CN1814305A - Gene-ordrug-carrying-carrying ultrasonic microvesicle contrast-media and preparing method thereof - Google Patents
Gene-ordrug-carrying-carrying ultrasonic microvesicle contrast-media and preparing method thereof Download PDFInfo
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Abstract
This invention discloses an ultrasonic microbubble contrast agent of carrier gene, it includes microbubble, fluorocarbon gas, gene or medicine. Microbubble wall is made up by lipid bielement, fluorocarbon gas is encapsulated in it, gene or medicine entrapped in microbubble envelope. This invention also discloses its preparation method, two stearyl phosphatidylcholine, two palmitoyl phosphatidyl ethanolamine, sapn-60, glycerol, phosphate buffer solution and gene or medicine is mixed as proper proportion for mechanical oscillations. The property of carry gene or medicine microbubble is stable, particle diameter disperses equally, local tissue medicine concentration can be obviously advanced though effect of transonic breaking microbubble, and curative effect is advanced and side effect can be reduced. Flushing and dilution of blood to gene and medicine can be prevented when used in body, and also prevent gene or medicine from degrading by nuclease in cycle to ensure that lot of gene and medicine can be transported to target tissue. And it is linear increase follow gene or medicine input amount in certain range.
Description
Technical field
The present invention relates to a kind ofly can efficiently carry ultrasound microbubble contrast agent of gene or medicine and preparation method thereof, for the treatment of gene or drug targeting provides a kind of new and effective transport vehicle that can inject by vein, not only be suitable for the disease of ultrasonic ruptured microbubbles targeting transmission gene and Drug therapy cardiovascular system, the targeted therapy that also can be the substantial viscera disease provides an effective vehicle.
Background technology
1, acoustic contrast agent development present situation
Acoustic contrast is the gassiness microvesicle by similar erythrocyte size, can arrive the position that erythrocyte can arrive after injecting vein, under action of ultrasonic waves, have linear scattering and nonlinear scattering characteristic, thereby can make the abundant organa parenchymatosum of blood flow when the two-dimensional ultrasound instrument detects, produce strong contrast development as cardiac muscle, liver, kidney etc.With other shadowgraph technique such as X line relatively, have noinvasive, in good time, bed is other and advantage such as inexpensive.Acoustic contrast agent is succeeded in developing, and has broad application prospects for the non-invasive diagnosis of cardiovascular disease and tumor.
The acoustic contrast agent of the clinical practice of drugs approved by FDA all is the sound albumin that shakes as Albunex and Optison.At the Levovist of Europe approval clinical practice, belong to the 1st and the 2nd generation product.In recent years developing lipid, macromolecule polymer filmogen and flexible shell the 3rd generation acoustic contrast agent, it is good that cardiac muscle and liver are developed, and with the research of clinical phase, demonstrated good prospects for application by experiment.And the Sonovue of domestic listing is unique by the acoustic contrast agent of China's Ministry of Public Health approval clinical practice, but costs an arm and a leg because of it, causes it to be difficult for promoting the use of.The Hospital of Southern Medical University Ultrasonography, the sound albumin fluorocarbon gas acoustic contrast agent of developing that shakes, belong to the 2nd generation product, use does not at present yet put goods on the market.So far, still there is not the report of homemade acoustic contrast agent clinical practice.Therefore, developing a kind of stable, reliable, inexpensive homemade acoustic contrast agent, is the urgent task that China's medical science and correlative technology field face.
2, the acoustic contrast agent development present situation of double gene or drug targeting carrier
(1) genophore development present situation
The continuous discovery with Disease-causing gene finished along with the Human Genome Project, fact proved that human a lot of diseases are all relevant with gene, relevant gene therapy research also obtained significant progress, at present, cardiovascular and cerebrovascular disease and genetic treatment of tumor have become one of most active fields in the gene therapy research.
Vectors in Gene Therapy mainly comprises non-virus carrier and viral vector.In non-virus carrier, the application of uncorrected gene expression occupies an important position, but that its shortcoming is a transfection efficiency is lower, lack targeting, can only local action, may also need carry out surgical operation sometimes to expose target organ, and cause its clinical practice to be subjected to certain limitation.In the status of affirming gymnoplasm grain carrier, to recognize that also viral vector still takes advantage in gene therapy.The transfection efficiency height is the distinguishing feature of viral vector, but owing to after viral gene is integrated into host cell, the danger that causes the slot gene mutation is arranged, also may activated oncogene, so its safety is an an open question still.Liposome also is a kind of gene therapy carrier commonly used, though behind its parcel DNA, direct injection safety, non-immunogenicity, liposome exist shortcomings such as no targeting and specificity, vivo gene transfer efficient are low.Though many new targeting specific gene therapies are arranged, and successful gene therapy is still challenging, wherein topmost obstacle be lack noinvasive, effectively, have a carrier delivery system of targeting.
(2) the development present situation of pharmaceutical carrier
In tumor pharmacother, still there is not pharmaceutical carrier delivery system specific, targeting at present.Anticarcinogen is kill cancer cell not only, and destroys normal histiocyte.To the research of immunoliposome, treatment brings new hope to tumor-targeting drug, but antibody is used for the breakthrough that the targeted therapy of tumor has benefited from two guardian techniques, and 1. the cell of solid tumor is by the substrate of densification parcel, and antibody is difficult to penetrate this barrier; 2. all there is the lymphatic return obstacle in most of solid tumors, cause a matter internal pressure to raise, hinder antibody and entered tumor epithelial cell, and fraction enters the antibody of solid tumor inside, what at first run into is circumvascular tumor cell and combined, makes antibody can't arrive the tumor cell far away apart from blood vessel.Therefore, it is still undesirable to use the effect of antibody drug treatment large volume solid tumor at present.In recent years, along with the research of nanotechnology and medical macromolecular materials, magnetic nano-particle has become the popular research direction in this field, but its poor stability, complicated operation is still a still unsolved difficult problem.
Discover that the ultrasound wave of certain energy can make the microbubble destruction of carrying gene or medicine, makes entrained gene or medicine be released in the particular organization position.But the present year gene microvesicle that does not still have both at home and abroad as specialization transmission gene or medicine.For carrying the gene microvesicle, present stage is utilized albuminous adhesiveness or lipid microbubble surface charge characteristic more, simply gene is adhered to microvesicle surface and the preparation gene sticks microvesicle, not only it carries the limited amount of gene, and variability is big, repeatability and poor stability have a strong impact on gene transfection efficient.What is more important because gene exposes on the microvesicle surface, when gene is used in vivo, can not prevent the degraded of blood amplifying nucleic acid enzyme to gene, so microvesicle can't guarantee a large amount of genes is delivered to target tissue.Thereby how under the stable impregnable prerequisite of performance that guarantees gene and microvesicle, by simple and effective technology, gene or medicine be integrated on the microvesicle wall and increase the amount of carrying of gene, and will carry gene or medicine microvesicle and be one as new transport vehicle key issue to be solved is arranged.
Therefore, study a kind of novel acoustic contrast agent, and can be used as the carrier that carries several genes or medicine, realize gene transfection or drug release at the particular organization position, fill up the blank of gene importing and drug targeting release by ultrasonic mediation.It will have broad application prospects and huge economic benefit undoubtedly.
Summary of the invention
At defective or the deficiency that prior art exists, the purpose of this invention is to provide a kind of stable in properties and can be with a large amount of genes or drug conveying the ultrasound microbubble contrast agent that carries gene or medicine to target tissue.
Another object of the present invention provides the method that the ultrasound microbubble contrast agent of gene or medicine is carried in a kind of preparation.
For achieving the above object, the ultrasound microbubble contrast agent of a kind of year gene of the present invention or medicine comprises microvesicle, fluorocarbon gas, gene or medicine; The microvesicle wall of this microvesicle is made of lipid bilayer, parcel fluorocarbon gas in the microvesicle, and gene or pharmaceutical pack are embedded in the microvesicle peplos.
Further, described fluorocarbon gas is a perfluoropropane gas;
Further, described fluorocarbon gas is a HFC-236fa gas;
For achieving the above object, the method for the ultrasound microbubble contrast agent of a kind of year gene of the present invention or medicine may further comprise the steps:
1, take by weighing following raw materials according by weight percentage:
Distearoyl phosphatidylcholine DSPC 0.8-1.2%
Two palmityl PHOSPHATIDYL ETHANOLAMINE DPPE 0.2-0.5%
Arlacel-60 span60 0.18-0.24%
Glycerol 8-15%
Gene or medicine 0.2-0.5%
Phosphate buffer PBS 82.56-90.62%;
2, adopt conventional method to mix above-mentioned raw materials after, place to include in the container that purity is not less than 99% fluorocarbon gas, the ratio between the volume of this container and the raw material volume sum is 1: 3;
3, container is placed 37-40 ℃ water water-bath 20-30 minute;
4, container is fixed on vibration 30-35 second in the argental mercury dispenser, the operating frequency of argental mercury dispenser is 4500 times/minute;
5, leave standstill 3-5 minute, add the phosphate buffer PBS dilution with the same volume of raw material again, can obtain the ultrasound microbubble contrast agent of described year gene or medicine.
Wherein, in the above-mentioned preferred version, the described container in the step 2 is a phial, and this container places 40 ℃ water water-bath 30 minutes in the described step 3; Argental mercury dispenser in the step 4 is the dentistry shaker, and duration of oscillation is 35 seconds; The time of leaving standstill in the step 5 is 5 minutes.
Further, the preferred percentage by weight of raw material is in the above-mentioned steps 1:
Two palmityl PHOSPHATIDYL ETHANOLAMINE DPPE 0.4%
Arlacel-60 0.2%
Glycerol 10%
Gene 0.2%
Phosphate buffer PBS 88.2%.
Further, above-mentioned described gene is VEGF VEGF or basic fibroblast
Cell growth factor bFGF or hepatocyte growth factor HGF or P53 wherein any one.
Wherein, the preferred percentage by weight of above-mentioned raw materials is:
Two palmityl PHOSPHATIDYL ETHANOLAMINE DPPE 0.4%
Arlacel-60 0.2%
Glycerol 10%
Medicine 0.4%
Phosphate buffer PBS 88%.
Further, described medicine is a fat-soluble medicine.
Compared with prior art, the ultrasound microbubble contrast agent of of the present invention year gene or medicine, on the microvesicle wall, this novel year gene microvesicle be stable in properties not only with gene or medicine stable integration, and in the time of improving gene transfection, the concertedness of gene---microvesicle on the time and space; And because gene or pharmaceutical pack are embedded in the microvesicle peplos, can prevent blood when using in vivo to the washing away and dilute of gene or medicine, can also protecting group because of or medicine avoid the circulating degraded of amplifying nucleic acid enzyme, assurance with a large amount of genes or drug conveying to target tissue; This novel year simultaneously gene or medicine microvesicle carry the mode that gene dosage or medication amount obviously are better than adhering to microvesicle, and are linear with the increase of gene or medicine input amount within the specific limits and increase.The character of this novel year gene or medicine microvesicle is also very stable, and particle size distribution is even, can obviously improve the drug level of local organization by the effect of ultrasound destruction microvesicle, increases curative effect, reduces side effect.
Description of drawings
Fig. 1 is the particle size distribution figure of ultrasound microbubble contrast agent under the light microscopic;
Fig. 2 carries the particle size distribution figure of the ultrasound microbubble contrast agent of gene for the present invention under the light microscopic;
Fig. 3 is the microbubble fine structure sketch map of the ultrasound microbubble contrast agent of medicine carrying thing;
Fig. 4 is incorporated into the dose-effect relationship figure of microvesicle for plasmid;
Fig. 5 shows the echo sketch map of the preceding mouse liver of radiography;
Fig. 6 shows the echo sketch map of mouse liver behind the radiography.
The specific embodiment
The present invention be adopt the mechanical oscillation method but not method that sound shakes prepare can intravenous ultrasound microbubble contrast agent.The bubble wall of this microbubble is made up of lipid, and microbubble hollow includes fluorocarbon gas, in the presence of surfactant, regulates the surface tension of gas phase and liquid phase, by adopting the method for machinery concussion, gas phase is entered in the microvesicle.The ultrasound microbubble contrast agent that adopts this method to prepare, its concentration diameter Distribution scope is 0.8～6 μ m, and the microvesicle diameter more than 90% is 2～6 μ m, and the half-life of microvesicle is 32.1～33.2min.As shown in Figure 1, visible ultrasound microbubble contrast agent particle size distribution is even under light microscopic, and good dispersion degree meets the characteristic of desirable contrast agent:
1, its concentration, diameter Distribution scope and stability reach the hemorheology and the acoustic characteristic requirement of using in the body fully;
2, its synthesis material is pharmacology's acceptable material, safety non-toxic;
3, it can be used for heart and myocardium ultrasonic contrast and organa parenchymatosum's development:
1) can be used for the early diagnosis of acute myocardial infarction, infarction size and pour into the curative effect damage again and estimate; And the diagnosis of chronic ischemia cardiac muscle;
2) be used for the reperfusion injury of cardiac muscle scoring;
3) strengthen the definition at endocardium interface, improved the accuracy that ultrasoundcardiogram is estimated cardiac function;
Particularly thrombosis diagnosis in the transthoracic echocardiography auricle that is difficult for detecting in trunk intracavity that 4) can detect such as neck and burst arteriovenous etc., the chambers of the heart;
5) can be used for the development of parenchymal visceras such as liver, kidney, spleen, prostate, mammary gland and tumor.
If ultrasound microbubble contrast agent used film forming material is mixed stirring by a certain percentage with fat-soluble medicine, just can be prepared into the acoustic contrast agent of the gassiness microvesicle that carries curative drug, shown in Fig. 3 medicine carrying lipid microbubble simulation drawing, the outer wall of microbubble is a lipid bilayer 1, inside is perfluoropropane gas 3, and paclitaxel 2 is dissolved in the lipid layer; If ultrasound microbubble contrast agent used film forming material is mixed stirring with plasmids such as VEGF (VEGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF) or P53 with certain proportion, just can be prepared into the acoustic contrast agent of the gassiness microvesicle that carries therapeutic gene.The hold concurrently acoustic contrast agent of medicine or gene targeting vector, this year the gene microvesicle on the basis of the general characteristic that does not change microvesicle, its envelop rate can reach 15%, carries a fat-soluble entrapment efficiency and can reach 99.8%.Be the carrier of ideal medicine and gene:
1) this acoustic contrast agent can be efficiently in conjunction with gene;
2), can realize the organa parenchymatosum and directed transfer of gene in conjunction with ultrasound destruction microvesicle location release tech;
3) carry specific antibody or antitumor drug, under ultrasonication, in organa parenchymatosums' such as liver, kidney tumor locus release;
4) carry polygenic acoustic contrast agents such as VEGF, bFGF, HGF, under ultransonic effect, in myocardial ischemia slough position microbubbles rupture, polygenes such as VEGF are released in the ischemic myocardium part by targeting, transfection cardiac muscle endotheliocyte, promote myocardium blood capillary and collateral blood vessels logical again, promptly the noinvasive therapeutic transgene is realized blood capillary " molecule bridging " in the non-invasive cardiac muscle;
5) carry specific antibody or gene or specificity antineoplastic medicine, targeting organa parenchymatosum's tumor, organa parenchymatosum's tumors such as specific treatment liver, kidney, spleen, prostate, mammary gland are avoided the toxic and side effects of systemic administration;
6) introducing of synthesizing formula that microvesicle is new and surfactant, avoid ultrasonic sound to shake to the destruction of gene, strengthened simultaneously and carried gene or the stability of medicine microvesicle and the uniformity of particle size distribution, made it be more suitable for application in ultrasonic ruptured microbubbles targeting transmission gene or drug system.
The present invention is described in further detail below in conjunction with specific embodiment, experiment and accompanying drawing:
[embodiment 1] preparation ultrasound microbubble contrast agent
1, take by weighing raw material:
Distearoyl phosphatidylcholine DSPC 5mg
Two palmityl PHOSPHATIDYL ETHANOLAMINE DPPE 2mg
span60 1mg
2, above-mentioned raw materials is mixed after, place the including in the phial that purity is 100% perfluoropropane of 1.5ml;
3, phial is placed 40 ℃ of water water-baths 30 minutes;
4, phial was vibrated 35 seconds with the vibration of dentistry shaker;
5, leave standstill 5 minutes, add the PBS dilution of 0.5ml, can obtain ultrasound microbubble contrast agent.
The ultrasound microbubble contrast agent of VEGF VEGF is carried in [embodiment 2] preparation
1, take by weighing raw material:
Distearoyl phosphatidylcholine DSPC 5mg
Two palmityl PHOSPHATIDYL ETHANOLAMINE DPPE 2mg
span60 1mg
PBS 450μl
VEGF VEGF 1mg;
2, adopt oscillation method to mix above-mentioned raw materials after, place a 1.5ml to include in the phial that purity is 99.99% perfluoropropane;
3, phial is placed 40 ℃ of water water-baths 30 minutes;
4, phial was vibrated 35 seconds with the vibration of dentistry shaker;
5, leave standstill 5 minutes, add 0.5ml PBS dilution, can obtain carrying the ultrasound microbubble contrast agent of gene.
The ultrasound microbubble contrast agent of basic fibroblast growth factor bFGF is carried in [embodiment 3] preparation
1, take by weighing raw material by weight percentage:
Distearoyl phosphatidylcholine DSPC 4mg
Two palmityl PHOSPHATIDYL ETHANOLAMINE DPPE 2.5mg
span60 0.9mg
Glycerol 40 μ l
PBS 460μl
Basic fibroblast growth factor bFGF 2.5mg;
2, above-mentioned raw materials is adopted the method for vibration mix after, place the including in the phial that purity is 99% perfluoropropane of 1.5ml;
3, phial is placed 40 ℃ of water water-baths 30 minutes;
4, phial was vibrated 35 seconds with the vibration of dentistry shaker;
5, leave standstill 5 minutes, add the PBS dilution of 0.5ml, can obtain carrying the ultrasound microbubble contrast agent of gene.
The ultrasound microbubble contrast agent of hepatocyte growth factor HGF is carried in [embodiment 4] preparation
1, take by weighing raw material by weight percentage:
Distearoyl phosphatidylcholine DSPC 6mg
Two palmityl PHOSPHATIDYL ETHANOLAMINE DPPE 1mg
span60 1.2mg
Glycerol 75 μ l
PBS 425μl
Hepatocyte growth factor HGF 1mg;
2, above-mentioned raw materials is adopted the method for vibration mix after, place the including in the phial that purity is 99.9% perfluoropropane of 1.5ml;
3, phial is placed 40 ℃ of water water-baths 30 minutes;
4, phial was vibrated 35 seconds with shaker;
5, leave standstill 5 minutes, add the PBS dilution of 0.5ml, can obtain carrying the ultrasound microbubble contrast agent of gene.
The ultrasound microbubble contrast agent of paclitaxel is carried in [embodiment 5] preparation
1, take by weighing raw material by weight percentage:
Distearoyl phosphatidylcholine DSPC 5mg
Two palmityl PHOSPHATIDYL ETHANOLAMINE DPPE 2mg
span60 1mg
PBS 450μl
Paclitaxel 2mg;
2, adopt oscillation method to mix above-mentioned raw materials after, place the including in the phial that purity is 99.99% perfluoropropane of 1.5ml;
3, phial is placed 40 ℃ of water water-baths 30 minutes;
4, phial was vibrated 35 seconds with the vibration of dentistry shaker;
5, leave standstill 5 minutes, add the PBS dilution of 0.5ml, can obtain the ultrasound microbubble contrast agent of medicine carrying thing.
The ultrasound microbubble contrast agent of amycin is carried in [embodiment 6] preparation
1, take by weighing raw material by weight percentage:
Distearoyl phosphatidylcholine DSPC 6mg
Two palmityl PHOSPHATIDYL ETHANOLAMINE DPPE 1mg
span60 1.2mg
Glycerol 40 μ l
PBS 460μl
Amycin 1mg;
2, above-mentioned raw materials is adopted the method for vibration mix after, place the including in the phial that purity is 99% perfluoropropane of 1.5ml;
3, phial is placed 40 ℃ of water water-baths 30 minutes;
4, phial was vibrated 35 seconds with the vibration of dentistry shaker;
5, leave standstill 5 minutes, add the PBS dilution of 0.5ml, can obtain the ultrasound microbubble contrast agent of medicine carrying thing.
The ultrasound microbubble contrast agent of fluorouracil is carried in [embodiment 7] preparation
1, take by weighing raw material by weight percentage:
Distearoyl phosphatidylcholine DSPC 4mg
Two palmityl PHOSPHATIDYL ETHANOLAMINE DPPE 2.5mg
span60 0.9mg
Glycerol 75 μ l
PBS 425μl
Fluorouracil 2.5mg;
2, adopt oscillation method to mix above-mentioned raw materials after, place the including in the phial that purity is 99.99% perfluoropropane of 1.5ml;
3, phial is placed 40 ℃ of water water-baths 30 minutes;
4, phial was vibrated 35 seconds with shaker;
5, leave standstill 5 minutes, add the PBS dilution of 0.5ml, can obtain the ultrasound microbubble contrast agent of medicine carrying thing.
The gene mapping detection of the ultrasound microbubble contrast agent of [experiment 1] year gene and the optimization of dose-effect relationship
In embodiment 3, the pVEGF plasmid DNA that in step 1, can also add different components respectively, for example the component of pVEGF plasmid DNA is respectively: 0.125,0.25,0.5,1,2,4,6 and 8mg, all the other raw materials are joined according to attach ratios and are got, according to the same method among the embodiment 3, make eight kinds of ultrasound microbubble contrast agents that carry gene respectively then.With eight kinds of ultrasound microbubble contrast agents that carry gene, with PBS washing 3 times, removing unconjugated DNA respectively then, is the ultraviolet spectrophotometer of 260nm with wavelength, detects the content of DNA in the microvesicle solution.As shown in Figure 4, within the specific limits, the lipid soln dna content becomes positive correlation with dna content on the microvesicle film, and when DNA concentration was 4mg/ml, the dna content in the microvesicle reached capacity, and DNA embeds that amount can reach 0.33pg/microbubble on the microvesicle film.Draw the following dyeing of propidium iodide (PI) the room temperature 30min that 100 μ l microvesicles add 100 μ l, 50 μ g/ml, under fluorescence microscope, detect, as shown in Figure 2, fluorescence microscope shows that down the pVEGF plasmid DNA is embedded on the wall of microvesicle, and the particle diameter and the dispersion that send green fluorescence and take the gene microvesicle after propidium iodide (PI) dyeing there is no obvious change.Fig. 4 is incorporated into the dose-effect relationship figure of microvesicle for plasmid, as shown in the figure, within the specific limits, the lipid soln dna content becomes positive correlation with dna content on the microvesicle film, nearly 15% plasmid DNA is incorporated on the microvesicle film, when the lipid soln dna content was 4000 μ g/ml, the dna content on the microvesicle film can reach capacity.The result shows that the microvesicle wall can send red fluorescence, proves that plasmid DNA is embedded in the wall of microvesicle.
Video picture is observed in [experiment 2] ultrasound microbubble contrast agent Mouse Liver
Use GE Vivid7 colorful ultrasonic diagnostic apparatus, the 12L linear array probe, second harmonic tranmitting frequency 5.2MHz, receive frequency 10.5MHz, the instrument all conditions is set to same standard, and mechanical index during second harmonic (MI) is set at 0.4, gains to be-14dB, the video picture degree of depth is fixed as 4cm, and parameters such as TGC, focusing range all transfer to optimum state.With Ultrasound Instrument internal work station storage sound attitude radiography data.Behind the white mice of 50mg/kg pentobarbital sodium intraperitoneal injection of anesthesia Kunming, dorsal position is fixed, and adopts self cross-reference method, and conventional first-harmonic ultrasonic scan obtains Mouse Liver tangent plane ultrasonogram before the radiography, and mouse liver is low echo as shown in Figure 5.Change imaging modality into second harmonic, with 0.3ml normal saline dilution 0.05ml ultrasound microbubble contrast agent, inject in the mice body through tail vein group, visual observations under the harmonic wave state is behind the injection microbubble 3s, the contrast agent filling appears in the liver blood vessel, the appearance of liver parenchyma echo strengthens behind the 5s, and liver parenchyma echo obviously strengthens behind the 1min, peaks behind the 30min, as shown in Figure 6, still weaken gradually behind the 60min.
Claims (9)
1, the ultrasound microbubble contrast agent of a kind of year gene or medicine is characterized in that, comprises microvesicle, fluorocarbon gas, gene or medicine; The microvesicle wall of this microvesicle is made of lipid bilayer, parcel fluorocarbon gas in the microvesicle, and gene or pharmaceutical pack are embedded in the microvesicle peplos.
2, according to the ultrasound microbubble contrast agent of described year gene of claim 1 or medicine, it is characterized in that described fluorocarbon gas is a perfluoropropane.
3, according to the ultrasound microbubble contrast agent of described year gene of claim 1 or medicine, it is characterized in that described fluorocarbon gas is a HFC-236fa.
4, a kind of method for preparing the ultrasound microbubble contrast agent of arbitrary described year gene of claim 1 to 3 or medicine may further comprise the steps:
(1) take by weighing following raw materials according by weight percentage:
Distearoyl phosphatidylcholine DSPC 0.8-1.2%
Two palmityl PHOSPHATIDYL ETHANOLAMINE DPPE 0.2-0.5%
Arlacel-60 span60 0.18-0.24%
Glycerol 8-15%
Gene or medicine 0.2-0.5%
Phosphate buffer PBS 82.56-90.62%;
(2) adopt conventional method to mix above-mentioned raw materials after, place to include in the container that purity is not less than 99% fluorocarbon gas, the ratio between the volume of this container and the raw material volume sum is 1: 3;
(3) container is placed 37-40 ℃ water water-bath 20-30 minute;
(4) container is fixed on vibration 30-35 second in the argental mercury dispenser, the operating frequency of argental mercury dispenser is 4500 times/minute;
(5) leave standstill 3-5 minute, add the phosphate buffer PBS dilution with the same volume of raw material again, can obtain the ultrasound microbubble contrast agent of described year gene or medicine.
According to the described method of claim 4, it is characterized in that 5, the container in the described step (2) is a phial, this phial places 40 ℃ water water-bath 30 minutes in the described step (3); Argental mercury dispenser in the described step (4) is the dentistry shaker, and duration of oscillation is 35 seconds; The time of leaving standstill in the described step (5) is 5 minutes.
6, method according to claim 5 is characterized in that, raw material is in the step (1):
Distearoyl phosphatidylcholine DSPC 1%
Two palmityl PHOSPHATIDYL ETHANOLAMINE DPPE 0.4%
Arlacel-60 0.2%
Glycerol 10%
Gene 0.2%
Phosphate buffer PBS 88.2%.
7, method according to claim 5 is characterized in that, raw material is in the step (1):
Distearoyl phosphatidylcholine DSPC 1%
Two palmityl PHOSPHATIDYL ETHANOLAMINE DPPE 0.4%
Arlacel-60 0.2%
Glycerol 10%
Medicine 0.4%
Phosphate buffer PBS 88%.
8, according to the described method of claim 6, it is characterized in that, described gene be VEGF VEGF or basic fibroblast growth factor bFGF or hepatocyte growth factor HGF or P53 wherein any one.
9, method according to claim 7 is characterized in that, described medicine be paclitaxel or amycin or fluorouracil wherein any one.
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| CN1943541B (en) * | 2006-10-18 | 2010-05-12 | 许川山 | Supersonic micro bubble skin permeate promotor |
| CN101120921B (en) * | 2007-07-10 | 2011-01-19 | 中国人民解放军第二军医大学 | Target preparation consisting of liposome and nucleic acid coating contrast agent |
| CN101711736B (en) * | 2009-12-17 | 2011-09-14 | 重庆医科大学 | Method for preparing medicine-carrying microvesicle |
| CN101528268B (en) * | 2006-09-05 | 2012-07-04 | 博莱科瑞士股份有限公司 | Gas-filled microvesicles with polymer-modified lipids |
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| CN105214221A (en) * | 2015-10-16 | 2016-01-06 | 中国人民解放军第三军医大学第三附属医院 | The apparatus for ultrasonic therapeutic treatment of liver trauma hemostasis |
| CN106267246A (en) * | 2016-08-10 | 2017-01-04 | 杭州师范大学附属医院 | A kind of ultrasonic in combination targeted microbubble contrast medium and preparation method thereof |
| CN111701035A (en) * | 2020-06-28 | 2020-09-25 | 南京超维景生物科技有限公司 | Ultrasonic contrast agent composition, ultrasonic contrast agent, preparation method of ultrasonic contrast agent and application of acoustic deformation material |
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2005
- 2005-12-09 CN CNA2005101279963A patent/CN1814305A/en active Pending
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