the wall material adopts a composite system of natural phospholipids, targeted modified phospholipids, stabilizers, membrane elasticity modifiers, and antioxidants. Among them, natural phospholipids ensure biocompatibility; targeted modified phospholipids achieve specific enrichment of diseased tissues; stabilizers enhance membrane rigidity and reduce gas leakage; membrane elasticity modifiers optimize membrane flexibility and can buffer membrane damage caused by the impact of ultrasonic cavitation effect, ensuring that vesicles expand without rupture under dual ultrasonic-temperature triggering; and antioxidants prevent phospholipid oxidation and improve storage stability.

[0025] The core material employs a dual-liquid phase-change gas synergistic design: perfluorobutane (low phase change temperature) is prepared in a liquid state and undergoes a rapid phase change within the body, serving as a framework gas to maintain the microbubble structure; perfluoropentane (high phase change temperature) is prepared in a liquid state and serves as a driving gas to provide expansion power; the molar ratio of the two gases is (1:9) to (9:1), and they are stably co-loaded through intermolecular hydrophobic interactions. This ratio directly controls the phase change temperature, adapting to different disease lesion temperature environments. The liquid fluorocarbons mentioned are two of perfluorobutane, perfluoropentane, perfluorohexane, perfluorooctane, perfluoronaphthane, and perfluoropolyether.

[0026] Dual-trigger performance parameters: To meet vascular penetration requirements, the initial particle size is 100-800nm; to meet ultrasound scattering requirements, the particle size expands to 1-10μm microbubbles after phase transition; among the ultrasound trigger parameters, the frequency is 1-15MHz, which is a commonly used frequency band for clinical ultrasound contrast imaging, and the sound pressure is within the safe sound pressure range of 0.1-1.0MPa; the phase transition response time under dual triggering is ≤3s.

[0027] The preparation method adds an ultrasonic response calibration step to the original process: Dual-trigger performance calibration: By changing the temperature and using an ultrasound triggering test system to simulate clinical ultrasound parameters to detect the response time, the molar ratio of the two gases is dynamically adjusted. If the response time is too long, the proportion of perfluorobutane is appropriately increased. If the phase transition temperature deviates, the ratio is adjusted according to quantitative correlation to ensure that the performance meets the standards under dual-trigger conditions. Ultrasound parameter matching: Determine the ultrasound frequency as 1-15MHz (preferably 2-15MHz, adapting to the imaging needs of different organs: use high frequency 5-15MHz for superficial organs and low frequency 1-5MHz for internal organs), and sound pressure as 0.1-1.0MPa to ensure clinical compatibility; Low-temperature process optimization: Low-temperature control throughout the process (≤15℃) to avoid premature phase change of gas before ultrasonic calibration and ensure test accuracy.

[0028] During application, after intravenous injection, the contrast agent, with a nanoparticle size of 100-800nm, penetrates the interendothelial space of blood vessels and accumulates in the lesion tissue. When the dual conditions of lesion temperature (25℃~37℃) and ultrasound (1-15MHz, 0.1-1.0MPa) are met, the cavitation effect of ultrasound disrupts the local molecular forces on the vesicle membrane surface, and the thermal effect rapidly replenishes the phase transition heat. The temperature-induced phase transition of the two liquid gases is triggered simultaneously: perfluorobutane rapidly changes to gas to form a stable core, and perfluoropentane changes to gas to generate a strong expansion force, driving the nanovesicles to expand into 1-10μm micron-sized microbubbles within ≤3s. The micron-sized microbubbles generate a strong scattering effect in the ultrasound field, enhancing the ultrasound signal in the lesion area and achieving precise and rapid imaging. The dual gases work together to maintain the stability of the microbubble structure.

[0029] Beneficial effects of the present invention Compared with the prior art, the present invention has the following beneficial effects: The ultrasound-temperature dual-triggered dual liquid-gas phase change contrast agent of the present invention adopts dual triggering for precise and rapid response. Ultrasound and temperature are synergistically triggered, and the phase change response time is ≤3s, which is significantly faster than the single temperature triggering time of >5s. The phase change only occurs under the dual conditions of lesion temperature and ultrasound irradiation, reducing the risk of non-specific imaging. Unlike traditional contrast agents with a single phase transition temperature, the phase transition temperature of the dual liquid-gas phase transition contrast agent in this application is precisely adjustable over a wide range. The molar ratio of the two gases is quantitatively correlated with the phase transition temperature, covering 25℃~39℃, which is suitable for different temperature environments such as superficial lesions and internal organs. The targeted modified phospholipids in the composite phospholipid wall material can achieve specific enrichment of diseased tissues, and the strong targeting can reduce non-specific imaging; the phospholipid material has good biocompatibility, is biodegradable, has no obvious toxic side effects, and meets the requirements for clinical application. The synergistic effect of the composite phospholipid membrane and stabilizer reduces gas leakage and has high stability. The co-carrying system of the two gases prolongs the in vivo circulation time of the contrast agent, resulting in long-lasting imaging with a duration of 15-30 minutes, which meets the operational requirements of clinical diagnosis. The ultrasound-temperature dual-triggered dual liquid-gas phase change contrast agent of the present invention can be prepared using conventional laboratory equipment: rotary evaporator and high-pressure reactor. The process steps are clear and the parameters are controllable, making it suitable for large-scale production. Furthermore, the phase change temperature and targeting ligand can be flexibly adjusted according to clinical needs. Attached Figure Description

[0030] Figure 1 Comparison of emulsions containing two liquid perfluorinated carbons in ultrasound contrast agents before and after phase transition; Figure 2 The particle size potential distribution of a nanoscale ultrasound contrast agent encapsulating two types of liquid perfluorinated carbon. Figure 3 Storage stability of nanoscale ultrasound contrast agents encapsulated with two liquid perfluorinated carbons; Figure 4 Transmission electron microscopy image of a nanoscale ultrasonic contrast agent encapsulating two liquid perfluorinated carbons before phase transition; Figure 5 Microscopic imaging of an ultrasonic contrast agent encapsulating two liquid perfluorinated carbons after a phase transition; Figure 6 An in vitro imaging image of an ultrasound contrast agent encapsulating two liquid perfluorocarbons; Figure 7 An in vitro imaging image of degassed saline as an ultrasound contrast agent. Detailed Implementation

[0031] The following examples are used to illustrate preferred embodiments of the invention. Those skilled in the art will understand that the techniques disclosed in the examples represent techniques discovered by the inventors that can be used to implement the invention, and therefore can be considered preferred embodiments for implementing the invention. However, those skilled in the art should understand from this specification that many modifications can be made to the specific embodiments disclosed herein, still yielding the same or similar results, without departing from the spirit or scope of the invention.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and all materials disclosed herein are incorporated herein by reference. Many equivalent techniques of specific embodiments of the invention described herein will be recognized or can be understood by those skilled in the art through conventional experimentation. These equivalents will be included in the claims.

[0033] The technical solution of this application will be further described in detail below with reference to specific embodiments.

[0034] Example 1: Ultrasound contrast agent targeting liver tumors with a phase transition temperature of 37°C 1. Preparation of composite phospholipid films Take 1.0g egg yolk lecithin, 0.3g galactose-modified phospholipid (liver tumor targeting ligand) and 0.05g cholesterol, dissolve them in 25mL chloroform-methanol mixture (chloroform / methanol volume ratio 2:1), and rotary evaporate at 40℃ for 25min to remove organic solvent, forming a uniform and dense composite phospholipid film. 2. Targeted vesicle hydration Add 50 mL of PBS buffer solution to the composite phospholipid film, sonicate at 200 W and 25 kHz for 30 min at 37 °C, and then shake at 150 r / min for 2 h on a constant temperature shaker to obtain a targeted phospholipid vesicle dispersion. 3. Liquid-liquid dual-gas co-carrying The dispersion was transferred to a high-pressure reactor. Liquid perfluorohexane (phase change temperature 37℃) was first introduced, and the pressure was gradually increased to 2.5 MPa at a rate of 0.3 MPa / 20 min. The temperature was controlled at 25℃ (40℃ below the atmospheric boiling point of perfluorohexane) and kept at this temperature for 1.5 h. Then, liquid perfluorobutane was introduced, and the total pressure was adjusted to 4 MPa and the temperature to 1-3℃. The mixture was stirred at 600 r / min for 3 h. 4. Phase transition temperature calibration The initial phase transition temperature was determined to be 39℃ by particle size analysis. The molar ratio of perfluorobutane to perfluorohexane was adjusted to 2:8, and step (3) was repeated. The final phase transition temperature was 37℃. 5. Contrast agent purification and stabilization The dispersion was filtered through a 5 μm filter membrane, centrifuged at 4000 rpm for 10 min, and the precipitate was collected. The precipitate was washed twice with PBS buffer, and a protective solution containing 3% mannitol was added. The mixture was then sealed and stored at 4°C to obtain the ultrasound contrast agent. Analysis showed that the particle size of this contrast agent was 300-800 nm, and after phase transition, the particle size expanded to the micrometer level. It was stored at 4°C.

[0035] Example 2: Ultrasound contrast agents targeting cardiovascular stenosis 1. Preparation of composite phospholipid films Take 0.8g of soybean lecithin, 0.2g of RGD peptide-modified phospholipid (vascular endothelial targeting ligand) and 0.03g of polyethylene glycol-cholesterol succinate, dissolve them in 20mL of ethanol-dichloromethane mixture (ethanol / dichloromethane volume ratio 3:1), and vacuum rotary evaporate at 35℃ for 20min to form a composite phospholipid film; 2. Targeted vesicle hydration Add 40 mL of physiological saline (pH=7.4, osmotic pressure 310 mOsm / kg) to the membrane, disperse it at 30℃ with ultrasonication at 180 W and 20 kHz for 25 min, and then shake it in a constant temperature shaker at 120 r / min for 1.5 h to obtain the targeted vesicle dispersion; 3. Liquid-liquid two-phase gas co-carrying The dispersion was transferred to a high-pressure reactor. Liquid perfluorobutane (phase change temperature -2℃) was first introduced, and the pressure was gradually increased to 2.5 MPa at a rate of 0.3 MPa / 20 min. The temperature was controlled at 1℃ and kept at this temperature for 1.5 h. Then, liquid perfluoropentane was introduced, and the total pressure was adjusted to 4 MPa and the temperature to 15℃. The mixture was stirred at 600 r / min for 3 h. 4. Phase transition temperature calibration The initial phase transition temperature was determined to be 39℃ by particle size analysis. The molar ratio of perfluorobutane to perfluoropentane was adjusted to 2:8. Step (3) was repeated, and the final phase transition temperature was determined to be 37℃. 5. Contrast agent purification and stabilization The dispersion was filtered through a 5 μm filter membrane, centrifuged at 4000 rpm for 10 min, and the precipitate was collected. The precipitate was washed twice with PBS buffer, and a protective solution containing 3% mannitol was added. The mixture was then sealed and stored at 4°C to obtain the ultrasound contrast agent. Analysis showed that the particle size of this contrast agent was 300-800 nm, and after phase transition, the particle size expanded to the micrometer level. It was stored at 4°C.

[0036] Example 3: Preparation of ultrasound contrast agent 1. Preparation of composite phospholipid films Take 0.9 mg DPPA, 8 mg DPPC and 6 mg PEG5K-DPPE, dissolve them in 4 mL chloroform-methanol solution (chloroform / methanol volume ratio of 3:1), and evaporate under vacuum at 35 °C for 20 min to form a composite phospholipid film; 2. Vesicle hydration Add 8 mL of physiological saline to the membrane, hydrate the membrane at 50 °C, and then disperse it by ultrasonication at 200 W for 6 seconds, followed by a 6-second interval, for a total duration of 6 minutes to obtain the vesicle dispersion. 3. Liquid-liquid two-phase gas co-carrying The dispersion was transferred to an ice-water bath at 0-1℃, and liquid perfluorobutane and liquid perfluoropentane were added successively. The speed of the high-speed shear was adjusted to 20000 r / min and sheared for 10 min. 4. Phase transition temperature calibration By comparing the emulsion state and particle size, the initial phase transition temperature was 39℃. The molar ratio of perfluorobutane to perfluoropentane was adjusted to 3:7, and the previous steps were repeated. The final phase transition temperature was measured to be 37℃. 5. Contrast agent purification and stabilization The dispersion was filtered through a 5 μm filter membrane, centrifuged at 4000 rpm for 10 min, and the precipitate was collected. The precipitate was washed twice with PBS buffer, and a protective solution containing 3% mannitol was added. The mixture was then sealed and stored at 4°C to obtain the ultrasound contrast agent. Analysis showed that the particle size of this contrast agent was 200-800 nm, and after phase transition, the particle size expanded to the micrometer level. It was stored at 4°C.

[0037] Example 4: Ultrasound Contrast Agent 1. Preparation of phospholipid membranes Accurately weigh 100 mg of soybean lecithin and 20 mg of DSPE-PEG2000, place them in a 50 mL round-bottom flask, add 10 mL of anhydrous ethanol, and stir to dissolve in a 60℃ constant temperature water bath to form a homogeneous phospholipid ethanol solution. Then, turn on a rotary evaporator and evaporate the ethanol at 60℃, 100 r / min, and vacuum to form a uniform and transparent phospholipid film on the inner wall of the flask. Vacuum dry for 12 h to completely remove residual ethanol.

[0038] 2. Aqueous phase dispersion and pre-emulsification Add 20 mL of physiological saline containing 5% glycerol to the round-bottom flask containing the phospholipid film. Place the flask in a 37°C constant temperature water bath and hydrate it with magnetic stirring at 300 r / min for 30 min, allowing the phospholipid film to fully swell and detach, forming a homogeneous phospholipid vesicle suspension. Then transfer the suspension to an ultrasonic cell disruptor and sonicate it under ice bath conditions. The ultrasonic power is 200 W, with a 3-second operation followed by a 5-second interval, for a total duration of 10 min, yielding a primary emulsion.

[0039] 3. Dual-liquid phase change gas loading and high-pressure homogenization The primary emulsion was transferred to a sealed reactor in a high-pressure homogenizer. Perfluoropentane (PFP) and perfluorohexane (PFH) gases were introduced into the reactor at a volume ratio of 1:1 to maintain the pressure at 0.8 MPa. The reactor was stirred at 30°C and 500 r / min for 30 minutes to ensure that the two liquid phase change gases were fully dissolved and incorporated into the phospholipid vesicles. Subsequently, the high-pressure homogenizer was turned on and homogenized 15 times at 80 MPa to homogenize the vesicle particle size and further encapsulate the gas.

[0040] 4. Nano-to-micron conversion regulation and purification After homogenization, the reaction system was slowly cooled to 4°C and kept at this temperature for 2 hours to induce structural reconstruction of phospholipid vesicles, forming a nanoparticle suspension with nano-to-micron conversion potential. The suspension was then centrifuged at 4°C and 3000 r / min for 10 minutes to remove unencapsulated free gas and a small amount of large-particle impurities from the upper layer. The lower precipitate was collected and resuspended in physiological saline at 4°C. This centrifugation-resuspending process was repeated twice to obtain a dual-liquid phase change gas co-loaded nano-to-micron phospholipid ultrasound contrast agent suspension triggered by both ultrasound and temperature. This suspension was then sealed and stored at 4°C for later use.

[0041] Example 5 Product Characterization 1. Comparison of the ultrasound contrast agent suspension prepared in Example 3 before and after phase transition. Observation shows that the solution becomes turbid after the phase transition. (See attached image) Figure 1 .

[0042] 2. Particle Size and Potential Analysis: The contrast agent suspension from Example 3 was diluted 100 times with physiological saline. The particle size distribution, average particle size, and Zeta potential were measured using a dynamic light scattering (DLS) instrument at 25°C. The results showed that the average particle size of the contrast agent was 200-800 nm, the particle size distribution index (PDI) was 0.1-0.3, and the Zeta potential was -42.56 mV, indicating that the product had uniform particle size and good dispersibility. (See Appendix) Figure 2 .

[0043] 3. Stability Observation: The prepared ultrasound contrast agent of Example 3 was sampled at 4℃ on days 0, 1, 2, 3, 4, 5, 6, and 7 for particle size potential detection. The particle size and potential were stable, with no significant changes observed. The results are shown in the appendix. Figure 3 .

[0044] 4. Morphological observation: A suitable amount of contrast agent suspension was dropped onto a copper grid, stained with phosphotungstic acid, and then observed under a transmission electron microscope (TEM). The results showed that the contrast agent was a spherical or near-spherical vesicle structure with clear vesicle walls. The particle size was basically consistent with the DLS measurement results. (See attached image) Figure 4 .

[0045] Example 6: Performance Verification of Dual Triggering of Ultrasonic and Temperature 1. Experimental Grouping The contrast agent liquid prepared in Example 3 and the degassed physiological saline were placed in latex finger cots, respectively. Three samples were set up in each group.

[0046] 2. Trigger condition settings Temperature trigger: Place the sample in a heating bath and incubate at a constant temperature of 37°C for 30 minutes.

[0047] Ultrasonic triggering: A diagnostic-grade ultrasound probe was placed on the surface of a latex finger cot, and the ultrasound parameters were set as follows: frequency 15MHz, irradiation time 30s.

[0048] 3. Detection Indicators and Results Ultrasonic signal intensity: The ultrasonic echo signal intensity of each group of samples was measured using an ultrasonic diagnostic instrument. Microscopic imaging of the ultrasonic contrast agents encapsulating two liquid perfluorocarbons after phase transition is shown below. Figure 5 As shown; in vitro imaging of ultrasound contrast agents encapsulating two types of liquid perfluorinated carbon. Figure 6 As shown; in vitro imaging of degassed saline as an ultrasound contrast agent is shown below. Figure 7 As shown.

[0049] The results showed that the ultrasound signal intensity was significantly increased in the ultrasound-temperature dual-trigger group with contrast agent, while no ultrasound signal intensity was observed in the saline group, indicating that the ultrasound imaging effect of the contrast agent was significant under dual-trigger conditions.

[0050] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by this application.

Claims

1. A method for preparing a dual-liquid-gas phase change contrast agent triggered by both ultrasound and temperature, characterized in that, Includes the following steps: S1. A composite phospholipid composition consisting of natural / synthetic phospholipids, targeted modified phospholipids, stabilizers, and membrane elasticity modifiers is dissolved in a mixed organic solvent, and the organic solvent is removed by rotary evaporation at 35-50 °C to form a uniform and dense composite phospholipid film. S2. Add an isotonic buffer solution to the composite phospholipid film, disperse it by ultrasonication, and then oscillate to obtain a targeted phospholipid vesicle dispersion. S3. Transfer the targeted phospholipid vesicle dispersion to a high-pressure reactor. First, introduce liquid high phase change temperature gas and gradually increase the pressure to 2.5 MPa at a rate of 0.3 MPa / 20 min, while controlling the temperature to not exceed 25 ℃. Keep it at this temperature and let it stand. Then, introduce liquid low phase change temperature gas, adjust the total pressure to 4 MPa, control the temperature to 1-3 ℃, and stir. S4. The product generated in step S3 is subjected to particle size detection, and the molar ratio of perfluorobutane to perfluorohexane is repeatedly adjusted to calibrate the phase transition temperature. S5. Filter the dispersion using a filter membrane, collect the precipitate after centrifugation, wash with PBS buffer solution, add protective solution, seal and store to obtain the dual liquid-gas phase change contrast agent.

2. The method for preparing a dual-liquid-gas phase change contrast agent triggered by ultrasound and temperature according to claim 1, characterized in that, In step S1, the mass ratio of natural phospholipids / synthetic phospholipids, targeted modified phospholipids, stabilizers, and membrane elasticity modifiers in the composite phospholipid composition is 1:(0.2-0.6):(0.01-0.04):(0.001-0.005).

3. The method for preparing a dual-liquid-gas phase change contrast agent triggered by ultrasound and temperature according to claim 2, characterized in that, In step S1, an antioxidant with a mass fraction of 0.05%-0.3% is added to the complex phospholipid composition. The antioxidant is selected from one or more of ascorbyl palmitate and glutathione.

4. The method for preparing a dual-liquid-gas phase change contrast agent triggered by ultrasound and temperature according to claim 2, characterized in that, In step S1, the mixed organic solvent is a chloroform-methanol mixture with a volume ratio of (1-6):1 or an ethanol-dichloromethane mixture with a volume ratio of (2-8):1; the mass-volume ratio of the composite phospholipid composition to the mixed organic solvent is 1 g: (25-80) mL; the temperature of the mixed organic solvent is maintained at ≤10℃ by an ice bath or a low-temperature constant temperature bath.

5. The method for preparing a dual-liquid-gas phase change contrast agent triggered by ultrasound and temperature according to claim 2, characterized in that, The natural phospholipid is one or more of soybean lecithin and egg yolk lecithin; the synthetic phospholipid is one or more of DPPA, DPPC, DSPA, DSPC, DOPC, POPC, DSPE-PEG, and DPPE-PEG.

6. The method for preparing a dual-liquid-gas phase change contrast agent triggered by ultrasound and temperature according to claim 2, characterized in that, The targeted modified phospholipids are one or more of the following: folic acid-modified phospholipids, RGD peptide-modified phospholipids, galactose-modified phospholipids, and transferrin-modified phospholipids.

7. The method for preparing a dual-liquid-gas phase change contrast agent triggered by ultrasound and temperature according to claim 2, characterized in that, The stabilizer is one or more of cholesterol, polyethylene glycol, and vitamin E succinate; the membrane elasticity modifier is one or more of palmitic acid, stearic acid, and oleic acid.

8. The method for preparing a dual-liquid-gas phase change contrast agent triggered by ultrasound and temperature according to claim 1, characterized in that, In step S2, the isotonic buffer solution is one of phosphate buffer solution, physiological saline or HEPES buffer solution, with a pH value of 7.2-7.5, an osmotic concentration of 280-330 mOsm / kg, and a mass-to-volume ratio of composite phospholipid membrane to isotonic buffer solution of 1 g: (50-120) mL.

9. The method for preparing a dual-liquid-gas phase change contrast agent triggered by ultrasound and temperature according to claim 1, characterized in that, In step S5, the protective solution contains 3%-8% mannitol, 1%-2% trehalose, and 0.5%-1% proline by mass.

10. A dual-liquid-gas phase change contrast agent triggered by both ultrasound and temperature, characterized in that: Prepared by the method according to any one of claims 1-9.

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