CN1895678A - Microvesicle ultrasonic biologically-degradable contrast medium containing polymer and its preparation - Google Patents

Abstract

The invention discloses a biodegradable polymer gas-containing microbubble ultrasonic contrast agent and a preparation method thereof. It is characterized by microbubbles formed by biodegradable polymer materials, which contain air, nitrogen or fluorine-containing gases, with an average particle size of 0.5-8 μm and a wall thickness of 20-5000 nm. Dissolve the biodegradable polymer in an organic solvent to form a 10-400 mg/ml polymer solution, add 0-8% sublimable substances to the solution, and add 0-100% inorganic salts to the solution according to 100:1-100:40 Add 6% distilled water to the polymer solution, add 0.01~1g of emulsifier to emulsify to form W ₁ /O initial emulsion, add the initial emulsion to distilled water containing 0.1~10% stabilizer, and form W after rapid stirring The ₁ /O/W ₂ emulsion was stirred at a low speed to evaporate the solvent, then centrifuged, washed, freeze-dried, and finally the corresponding gas was gradually introduced to obtain a biodegradable polymer gas-containing microbubble ultrasonic contrast agent.

Description

Biodegradable polymer gas-containing microbubble ultrasound contrast agent and preparation method thereof

technical field

The invention relates to a biodegradable polymer gas-containing microbubble ultrasonic contrast agent, which belongs to the field of biomedical diagnosis and treatment.

Background technique

Ultrasound contrast agents can enhance the contrast between lesion and normal tissue ultrasound images, and improve the accuracy, accuracy and sensitivity of ultrasound diagnosis. The research on ultrasound contrast agents has developed from the initial free bubbles to natural materials such as gelatin, albumin, lipids and other wrapped gases (O ₂ , N ₂ , air, fluorocarbon gases, etc.), to the current gas-containing polymers particle. US Pat. No. 4,774,958, Chinese patents CN1156626 and CN1194161 disclose microvesicles encapsulated with human albumin and carbohydrates. The use of this class of protein-derived contrast agents raises the issue of potential proteinaceous hypersensitivity reactions. World patent WO80/02365 and Chinese patent 1369311 use natural biological material gelatin or dextran as a contrast agent encapsulation material. Due to the limitations of the material itself, this type of contrast agent has disadvantages such as poor stability and uneven particle size distribution. European patent EP-A-0327490 using biodegradable polymers to wrap gas as a contrast agent uses hydroxycarboxylic acid polyesters, polycyanoacrylates, polyamino acids, polyamides, polyacrylated polysaccharides, and polyorthoesters As a particulate material; EP-A-0441468 uses polyoxymethylene, EP-A-0458079 uses polyamino acid-polycyclic imine to prepare microparticles; use emulsification method to wrap air with polyamino acid, polylactide, polyglycolide, etc. as materials or Microbubbles of other gases are EP-A-0458745, WO93/17718. The contrast agent disclosed in Chinese patent CN1082924 is a gas-containing polycyanoacrylate or α-, β-, and γ-hydroxyacid polyester particles; similarly, the CN1157573 patent is a gas-containing polymer particle composed of a biodegradable polymer and and/or microspheres of contrast agents. Both patents are patents applied by foreign companies in China. The other is CN1398640, which was applied by the Chinese themselves, using biodegradable polymer materials to prepare contrast agents. It was disclosed in 2003, using biodegradable aliphatic polylactone as the material, which is hollow microcapsule particles. In these patents, the toxicity of the degradation products of polycyanoacrylate is still under study; the biodegradable polymer material contrast agent prepared from polyester is only hollow microcapsule type and does not contain other types of gases.

Contents of the invention

The object of the present invention is to provide a biodegradable polymer material ultrasound contrast agent and a preparation method thereof in view of the deficiencies of the current technology.

The average particle size of the biodegradable macromolecule gas-containing microbubble ultrasound contrast agent is 0.5-8 μm, the particles with an average particle size greater than 8 μm are less than 3%, and the wall thickness is 20-5000 nm.

The biodegradable polymer gas-containing microbubble ultrasound contrast agent is synthetic biodegradable polyester and its block copolymer or graft copolymer, and the gas contained is air, nitrogen or fluorine-containing gas with low blood solubility and low diffusivity. This contrast agent has excellent storage stability, good stability in the circulation in the body and better contrast effect, and can also be controlled by the molecular weight of the material, the properties of each segment of the block or graft copolymer and The size is used to control the stability, dispersion and physiological properties of contrast agents.

Biodegradable polymers are aliphatic polyesters including two-component polyesters such as polyethylene adipate, polybutylene adipate, polyhexamethylene adipate, or polyesters formed from hydroxy acids such as polyethylene Lactide, polylactide, polycaprolactone; or binary or ternary random or block copolymers, graft copolymers; or polyethers with a molecular weight of less than 20,000 such as polyethylene glycol and polypropylene glycol, Any of its binary or ternary random or block copolymers and graft copolymers formed.

Preparation method of biodegradable ultrasound contrast agent

(1) Preparation of air and nitrogen-containing ultrasound contrast agents

Dissolve biodegradable polymers with a molecular weight of 3,000 to 300,000 in an organic solvent to form a 10 to 400 mg/ml polymer solution. After adding 0 to 8% sublimable substances to the solution, the volume ratio of the solution to water is 100 : 1~100:40 Add distilled water containing 0~6% inorganic salt to the polymer solution, and add emulsifier, the amount of emulsifier is 10ml polymer solution plus 0.01~1g emulsifier; the second step, magnetic stirring , mechanical stirring or ultrasonic emulsification for 0.5 to 30 minutes to form a W ₁ /O primary emulsion; in the third step, the primary emulsion is added to distilled water containing 0.1 to 10% stabilizer at a temperature of 0 to 40°C, and the temperature is 8000 to 25000 Stir at a high speed of rpm for 1 to 5 minutes, and then stir at a lower speed of 300 to 1500 rpm for 2 to 24 hours. The operating temperature is room temperature to 60°C. Washing with distilled water; the fifth step, freeze-drying, temperature -25 ° C ~ -80 ° C, time 8 ~ 24 hours, if necessary, pre-freeze and add 0 ~ 10% cryoprotectant such as sugar or polyvinyl alcohol, polyethylene glycol , glycerol; the sixth step, gradually introduce air or nitrogen;

(2) Preparation of fluorine-containing gas ultrasound contrast agent

Dissolve biodegradable polymers with a molecular weight of 3,000 to 300,000 in an organic solvent to form a 10 to 400 mg/ml polymer solution. After adding 0 to 8% sublimable substances to the solution, the volume ratio of the solution to water is 100 : 1~100:40 Add distilled water containing 0~6% inorganic salt to the polymer solution, and add emulsifier, the amount of emulsifier is 10ml polymer solution plus 0.01~1g emulsifier; the second step, magnetic stirring , mechanical stirring or ultrasonic emulsification for 0.5 to 30 minutes to form W ₁ /O primary emulsion; the third step, the primary emulsion is added to distilled water containing 0.1 to 10% stabilizer at a temperature of 0 to 40 ° C, at 8000 to 25000 rpm Stirring at a high speed per minute for 1-5 minutes, and then stirring at a lower speed of 300-1500 rpm for 2-24 hours. Washing with water; the fifth step, freeze drying, temperature -25 ° C ~ -80 ° C, time 8 ~ 24 hours, if necessary, pre-freeze and add 0 ~ 10% cryoprotectant such as sugar or polyvinyl alcohol, polyethylene glycol, Glycerin; the sixth step, maintain a vacuum of 15-250Pa, connect with the fluorine-containing gas, and gradually introduce the fluorine-containing gas;

The air-containing or fluorine-containing gas polyester microbubble particles prepared by the above-mentioned process are all white powder-like microbubble particles, which are resuspended in water for injection, or suspended in a mixture of Water for injection of any one of lactose, glycerin, and polyethylene glycol.

The solvent forming the polymer solution is dichloromethane, chloroform, ethyl chloride, 1,2-dichloroethane, 1,1,1-trichloroethane, 1,1,2-trichloroethane, At least one of 1,1,1,2-tetrachloroethane, 1,1,2,2-tetrachloroethane, acetone, ethyl acetate, and tetrahydrofuran.

The sublimable substance is any one of camphene, cyclohexane, cyclooctane, and camphor.

The inorganic salt is any one of ammonium bicarbonate, ammonium carbonate, ammonium bisulfate, ammonium sulfate, sodium bicarbonate, and sodium carbonate.

The substance as an emulsifier can be at least one of phosphatidylcholine, Span, Tween, sucrose, palmitic acid, and stearate.

The stabilizer is at least one of polyvinyl alcohol, polyethylene glycol, denatured protein, gelatin, polyvinylpyrrolidone, sugar alcohol and sugar.

The fluorine-containing gas is any of SF ₆ (sulfur hexafluoride), C ₃ F ₈ (perfluoropropane), C ₄ F ₁₀ (perfluorobutane), and C ₆ F ₁₄ (perfluorohexane).

The present invention has the following advantages:

1) The material used in the invention is a synthetic biodegradable polymer, which is degraded into glycol or/and dibasic acid or hydroxy acid or/and polyether with molecular weight less than 20,000 in a physiological environment, which is non-toxic to the human body and can Participate in metabolism in the body or be eliminated from the body.

2) The biodegradable material used in the invention can adjust the properties of the material by adjusting the components, block or graft structure, and molecular weight, so as to prepare a contrast agent with proper degradability and suitable elastic microbubble walls.

3) The contrast agent preparation technology used in the present invention can not only use ultrasonic emulsification, but also use mechanical stirring and magnetic stirring to make the preparation process easier to implement when forming the primary emulsion.

4) The present invention wraps different gases in microbubble particles, such as air, nitrogen, fluorocarbon gas, etc., to better improve the imaging effect.

5) The particle size of the microbubble particles prepared by the present invention is 0.5-8 μm, and the particle size distribution is greater than 8 μm, less than 3%.

6) The biodegradable ultrasound contrast agent prepared by the present invention is suspended in water for injection, or suspended in water for injection added with one or more of the following substances glucose, mannose, lactose, glycerin, polyethylene glycol, and enters in an appropriate way Measure the site in the body and improve the contrast effect.

Detailed ways

The present invention is specifically described below through the examples, it is necessary to point out that the present examples are only used to further illustrate the present invention, and can not be interpreted as limiting the protection scope of the present invention, those skilled in the art can according to the present invention Contents Some non-essential improvements and adjustments are made to the present invention.

Example 1:

Dissolve 50 mg of polylactide-polyethylene glycol-polylactide PLA-PEG-PLA with a molecular weight of 300,000 in a mixed solvent of 5ml 1,1,2-trichloroethane and ethyl acetate (10:1), add Mix 0.1g of emulsifier with Span 80 and Tuwin 60, add 2ml of distilled water, and emulsify on magnetic stirring for 10min to form W ₁ /O. The obtained W ₁ /O emulsion was injected into water containing ~4% polyvinyl alcohol as a stabilizer at a temperature of 20°C, stirred and emulsified at a high speed (8000 rpm) for 1 min, and then stirred at a low speed (400 rpm) for 20 h. After the solvent evaporates, it is centrifuged, washed three times with double distilled water, and freeze-dried to obtain white powder-like microbubble particles with a particle size of 0.5-2 μm and a narrow particle size distribution. Suspend it in water for injection containing a small amount of glycerin to form an ultrasound contrast agent containing 5×10 ⁸ microbubble particles/mL suspension. Dogs were used as model animals to measure the effect of contrast-enhanced ultrasound in vivo by intravenous infusion; at the same time, the effect of contrast-enhanced ultrasound in vivo was measured by the same method using Sonovue (USCA, which is allowed to be used clinically by the Ministry of Health of China) as a comparison. The results show that the PLA-PEG-PLA copolymer ultrasound contrast agent has a better contrast effect.

Example 2:

Dissolve 300 mg of PLA polymer with a molecular weight of 30,000 in 10 ml of chloroform solvent, add 0.8 g of cyclooctane, add 0.1 g of PEG2000 as an emulsifier, then add 1 ml of distilled water, and emulsify on a magnetic stirrer for 20 min to form W ₁ /O. The obtained W ₁ /O emulsion was poured into water containing ~2% polyvinyl alcohol as a stabilizer, stirred and emulsified at 15,000 rpm for 1 min, and then stirred at a low speed (1,000 rpm) for 8 h. After the solvent evaporates, it is centrifuged, washed three times with double distilled water, and freeze-dried to obtain white powder-like microbubble particles with a particle size of 1-6 μm, which are hollow microspheres under scanning electron microscope observation. Suspend it in water for injection containing a small amount of polyethylene glycol to form an ultrasound contrast agent containing 2×10 ⁹ microbubble particle suspensions/mL. Dogs were used as model animals to measure the effect of contrast-enhanced ultrasound in vivo by intravenous infusion; at the same time, the effect of contrast-enhanced ultrasound in vivo was measured by the same method using Sonovue (USCA, which is allowed to be used clinically by the Ministry of Health of China) as a comparison. The results show that the PLA-PEG-PLA copolymer ultrasound contrast agent has a better contrast effect.

Example 3:

A solution of 300 mg of poly(lactide-caprolactone-polyethylene glycol) copolymer with a molecular weight of 20,000 in dichloromethane and acetone, Gasban 60 was used as an emulsifier, then 0.5 ml of distilled water was added, and ultrasonic emulsification was performed for 5 min W ₁ /O is formed. Pour the obtained W ₁ /O emulsion into water containing ~1% polyvinyl alcohol as a stabilizer, add 0.02g of Tween 60, stir and emulsify at 10,000 rpm for 3 minutes, and then stir at a low speed (9,000 rpm) for 9 hours. After the solvent evaporates, it is centrifuged, washed three times with double distilled water, freeze-dried, and then gradually introduces fluorine-containing gas to obtain white powder particles with a particle size of 0.9-5 μm. Suspend it in glucose-containing water for injection to form an ultrasound contrast agent containing 3×10 ⁸ microbubble particles/mL suspension. Taking dogs as model animals, the effect of contrast-enhanced ultrasound in vivo was measured by intravenous infusion. The contrast effect is better than that of Examples 1 and 2.

Example 4:

Dissolve 2 g of polyethylene adipate with a molecular weight of 3000 in a mixed solvent of 5 ml of methylene chloride and ethyl acetate, add 0.1 g of a phosphatidylcholine emulsifier, and then add 2 ml of an aqueous solution containing 4% ammonium bicarbonate , emulsified with mechanical stirring for 10min to form W ₁ /O. The obtained W ₁ /O emulsion was injected into water containing ~4% polyvinyl alcohol as a stabilizer at a temperature of 10°C, stirred and emulsified at a high speed (8000 rpm) for 1 min, and then stirred at a low speed (600 rpm) for 15 h. After the solvent evaporates, centrifuge, wash with double distilled water three times, freeze-dry, and then gradually introduce nitrogen-containing gas to obtain white powder-like microbubble particles with a particle size of 0.5-3 μm and a narrow particle size distribution. Suspend it in water for injection containing a small amount of glycerin, and measure the effect of contrast-enhanced ultrasound in vivo by intravenous infusion. The contrast effect is similar to Example 1.

Example 5:

The poly(glycolide-lactide) copolymer 400mg that molecular weight is 20000 is dissolved in the 5ml dichloromethane, adds 0.05g amphene, and adds Span 60 and Tween 60 mixed emulsifier 0.03g, then adds containing 2ml aqueous solution of 1% sodium bicarbonate, ultrasonic emulsification for 5min to form W ₁ /O. The obtained W ₁ /O emulsion was injected into water containing ~1% stabilizer gelatin at a temperature of 5°C, stirred and emulsified at a high speed (12000 rpm) for 1 min, and then stirred at a low speed (700 rpm) for 12 hours. After the solvent evaporates, centrifuge, wash with double distilled water for 3 times, pre-freeze and add cryoprotectant polyvinyl alcohol to freeze-dry to obtain white powder-like microbubble particles with a particle size of 1-5 μm. Suspend it in water for injection containing a small amount of glycerin, inject it into the body, and measure the contrast-enhancing effect of ultrasound. The contrast effect is similar to Example 3.

The biodegradable polymer materials used in the present invention are provided by the Polymer Institute of Sichuan University, and other chemical reagents are commercially available analytically pure preparations.

Claims (10)

1. the acoustic contrast agent of a Biodegradable high molecular gassiness microvesicle, the mean diameter that it is characterized in that this gassiness microcapsular ultrasound contrast agent is 0.5～8 μ m, and mean diameter is less than 3% greater than the particle of 8 μ m, and wall thickness is 20～5000nm.

2. microvesicle ultrasonic biologically-degradable contrast medium containing polymer according to claim 1, the acoustic contrast agent that it is characterized in that the gassiness microvesicle is synthetic Biodegradable polyester and block copolymer or graft copolymer, contains gas and be in air, nitrogen or the fluoro-gas any.

3. by the acoustic contrast agent of claim 1 and 2 described Biodegradable high molecular gassiness microvesicles, it is characterized in that Biodegradable polyester is that aliphatic polyester comprises that polyester that bi-component polyester forms as poly-adipic acid second diester, poly-adipic acid fourth diester, polyhexamethylene adipate or hydroxy acid is as poly-Acetic acid, hydroxy-, bimol. cyclic ester, polylactide, polycaprolactone; Or its binary or ternary is random or block copolymer, graft copolymer; Or itself and molecular weight be less than 20000 polyethers such as Polyethylene Glycol, polypropylene glycol, its binary of formation ternary is random or block copolymer, graft copolymer in any.

4. press the preparation method of the acoustic contrast agent of one of claim 1～3 described Biodegradable high molecular gassiness microvesicle, it is characterized in that this method may further comprise the steps:

(1) contains the preparation of air, nitrogen acoustic contrast agent

With molecular weight is that 3000～300000 Biodegradable high moleculars are dissolved in the organic solvent, form 10～400mg/ml polymer solution, add in this solution 0～8% have the material of sublimability after, volume ratio by solution and water is that the distilled water that will contain 0～6% inorganic salt in 100: 1～100: 40 joins in the solution of polymer, and the adding emulsifying agent, the emulsifying agent consumption adds 0.01～1g emulsifying agent for the 10ml polymer solution; Second step, magnetic agitation, mechanical agitation or ultrasonic emulsification 0.5～30min, the colostric fluid of formation W1/O; The 3rd step, it is in 0～40 ℃ of distilled water that contains stabilizing agent 0.1～10% that this colostric fluid is joined temperature, stir 1～5min with 8000～25000 rev/mins of fast speeds, after this stir 2～24h with 300～1500 rev/mins than low velocity again, operative temperature is room temperature～60 ℃; In the 4th step, after solvent evaporates was intact, centrifugalize was washed with redistilled water; The 5th step, lyophilization, temperature-25 ℃～-80 ℃, time 8～24h, precooling and add 0～10% cryoprotective agent such as saccharide or polyvinyl alcohol, Polyethylene Glycol, glycerol in case of necessity; In the 6th step, progressively introduce air or nitrogen;

(2) preparation of fluoro-gas acoustic contrast agent

With molecular weight is that 3000～300000 Biodegradable high moleculars are dissolved in the organic solvent, form 10～400mg/ml polymer solution, add in this solution 0～8% have the material of sublimability after, volume ratio by solution and water is that the distilled water that will contain 0～6% inorganic salt in 100: 1～100: 40 joins in the solution of polymer, and the adding emulsifying agent, the emulsifying agent consumption adds 0.01～1g emulsifying agent for the 10ml polymer solution; In second step, magnetic agitation, mechanical agitation or ultrasonic emulsification 0.5～30min form the W1/O colostric fluid; The 3rd step, it is in 0～40 ℃ of distilled water that contains stabilizing agent 0.1～10% that this colostric fluid is joined temperature, stir 1～5min with 8000～25000 rev/mins of fast speeds, after this stir 2～24h with 300～1500 rev/mins than low velocity again, operative temperature is room temperature～60 ℃; In the 4th step, after solvent evaporates was intact, centrifugalize was washed with redistilled water; The 5th step, lyophilization, temperature-25 ℃～-80 ℃, time 8～24h, precooling and add 0～10% cryoprotective agent such as saccharide or polyvinyl alcohol, Polyethylene Glycol, glycerol in case of necessity; The 6th step kept vacuum 15～250Pa, was connected with fluoro-gas, progressively introduced fluoro-gas;

By said process preparation contain air or fluoro-gas polyesters microbubble particles is white powder shape material, this white powder shape microbubble particles resuspending is in water for injection, or is suspended in any the water for injection that is added with in glucose, mannose, lactose, glycerol, the Polyethylene Glycol.

5. as the preparation method of microvesicle ultrasonic biologically-degradable contrast medium containing polymer as described in the claim 4, the solvent that it is characterized in that forming polymer solution is dichloromethane, chloroform, ethyl chloride, 1,2-dichloroethanes, 1,1,1-trichloroethane, vinyl trichloride, 1,1,1,2-sym-tetrachloroethane, 1,1,2, at least a in 2-sym-tetrachloroethane, acetone, ethyl acetate, the oxolane.

6. as the preparation method of microvesicle ultrasonic biologically-degradable contrast medium containing polymer as described in the claim 4, the material that it is characterized in that sublimability is any in camphene, cyclohexane extraction, cyclooctane, the Camphora.

7. as the preparation method of microvesicle ultrasonic biologically-degradable contrast medium containing polymer as described in the claim 4, it is characterized in that inorganic salt is any in ammonium bicarbonate, ammonium carbonate, ammonium hydrogen sulfate, ammonium sulfate, sodium bicarbonate, the sodium carbonate.

8. as the preparation method of microvesicle ultrasonic biologically-degradable contrast medium containing polymer as described in the claim 4, it is characterized in that material as emulsifying agent can be at least a in phosphatidylcholine, class of department, soil temperature, sucrose, Palmic acid, the stearate.

9. as the preparation method of microvesicle ultrasonic biologically-degradable contrast medium containing polymer as described in the claim 4 and claim 4, it is characterized in that stabilizing agent is at least a in polyvinyl alcohol, Polyethylene Glycol, Denatured protein, gelatin, polyvinylpyrrolidone, sugar alcohol, the sugar.

10. as the preparation method of microvesicle ultrasonic biologically-degradable contrast medium containing polymer as described in the claim 4, it is characterized in that fluoro-gas is SF ₆(sulfur hexafluoride), C ₃F ₈(perfluoropropane), C ₄F ₁₀(perfluorinated butane), C ₆F ₁₄In (perflexane) any.