Lipid microsphere composition and preparation method thereof

Abstract

The invention belongs to the field of pharmaceutical preparations, and particularly relates to a lipid microsphere composition and a preparation method thereof. The present invention relates to an article comprising a container and a combination comprising an octafluoropropane gas and a lipid mixed solution, the top space in the container containing the octafluoropropane gas and the lower layer being the lipid mixed solution, where the container is sealed by a rubber stopper which is free of silicone oil. The octafluoropropane lipid combination is prepared and placed in the container, the container is sealed through the silicone oil-free rubber plug, and the microspheres obtained after activation are high in number concentration, appropriate in particle size and good in stability.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of pharmaceutical preparations, and particularly relates to a lipid microsphere combination and a preparation method thereof. BACKGROUND

[0002] Octafluoropropane lipid microsphere injection is an ultrasound contrast agent, which has been approved for marketing in China, the United States and Europe, and the trade name is Sonovue. It is used for the left ventricular endocardial border recognition enhancement in the case of unclear conventional echocardiography. The product contains octafluoropropane gas and lipid solution, which is packaged in an injection bottle. The octafluoropropane is in the form of gas in the upper space of the injection bottle, and the lower layer contains a lipid mixture liquid containing a lipid mixture (DPPA, DPPC and MPEG5000 DPPE) and inactive ingredients sodium chloride, propylene glycol, glycerol, sodium dihydrogen phosphate monohydrate and disodium hydrogen phosphate heptahydrate. Before use, the clinical activation needs to be carried out by a mechanical shaking device such as to form lipid microspheres containing octafluoropropane, and then administered intravenously.

[0003] The number of microspheres, the concentration of microspheres and the size of microspheres will affect the imaging effect. It is disclosed in Niu Jinhai, Ultrasonic Principle and Biomedical Engineering Application, Shanghai Jiaotong University Press, 2017, that an ultrasound contrast agent is a substance used to enhance the contrast of images in ultrasonic imaging; generally, it is a micrometer-sized envelope microbubble, which is injected into the blood circulation system through intravenous injection to enhance the scattering intensity of ultrasonic waves, so as to achieve the purpose of ultrasonic contrast imaging. Its role is that after the ultrasonic contrast agent is injected into the blood vessel, it can change the ultrasonic properties of the tissue to produce a contrast effect, and the enhancement effect depends on the concentration, size and ultrasonic emission frequency of the ultrasonic contrast agent. The microbubbles will vibrate under the action of ultrasonic waves, and the scattered ultrasonic signal will be enhanced. When the frequency of the incident sound wave is consistent with the resonance frequency of the microbubble, the scattering signal of the microbubble is greatly enhanced, and the contrast effect is clearer. The resonance frequency of the microbubble is related to the size of the microbubble. The currently used ultrasonic frequency range is 1-20 MHz, and the specific frequency selection is based on the inspection requirements, such as low frequency (1-5 MHz) for deep penetration inspection of abdomen, heart and the like, and high frequency (5-15 MHz) for high-resolution imaging of shallow tissues.

[0004] CN109641068A discloses a method and device for preparing an ultrasonic contrast agent, wherein it is recorded that the activation needs to ensure that the formed microspheres have a proper size and concentration which are effective and safe for diagnosis of individuals. The very low concentration or improper size of the microspheres will affect the imaging effect. It can be seen that the number of microbubble generation is reduced, resulting in a reduction in the number of effective circulating microbubbles; the microbubble concentration is low, which cannot guarantee that a sufficient number of microbubbles enter the imaging site, resulting in a weakened signal or incomplete imaging, thereby reducing the imaging effect.

[0005] Silicone oil, i.e. polydimethylsiloxane, is a colorless and odorless, non-volatile substance. The production process of rubber stopper is recorded in "Pharmaceutical Excipients and Pharmaceutical Packaging Materials Inspection Technology", Zhang Zhijun, China Medical Science and Technology Press, 2019, including plastication, mixing, calendering, extrusion, molding, vulcanization, washing, cleaning and packaging processes. In the cleaning process, purified water or injection water is used to clean the stopper, and then siliconization and drying (sterilization) are performed. An appropriate amount of silicone oil is added during the cleaning process to make the stopper smooth and run smoothly. "Pharmaceutical Production Verification Guide", GMP.GSP Committee of the State Pharmaceutical Administration, China Medical Science and Technology Press, 1996, records that some methyl silicone oil is usually added in the last process of the processing technology to make the surface of the stopper siliconized. Excessive siliconization will negatively affect the clarity of the injection. It can be seen that silicone oil will be added during the cleaning process of the rubber stopper to distribute a layer of silicone oil on the surface of the stopper. The literature "Determination of Dimethyl Silicone Oil Residue on the Surface of Pharmaceutical Halogenated Butyl Rubber Stopper and Its Migration in Glucose Sodium Chloride Injection by FTIR-ATR Method", Journal of Pharmaceutical Analysis, 2019, 39(4):693-701" measured that the dimethyl silicone oil residue on the surface of the rubber stopper was 0-25.9 μg / cm 2 Therefore, silicone oil will exist in the stopper. The octafluoropropane lipid microsphere injection needs to be activated during use, and the microspheres are formed by shaking, so that the silicone oil in the stopper contacts the microspheres, which may affect the number and quality of the microspheres, thereby affecting the imaging effect. SUMMARY

[0006] In one aspect, the present application provides an article comprising a container and a combination comprising an octafluoropropane gas and a lipid mixed solution, wherein the top space of the container comprises the octafluoropropane gas, and the lower layer is the lipid mixed solution, wherein the container is sealed by a rubber stopper, and the rubber stopper does not contain silicone oil; and the lipid mixed solution comprises DPPA, DPPC, MPEG5000 DPPE, propylene glycol, glycerol, buffer salt, sodium chloride and water.

[0007] In another aspect, the present application provides a combination capable of forming octafluoropropane microspheres, comprising an octafluoropropane gas and a lipid mixed solution, wherein the combination is provided in a container, the top space of the container comprises the octafluoropropane gas, and the lower layer is the lipid mixed solution, wherein the container is sealed by a rubber stopper, and the rubber stopper does not contain silicone oil; and the lipid mixed solution comprises DPPA, DPPC, MPEG5000 DPPE, propylene glycol, glycerol, buffer salt, sodium chloride and water.

[0008] In some embodiments, the container is a syringe or a vial; preferably a vial; more preferably a glass vial; further preferably a borosilicate glass tube injection vial.

[0009] In some embodiments, the container has a volume capacity of 2-4 mL.

[0010] In some embodiments, the rubber stopper is made of halogenated butyl rubber stopper, preferably brominated butyl rubber stopper or chlorinated butyl rubber stopper; more preferably injection liquid brominated butyl rubber stopper or injection liquid chlorinated butyl rubber stopper; further preferably injection liquid chlorinated butyl rubber stopper.

[0011] In some embodiments, the rubber stopper is pressed on the bottle opening by an aluminum plastic combination cap.

[0012] In some embodiments, the lipid mixed solution is formed by mixing an aqueous solution and a lipid non-aqueous solution, wherein the aqueous solution comprises sodium chloride, buffer salt, glycerol and water; and the lipid non-aqueous solution comprises propylene glycol, DPPA, DPPC and MPEG5000 DPPE.

[0013] In some embodiments, the lipid mixed solution is prepared by a preparation method comprising the following steps: mixing sodium chloride, buffer salt, glycerol and water by stirring to prepare an aqueous solution; mixing propylene glycol, DPPA, DPPC and MPEG5000 DPPE by stirring to prepare a lipid non-aqueous solution; adding the lipid non-aqueous solution into the aqueous solution by stirring and mixing, and filtering, to obtain the lipid mixed solution.

[0014] In some embodiments, the lipid mixed solution comprises about 0.7 mg to about 0.8 mg of combined DPPA, DPPC and MPEG5000 DPPE per mL of solution, wherein the molar ratio of DPPA, DPPC and MPEG5000 DPPE is 5-15:77-90:5-15, respectively; preferably, the lipid mixed solution comprises about 0.75 mg of combined DPPA, DPPC and MPEG5000 DPPE per mL of solution; preferably, the molar ratio of DPPA, DPPC and MPEG5000 DPPE is about 10:82:8.

[0015] In some embodiments, the container contains 5.5-7 mg / mL of octafluoropropane; preferably 6.52 mg / mL of octafluoropropane.

[0016] In some embodiments, the buffer salt is selected from acetate or phosphate; preferably phosphate; more preferably one or more selected from sodium phosphate monobasic monohydrate, disodium phosphate dodecahydrate or disodium phosphate heptahydrate; preferably a combination of sodium phosphate monobasic monohydrate and disodium phosphate heptahydrate, or a combination of sodium phosphate monobasic monohydrate and disodium phosphate dodecahydrate; more preferably a combination of sodium phosphate monobasic monohydrate and disodium phosphate dodecahydrate.

[0017] In some embodiments, the lipid mixture solution contains about 0.045 mg DPPA, about 0.401 mg DPPC, about 0.304 mg MPEG5000 DPPE, about 103.5 mg propylene glycol, about 126.2 mg glycerol, about 2.34 mg sodium phosphate monobasic monohydrate, about 2.88 mg sodium phosphate dibasic dodecahydrate, about 4.87 mg sodium chloride, and the rest is water per milliliter of the solution; preferably, the pH value of the lipid solution is 6.2 to 6.8.

[0018] Another aspect of the present application provides a method for preparing an octafluoropropane lipid microsphere injection, comprising the following steps: mixing sodium chloride, buffer salt, glycerol and water to prepare an aqueous phase solution; mixing propylene glycol, DPPA, DPPC and MPEG5000 DPPE to prepare a lipid non-aqueous solution; adding the lipid non-aqueous solution into the aqueous phase and diluting with water; filtering to obtain a lipid mixture solution; filling the lipid mixture solution into a container, semi-pressing a rubber plug without silicone oil, vacuumizing the semi-pressed plug, filling with octafluoropropane and then pressing the plug, capping, sterilizing, and obtaining the product.

[0019] In some embodiments, the propylene glycol, DPPA, DPPC and MPEG5000 DPPE are mixed, and the specific steps include directly dissolving DPPA, DPPC and MPEG5000 DPPE in propylene glycol; or dissolving DPPA, DPPC and MPEG5000 DPPE in an organic solvent, rotary evaporation, washing with the organic solvent, drying to obtain a lipid mixture, and dissolving the lipid mixture in propylene glycol.

[0020] In some embodiments, the organic solvent is a mixed solvent of toluene and methanol, a mixed solvent of chloroform and methanol, or a mixed solvent of dichloromethane and methanol; preferably, the organic solvent is a mixed solvent of toluene and methanol; more preferably, the volume ratio of toluene to methanol is 3:1.

[0021] In some embodiments, the cap in the capping is an aluminum plastic combined cap.

[0022] Another aspect of the present application provides a method for preparing lipid-encapsulated octafluoropropane gas microspheres, which forms lipid-encapsulated octafluoropropane gas microspheres by activating the above-mentioned combination in a container.

[0023] In some embodiments, the activation is achieved by an activation device; more preferably, the device activation parameters are 4530±100 oscillations per minute, and the oscillation time is 45 seconds.

[0024] In some embodiments, the lipid-encapsulated octafluoropropane gas microspheres have an average particle size of 1 μm to 5 μm; preferably, the average particle size is 1 μm to 2 μm.

[0025] In some embodiments, the combination produces at least about 3.5 x 10 9 microspheres per milliliter of microspheres; preferably at least about 4 x 10 9 microspheres per milliliter of microspheres.

[0026] In some embodiments, the combination, after activation, is used in the preparation of a therapeutic or diagnostic medical imaging.

[0027] Technical effects

[0028] The product needs to be activated for use, and more microspheres are provided by activation. However, the applicant found that when the product is placed in a small vial of injection and sealed with a silicone oil plug, the octafluoropropane gas is introduced into the lipid solution to form octafluoropropane microspheres during activation, but the microspheres are in contact with the silicone oil in the rubber plug, resulting in a decrease in the concentration of microspheres after activation, thereby affecting the quality of the product and reducing the contrast effect of the microspheres. The applicant found that by preparing an octafluoropropane lipid combination, placing it in a container, and sealing the container with a rubber plug containing no silicone oil, the concentration of microspheres obtained after activation is high, the average particle size is smaller, the particle size is suitable, and the stability is good, thereby obtaining stable and good quality imaging effect.

[0029] Definitions

[0030] DPPA in the present application refers to dipalmitoyl phosphatidic acid (i.e. 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid sodium), DPPC refers to dipalmitoyl phosphatidylcholine (i.e. 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine), MPEG5000 DPPE refers to polyethylene glycol, N-(methoxypolyethylene glycol amine 5000 carbamoyl)-1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine monosodium salt.

[0031] About in the present application refers to ± 5%, for example, the molar ratio of DPPA, DPPC and MPEG5000 DPPE is about 10:82:8, which means that the molar ratio of DPPA, DPPC and MPEG5000 DPPE is about (9.5-10.5):(77.9-86.1):(7.6-8.4).

[0032] The volume of the container in the present application refers to the volume of the liquid placed in the container.

[0033] The activation in the present application refers to the vigorous stirring by swirling, left and right or up and down movement, etc. The lipid solution is stirred so that the gas is introduced into the lipid solution, resulting in the formation of microspheres structure by the lipid wrapping gas. The stirring can be carried out by oscillating the container containing the lipid solution, which can be carried out by hand or vortex or mechanical oscillator.

[0034] The microsphere size according to the present application refers to the average particle size of the microspheres. The microsphere particle size is typically measured using instruments known and available in the art.

[0035] The octafluoropropane gas concentration refers to the concentration under about one atmosphere of pressure.

[0036] The silicone oil-free according to the present application refers to the fact that the stopper passes the test for silicone oil on the surface of rubber closures according to the United States Pharmacopoeia 4th Edition, Chapter 4222, Test for Silicone Oil on the Surface of Rubber Closures. Suitable stoppers can be commercially available, provided that the stoppers are silicone oil-free.

[0037] v / v refers to volume ratio. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 Particle size distribution plot for the silicone oil-containing stopper sample after activation in Example 3;

[0039] Figure 2 Particle size distribution plot for the silicone oil-free stopper after activation in Example 3. DETAILED DESCRIPTION

[0040] The present application will be described in more detail by way of examples. However, the specific descriptions are only for illustrating the technical solutions of the present application, and do not constitute any limitation on the present application.

[0041] The reagents and raw materials used in the present application are commercially available. The experimental methods not specified with specific conditions are usually carried out according to the conventional conditions.

[0042] Example 1 Preparation of octafluoropropane lipid microsphere injection

[0043] The prescription composition of the octafluoropropane lipid microsphere injection is shown in Table 1.

[0044] Table 1 Prescription composition

[0045]

[0046] The specific preparation process is as follows: (1) sodium chloride, sodium dihydrogen phosphate monohydrate, disodium hydrogen phosphate dodecahydrate, glycerol and water for injection are stirred and mixed to prepare an aqueous solution; (2) DPPA, DPPC and MPEG5000 DPPE are directly dissolved in propylene glycol to prepare a lipid non-aqueous solution; (3) the lipid non-aqueous solution is added to the aqueous solution, stirred at 50°C, and diluted with water for injection to 1L to obtain a lipid mixture solution, so that the total phospholipid concentration is 0.75mg per mL of solution; (4) filtration, 1.76mL of the lipid mixture solution is added to a 2mL borosilicate glass tube injection vial, and a chlorobutyl rubber stopper for injection is used for half-pressure stoppering; (4) the half-pressure stoppered product is vacuumed, the air in the top space of the vial is replaced with octafluoropropane gas (the concentration of octafluoropropane in the top space of the injection vial is 6.52mg / mL) by a freeze dryer, and then stoppered, capped (aluminum-plastic combined cap), sterilized, and obtained.

[0047] The amount of surface silicone oil in the rubber stopper used is determined by the method for determining surface silicone oil of rubber sealing parts in the fourth part of the 2025 edition of the Pharmacopoeia to be undetectable.

[0048] Example 2 Preparation of octafluoropropane lipid microsphere injection

[0049] The specific prescription is the same as in Table 1 of Example 1. The specific preparation process is as follows: (1) DPPA, DPPC and MPEG5000 DPPE are dissolved in toluene / methanol (v / v 3:1), filtered through a 0.45μm microporous filter, and rotary evaporated into a dense gel at 45-55°C. After washing with methyl tert-butyl ether, the solid is collected and vacuum dried at 40-50°C to constant weight to obtain a phospholipid mixture. (2) The phospholipid mixture is dissolved in propylene glycol at 50-55°C to obtain a lipid non-aqueous solution. (3) Sodium chloride, sodium dihydrogen phosphate monohydrate, disodium hydrogen phosphate dodecahydrate, glycerol and water for injection are stirred and mixed to prepare an aqueous solution. (4) The lipid non-aqueous solution is added to the aqueous solution, stirred at 50°C, and diluted with water for injection to 1L to obtain a lipid mixture solution, so that the total phospholipid concentration is 0.75mg per mL of solution. (5) Filtration, 1.76mL of the lipid solution is added to a 2mL borosilicate glass tube injection vial, and a chlorobutyl rubber stopper for injection is used for half-pressure stoppering; (6) the half-pressure stoppered product is vacuumed, the air in the top space of the vial is replaced with octafluoropropane gas (the concentration of octafluoropropane in the top space of the injection vial is 6.52mg / mL) by a freeze dryer, and then stoppered, capped (aluminum-plastic combined cap), sterilized, and obtained. The amount of surface silicone oil in the rubber stopper used is determined by the method for determining surface silicone oil of rubber sealing parts in the fourth part of the 2025 edition of the Pharmacopoeia to be undetectable.

[0050] Example 3 Effect of ordinary silicone stopper and silicone-free stopper on the number concentration of activated microspheres

[0051] The octafluoropropane lipid microsphere injection prepared in Example 1 is activated to form a suspension of lipid-coated octafluoropropane microspheres before use. The specific activation method is to activate the product by using a shaking device (the built-in solidification parameters of the shaking device are: 4530 ± 100 oscillations per minute, oscillation time 45 seconds) to form lipid microspheres containing octafluoropropane.

[0052] The detection method of microsphere number concentration and particle size distribution chart and average particle size is as follows: an appropriate amount of activated product is diluted with buffer (COULTER ISOTON II diluent (purchased from BECKMAN company in the United States)) to about 0.13 μL of the product per 1 mL of solution, 200 mL is taken and placed in a measuring cup, and after stirring for 2-3 minutes, a Beckman Coulter counter is used for determination. The parameters of the Coulter counter are: the determination range is 1-30 μm, and the analysis volume is 200 μL.

[0053] A commercially available siliconized halogenated butyl rubber stopper for injection (brominated) for freeze-drying (from Jiangsu Boseng Medical New Material Co., Ltd., batch number 21070546) is used as a comparative example.

[0054] The activated microsphere number concentration comparison results of the silicone oil-containing stopper and the silicone oil-free stopper samples are shown in Table 2, and the particle size distribution charts of the silicone oil-containing stopper and the silicone oil-free stopper samples after activation are shown in Figure 1 and Figure 2 .

[0055] Table 2 Effect of stopper on microsphere number concentration

[0056]

[0057] The results show that the silicone oil on the surface of the stopper can contact the drug and affect the number concentration of the octafluoropropane microspheres after activation. It can be seen that the number concentration of the microspheres is significantly increased by using a silicone oil-free stopper. Figure 1 and Figure 2 The particle size distribution of the silicone oil-containing stopper and the silicone oil-free stopper after activation is obviously different, as can be seen from Figure 1 , the sample containing silicone oil lacks small particle size microbubbles; and Figure 2 , the particle size distribution characteristics of the microbubble population are polydisperse distribution, and both small particle size microbubbles and large particle size microbubbles have a certain distribution. The polydisperse distribution of microbubbles can be applied to a wider clinical ultrasound frequency, and can provide clearer and more stable contrast images in various application scenarios.

[0058] Example 4 Stability study

[0059] The sample obtained in Example 1, which did not contain silicone oil, was subjected to stability release, in which the long-term condition was 2°C to 8°C, and was activated. The sample after activation was left to stand, and the microsphere number concentration was measured at 0 h and 12 h, respectively. The activation method and the microsphere number concentration measurement method were the same as in Example 3. The results are shown in Table 3.

[0060] Table 3

[0061] Sample 0h (cells / mL) 12h (cells / mL) Long term 6 month release sample 4.3 x 10 9 ]] 3.4 x 10 9 ]] Long term 12 month release sample 5.1 x 10 9 ]]> 4.9 x 10 9 ]]>

[0062] As can be seen, the sample that did not contain silicone oil had good stability.

Claims

1. An article comprising a container and a combination comprising octafluoropropane gas and a lipid mixture solution, the container comprising octafluoropropane gas in the headspace and a lipid mixture solution in the lower layer, wherein the container is sealed by a rubber stopper, the rubber stopper being free of silicone oil; and the lipid mixture solution comprising DPPA, DPPC, MPEG5000 DPPE, propylene glycol, glycerol, buffer salt, sodium chloride and water.

2. A combination capable of forming octafluoropropane microspheres, comprising octafluoropropane gas and a lipid mixture solution, the combination being provided in a container, the container comprising octafluoropropane gas in the headspace and a lipid mixture solution in the lower layer, wherein the container is sealed by a rubber stopper, the rubber stopper being free of silicone oil; and the lipid mixture solution comprising DPPA, DPPC, MPEG5000 DPPE, propylene glycol, glycerol, buffer salt, sodium chloride and water.

3. A method of preparing an octafluoropropane lipid microsphere injection, comprising the steps of: The sodium chloride, buffer salt, glycerol and water are mixed to prepare an aqueous solution; the propylene glycol, DPPA, DPPC and MPEG5000 DPPE are mixed to prepare a lipid non-aqueous solution; the lipid non-aqueous solution is added to the aqueous solution and diluted with water; the mixture is filtered to obtain a lipid mixture solution; the lipid mixture solution is filled into a container and semi-pressurized by a rubber stopper, the rubber stopper being free of silicone oil; the semi-pressurized product is vacuumized, filled with octafluoropropane and pressurized, capped and sterilized to obtain an octafluoropropane lipid microsphere injection; preferably, the propylene glycol, DPPA, DPPC and MPEG5000 DPPE are mixed, and the specific steps include directly dissolving DPPA, DPPC and MPEG5000 DPPE in propylene glycol; or dissolving DPPA, DPPC and MPEG5000 DPPE in an organic solvent, rotary evaporation, washing with the organic solvent, drying to obtain a lipid mixture, and dissolving the lipid mixture in propylene glycol; more preferably, the organic solvent is a mixture of toluene and methanol, a mixture of chloroform and methanol or a mixture of dichloromethane and methanol; more preferably, the organic solvent is a mixture of toluene and methanol; further preferably, the volume ratio of toluene to methanol is 3:

1.

4. The article of claim 1 or the combination of claim 2 or the method of claim 3, wherein, The container is a syringe or a vial; preferably a vial; more preferably a glass vial; further preferably a borosilicate glass tube injection vial; And / or, the volume capacity of the container is 2-4 mL; And / or, the rubber stopper is made of halogenated butyl rubber, preferably brominated butyl rubber or chlorinated butyl rubber; more preferably brominated butyl rubber for injection or chlorinated butyl rubber for injection; further preferably chlorinated butyl rubber for injection.

5. The article of claim 1 or the combination of claim 2, wherein, The lipid mixed solution is formed by mixing an aqueous solution and a lipid non-aqueous solution, wherein the aqueous solution comprises sodium chloride, buffer salt, glycerol and water; the lipid non-aqueous solution comprises propylene glycol, DPPA, DPPC and MPEG5000 DPPE; preferably, the lipid mixed solution is prepared by a preparation method comprising the following steps: stirring and mixing sodium chloride, buffer salt, glycerol and water to prepare the aqueous solution; stirring and mixing propylene glycol, DPPA, DPPC and MPEG5000 DPPE to prepare the lipid non-aqueous solution; stirring and mixing the lipid non-aqueous solution into the aqueous solution, and filtering to obtain the lipid mixed solution.

6. The article of claim 1 or the combination of claim 2 or the method of making of claim 3, wherein, The lipid mixed solution comprises about 0.7 mg to about 0.8 mg of combined DPPA, DPPC and MPEG5000 DPPE per milliliter of the solution, wherein the molar ratio of DPPA, DPPC and MPEG5000 DPPE is 5-15:77-90:5-15, respectively; preferably, the lipid mixed solution comprises about 0.75 mg of combined DPPA, DPPC and MPEG5000 DPPE per milliliter of the solution; preferably, the molar ratio of DPPA, DPPC and MPEG5000 DPPE is about 10:82:8; and / or the container contains 5.5-7 mg / mL of octafluoropropane; preferably 6.52 mg / mL of octafluoropropane.

7. The article of claim 1 or the combination of claim 2 or the method of making of claim 3, wherein, The buffer salt is selected from acetate or phosphate; preferably phosphate; more preferably one or more selected from sodium phosphate monobasic monohydrate, disodium phosphate dodecahydrate or disodium phosphate heptahydrate; preferably a combination of sodium phosphate monobasic monohydrate and disodium phosphate heptahydrate, or a combination of sodium phosphate monobasic monohydrate and disodium phosphate dodecahydrate; more preferably a combination of sodium phosphate monobasic monohydrate and disodium phosphate dodecahydrate.

8. The article of claim 1 or the combination of claim 2 or the method of making of claim 3, wherein, The lipid mixed solution contains about 0.045 mg of DPPA, about 0.401 mg of DPPC, about 0.304 mg of MPEG5000 DPPE, about 103.5 mg of propylene glycol, about 126.2 mg of glycerol, about 2.34 mg of sodium phosphate monobasic monohydrate, about 2.88 mg of disodium phosphate dodecahydrate, about 4.87 mg of sodium chloride per milliliter of the solution, and the rest is water; preferably, the pH value of the lipid solution is 6.2 to 6.

8.

9. A method for preparing lipid encapsulated octafluoropropane gas microspheres, by activating the combination of octafluoropropane gas and lipid mixed solution in the product of claim 1 or the combination of claim 2 to form lipid encapsulated octafluoropropane gas microspheres; preferably, the activation is achieved by an activation device; more preferably, the device activation parameters are 4530±100 oscillations per minute for 45 seconds.

10. The lipid-encapsulated octafluoropropane gas microspheres of claim 9 having an average particle size of 1 μm to 5 μm; preferably having an average particle size of 1 μm to 2 μm; and / or, the octafluoropropane gas and lipid mixture solution combination of claim 1 or the combination of claim 2 or the octafluoropropane lipid microspheres injection solution produced by the method of claim 3 when activated produce at least about 3.5 x 10 9 microspheres per milliliter of microspheres; preferably produce at least about 4 x 10 9 microspheres per milliliter of microspheres.

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