Lipid microbubble freeze-dried powder composition and preparation method thereof

Abstract

The invention provides a lipid microbubble freeze-dried powder composition and a preparation method thereof, and the composition comprises 1 part by weight of distearoyl phosphatidylcholine, 1 part by weight of 1, 2-palmitoyl phosphatidyl sodium glycerol, 100-240 parts by weight of polyethylene glycol 4000 and 0.1-0.3 part by weight of palmitic acid. The freeze-dried powder composition and physiologically acceptable gas form a sealed penicillin bottle, and the penicillin bottle is used for ultrasonic contrast after being redissolved. The lipid microbubble freeze-dried powder composition has the advantages of narrow particle size distribution of redissolved microbubbles, high stability of the microbubbles, difficulty in breakage, good pressure resistance and the like by adjusting the proportions of the components and process parameters of the lipid microbubble freeze-dried powder composition, and has stronger image acoustic signals and longer retention time at a target part during radiography.

Description

Lipid microvesicle freeze-dried powder composition and preparation method thereof Technical Field

[0001] The present application belongs to the field of medicine and relates to a lipid microbubble freeze-dried powder composition and a preparation method thereof, and specifically to an ultrasound contrast agent for improving the contrast of ultrasound images. Background Art

[0002] Ultrasound contrast agents (UCAs) are a class of diagnostic drugs that, when injected into a peripheral vein, cause strong scattering in the blood, significantly enhancing ultrasound detection signals. With the rapid development of ultrasound medicine and clinical pharmacology, ultrasound contrast imaging has become one of the fastest-growing technologies in medical imaging.

[0003] Sulfur hexafluoride microbubbles for injection (trade name: ) is an ultrasound contrast agent that has been on the market for many years at home and abroad. It is used for ultrasound contrast diagnosis and has multiple indications such as liver angiography, cardiac angiography, and urinary system angiography. It is widely used.

[0004] However, with clinical application, clinical researchers have discovered that the Sonovue contrast agent leaves room for improvement. This can be addressed in several key areas: First, the stability of the microbubbles after reconstitution needs to be improved. During Sonovue use, if the initial contrast imaging is unclear, a secondary imaging session can be performed. However, the enhancement effect of the secondary imaging session is not as good as the initial one. This is because, during the reconstitution and imaging process, the volume concentration of the microbubbles in the reconstituted solution gradually decreases over time, hindering contrast imaging. According to the literature (Influence of Bubble Size Distribution on the Echogenicity of Ultrasound Contrast Agents, Investigative Radiology, Volume 35, Number 11, 661–671), the particle size of ultrasound contrast agent microbubbles is related to the resonant frequency. Within a certain range, the higher the volume concentration of the central particle size, the stronger the resonant frequency, meaning the stronger the reflected acoustic signal. Therefore, improving the stability of contrast agent microbubbles and thus maintaining their volume concentration is an urgent issue.

[0005] Furthermore, the contrast effect of the contrast agent can be enhanced by increasing the volume concentration of the contrast agent microbubbles.

[0006] In addition, the pressure resistance of microbubbles needs to be improved. As stated in the Chinese document "Experimental Study on the Effect of Pressure on the Backscattering Intensity of Contrast Agent Microbubbles" (paper, author: Pan Min, Class of 2004, Huazhong University of Science and Technology, majoring in Imaging Medicine and Nuclear Medicine), Sonovue is suitable for ultrasound probes with a center frequency of 2-7.5MHz. However, the linear array probes used for superficial organs such as the thyroid and breast generally have a frequency of more than 10MHz. Under high-frequency probes, Sonovue microbubbles are easy to rupture, resulting in a shorter residence time of microbubbles in organs such as the thyroid and breast, and poor development effect. Therefore, improving the pressure resistance of microbubbles and expanding their scope of application are issues that urgently need to be improved.

[0007] Therefore, how to overcome the above-mentioned defects of the existing technology and improve the use effect of contrast agents is still a problem to be solved urgently in this field.

[0008] Summary of the Invention

[0009] In order to solve the problems of poor stability of Sonovue microbubbles after reconstitution, gradual decrease in microbubble volume concentration over time, and poor pressure resistance of microbubbles, the present application provides a lyophilized powder composition of a lipid microbubble ultrasound contrast agent, thereby obtaining a contrast agent with good stability, high microbubble volume concentration, good acoustic response, and good pressure resistance.

[0010] In a first aspect, the present application provides a lyophilized powder composition for preparing a lipid microbubble ultrasound contrast agent, the composition comprising distearoylphosphatidylcholine (DSPC), sodium 1,2-palmitoylphosphatidylglycerol (DPPG-Na), polyethylene glycol 4000 (PEG4000) and palmitic acid; wherein, DSPC is 1 part by weight; DPPG-Na is 1 part by weight, PEG4000 is 150-200 parts by weight, and palmitic acid is 0.1-0.3 parts by weight.

[0011] Preferably, the PEG4000 is 170-185 parts by weight.

[0012] Preferably, the PEG4000 is 173-185 parts by weight.

[0013] In a second aspect, the present application provides a sealed vial for preparing a lipid microbubble ultrasound contrast agent, comprising the lyophilized powder composition of the first aspect and a physiologically acceptable gas.

[0014] In some embodiments, the gas may be a fluorinated gas, wherein the fluorinated gas may be any one or a combination of two of perfluoromethane, perfluoropropane, perfluorobutane, perfluoropentane, and perfluorohalogenated alkanes.

[0015] In other embodiments, the gas may be sulfur hexafluoride.

[0016] In other embodiments, the gas may also be an inert gas with low water solubility; preferably, the inert gas with low water solubility may be nitrogen, argon, etc.

[0017] In a third aspect, the present application provides a method for preparing a sealed vial of a lipid microbubble ultrasound contrast agent, comprising the following steps:

[0018] (1) dissolving DSPC, DPPG-Na, PEG4000 and palmitic acid in a solvent to form a solution;

[0019] (2) The solution was quickly filled into a vial and vacuum freeze-dried to remove the solvent;

[0020] (3) introducing a physiologically acceptable gas into the vial and sealing it; and

[0021] (4) Place the sealed vial in a thermostat for activation for 12-24 hours.

[0022] In some embodiments, the solvent in step (1) is a mixture of any one or more of n-hexane, isopropanol, cyclohexanol, 2-methyl-2-butanol, tert-butanol, or n-butanol.

[0023] Preferably, the solvent in step (1) is a mixture of tert-butanol and isopropanol, or a mixture of n-hexane and isopropanol.

[0024] During the research process, the inventors found that heating and activating the freeze-dried powder of lipid microbubble ultrasound contrast agent is more conducive to the concentrated particle size distribution of the microbubbles formed after the microbubbles are re-dissolved, resulting in a higher volume concentration of the microbubbles, better acoustic response, and better pressure resistance.

[0025] Preferably, the temperature of the thermostat in step (4) is 35-50°C, more preferably 45°C.

[0026] Preferably, the activation time in step (4) is 18-22 hours.

[0027] In a fourth aspect, the present application provides a method for preparing a lipid microbubble ultrasound contrast agent, which comprises: dispersing the freeze-dried powder composition described in the first aspect in physiological saline (0.9% sodium chloride aqueous solution) in the presence of a physiologically acceptable gas to form a microbubble suspension, namely a lipid microbubble ultrasound contrast agent.

[0028] Preferably, the volume concentration of the microbubble suspension is ≥7 μL / mL; more preferably, the volume concentration of the microbubble suspension is ≥8 μL / mL.

[0029] Preferably, the concentration of the microbubble suspension is ≥5×10 8 pieces / cm 3More preferably, the concentration of the microbubble suspension is ≥7×10 8 pieces / cm 3 .

[0030] In a fifth aspect, the present application provides the use of the freeze-dried powder composition described in the first aspect as a blood pool ultrasound contrast agent and a cavity ultrasound contrast agent.

[0031] In a sixth aspect, the present application provides a method for performing ultrasound imaging using the lipid microbubble ultrasound contrast agent prepared by the method described in the fourth aspect, comprising the following steps:

[0032] administering to the patient an effective amount of a lipid microbubble ultrasound contrast agent;

[0033] transmitting an ultrasound signal to a body part of the patient; and

[0034] Echographic signals are collected from the body part.

[0035] Compared with the prior art, this application has the following beneficial effects:

[0036] The lyophilized powder composition of the lipid microbubble ultrasound contrast agent of the present application has multiple advantages such as high stability of reconstituted microbubbles, good acoustic response, and good pressure resistance. In addition, during ultrasound contrast imaging, the image acoustic signal is stronger, the target site residence time is longer, and the pressure resistance is better, making it suitable for various organs and probe contrast imaging modes. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] FIG1 is the particle size distribution of the lipid microbubble ultrasound contrast agent of batch 4-D sample in Example 4.

[0038] FIG2 is an ultrasound contrast image of the rabbit kidney obtained by performing ultrasound contrast imaging on the 4-D sample of Example 8.

[0039] FIG3 is an ultrasound contrast image of the rabbit kidney obtained after contrast-enhanced ultrasound of the SonoVue sample of Example 8.

[0040] FIG4 is an ultrasound contrast image of the rabbit fallopian tube obtained after contrast-enhanced ultrasound of the 4-D sample of Example 8. DETAILED DESCRIPTION

[0041] Abbreviation meaning

[0042] DSPC: Distearoylphosphatidylcholine

[0043] DPPG-Na: 1,2-palmitoylphosphatidylglycerol sodium

[0044] PEG4000: polyethylene glycol 4000

[0045] Terminology

[0046] SonoVue: Generic name: Sulfur hexafluoride microbubbles for injection. This drug is an ultrasound contrast agent developed by the Italian company Bracco Imaging. According to the instructions, each vial contains 59 mg of sulfur hexafluoride, 0.19 mg of distearoylphosphatidylcholine (DSPC), 0.19 mg of sodium 1,2-dipalmitoylphosphatidylglycerol (DPPG), 0.04 mg of palmitic acid, and 24.5 mg of polyethylene glycol 4000.

[0047] Polyethylene glycol 4000: Polyethylene glycol is a high molecular weight polymer with the chemical formula HO(CH2CH2O) n H, its properties vary depending on its molecular weight, ranging from a colorless, odorless, viscous liquid to a waxy solid. PEG4000 is a polyethylene glycol polymer with an average molecular weight of 4000, n=70-85, and a melting point of 53-56°C. The average molecular weight of polyethylene glycol 4000 is approximately 4000, and its structure is:

[0048] Microbubbles: Microbubbles are a class of pharmaceutical agents typically composed of a gas core and a stable shell. The bubble coating is typically composed of lipids, polymers, or proteins, which have good biocompatibility and are relatively safe for intravenous injection. Phospholipids, as the shell of microbubbles, are more flexible than cross-linked polymer shells. Under ultrasound, they are more susceptible to contraction, rupture, bending, and redispersion, thereby enhancing the microbubble's sensitivity to sound waves.

[0049] Microbubble volume concentration: referred to as "volume concentration", refers to the ratio of the total volume of all microbubbles in the suspension to the total volume of the suspension, that is, the total volume concentration of microbubbles in unit volume of suspension (μL / mL).

[0050] Microbubble number: referred to as "number", refers to the ratio of the total number of microbubbles in the suspension to the total volume of the suspension, that is, the number of microbubbles in unit volume of the suspension.

[0051] Reconstitution: refers to the process of adding physiological saline to the lyophilized powder preparation of ultrasound contrast agent and shaking it to dissolve it before clinical use to obtain a uniformly distributed microbubble suspension.

[0052] Residual ratio: refers to the percentage of the volume concentration of the microbubble suspension obtained after reconstitution to the initial volume concentration before treatment after being treated at room temperature or under pressure during stability investigation or pressure resistance test.

[0053] Blood pool ultrasound contrast imaging: refers to an ultrasound contrast imaging method in which a reconstituted ultrasound contrast agent microbubble suspension is injected intravenously into the systemic circulation to obtain contrast-enhanced ultrasound imaging images of organs in the body, including liver and abdominal organ ultrasound imaging, cardiac ultrasound imaging, vascular Doppler, etc.

[0054] Cavity ultrasound contrast imaging: refers to an ultrasound contrast imaging method in which the reconstituted ultrasound contrast agent is injected into the uterus, gallbladder, bladder and other cavity organs through a syringe / catheter to obtain organ contrast-enhanced ultrasound imaging images.

[0055] The present application is described in more detail below with reference to specific embodiments. It should be noted that the present application is not limited to the following embodiments.

[0056] Example 1 Preparation of a freeze-dried composition for direct freeze drying

[0057] DSPC, DPPG, palmitic acid and polyethylene glycol-4000 were weighed and dissolved in an organic solvent, and the mixture was dispensed into vials according to a ratio of (1:1:(0.1-0.3):(100-240)). The vials were half-stoppered with bromobutyl rubber stoppers, pushed onto the partition of a freeze dryer, and vacuum-frozen for 24 hours. Sulfur hexafluoride gas was then introduced, and the vials were stoppered and capped to prepare a sealed freeze-dried composition.

[0058] Preparation method of microbubbles: Before use, inject physiological saline into the vial and shake until the freeze-dried powder is completely dispersed into a uniform white milky liquid.

[0059] Example 2 Preparation of freeze-dried compositions with varying amounts of PEG4000 and parameter testing

[0060] The preparation method of Reference Example 1 was used with different weight portions of PEG4000.

[0061] Before detection, 5 mL of 0.9% sodium chloride aqueous solution was injected for reconstitution. Part of the sulfur hexafluoride was encapsulated in the milky white suspended microbubbles. The number and volume concentration of microbubbles were analyzed using a Multisizer 4e Coulter counter (Beckman Coulter, Inc.). The results are shown in Table 1 below.

[0062] Table 1

[0063] The results showed that as the amount of PEG4000 in the formulation increased, the microbubble volume concentration also increased. However, after the PEG4000 dosage increased to a certain weight, the volume concentration actually decreased. Based on the SonoVue quality standard volume concentration range of 2-10 μL / mL, the formulations in this application with a microbubble volume concentration above 10 μL / mL were all more effective than SonoVue, namely batches 3-A, 4-A, 5-A, 6-A, 7-A, and 8-A. Therefore, the optimal volume concentration of PEG4000 was achieved at 135-200 parts by weight.

[0064] Example 3 Selection of optimal activation temperature

[0065] According to the preparation method of Example 1, 40 mg of DSPC, 40 mg of DPPG, 8 mg of palmitic acid, and 6.90 g of polyethylene glycol 4000 (this amount is 173 parts by weight of the amount used in the highest volume concentration in Example 2, the amount used in batch 6-A) were weighed, dissolved and packaged, and then freeze-dried. Sulfur hexafluoride gas was added to form a freeze-dried composition sealed in a vial. The composition was divided into four groups and heated at 35°C, 40°C, 45°C, and 50°C for 24 hours. The specific design is shown in Table 2 below:

[0066] Table 2

[0067] Before detection, 5 mL of 0.9% sodium chloride aqueous solution was injected for reconstitution. Some sulfur hexafluoride was encapsulated in the milky white suspended microbubbles. The number and volume concentration of microbubbles were analyzed using a Multisizer 4e Coulter counter (Beckman Coulter, Inc.). The results are shown in Table 3 below.

[0068] Table 3

[0069] The results showed that the volume concentration of heat treatment at 35℃ was the lowest, and the volume concentration of heat treatment at 45℃ was the highest.

[0070] Example 4 Selection of optimal activation time

[0071] According to the preparation method of Example 1, 40 mg of DSPC, 40 mg of DPPG, 8 mg of palmitic acid, and 6.90 g of polyethylene glycol 4000 (this amount is the highest volume concentration amount in Example 2) were weighed and filled with sulfur hexafluoride gas to form a freeze-dried composition sealed in a vial. The sealed vial was placed in a 45°C thermostat and activated for 0 h, 6 h, 12 h, 24 h, and 48 h. Before detection, 5 mL of 0.9% sodium chloride aqueous solution was injected for redissolution. Some sulfur hexafluoride was encapsulated in the milky white suspended microbubbles. The number and volume concentration of microbubbles were analyzed using a Multisizer 4e Coulter counter (Beckman Coulter, Inc.). The results are shown in Table 4 below.

[0072] Table 4

[0073] It shows that the volume concentration reaches the peak value when the heat treatment time is more than 12 hours. After more than 12 hours, the volume concentration basically does not change with the increase of heat treatment time. TM, 203684Orig1s000, Chemistry Review(s)) shows that the number of microbubbles per ml is (1.5-5.6)×10 8 , the number of contrast agent microbubbles in all the prescriptions in the above table is better than that of SonoVue.

[0074] Note: Lumason TM SonoVue and SonoVue are the trade names of the same product launched in different countries.

[0075] Figure 1 shows the 24-hour sampling test results of batch 4-D samples.

[0076] To further explore the optimal heating process for more than 12 hours, the above experimental protocol was used to further study the number and concentration of contrast agent microbubbles after heating for 12 hours, 16 hours, 18 hours, 20 hours, and 22 hours. The results are shown in Table 5 below.

[0077] Table 5

[0078] The above research shows that further heat treatment investigation results show that the volume concentration basically does not change after heat treatment time of 12-22 hours.

[0079] Comparison of Example 5 and SonoVue Stability

[0080] Three commercially available SonoVue products were prepared according to the instructions as a control group, and three samples from batch 4-D in Example 4 were taken as a test group. Three samples from the control group and the test group were taken, and three samples were dissolved in 5 mL of a 0.9% sodium chloride aqueous solution using volume concentration as a parameter. The samples were analyzed using a Multisizer 4e Coulter counter (Beckman Coulter, Inc.), and the volume concentration at 0 min was recorded. The samples were then allowed to stand at room temperature and pressure for 10 min, 30 min, and 60 min, respectively, and the particle size distribution of the microbubbles was re-tested. The results are shown in Table 6 below.

[0081] Table 6

[0082] As can be seen from the above table, the stability of the test group is significantly better than that of the control group. There is no significant difference between the two at 10 minutes, and the remaining volume concentration ratio is more than 90%. After 60 minutes, the remaining volume concentration of the test group reaches 86.93%, but that of the control group decreases significantly, with only 68.16% remaining. At 60 minutes, the remaining volume concentration ratio of the test group is about 20% higher than that of the control group.

[0083] Furthermore, the above scheme was adopted, and three samples from batch 7-D in Example 3 were taken as test group 2, and the microbubble stability test was performed again. The test results are shown in Table 7 below.

[0084] Table 7

[0085] The results showed that the stability of experimental group 2 was basically consistent with that of the experimental group and was significantly better than that of the control group. At 10 minutes, there was no difference between the three, and the remaining volume concentration was more than 90%. After 60 minutes, the volume concentrations of the experimental group and experimental group 2 dropped to 86.93% and 83.56%, respectively, but only 68.16% remained in the control group.

[0086] It can be seen that the stability of the microbubbles prepared using the solution of Example 4 is better than that of the control group, SonoVue.

[0087] Example 6 Study on the pressure resistance of microbubbles

[0088] Pressure testing method: Using a simple homemade pressure device, a filled, sealed vial is injected with air via a syringe to pressurize the sample. The sample is exposed to overpressures of 100 mmHg, 120 mmHg, 140 mmHg, and 180 mmHg for 90 seconds. The overpressure is then released, and the concentration and average particle size of the microbubbles are measured. (Pressure device: One end of a mercury sphygmomanometer is connected via a rubber tube to a cork-sealed wide-mouth bottle containing a small vial. The vial contains the test contrast agent, and air is injected through the cork to pressurize the vial. The scale of the mercury sphygmomanometer indicates the air pressure within the bottle.)

[0089] Four bottles of SonoVue were prepared according to the instructions as a control group, and four samples from batch 4-D in Example 4 were used as a test group. Samples from the control group and the experimental group were taken, and four samples were added to 5 mL of 0.9% sodium chloride aqueous solution using volume concentration as a parameter for reconstitution. The samples were analyzed using a Multisizer 4e Coulter counter (Beckman Coulter, Inc.), and the volume concentration at 0 min was recorded. The samples were placed under a pressure of 120 mmHg and allowed to stand for 1 min, 3 min, 5 min, and 10 min, respectively. After returning to normal pressure and shaking evenly, the particle size distribution of the microbubbles was detected again. The test results are shown in Table 8 below.

[0090] Table 8

[0091] The results showed that as time went on within 10 minutes, both the test group and the control group showed a decreasing trend. After 10 minutes of 120 mmHg pressure test, 65.18% of the test group remained and 48.96% of the control group remained, indicating that the pressure resistance of the test group was better than that of the control group.

[0092] The above scheme was adopted, except that the heating temperature was 40°C and the heating time was 24 hours. Four samples from batch 7-D in Example 3 were taken as test group 2, and the microbubble pressure resistance test was performed again. The results are shown in Table 9 below.

[0093] Table 9

[0094] The results showed that as time went on within 10 minutes, the microbubble volume concentration in all groups showed a decreasing trend. After 10 minutes of 120 mmHg pressure test, more than 60% of the microbubble remained in the test group and test group 2, and 48.96% remained in the control group, indicating that the pressure resistance of the test group and test group 2 was better than that of the control group.

[0095] It can be seen that the pressure resistance of the microbubbles prepared using the solution of Example 4 is better than that of the control group, SonoVue.

[0096] Example 7 Study on the Effect of Ultrasound Contrast Imaging

[0097] Experimental animals: Female New Zealand white rabbits, weighing 2-2.5 kg, 4-5 months old, in good health.

[0098] Equipment model: GE E8 female probe RIC5-9-D, MI: 0.19, frequency: 4-9 MHz, using 3D real-time scanning to monitor the perfusion of contrast agent.

[0099] Test group sample: 1 tube of batch 4-D sample was injected with 5 mL of normal saline and shaken to reconstitute to obtain a uniform white microbubble suspension.

[0100] Control group sample: 1 vial of SonoVue was injected with 5 mL of normal saline and shaken to reconstitute to obtain a uniform white microbubble suspension.

[0101] Blood pool angiography results

[0102] Rabbit renal angiography: 0.1 mL of contrast agent was injected into the rabbit's ear vein in both the experimental and control groups. The renal imaging was monitored using the above protocol. The imaging data were evaluated after the contrast agent was injected until the enhancement effect completely disappeared. The images were analyzed using Image J software. Comparisons were made between various indicators.

[0103] Representative images from the experimental group are shown in Figure 2, and representative images from the control group are shown in Figure 3. Both images clearly demonstrate the characteristics of the rabbit kidney. A comparison reveals that the image in Figure 2 is brighter, fuller, and more uniform, with smooth, complete edges. Figure 3 exhibits slightly lower brightness, poorer uniformity, and incomplete edges.

[0104] Cavity angiography results

[0105] Rabbit salpingography: 0.5 mL of the microbubble suspension from the test group was diluted to 20 mL and slowly introduced into the uterine cavity of the female rabbit via the salpingography catheter. Transabdominal ultrasound was performed. The resulting imaging results are shown in Figure 4. The image brightness was high, the entire uterus was evenly visualized, and the contour edges were clear and regular, showing the normal physiological curvature structure of the rabbit uterus.

[0106] Therefore, the imaging effect of the microbubbles prepared by the scheme of Example 4 is better than that of the control group SonoVue.

[0107] In summary, the lipid microbubble suspension prepared using the lipid contrast agent lyophilized powder composition of the present application has a high volume concentration after reconstitution, good pressure resistance and stability of the microbubbles, and better contrast effect of the microbubbles.

[0108] Finally, it should be noted that the above description is merely a preferred embodiment of the present application and is intended only to illustrate the technical solution of the present application and is not intended to limit the scope of protection of the present application. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present application are included within the scope of protection of the present application.

Claims

1. A lyophilized powder composition for preparing a lipid microbubble ultrasound contrast agent, comprising distearoylphosphatidylcholine, sodium 1,2-palmitoylphosphatidylglycerol, polyethylene glycol 4000, and palmitic acid; in, The distearoylphosphatidylcholine is 1 part by weight; the 1,2-palmitoylphosphatidylglycerol sodium is 1 part by weight; the polyethylene glycol 4000 is 150-200 parts by weight; and the palmitic acid is 0.1-0.3 parts by weight.

2. The freeze-dried powder composition according to claim 1, wherein The polyethylene glycol 4000 is 170-185 parts by weight.

3. The freeze-dried powder composition according to claim 1, wherein The amount of polyethylene glycol 4000 is 173-185 parts by weight. 4 . A sealed vial for preparing a lipid microbubble ultrasound contrast agent, comprising the lyophilized powder composition according to claim 1 and a physiologically acceptable gas.

5. The sealed vial according to claim 4, wherein: The gas may be a fluorinated gas; The fluorinated gas is any one of perfluoromethane, perfluoropropane, perfluorobutane, perfluoropentane and perfluorohalogenated alkanes, or a combination of two of the two.

6. The sealed vial according to claim 4, wherein: The gas is sulfur hexafluoride.

7. The sealed vial according to claim 4, wherein: The gas is an inert gas with low water solubility; Preferably, the low water-soluble inert gas is nitrogen or argon.

8. A method for preparing the sealed vial according to any one of claims 4 to 7, comprising the following steps: (1) dissolving distearoylphosphatidylcholine, 1,2-palmitoylphosphatidylglycerol sodium, polyethylene glycol 4000, and palmitic acid in a solvent to form a solution; (2) The solution was quickly filled into a vial and vacuum freeze-dried to remove the solvent; (3) introducing a physiologically acceptable gas into the vial and sealing it; and (4) Place the sealed vial in a thermostat for activation for 12-24 hours.

9. The method of claim 8, wherein: The solvent described in step (1) is a mixture of any one or more of n-hexane, isopropanol, cyclohexanol, 2-methyl-2-butanol, tert-butanol or n-butanol.

10. The method according to claim 8 or 9, wherein The solvent described in step (1) is a mixture of tert-butyl alcohol and isopropyl alcohol, or a mixture of n-hexane and isopropyl alcohol.

11. The method of claim 8, wherein: The temperature of the thermostat is 35-50°C, preferably 45°C.

12. The method of claim 8, wherein: The activation time in step (4) is 18-22 hours.

13. A method for preparing a lipid microbubble ultrasound contrast agent, comprising: In the presence of a physiologically acceptable gas, the lyophilized powder composition according to any one of claims 1 to 3 is dispersed in physiological saline to form a microbubble suspension.

14. The method of claim 13, wherein: The volume concentration of the microbubble suspension is ≥7 μL / mL.

15. The method of claim 14, wherein: The volume concentration of the microbubble suspension is ≥8 μL / mL.

16. The method of claim 13, wherein: The concentration of the microbubble suspension is ≥5×10 8 pieces / cm 3 .

17. The method of claim 16, wherein: The concentration of the microbubble suspension is ≥7×10 8 pieces / cm 3 .

18. Use of the freeze-dried powder composition according to any one of claims 1 to 3 for blood pool angiography and cavity angiography.

19. A method of ultrasonic imaging, comprising the following steps: administering to a patient an effective amount of the lipid microbubble ultrasound contrast agent prepared by the method of any one of claims 14 to 17; transmitting an ultrasound signal to a body part of the patient; and Echographic signals are collected from the body part.

20. Use of the lyophilized powder composition according to any one of claims 1 to 3 in preparing reconstituted lipid ultrasound microbubbles with increased volume concentration, enhanced stability, and / or improved acoustic response signal and pressure resistance.

Images