CN103432602A - Micro-capsule ultrasonic contrast agent and preparation method thereof - Google Patents
Micro-capsule ultrasonic contrast agent and preparation method thereof Download PDFInfo
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- CN103432602A CN103432602A CN2013103784783A CN201310378478A CN103432602A CN 103432602 A CN103432602 A CN 103432602A CN 2013103784783 A CN2013103784783 A CN 2013103784783A CN 201310378478 A CN201310378478 A CN 201310378478A CN 103432602 A CN103432602 A CN 103432602A
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Abstract
The invention discloses a micro-capsule ultrasonic contrast agent and a preparation method thereof. The preparation method comprises the following steps of (1) dissolving biodegradable high polymer materials into an organic solvent to obtain a solution marked as a solution A, and dissolving a stabilizer into water to obtain a solution marked as a solution B; (2) mixing the solution A with water, and carrying out ultrasonic treatment to obtain a water-in-oil primary emulsion; (3) adding the primary emulsion into the solution B, and stirring to obtain an oil-in-water-in-water pre-composite emulsion; (4) adding the pre-composite emulsion into a membrane emulsification device for passing through the membrane to obtain an oil-in-water-in-water composite emulsion; (5) stirring and centrifuging the oil-in-water-in-water composite emulsion, and collecting the precipitate to obtain the micro-capsule ultrasonic contrast agent. The ultrasonic contrast agent is sent into the part of the human body to be detected through injection and intravenous perfusion, vibrates under the action of the ultrasonic so as to change the ultrasound characters of the tissue, and increases the contrast ratio between the lesion tissue and the normal tissue as well as between the motion part and the static part, thereby playing an ultrasonic developing effect.
Description
Technical field
The present invention relates to a kind of microcapsule formula acoustic contrast agent and preparation method thereof.
Background technology
At present medical imaging diagnosis technology commonly used comprises nuclear magnetic resonance, NMR, X ray and ultrasonic three major types clinically.Because ultrasonic diagnosis has that safe, widely applicable, directly perceived, sensitivity is strong, low cost, noinvasive, the advantage such as be easy to carry, so to become means the most commonly used in the Medical diagnostic imaging.The ultrasonic diagnosis technology is that reflection and the strong and weak difference of scattered signal of utilizing ultrasound wave to occur at the tissue interface show the normal and abnormal information of organism interior tissue organ structure function, thereby reaches the technology of medical diagnosis on disease purpose.Although the changes in microstructure that ultrasonic imaging method causes for some diseases has good diagnosis and locating effect, work as pathological tissues and normal structure acoustic properties and differ not obvious, make organ structure develop not good, easily cause mistaken diagnosis or fail to pinpoint a disease in diagnosis.In addition, during the mobility status of the little vessel inner blood of conventional ultrasonic diagnosis in detecting body deep tissues organ, can the use of this method be restricted due to acoustic attenuation and the stronger echo effect of peripheral static tissue.Therefore, extremely needing, by introducing certain medium, increases the contrast between pathological tissues and normal structure ultrasonoscopy, thereby improves correctness, degree of accuracy and the sensitivity of diagnosis.
Acoustic contrast agent, after vein is injected in vivo, can strengthen the contrast of ultrasonic image, can distinguish the slight change of normal structure and pathological tissues blood perfusion, can detect the medium and small vessel inner blood mobility status of body deep tissues organ.Current widely used acoustic contrast agent typically refers to the microcapsule bubble that the diameter that includes gas is 2 microns to 8 microns, case material can adopt albumen, lipid, surfactant, saccharide or polymer, and gas can adopt carbon dioxide, air, oxygen and fluorocarbon gas etc.The gas of microbubble internal package can change histoorgan to ultrasonic scattering, decay and ultrasonic spread speed in tissue and nonlinear effect etc., significantly strengthen its echological picture signal, greatly improve the sensitivity and specificity of diagnosis, make up the shortcomings and deficiencies of conventional Ultrasound diagnosis, make the ultrasonic diagnosis field that larger development arranged.
The size of acoustic contrast agent particle diameter and particle size distribution have important impact to its ultrasonic development effect and safety.Acoustic contrast agent is the meeting vibration-generating under ultrasonic effect, improves backscatter signals and produces the radiography effect.The ultrasound intensity received is the function of the scattering section of incident intensity and reflector.Scattering section is to be directly proportional to the biquadratic of frequency and six powers of scattering object radius, so size affects acoustic contrast agent radiography development effect.In order to be free to after being injected in vivo by pulmonary circulation, do not cause pulmonary infarction, the particle diameter of acoustic contrast agent requires to be less than 8 microns.Can produce very wide echo frequency during the particle size distribution heterogeneity, and clinical ultrasound often is defined in the certain frequency scope, this will cause only having the part microcapsule to play the development effect, and efficiency is not high.Therefore, desirable acoustic contrast agent requires particle diameter between 2 microns to 8 microns, the particle size distribution homogeneous.
The method for preparing at present acoustic contrast agent also has a lot, comprises hand agitation, sound shake cavitation method, freeze-drying, liquid-gas phase transition method, thin film aquation method, jet atomization method, seasoning etc.Yet microcapsule size prepared by above method is not concentrated, be difficult to control and regulate size and the particle size distribution of microcapsule, thereby urgently need to invent a kind of can the grain-size size and optimize the preparation method of particle size distribution.
Summary of the invention
The purpose of this invention is to provide a kind of microcapsule formula acoustic contrast agent and preparation method thereof, preparation method provided by the invention can overregulate film emulsion process parameter control preparation contrast agent particle diameter size and optimize the particle size distribution of microcapsule, finally obtain the microcapsule acoustic contrast agent of particle size distribution homogeneous.
The preparation method of a kind of microcapsule formula acoustic contrast agent provided by the present invention, comprise the steps:
(1) biodegradable macromolecular material is dissolved in organic solvent, obtains solution, be designated as solution A;
Stabilizing agent is dissolved in the water, obtains solution, be designated as solution B;
(2) described solution A is mixed with water, and obtained water in oil elementary emulsion through ultrasonic;
(3) described elementary emulsion is joined in described solution B, obtain the pre-double emulsion of W/O/W through stirring;
(4) described pre-double emulsion is added in the film emulsifier unit and carried out film, obtain the double emulsion of W/O/W;
(5) double emulsion of described W/O/W stirs and is centrifugal, and collecting precipitation obtains described microcapsule formula acoustic contrast agent.
In above-mentioned preparation method, described biodegradable macromolecular material can be the biodegradable macromolecular material of modified natural and synthesizing biological degradable macromolecular material;
The biodegradable macromolecular material of described modified natural is oil-soluble material modified, comprises acetate starch, succinic acid esterification starch, acylation chitosan, Nmaleoyl chitosan, Glycerolchitosan, alkylated chitosan, butyryl chitosan and hydroxypropyl chitosan;
Described alkylated chitosan specifically comprises N-butyl alkylated chitosan, N-octyl group alkylated chitosan, N-palmityl alkylated chitosan, N, the two octyl group alkylated chitosans of N-, N, the two decyl chitosans of N-and N, the two dodecyl chitosans of N-;
Described synthesizing biological degradable macromolecular material can be the aliphatic poly lactone.
In above-mentioned preparation method, described aliphatic poly lactone is polylactic acid (PLA), poly-(lactide-Acetic acid, hydroxy-, bimol. cyclic ester) copolymer (PLGA), poly-(lactide-caprolactone) copolymer (PLC), poly-(Acetic acid, hydroxy-, bimol. cyclic ester-caprolactone) copolymer (PGC), poly-(Acetic acid, hydroxy-, bimol. cyclic ester-lactide-caprolactone) copolymer (PGLC), PLE copolymer (PEG-b-PLA), poly-(Acetic acid, hydroxy-, bimol. cyclic ester-polyglycol ether) copolymer (PEG-b-PGA), poly-(own lactide-polyglycol ether) copolymer (PEG-b-PCL), poly-(lactide-Acetic acid, hydroxy-, bimol. cyclic ester-polyglycol ether) copolymer (PEG-b-PLGA), gather at least one in (lactide-caprolactone-polyglycol ether) copolymer (PEG-b-PLC) and poly-(Acetic acid, hydroxy-, bimol. cyclic ester-caprolactone-polyglycol ether) copolymer (PEG-b-PGC),
The number-average molecular weight of described aliphatic poly lactone can be 5,000～500, and 000,
The number-average molecular weight of the polyglycol ether section in described PLE copolymer, described poly-(Acetic acid, hydroxy-, bimol. cyclic ester-polyglycol ether) copolymer, described poly-(own lactide-polyglycol ether) copolymer, described poly-(lactide-Acetic acid, hydroxy-, bimol. cyclic ester-polyglycol ether) copolymer, described poly-(lactide-caprolactone-polyglycol ether) copolymer and described poly-(Acetic acid, hydroxy-, bimol. cyclic ester-caprolactone-polyglycol ether) copolymer can be 200～20,000.
In above-mentioned preparation method, in step (1), described organic solvent can be dichloromethane, chloroform or dichloroethanes;
The mass body volume concentrations of described solution A can be 1～200mg/mL, specifically can be 1～150mg/mL, 1～120mg/mL, 1～100mg/mL, 10～5mg/mL, 1mg/mL, 2mg/mL, 4mg/mL, 5mg/mL, 7mg/mL, 10mg/mL, 20mg/mL, 50mg/mL, 100mg/mL, 120mg/mL, 150mg/mL or 200mg/mL;
Described stabilizing agent can be polyvinyl alcohol, gelatin or polyvinylpyrrolidone;
The quality percentage composition of described solution B can be 0.5～5.0%, specifically can be 0.5～4.5%, 0.5～3.5%, 0.5～3.0%, 0.5～2.5%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4.5% or 5%.
In above-mentioned preparation method, in step (2), the volume ratio of described solution A and described water can be 2～40:1, specifically can be 2～35:1,2～30:1,2～25:1,7～40:1,2:1,3:1,4:1,5:1,7:1,10:1,15:1,20:1,25:1,30:1,35:1 or 40:1;
Described ultrasonic power can be 50～500W, specifically can be 100～500W, 100W, 150W, 200W, 300W, 350W, 400W, 450W or 500W, time can be 5～240 seconds, specifically can be 10～240 seconds, 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, 70 seconds, 80 seconds, 90 seconds, 150 seconds, 200 seconds or 240 seconds.
In above-mentioned preparation method, in step (3), the volume ratio of described elementary emulsion and described solution B can be 1:5～50, specifically can be 1:5～40,1:5～35,1:12～50,1:5,1:7,1:8,1:10,1:12,1:15,1:20,1:25,1:30,1:35,1:40 or 1:50;
The speed of described stirring can be 60～2000 rev/mins, specifically can be 120～2000 rev/mins, 300～1000 rev/mins, 120 rev/mins, 200 rev/mins, 300 rev/mins, 400 rev/mins, 500 rev/mins, 600 rev/mins, 700 rev/mins, 900 rev/mins, 1000 rev/mins or 2000 rev/mins, time can be 0.5～30.0 minute, specifically can be 1～30.0 minute, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 8 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes or 30 minutes.
In above-mentioned preparation method, in step (4), the described film of crossing can carry out 1～15 time under 20～200 kPas of nitrogen pressures, specifically can be: under 20 kPas of nitrogen pressures, carry out 3 times, under 50 kPas of nitrogen pressures, carry out 3 times, under 60 kPas of nitrogen pressures, carry out 4 times, under 80 kPas of nitrogen pressures, carry out 5 times, under 90 kPas of nitrogen pressures, carry out 5 times, under 100 kPas of nitrogen pressures, carry out 5 times, under 110 kPas of nitrogen pressures, carry out 4 times, under 120 kPas of nitrogen pressures, carry out 6 times, under 130 kPas of nitrogen pressures, carry out 6 times, under 140 kPas of nitrogen pressures, carry out 7 times, carry out 1 time under 50 kPas of nitrogen pressures or carry out 10 times under 180 kPas of nitrogen pressures.
In above-mentioned preparation method, in step (5), the time of described stirring can be 1～24 hour, specifically can be 2～18 hours, 2 hours, 4 hours, 8 hours, 12 hours, 14 hours, 16 hours, 18 hours or 24 hours, the speed of described stirring can be 60～2000 rev/mins, specifically can be 60～1800 rev/mins, 60 rev/mins, 80 rev/mins, 100 rev/mins, 150 rev/mins, 200 rev/mins, 280 rev/mins, 300 rev/mins, 1500 rev/mins or 1800 rev/mins.
In above-mentioned preparation method, in step (5), described method also comprises the step that water is washed described precipitation;
Step (1), step (2), step (3), step (4) and step (5) all can be carried out under the condition of 0～50 ℃.
The present invention further provides the microcapsule formula acoustic contrast agent prepared by above-mentioned, it is hollow structure, and its particle diameter is 2 microns～8 microns.The size of microcapsule formula acoustic contrast agent of the present invention can be carried out regulation and control, the distribution homogeneous of particle diameter by regulating the preparation process parameter.
The present invention prepares acoustic contrast agent and sends into the detected position of human body by injection, venous perfusion, under ultrasonic effect, vibrate, change the ultrasonic characteristic (as backscattering coefficient, attenuation quotient, the velocity of sound and nonlinear effect) of tissue, increase between pathological tissues and normal structure, the contrast between motion parts and stationary part, play the effect of ultrasonic development.
The accompanying drawing explanation
The stereoscan photograph of the microcapsule formula contrast agent that Fig. 1 is embodiment 1, embodiment 3 and embodiment 8 preparations, Fig. 1 (A), Fig. 1 (B) and Fig. 1 (C) are respectively the stereoscan photograph of the contrast agent of embodiment 1, embodiment 3 and embodiment 8 preparations.
SEM picture and the particle size distribution figure of the microcapsule formula contrast agent that Fig. 2 is embodiment 1 preparation.
The hollow-core construction figure of the microcapsule formula contrast agent that Fig. 3 is embodiment 2 preparations.
The ultrasonic development design sketch of the microcapsule formula contrast agent that Fig. 4 is embodiment 6 preparations, wherein Fig. 4 (A) is the development effect figure while not adding contrast agent of the present invention, Fig. 4 (B) is the development effect figure while adding contrast agent of the present invention, and Fig. 4 (C) is the development effect figure while adding commercially available Sonovue contrast agent.
The ultrasonic development design sketch of the microcapsule formula contrast agent that Fig. 5 is embodiment 1,3,5 and 8 preparations, wherein Fig. 5 (A), Fig. 5 (B), Fig. 5 (C) and Fig. 5 (D) are respectively the ultrasonic development design sketch of the microcapsule formula contrast agent that adds embodiment 1,3,5 and 8 preparations.
The specific embodiment
The experimental technique used in following embodiment if no special instructions, is conventional method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
Embodiment 1, prepare microcapsule formula acoustic contrast agent
The PLA that is 5,000 by number-average molecular weight is dissolved in dichloromethane, is configured to the solution of 1mg/mL.After this solution mixes according to the volume ratio of 40:1 with water, under the power of 500 watts ultrasonic 240 seconds, make water in oil elementary emulsion.Under the mixing speed of 2000 rev/mins, elementary emulsion obtained above is splashed into to the polyvinyl alcohol water solution (5%, mass concentration) of 50 times of volumes of elementary emulsion, stir 1 minute.The gained mixed system is poured in the storage tank of fast film emulsifier unit (membrane aperture is 7 microns), crosses film 10 times under 180 kPas of nitrogen pressures.Under the gained W/O/W double emulsion speed that at room temperature 60 turn/films divide, stir 24 hours.Centrifugal collection product.Products therefrom with water washing after, lyophilization.
Obtain the microcapsule that mean diameter is 2 microns.Gained microcapsule lucifuge, drying, cryopreservation.
The stereoscan photograph of microcapsule prepared by the present embodiment is as shown in Fig. 1 (A).
The PLGA that is 20,000 by number-average molecular weight is dissolved in dichloromethane, is configured to the solution of 2mg/mL.After this solution mixes according to the volume ratio of 35:1 with water, under 350 watts of power ultrasonic 70 seconds, make water in oil elementary emulsion.Under the mixing speed of 1000 rev/mins, elementary emulsion is splashed into to the polyvinyl alcohol water solution (5%, mass concentration) of 40 times of volumes of elementary emulsion, stir 25 minutes.The gained mixed system is poured in the storage tank of fast film emulsifier unit (membrane aperture is 7 microns), crosses film 1 time under 50 kPas of nitrogen pressures.Gained W/O/W double emulsion at room temperature stirs 9 hours under the speed of 150 rev/mins.Centrifugal collection product.Products therefrom with water washing after, lyophilization.
The microcapsule that the acquisition mean diameter is 2.5 microns.Gained microcapsule lucifuge, drying, cryopreservation.
Stereoscan photograph when microcapsule prepared by the present embodiment is cut open as shown in Figure 3, can be learnt by this figure, and method provided by the invention can make the microcapsule of hollow structure.
The PLC that is 50,000 by number-average molecular weight is dissolved in chloroform, is configured to the solution of 4mg/mL.After this solution mixes according to the volume ratio of 30:1 with water, under 450 watts of power ultrasonic 90 seconds, make water in oil elementary emulsion.Under the mixing speed of 120 rev/mins, elementary emulsion is splashed into to the polyvinyl alcohol water solution (4.5%, mass concentration) of 35 times of volumes of elementary emulsion, stir 30 minutes.The gained mixed system is poured in the storage tank of fast film emulsifier unit (membrane aperture is 8 microns), crosses film 7 times under 140 kPas of nitrogen pressures.Gained W/O/W double emulsion at room temperature stirs 18 hours under the speed of 80 rev/mins.Centrifugal collection product.Products therefrom with water washing after, lyophilization.
The microcapsule that the acquisition mean diameter is 3 microns.Gained microcapsule lucifuge, drying, cryopreservation.
The stereoscan photograph of microcapsule prepared by the present embodiment is as shown in Fig. 1 (B).
Embodiment 4, prepare microcapsule formula acoustic contrast agent
The PGC that is 80,000 by number-average molecular weight is dissolved in dichloroethanes, is configured to the solution of 5mg/mL.After this solution mixes according to the volume ratio of 25:1 with water, under 450 watts of power ultrasonic 90 seconds, make water in oil elementary emulsion.Under the mixing speed of 2000 rev/mins, elementary emulsion is splashed into to the polyvinyl alcohol water solution (4.5%, mass concentration) of 30 times of volumes of elementary emulsion, stir 2 minutes.The gained mixed system is poured in the storage tank of fast film emulsifier unit (membrane aperture is 8 microns), crosses film 6 times under 130 kPas of nitrogen pressures.Gained W/O/W double emulsion at room temperature stirs 18 hours under the speed of 80 rev/mins.Centrifugal collection product.Products therefrom with water washing after, lyophilization.
The microcapsule that the acquisition mean diameter is 3.5 microns.Gained microcapsule lucifuge, drying, cryopreservation.
The PGLC that is 100,000 by number-average molecular weight is dissolved in dichloromethane, is configured to the solution of 7mg/mL.After this solution mixes according to the volume ratio of 20:1 with water, under 100 watts of power ultrasonic 10 seconds, make water in oil elementary emulsion.Under the mixing speed of 200 rev/mins, elementary emulsion is splashed into to the polyvinyl alcohol water solution (4.5%, mass concentration) of 25 times of volumes of elementary emulsion, stir 3 minutes.The gained mixed system is poured in the storage tank of fast film emulsifier unit (membrane aperture is 9 microns), crosses film 6 times under 120 kPas of nitrogen pressures.Gained W/O/W double emulsion at room temperature stirs 16 hours under the speed of 300 rev/mins.Centrifugal collection product.Products therefrom with water washing after, lyophilization.
The microcapsule that the acquisition mean diameter is 4 microns.Gained microcapsule lucifuge, drying, cryopreservation.
The PEG that is 150,000 by number-average molecular weight
200-b-PLA is dissolved in dichloromethane, is configured to the solution of 10mg/mL.After this solution mixes according to the volume ratio of 15:1 with water, under 300 watts of power ultrasonic 20 seconds, make water in oil elementary emulsion.Under the mixing speed of 400 rev/mins, elementary emulsion is splashed into to the polyvinyl alcohol water solution (3.5%, mass concentration) of 20 times of volumes of elementary emulsion, stir 5 minutes.The gained mixed system is poured in the storage tank of fast film emulsifier unit (membrane aperture is 9 microns), crosses film 4 times under 110 kPas of nitrogen pressures.Gained W/O/W double emulsion at room temperature stirs 14 hours under the speed of 100 rev/mins.Centrifugal collection product.Products therefrom with water washing after, lyophilization.
The microcapsule that the acquisition mean diameter is 4.5 microns.Gained microcapsule lucifuge, drying, cryopreservation.
Embodiment 7, prepare microcapsule formula acoustic contrast agent
The PEG that is 20,000 by number-average molecular weight
2000-b-PGA is dissolved in dichloromethane, is configured to the solution of 20mg/mL.After this solution mixes according to the volume ratio of 10:1 with water, under 200 watts of power ultrasonic 80 seconds, make water in oil elementary emulsion.Under the mixing speed of 600 rev/mins, elementary emulsion is splashed into to the polyvinyl alcohol water solution (3%, mass concentration) of 15 times of volumes of elementary emulsion, stir 6 minutes.The gained mixed system is poured in the storage tank of fast film emulsifier unit (membrane aperture is 9 microns), crosses film 5 times under 100 kPas of nitrogen pressures.Gained W/O/W double emulsion at room temperature stirs 12 hours under the speed of 1000 rev/mins.Centrifugal collection product.Products therefrom with water washing after, lyophilization.The microcapsule that the acquisition mean diameter is 5 microns.Gained microcapsule lucifuge, drying, cryopreservation.
Embodiment 8, prepare microcapsule formula acoustic contrast agent
The PEG that is 200,000 by number-average molecular weight
4000-b-PCL is dissolved in dichloromethane, is configured to the solution of 50mg/mL.After this solution mixes according to the volume ratio of 7:1 with water, under 150 watts of power ultrasonic 150 seconds, make water in oil elementary emulsion.Under the mixing speed of 300 rev/mins, elementary emulsion is splashed in the polyvinyl alcohol water solution (2.5%, mass concentration) of 12 times of volumes of elementary emulsion, stir 10 minutes.The gained mixed system is poured in the storage tank of fast film emulsifier unit (membrane aperture is 9 microns), crosses film 5 times under 90 kPas of nitrogen pressures.Gained W/O/W double emulsion at room temperature stirs 12 hours under the speed of 150 rev/mins.Centrifugal collection product.Products therefrom with water washing after, lyophilization.
The microcapsule that the acquisition mean diameter is 5 microns.Gained microcapsule lucifuge, drying, cryopreservation.
The stereoscan photograph of microcapsule prepared by the present embodiment is as shown in Fig. 1 (C).
Embodiment 9, prepare microcapsule formula acoustic contrast agent
The PEG that is 50,000 by number-average molecular weight
6000-b-PLGA is dissolved in dichloromethane, is configured to the solution of 100mg/mL.After this solution mixes according to the volume ratio of 5:1 with water, under 300 watts of power ultrasonic 30 seconds, make water in oil elementary emulsion.Under the mixing speed of 700 rev/mins, elementary emulsion is splashed into to the polyvinyl alcohol water solution (2.0%, mass concentration) of 10 times of volumes of elementary emulsion, stir 15 minutes.The gained mixed system is poured in the storage tank of fast film emulsifier unit (membrane aperture is 9 microns), crosses film 5 times under 80 kPas of nitrogen pressures.Gained W/O/W double emulsion at room temperature stirs 8 hours under the speed of 1800 rev/mins.Centrifugal collection product.Products therefrom with water washing after, lyophilization.
The microcapsule that the acquisition mean diameter is 6 microns.Gained microcapsule lucifuge, drying, cryopreservation.
The PEG that is 90,000 by number-average molecular weight
20000-b-PGC is dissolved in dichloromethane, is configured to the solution of 120mg/mL.After this solution mixes according to the volume ratio of 4:1 with water, under 250 watts of power ultrasonic 50 seconds, make water in oil elementary emulsion.Under the mixing speed of 500 rev/mins, elementary emulsion is splashed into to the polyvinyl alcohol water solution (1.5%, mass concentration) of 8 times of volumes of elementary emulsion, stir 8 minutes.The gained mixed system is poured in the storage tank of fast film emulsifier unit (membrane aperture is 9 microns), crosses film 4 times under 60 kPas of nitrogen pressures.Gained W/O/W double emulsion at room temperature stirs 8 hours under the speed of 250 rev/mins.Centrifugal collection product.Products therefrom with water washing after, lyophilization.
The microcapsule that the acquisition mean diameter is 6.5 microns.Gained microcapsule lucifuge, drying, cryopreservation.
Embodiment 11, prepare microcapsule formula acoustic contrast agent
The PEG that is 500,000 by number-average molecular weight
8000-b-PCL is dissolved in dichloromethane, is configured to the solution of 200mg/mL.After this solution mixes according to 2/1 volume ratio with water, under 200 watts of power ultrasonic 40 seconds, make water in oil elementary emulsion.Under the mixing speed of 1000 rev/mins, elementary emulsion is splashed into to the polyvinyl alcohol water solution (0.5%, mass concentration) of 5 times of volumes of elementary emulsion, stir 4 minutes.The gained mixed system is poured in the storage tank of fast film emulsifier unit (membrane aperture is 9 microns), crosses film 3 times under 20 kPas of nitrogen pressures.Gained W/O/W double emulsion at room temperature stirs 2 hours under the speed of 200 rev/mins.Centrifugal collection product.Products therefrom with water washing after, lyophilization.
The microcapsule that the acquisition mean diameter is 8 microns.Gained microcapsule lucifuge, drying, cryopreservation.
Embodiment 12, prepare microcapsule formula acoustic contrast agent
The acetate starch that is 10,000 by number-average molecular weight is dissolved in dichloromethane, is configured to the solution of 150mg/mL.After this solution mixes according to the volume ratio of 3:1 with water, under 400 watts of power ultrasonic 200 seconds, make water in oil elementary emulsion.Under the mixing speed of 900 rev/mins, elementary emulsion is splashed into to the polyvinyl alcohol water solution (1%, mass ratio) of 7 times of volumes of elementary emulsion, stir 20 minutes.The gained mixed system is poured in the storage tank of fast film emulsifier unit (membrane aperture is 9 microns), crosses film 3 times under 50 kPas of nitrogen pressures.Gained W/O/W double emulsion at room temperature stirs 4 hours under the speed of 1500 rev/mins.Centrifugal collection product.Products therefrom with water washing after, lyophilization.
The microcapsule that the acquisition mean diameter is 7 microns.Gained microcapsule lucifuge, drying, cryopreservation.
Fig. 1 shows the stereoscan photograph of the microcapsule that mean diameter is 2 microns, 3 microns and 5 microns, can learn, concentration by figure by oil phase polymer in the change preparation method, cross pressure and the number of times of film, outer water, oil phase, interior water amount ratio, the microcapsule of different-grain diameter size can be obtained, therefore the size of microcapsule can be freely adjusted as required.And from each picture, can find out, the size ratio of microcapsule prepared by the present invention is than homogeneous.
Fig. 2 shows SEM picture and the particle size distribution figure of the microcapsule formula contrast agent of embodiment 1 preparation, can be learnt the microcapsule diameter distribution homogeneous that the present invention makes by Fig. 2.
Fig. 4 shows the ultrasonic development design sketch of the microcapsule formula contrast agent of embodiment 6 preparations, wherein Fig. 4 (A) is the development effect figure while not adding contrast agent of the present invention, Fig. 4 (B) is the development effect figure while adding contrast agent of the present invention, and Fig. 4 (C) is for adding contrast agent (manufacturing enterprise: the development effect figure in the time of Bracco Suisse SA) of commercially available sound Novi (Sonovue).By this, can be found out, under test condition, contrast agent prepared by the present invention can reach the ultrasonic development effect that even is better than commercially available Sonovue contrast agent in vitro.
Fig. 5 shows the ultrasonic development design sketch of the prepared microcapsule formula contrast agent of embodiment 1,3,5 and 8, and as seen from the figure, microcapsule formula contrast agent prepared by the present invention has all reached ultrasonic development effect preferably.
Above result shows that the present invention can prepare the microcapsule of different Biodegradable materials, by regulating film emulsion process parameter, can control the size of microcapsule diameter and optimize its particle size distribution.The microcapsule of hollow structure can ultrasonic development under ultrasonication, and the hydrophilic and oleophilic of size, material category, material affects the development effect of microcapsule.Therefore, can, by changing preparation condition and material type, meet the demand of different ultrasonic contrast situations.
Claims (10)
1. the preparation method of a microcapsule formula acoustic contrast agent, comprise the steps:
(1) biodegradable macromolecular material is dissolved in organic solvent, obtains solution, be designated as solution A;
Stabilizing agent is dissolved in the water, obtains solution, be designated as solution B;
(2) described solution A is mixed with water, and obtained water in oil elementary emulsion through ultrasonic;
(3) described elementary emulsion is joined in described solution B, obtain the pre-double emulsion of W/O/W through stirring;
(4) described pre-double emulsion is added in the film emulsifier unit and carried out film, obtain the double emulsion of W/O/W;
(5) double emulsion of described W/O/W stirs and is centrifugal, and collecting precipitation obtains described microcapsule formula acoustic contrast agent.
2. preparation method according to claim 1, it is characterized in that: described biodegradable macromolecular material is the biodegradable macromolecular material of modified natural and synthesizing biological degradable macromolecular material;
The biodegradable macromolecular material of described modified natural is oil-soluble material modified, described oil-soluble material modified be acetate starch, succinic acid esterification starch, acylation chitosan, Nmaleoyl chitosan, Glycerolchitosan, alkylated chitosan, butyryl chitosan or hydroxypropyl chitosan;
Described synthesizing biological degradable macromolecular material is the aliphatic poly lactone.
3. preparation method according to claim 2, it is characterized in that: described aliphatic poly lactone is polylactic acid, poly-(lactide-Acetic acid, hydroxy-, bimol. cyclic ester) copolymer, poly-(lactide-caprolactone) copolymer, poly-(Acetic acid, hydroxy-, bimol. cyclic ester-caprolactone) copolymer, poly-(Acetic acid, hydroxy-, bimol. cyclic ester-lactide-caprolactone) copolymer, the PLE copolymer, poly-(Acetic acid, hydroxy-, bimol. cyclic ester-polyglycol ether) copolymer, poly-(own lactide-polyglycol ether) copolymer, poly-(lactide-Acetic acid, hydroxy-, bimol. cyclic ester-polyglycol ether) copolymer, gather at least one in (lactide-caprolactone-polyglycol ether) copolymer and poly-(Acetic acid, hydroxy-, bimol. cyclic ester-caprolactone-polyglycol ether) copolymer,
The number-average molecular weight of described aliphatic poly lactone is 5,000～500,000,
The number-average molecular weight of the polyglycol ether section in described PLE copolymer, described poly-(Acetic acid, hydroxy-, bimol. cyclic ester-polyglycol ether) copolymer, described poly-(own lactide-polyglycol ether) copolymer, described poly-(lactide-Acetic acid, hydroxy-, bimol. cyclic ester-polyglycol ether) copolymer, described poly-(lactide-caprolactone-polyglycol ether) copolymer and described poly-(Acetic acid, hydroxy-, bimol. cyclic ester-caprolactone-polyglycol ether) copolymer is 200～20,000.
4. according to the described preparation method of any one in claim 1-3, it is characterized in that: in step (1), described organic solvent is dichloromethane, chloroform or dichloroethanes;
The mass body volume concentrations of described solution A is 1～200mg/mL;
Described stabilizing agent is polyvinyl alcohol, gelatin or polyvinylpyrrolidone;
The quality percentage composition of described solution B is 0.5～5.0%.
5. according to the described preparation method of any one in claim 1-4, it is characterized in that: in step (2), the volume ratio of described solution A and described water is 2～40:1;
Described ultrasonic power is 50～500W, and the time is 5～240 seconds.
6. according to the described preparation method of any one in claim 1-5, it is characterized in that: in step (3), the volume ratio of described elementary emulsion and described solution B is 1:5～50;
The speed of described stirring is 60～2000 rev/mins, and the time is 0.5～30.0 minute.
7. according to the described preparation method of any one in claim 1-6, it is characterized in that: in step (4), the described film of crossing carries out 1～15 time under 20～200 kPas of nitrogen pressures.
8. according to the described preparation method of any one in claim 1-7, it is characterized in that: in step (5), the time of described stirring is 1～24 hour, and the speed of described stirring is 60～2000 rev/mins.
9. according to the described preparation method of any one in claim 1-8, it is characterized in that: in step (5), described method also comprises the step that water is washed described precipitation;
Step (1), step (2), step (3), step (4) and step (5) are all carried out under the condition of 0～50 ℃.
10. the microcapsule formula acoustic contrast agent that in claim 1-9 prepared by the described method of any one.
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