Lipid microbubble lyophilized powder compositions and methods for their preparation

Abstract

Provided are a lipid microbubble lyophilized powder composition and a preparation method thereof, the composition comprising 1 part by weight of distearoyl phosphatidylcholine, 1 part by weight of 1,2-palmitoyl phosphatidylglycerol sodium, 100-240 parts by weight of polyethylene glycol 4000, and 0.1-0.3 parts by weight of palmitic acid. The lyophilized powder composition is sealed in a physiological gas forming vial, and is used for ultrasonic imaging after reconstitution. By adjusting the proportion of each component of the lipid microbubble lyophilized powder composition and the process parameters, the reconstituted microbubble particle size distribution is narrow, the microbubble stability is high, the microbubble is not easy to break, the pressure resistance is good, and the like, the acoustic signal of the image is stronger during imaging, and the target site stays longer.

Description

Technical Field

[0001] This application belongs to the pharmaceutical field and relates to a lipid microbubble lyophilized powder composition and its preparation method, specifically to an ultrasound contrast agent for improving the contrast of ultrasound images. Background Technology

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

[0003] Sulfur hexafluoride microbubbles for injection (trade name: It is an ultrasound contrast agent that has been on the market both domestically and internationally for many years. It is used for ultrasound contrast imaging diagnosis and has a wide range of indications, including liver contrast imaging, cardiac contrast imaging, and urinary system contrast imaging.

[0004] However, with clinical application, clinical researchers have found room for improvement in Sonovue contrast agent. This can be addressed in several areas: First, the microbubble stability after reconstitution needs improvement. During Sonovue use, if the initial imaging is unclear, a second imaging session can be performed. However, the enhancement effect of the second session is not as good as the first. This is because, during the imaging process after reconstitution of Sonovue, the volume concentration of the microbubbles in the prepared solution gradually decreases over time, which is detrimental to 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 its resonant frequency, i.e., the stronger the reflected acoustic signal. Therefore, improving the stability of the contrast agent microbubbles, thereby maintaining their volume concentration, is an urgent problem to be solved.

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

[0006] In addition, the pressure resistance of the microbubbles needs improvement. The Chinese literature, "Experimental Study on the Influence 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), states that Sonovue is suitable for ultrasound probes with a center frequency of 2-7.5 MHz. However, linear array probes used for superficial organs such as the thyroid and breast typically have frequencies above 10 MHz. Under high-frequency probes, Sonovue microbubbles are prone to rupture, resulting in shorter microbubble residence times in organs such as the thyroid and breast, leading to poor imaging effects. Therefore, improving the pressure resistance of the microbubbles to expand their application range is an urgent issue that needs to be addressed.

[0007] Therefore, how to overcome the aforementioned defects in existing technologies and improve the effectiveness of contrast agents remains an urgent problem to be solved in this field. Summary of the Invention

[0008] To address the issues of poor stability after reconstitution of Sonovue microbubbles, gradual decrease in microbubble volume concentration over time, and poor microbubble pressure resistance, this 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.

[0009] In a first aspect, this application provides a lyophilized powder composition for preparing a lipid microbubble ultrasound contrast agent, the composition comprising distearate phosphatidylcholine (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.

[0010] Preferably, the PEG4000 is 170-185 parts by weight.

[0011] Preferably, the PEG4000 is in the form of 173-185 parts by weight.

[0012] Secondly, this application provides a sealed vial containing the lyophilized powder composition described in the first aspect and a physiologically acceptable gas for preparing a lipid microbubble ultrasound contrast agent.

[0013] 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 perfluorohaloalkanes.

[0014] In other embodiments, the gas may be sulfur hexafluoride.

[0015] In other embodiments, the gas may be a low-water-soluble inert gas; preferably, the low-water-soluble inert gas may be nitrogen, argon, etc.

[0016] Thirdly, this application provides a method for preparing a sealed vial of a lipid microbubble ultrasound contrast agent, comprising the following steps:

[0017] (1) Dissolve DSPC, DPPG-Na, PEG4000 and palmitic acid in a solvent to form a solution;

[0018] (2) Quickly fill the solution into vials and freeze-dry under vacuum to remove the solvent;

[0019] (3) Introduce a physiologically acceptable gas into the vial and seal it; and

[0020] (4) Place the sealed vial in a constant temperature incubator for 12-24 hours to activate it.

[0021] In some embodiments, the solvent in step (1) is any one or a mixture of n-hexane, isopropanol, cyclohexanol, 2-methyl-2-butanol, tert-butanol or n-butanol.

[0022] Preferably, the solvent in step (1) is a mixture of tert-butanol and isopropanol, or a mixture of n-hexane and isopropanol.

[0023] During their research, the inventors discovered that heating and activating the lyophilized powder of the lipid microbubble ultrasound contrast agent is more conducive to the concentrated particle size distribution of the microbubbles formed after microbubble reconstitution, resulting in a larger microbubble volume concentration, better acoustic response, and better pressure resistance.

[0024] Preferably, the temperature of the constant temperature chamber in step (4) is 35-50℃, more preferably 45℃.

[0025] Preferably, the activation time in step (4) is 18-22 hours.

[0026] Fourthly, this application provides a method for preparing a lipid microbubble ultrasound contrast agent, comprising: dispersing the lyophilized powder composition described in the first aspect in physiological saline (0.9% sodium chloride aqueous solution) to form a microbubble suspension, i.e., a lipid microbubble ultrasound contrast agent, in the presence of a physiologically acceptable gas.

[0027] 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.

[0028] Preferably, the concentration of the microbubble suspension is ≥5×10⁻⁶. 8 pcs / cm 3More preferably, the concentration of the microbubble suspension is ≥7×10⁻⁶. 8 pcs / cm 3 .

[0029] Fifthly, this application provides the use of the lyophilized powder composition described in the first aspect as a blood pool ultrasound contrast agent and a cavity ultrasound contrast agent.

[0030] Sixthly, this application provides a method for ultrasound imaging using a lipid microbubble ultrasound contrast agent prepared by the method described in the fourth aspect, comprising the following steps:

[0031] Administer an effective amount of lipid microbubble ultrasound contrast agent to the patient;

[0032] Transmitting ultrasound signals to the patient's body parts; and

[0033] Echocardiogram signals are collected from the body parts.

[0034] Compared with the prior art, this application has the following beneficial effects:

[0035] The lyophilized powder composition of the lipid microbubble ultrasound contrast agent of this application has many advantages such as high stability of reconstituted microbubbles, good acoustic response, and good pressure resistance. In addition, the image acoustic signal is stronger, the residence time at the target site is longer, and the pressure resistance is better during ultrasound contrast imaging, making it suitable for various organ and probe contrast modes. Attached Figure Description

[0036] Figure 1 This is the particle size distribution of the lipid microbubble ultrasound contrast agent in batch 4-D of Example 4.

[0037] Figure 2 The image shows a rabbit kidney contrast image obtained after ultrasound contrast imaging of the 4-D sample in Example 8.

[0038] Figure 3 The image shows a rabbit kidney contrast image obtained after ultrasound contrast imaging of the SonoVe sample from Example 8.

[0039] Figure 4 The image is a rabbit oviduct imaging image obtained after ultrasound contrast imaging of the 4-D sample of Example 8. Detailed Implementation

[0040] Abbreviation Explanation

[0041] DSPC: Distearylphosphatidylcholine

[0042] DPPG-Na: Sodium 1,2-palmitoylphosphatidylglycerol

[0043] PEG4000: Polyethylene Glycol 4000

[0044] Terminology Explanation

[0045] SonoVe: Generic name Sulfur hexafluoride microbubbles for injection. This drug is an ultrasound contrast agent developed by the Italian company Bolaco Imaging. According to the instructions, each vial contains 59 mg of sulfur hexafluoride, 0.19 mg of distearylphosphatidylcholine (DSPC), 0.19 mg of sodium 1,2-dipalmitoylphosphatidylglycerol (DPPG), 0.04 mg of palmitic acid, and 24.5 mg of polyethylene glycol 4000.

[0046] Polyethylene glycol 4000: Polyethylene glycol is a high molecular weight polymer with the chemical formula HO(CH2CH2O). n H, depending on its molecular weight, varies in properties, 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℃. The average molecular weight of polyethylene glycol 4000 is approximately 4000, and its structure is as follows:

[0047] Microbubbles: Microbubbles are a class of drug formulations, generally composed of a gas core and a stable shell. The coating material of the bubbles is usually composed of lipids, polymers, or proteins, which have good biocompatibility and are relatively safe for intravenous injection. Among them, phospholipids, as the shell of microbubbles, are more flexible than cross-linked polymer hard shells. Under the action of ultrasound, they are more likely to shrink, rupture, bend, or re-spread, thereby enhancing the sensitivity of microbubbles to sound waves.

[0048] Microbubble volume concentration: Abbreviated as "volume concentration", it refers to the ratio of the total volume of all microbubbles in a suspension to the total volume of the suspension, that is, the total volume concentration of microbubbles per unit volume of suspension (μL / mL).

[0049] Microbubble count: Abbreviated as "count", it refers to the ratio of the total number of microbubbles in a suspension to the total volume of the suspension, that is, the number of microbubbles per unit volume of suspension.

[0050] Reconstitution: refers to the process of adding physiological saline to the lyophilized powder of ultrasound contrast agent and shaking it to dissolve it before clinical use, so as to obtain a uniformly distributed microbubble suspension.

[0051] Residual percentage: refers to the percentage of the volume concentration of the microbubble suspension obtained after reconstitution, after being subjected to room temperature or pressure treatment during stability studies or pressure tests, compared to the initial volume concentration before treatment.

[0052] Blood pool contrast ultrasound: This 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 images of organs in the body. It includes contrast ultrasound of liver and abdominal organs, contrast ultrasound of the heart, and vascular Doppler.

[0053] Cavity contrast ultrasound: This refers to an ultrasound imaging method in which a reconstituted ultrasound contrast agent is injected into cavity organs such as the uterus, gallbladder, and bladder through a syringe / catheter to obtain contrast-enhanced ultrasound images of the organs.

[0054] The present application will be 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.

[0055] Example 1: Preparation of a freeze-dried composition for direct freeze-drying

[0056] Weigh out DSPC, DPPG, palmitic acid and polyethylene glycol-4000 and dissolve them in an organic solvent. Dispense them into vials in a ratio of (1:1:(0.1-0.3):(100-240)). Seal the vials with brominated butyl rubber stoppers and push them into the freeze dryer. Freeze-dry under vacuum for 24 hours. Purge with sulfur hexafluoride gas, seal and cap the vials to obtain a sealed freeze-dried composition.

[0057] Preparation method of microbubbles: Before use, inject physiological saline into the vial and shake until the lyophilized powder is completely dispersed into a uniform white emulsion.

[0058] Example 2: Preparation and parameter detection of lyophilized compositions with varying PEG4000 dosage

[0059] Following the preparation method of Example 1, different weight parts of PEG4000 were used.

[0060] Before detection, 5 mL of 0.9% sodium chloride aqueous solution was injected for reconstitution. Some of the sulfur hexafluoride was encapsulated in the milky white suspension 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.

[0061] Table 1

[0062]

[0063]

[0064] The results showed that the microbubble volume concentration increased with the increase of PEG4000 dosage in the formulation. However, after the PEG4000 dosage increased to a certain weight, the volume concentration decreased. Based on the SonoVe quality standard volume concentration range of 2-10 μL / mL, the formulations in this application with microbubble volume concentrations above 10 μL / mL were superior to SonoVe formulations, specifically batches 3-A, 4-A, 5-A, 6-A, 7-A, and 8-A. Therefore, the optimal volume concentration of PEG4000 was achieved at a weight of 135-200 parts.

[0065] Example 3: Selection of Optimal Activation Temperature

[0066] Following 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 of 173 parts by weight in Example 2, the amount used in batch 6-A) were weighed, dissolved, dispensed, and freeze-dried. Sulfur hexafluoride gas was then introduced to form a freeze-dried composition sealed in vials. The mixture 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.

[0067] Table 2

[0068]

[0069] Before detection, 5 mL of 0.9% sodium chloride aqueous solution was injected for reconstitution. Some sulfur hexafluoride was encapsulated in the milky white suspension 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.

[0070] Table 3

[0071]

[0072]

[0073] The results showed that the volume concentration was lowest at 35℃ and highest at 45℃.

[0074] Example 4: Selection of Optimal Activation Time

[0075] Following 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 used in Example 2) were weighed, and sulfur hexafluoride gas was introduced to form a lyophilized composition sealed in a vial. The sealed vial was placed in a 45°C incubator for activation at 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 of the sulfur hexafluoride was encapsulated in milky white suspended microbubbles. The number and volume concentration of microbubbles were analyzed using a Multisizer 4e Coulter counter (Beckman Coulter, Inc.), and the results are shown in Table 4 below.

[0076] Table 4

[0077]

[0078] This indicates that the volume concentration reaches its peak after a heat treatment time of 12 hours or more. Beyond 12 hours, the volume concentration essentially stops changing with further heat treatment time. (Based on SonoVe's FDA application materials (FDA, Center for Drug Valuation and Research, Lumason)). TM According to data from 203684Orig1s000, Chemistry Review(s), the number of microbubbles per milliliter is (1.5-5.6) × 10⁻⁶. 8 The number of contrast agent microbubbles in all prescriptions listed in the table above is superior to that of SonoVe.

[0079] Note: Lumason TM It is the same product sold under different brand names in different countries as SonoVe.

[0080] Figure 1 The results are from the sampling and testing of the 4-D batch of samples over 24 hours.

[0081] To further explore the optimal process for heating for more than 12 hours, the above experimental scheme was used to further study the number and concentration of contrast agent microbubbles at heating times of 12h, 16h, 18h, 20h, and 22h. The results are shown in Table 5 below.

[0082] Table 5

[0083]

[0084] The above studies indicate that further heat treatment results show that the volume concentration basically no longer changes after 12-22 hours of heat treatment.

[0085] Comparison of Example 5 and SonoVe stability

[0086] Three vials of commercially available SonoVe medicine were prepared according to the instructions and used as the control group. Three vials of sample 4-D from Example 4 were used as the experimental group. Samples from both the control and experimental groups were taken separately. Using volume concentration as the parameter, three samples were dissolved in 5 mL of 0.9% sodium chloride aqueous solution. Analysis was performed using a Multisizer4e 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 atmospheric pressure for 10 min, 30 min, and 60 min, respectively, and the particle size distribution of the microbubbles was measured again. The results are shown in Table 6 below.

[0087] Table 6

[0088]

[0089] As can be seen from the table above, the stability of the experimental group is significantly better than that of the control group. At 10 min, there is no significant difference between the two, and the remaining volume concentration is over 90%. After 60 min, the remaining volume concentration of the experimental group is 86.93%, while that of the control group is significantly reduced to only 68.16%. At 60 min, the remaining volume concentration of the experimental group is about 20% higher than that of the control group.

[0090] Furthermore, using the above scheme, three samples from batch 7-D in Example 3 were taken as test group 2 and microbubble stability tests were conducted again. The test results are shown in Table 7 below.

[0091] Table 7

[0092]

[0093] The results showed that the stability of experimental group 2 was basically the same as that of the experimental group, and significantly better than that of the control group. At 10 min, there was no difference among the three groups, and the volume concentration remained above 90%. After 60 min, the volume concentrations of experimental group and experimental group 2 decreased to 86.93% and 83.56% respectively, but the control group had only 68.16% remaining.

[0094] Therefore, it can be seen that the microbubbles prepared using the method in Example 4 have better stability than the control group, SonoVe.

[0095] Example 6: Study on the pressure resistance of microbubbles

[0096] Pressure testing method: Using a homemade simple pressurization device, a sealed vial containing the test contrast agent was filled and pressurized with air by injecting air through a syringe. The sample was then exposed to overpressures of 100 mmHg, 120 mmHg, 140 mmHg, and 180 mmHg for 90 seconds, respectively. After releasing the pressure, the concentration and average particle size of the microbubbles were measured. (Pressurization device: One end of a mercury sphygmomanometer was connected with a rubber tube to a corked, wide-mouthed bottle containing a small vial. The vial contained the test contrast agent, and air was injected through the stopper to pressurize it. The air pressure inside the vial was displayed according to the scale of the mercury sphygmomanometer.)

[0097] Four vials of SonoVe drug were prepared according to the instructions and used as the control group. Four vials of sample 4-D from Example 4 were used as the experimental group. Samples from both the control and experimental groups were taken separately. Using volume concentration as the parameter, four samples were added to 5 mL of 0.9% sodium chloride aqueous solution for redissolution. Analysis was performed using a Multisizer 4e Coulter counter (Beckman Coulter, Inc.), and the volume concentration at 0 min was recorded. The samples were then placed under 120 mmHg pressure for 1 min, 3 min, 5 min, and 10 min, respectively. After returning to normal pressure and shaking thoroughly, the particle size distribution of the microbubbles was measured again. The results are shown in Table 8 below.

[0098] Table 8

[0099]

[0100]

[0101] The results showed that both the experimental group and the control group showed a decreasing trend over time within 10 minutes. After 10 minutes of 120 mmHg pressure testing, the experimental group had 65.18% remaining pressure, while the control group had 48.96% remaining pressure, indicating that the pressure resistance of the experimental group was better than that of the control group.

[0102] The above scheme was adopted, except that the heating temperature was 40℃ and the heating time was 24 hours. Four samples from batch 7-D in Example 3 were taken as test group 2 and microbubble pressure resistance test was performed again. The results are shown in Table 9 below.

[0103] Table 9

[0104]

[0105] The results showed that the microbubble volume concentration of all groups decreased over time within 10 minutes. After 10 minutes of 120 mmHg pressure testing, the experimental group and experimental group 2 had more than 60% remaining, while the control group had 48.96% remaining. This indicates that the pressure resistance of the experimental group and experimental group 2 was better than that of the control group.

[0106] Therefore, it can be seen that the microbubbles prepared using the scheme of Example 4 have better pressure resistance than the control group, SonoVe.

[0107] Example 7 Study on the Effect of Ultrasound Contrast Imaging

[0108] Experimental animals: female New Zealand white rabbits, weighing 2-2.5 kg, 4-5 months old, in good health.

[0109] Equipment model: GE E8 negative probe RIC5-9-D, MI: 0.19, frequency: 4-9MHz, using 3D real-time scanning to monitor the perfusion of contrast agent.

[0110] Experimental group sample: 1 vial of 4-D batch sample, injected with 5mL of physiological saline and shaken to reconstitute, resulting in a uniform white microbubble suspension.

[0111] Control group sample: 1 vial of SonoVe was injected with 5 mL of physiological saline and shaken to reconstitute, resulting in a uniform white microbubble suspension.

[0112] Blood pool imaging results

[0113] Rabbit nephrography: 0.1 mL of contrast agent was injected into the marginal ear vein of the rabbits in both the experimental and control groups. The contrast agent was injected until the enhancement effect completely disappeared. The image data was evaluated, and the images were analyzed using ImageJ software. Various indicators were compared and observed.

[0114] Representative images obtained from the experimental group are as follows: Figure 2 As shown, the representative images obtained from the control group are as follows: Figure 3 As shown, both methods effectively display the characteristics of rabbit kidneys. A comparison reveals that... Figure 2 The image has high brightness, is full, develops evenly, and has complete and smooth edges and contours. Figure 3 The brightness is slightly low, the uniformity is poor, and the edge contours are incomplete.

[0115] results of endoscopic imaging

[0116] Rabbit oviposition imaging: 0.5 mL of the experimental group microbubble suspension was diluted to 20 mL and slowly injected into the uterine cavity of the female rabbit through the imaging catheter. Abdominal ultrasound scan was then performed. The resulting images are as follows: Figure 4 As shown, the image has high brightness, the entire uterus is uniformly visualized, and the outline edges are clear and regular, which can show the normal physiological curvature of the rabbit uterus.

[0117] Therefore, the microbubbles prepared using the method in Example 4 showed better contrast effects than the control group, SonoVe.

[0118] In summary, the lipid microbubble suspension prepared using the lipid contrast agent lyophilized powder composition of this application has a high volume concentration after reconstitution, good pressure resistance and stability of the microbubbles, and better contrast effect of the microbubbles.

[0119] Finally, it should be noted that the above description is merely a preferred embodiment of this application and is only used to illustrate the technical solution of this application, and is not intended to limit the scope of protection of this application. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application are included within the scope of protection of this application.

Claims

1. A sealed vial for preparing a lipid microbubble ultrasound contrast agent, comprising a lyophilized powder composition for preparing a lipid microbubble ultrasound contrast agent and a physiologically acceptable gas, wherein, The lyophilized powder composition for preparing lipid microbubble ultrasound contrast agent consists of 1 part by weight of distearate phosphatidylcholine, 1 part by weight of sodium 1,2-palmitoylphosphatidylglycerol, 170-173 parts by weight of polyethylene glycol 4000, and 0.1-0.3 parts by weight of palmitic acid. The method for preparing the sealed vial includes the following steps: (1) Distearate phosphatidylcholine, sodium 1,2-palmitoylphosphatidylglycerol, polyethylene glycol 4000 and palmitic acid are dissolved in a solvent to form a solution; (2) Quickly fill the solution into vials and freeze-dry under vacuum to remove the solvent; (3) Introduce a physiologically acceptable gas into the vial and seal it; and (4) Place the sealed vial in a constant temperature chamber for 12-24 hours to activate it, wherein the temperature of the constant temperature chamber is 40-45℃; The solvent mentioned in step (1) is a mixture of tert-butanol and isopropanol, or a mixture of n-hexane and isopropanol.

2. The sealed vial as described in claim 1, wherein, The polyethylene glycol 4000 is 173 parts by weight.

3. The sealed vial as described in claim 1, wherein, The gas is a fluorinated gas; The fluorinated gas is any one or a combination of two of perfluoromethane, perfluoropropane, perfluorobutane, perfluoropentane, and perfluorohaloalkanes.

4. The sealed vial as described in claim 1, wherein, The gas is sulfur hexafluoride.

5. The sealed vial as described in claim 1, wherein, The gas is a low-water-soluble inert gas.

6. A method for preparing a sealed vial according to any one of claims 1-5, comprising the following steps: (1) Distearate phosphatidylcholine, sodium 1,2-palmitoylphosphatidylglycerol, polyethylene glycol 4000 and palmitic acid are dissolved in a solvent to form a solution; (2) Quickly fill the solution into vials and freeze-dry under vacuum to remove the solvent; (3) Introduce a physiologically acceptable gas into the vial and seal it; and (4) Place the sealed vial in a constant temperature chamber for 12-24 hours to activate it, wherein the temperature of the constant temperature chamber is 40-45℃; The solvent mentioned in step (1) is a mixture of tert-butanol and isopropanol, or a mixture of n-hexane and isopropanol.

7. A method for preparing a lipid microbubble ultrasound contrast agent, comprising: The lyophilized powder composition of claim 1 is dispersed in physiological saline to form a microbubble suspension in the presence of a physiologically acceptable gas.

8. The method of claim 7, wherein, The concentration of the microbubble suspension is ≥5×10 8 pcs / cm 3 .

9. Use of the lyophilized powder composition of claim 1 in the preparation of reconstituted lipid ultrasonic microbubbles having increased volume concentration, improved stability, and / or improved acoustic response signal and pressure resistance.

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