CN102772808A - Multi-modality imaging microbubble structure, preparation method and applications - Google Patents
Multi-modality imaging microbubble structure, preparation method and applications Download PDFInfo
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Abstract
The invention relates to the cross field of chemical engineering, materials and nanomedicines, and specifically discloses a multi-modality imaging probe structure and a preparation method thereof. A multi-modality imaging probe is a microbubble with a putamen structure, a shell membrane material is composed of a high polymer material and a phospholipid material which are biodegradable and good in biocompatibility, water-solubility quantum dot solutions with different fluorescent characteristics are embedded at the center, a perfluor carbon alkyl gas is fed, and the multi-modality probe which is controllable in grain diameter, good and stable in dispersion and easy to store are prepared by a double-emulsion-freeze-drying gas feeding method. According to the multi-modality imaging probe structure the preparation method thereof, the microbubble is a reticuloendothelial system specificity contrast agent, the fluorescent imaging is achieved, the enhancement for ultrasonoscopy and magnatic resonance imaging (MRI) display and the microbubble multi-mode imaging can be achieved, and the multi-modality imaging probe structure the preparation method thereof have a wide application prospect.
Description
Technical field
The present invention relates to the crossing domain of biomedicine, material and nanometer medicine, be specifically related to a kind of multi-modal targeted imaging microbubble structure, Preparation method and use.
Background technology
Along with the development of Medical Imaging, the application of all kinds of contrast agent in clinical also more and more widely.Contrast agent can increase contrast in tissue, improves the qualitative localized ability of image, improves accuracy of diagnosis.At present, various imaging techniques all have contrast agent separately, as are used for the microbubble contrast agent of ultra sonic imaging, are used for the diodone of CT imaging, are used for Gd-DTPA and the SPIO of nuclear magnetic resonance etc.Same patient is for clarifying a diagnosis; Usually need accept a large amount of, multiple contrast agent in a short time; Not only increase the weight of the risk that organism metabolism burden and adverse reactions to contrast medium take place, also increased medical expense, thereby; Press for a kind of contrast agent that can be used for multiple imaging technique simultaneously clinically, promptly multi-functional contrast agent.Chinese scholars has been carried out correlational study to multi-functional contrast agent; The contrast agent of ultra sonic imaging and nuclear magnetic resonance, radionuclide imaging, fluorescence imaging associating and fluorescence imaging and radionuclide imaging associating is in the news successively, but the contrast agent that can be used for ultrasonic, fluorescence and nuclear magnetic resonance does not simultaneously appear in the newspapers as yet.
Ultra sonic imaging is a kind of methods for clinical diagnosis commonly used, with its fast, safety, cheapness, characteristic such as portable, worldwide be widely used.But because the restriction of ultrasonic diagnosis method itself, the resolution of ultra sonic imaging and accuracy can not satisfy the requirement of advanced clinical diagnosis far away.The ultrasonic contrast medium is Enhancement of Medical ultrasonic diagnosis signal significantly, obtains, more accurately diagnostic message abundanter than common ultra sonic imaging, for the diagnoses and treatment of disease provides more foundations.Being applied to scientific research and maximum clinically acoustic contrast agents is microvesicle (MBs) contrast medium, and it is made up of the gas (air, nitrogen, perfluocarbon, sulfur hexafluoride etc.) of outer peplos shell (phospholipid, surfactant, macromolecule etc.) and internal package.
Gd-DTPA (Magnevist) is a kind of nuclear magnetic resonance agent of clinical use, good stability, and magnetic property is good.Yet discovered in recent years Gd-DTPA can cause the hepatocyte chronic fibrosis, and some country stops using.Can when keeping the Gd-DTPA premium properties can discharge smoothly in the body again be new strategy, as: the Gd-DTPA molecule is connected with albumen, saccharide and, obtains preferably and make progress with modifications such as suitable polymer microsphere parcels.
Quantum dot (quantumn dots QDs) is as a kind of semiconductor fluorescence nano-particle, because its good optical characteristics is a research focus of nano biological medical domain in recent years.Generally constitute by second family and the 6th family's element or three races and pentels.Quantum dot has very wide absworption peak scope, narrow and symmetric emission wavelength, and bigger Stokes shift, higher quantum yield and very strong anti-photobleaching property have the incomparable optical characteristics of traditional organic fluorescent substance.More international in recent years famous publications are constantly reported the applied research of quantum dot at biomedicine and nanometer field of medicaments; Quantum dot is used and has been penetrated into biomedical every field gradually; Comprise cytobiology; Molecular biology, protein science and nano-probe become a kind of ideal external and cells in vivo/histofluorescence image-forming diagnose reagent.How quick, obtain cheaply, high yield, the quantum dot microsphere that toxicity is little are biomedical applications field problems anxious to be solved.Different qualities, difference in functionality quantum dot microsphere and microbubble have high researching value and application prospect.
Summary of the invention
The present invention is directed to different imaging techniques and need use different contrast agent; Not only increase the organism metabolism burden, and the problem of adverse reactions to contrast medium, a kind of multi-modal ultrasonic imaging agent is provided; In the hope of realizing the purpose of " probe is multi-functional ", the present invention also provides the method for the said microbubble of preparation.
For achieving the above object, technical scheme provided by the present invention is such:
A kind of multi-modality imaging microbubble; Be core-shell structure, the shell membrane material of said core-shell structure is made up of high molecular polymerization nano material and phospholipid, and nuclear material is water-soluble quantum dot and noble gas; Said water-soluble quantum dot is the cadmium telluride that mercaptopropionic acid is modified, and the particle diameter radius is 3.2-9.0nm.
Prepare the method for said multi-modality imaging microbubble, may further comprise the steps:
Step 1, with phospholipid with contain hold carboxyl macromolecule polymeric material by 1: the mol ratio of 8.75-9.25 is dissolved in the dichloromethane solvent, adds water-soluble quantum dot then, and the 35-45s that shakes obtains colostric fluid; With said colostric fluid and concentration is that the poly-vinyl alcohol solution of 3%-5% mixes; And be homogeneous dispersion effect 5min in the homogenizer of 2000-10000r/min at rotating speed, obtain microsphere and redissolve suspension, add the aqueous isopropanol that concentration is 2%-4% again; At room temperature stir 2-5h; Through centrifugal rinsing, collect lower floor's microsphere, and the microsphere of collecting is charged into noble gas promptly obtain being lyophilized powder multi-modality imaging microbubble after lyophilization.
Further, said noble gas is an octafluoropropane.
Further, said shell membrane is provided with the selectively targeted part that connects through chemical e-amido link.
Further, said macromolecule polymeric material be end group be carboxyl by homopolymer or copolymer after lactic acid, the hydroxyacetic acid polymerization.
Further, said selectively targeted part comprises antibody, micromolecule complex or the adaptive son with free amine group.
Further, said phospholipid is phosphoglyceride.
Further; Also comprise step 2, get step 1 and make multi-modality imaging microbubble lyophilized powder and in the MES-TRIS of deoxyribonuclease DNase aqueous solution or PH=6.0 buffer, dissolve; And adding coupling activator EDC/NHS; Adopt ice bath or shaking table constant temperature to hatch 30～45min; Then with the centrifugal rinsing of the MES-TRIS buffer of said PH=6.0 three times, and to limit centrifugal rotational speed be 3000r/min, in order to remove unreacted EDC/NHS in the microbubble; The microbubble of gained after the centrifugal rinsing is dissolved in the MES-TRIS buffer of deoxyribonuclease DNase aqueous solution or PH=8.0, and adding target ligand mixing low temperature is hatched 2-4h; The centrifugal rinsing of the MES-TRIS buffer of reuse said deoxyribonuclease DNase aqueous solution or PH=8.0 three times, the microbubble of collection is multi-modal targeted imaging microbubble.
Said multi-modality imaging microbubble has the purposes that is used to prepare the MRI contrast medium.
The present invention successfully prepares the embedded quantum dots microbubble with two emulsifying freeze-dryings, and its stable in properties is easy to store; Synthesis technique is simple and easy, and material requested is simple, and is cheap, consuming time few, is convenient to produce in batches.
Multi-modality imaging microbubble of the present invention; Cytotoxicity is little; Possess the fluorescence imaging function, simultaneously can not only strengthen ultrasonoscopy, under the prerequisite of not adding existing disclosed MRI image forming material but also can be used as the MRI contrast medium; The inside and outside all has imaging results, and the external multi-modality imaging microbubble imaging results that is added with targeting antibodies or micromolecule complex is particularly remarkable.
Multi-modality imaging microbubble of the present invention can tentatively be realized the video picture of target tumor.
Be different from conventional ultrasound microbubble contrast agent, magnetic resonance Gd-DTPA contrast agent; The fluorescence imaging agent, multi-modality imaging microbubble of the present invention, circulating half-life is long in vivo; After intravenous injection; By the reticuloendothelial system cytophagy, thereby rechecking, dynamic monitoring and the curative effect assessment etc. of pathological changes are convenient in the lasting passive target reinforcement of internal organs such as realization liver, spleen.But the multi-modality imaging targeted micro-bubble of coupling target ligand more makes the early diagnosis of tumor, treatment in time become the thing of phase.
Target ligand chemistry covalency sent out be coupled on the shell membrane, strengthened the anti-blood flow shear ability of targeted microbubble, can utilize ultrasonic distinctive cavitation effect simultaneously in video picture like this, realize the clear location of target area tumor, packaging medicine targeting location is discharged.
The shell membrane material is made up of high molecular polymerization nano material and phospholipid; Though the ultrasonic contrast effect of phospholipid material obviously is superior to the high molecular polymerization nano material; But structural stability is poorer relatively than high molecular polymerization nano material; Because water-soluble quantum dot of the present invention is a toxicant, from security consideration, so in membrane material, added metastable high molecular polymerization nano material.But this needs certain proportion control; Should be that the master (can not be again the high molecular polymerization nano material fully with the high molecular polymerization nano material; The ultrasonic development effect is relatively poor like this; The lipid amount is many, can influence the stability of monobloc container membrane structure again), the mol ratio of high molecular polymerization nano material and lipid is controlled at 8.75-9.25 in the application of the present invention: 1.
Description of drawings:
Fig. 1 Malvern laser analyzer detects the grain-size graph of multifunction supersonic contrast agent;
Images of transmissive electron microscope of the multi-modal microbubble of Fig. 2 (80KV * 9800) and scanning electron microscope image (1.0Kv-D7.9mm x10.0k) map;
MRI video picture map in the multi-modal microbubble body of Fig. 3.SD rat kidney radiography behind the multi-modal non-targeted micro-bubble 1min of left figure injection the present invention, the common microvesicle 1min contrast of right figure injection, middle figure is the 1min contrast of only injecting quantum dot solution, the position of arrow indication is the kidney position of SD rat among the figure;
The external MRI video picture of the multi-modal microbubble of Fig. 4 map, MB representes common microvesicle, MB among the figure
QDSThe expression non-targeted microbubble contrast medium MB that is encapsulated with quantum dot that the present invention protected
QDS-T representes the targeted microbubble contrast medium that is encapsulated with quantum dot that the present invention protects;
The multi-modal microbubble of Fig. 5 strengthens nude mice lotus tumor acoustic image map, and Fig. 5 a is injection of contrast medium pre-neoplastic ultra sonic imaging figure, and Fig. 5 b is ultra sonic imaging figure behind the non-targeted micro-bubble contrast agent 1min of injection; Fig. 5 c is ultra sonic imaging figure behind the injection targeted micro-bubble contrast agent 1min;
The multi-modal microbubble fluorescence imaging of Fig. 6 map, fluorescence imaging under the left figure toy live body white light pattern, right figure is a fluorescence imaging under toy live body gold-tinted (wavelength 485nm) pattern;
Fig. 7 is multi-modal micro air bubble ultrasonic imaging map; Ultrasonoscopy figure when left side figure is entirely high molecular polymerization nano material PLGA for the shell membrane material, the ultrasonoscopy figure the when mol ratio that right figure is high molecular polymerization nano material and lipid was formed the shell membrane material at 9: 1 o'clock.
The specific embodiment:
Below in conjunction with the specific embodiment the present invention is done further explain, but content of the present invention is not limited to the embodiment that lifted
The used high molecular polymerization nano material of the following instance of the present invention PLGA is lactic acid: hydroxyacetic acid=50: 50; Molecular weight is 12; 000 high molecular polymer; The mol ratio of high molecular polymerization nano material and lipid 9: 1, water-soluble quantum dot are the cadmium telluride that mercaptopropionic acid is modified, and the particle diameter radius is 5.1nm.
The preparation of the multi-modal microbubble of embodiment 1 the present invention
Electricity divides balance to take by weighing 50mg PLGA (50: 50, MW 12,000), and 0.15mg PE is dissolved in the 2ml dichloromethane solvent, and the full back of dissolving adds 100 μ l water-soluble quantum dots, and the sound 35-45s that shakes gets colostric fluid (W/O); Colostric fluid is mixed with 6ml 4% poly-vinyl alcohol solution, and in the homogeneous dispersion equipment of 9600r/min, act on 5min, obtain microsphere (W/O/W); Add 5ml 2% aqueous isopropanol; The room temperature magnetic stirrer at the uniform velocity stirs 2-5h, and microsphere surface is solidified, the dichloromethane nature volatilization of trying one's best.The aforesaid liquid branch is packed in the 5ml centrifuge tube, and (3500rpm 5min) abandons supernatant to high speed centrifugation.Again add an amount of distilled water,, abandon supernatant with the abundant mixing of vortex mixed instrument, washing, centrifugal.Altogether washing, centrifugal 5 times.Collect the about 2ml of lower floor's liquid.The centrifuge tube that fills the PLGA microsphere is added an amount of distilled water, and the abundant mixing of vortex mixed instrument is put-20 ℃ of refrigerator and cooled and is frozen 30min.Put vacuum lyophilization 48h in-50 ℃ of vacuum freeze driers, charge into the PLGA that perfluoropropane gas gets white powder parcel quantum dot
QDsMicrobubble.After weighing it is put preservation, subsequent use in 4 ℃ of refrigerators.
It is spherical in shape that light microscopic is observed visible this nanoparticle down, evenly big or small, and the form rule is disperseed better; It is (662 ± 20.5) nm that the Malvern laser analyzer records the nanoparticle particle diameter, narrowly distributing (Fig. 1); The visible CdTe nano-particle of transmission electron microscope is distributed in (Fig. 2 left side) in the nanoparticle nuclear.Scanning electron microscope shows that two emulsifying-freeze-dryings have made porous nano grain (Fig. 2 is right).The lyophilized powder sample is placed 4 ℃ of freezer storages, redissolves after several weeks, and light microscopic is observed form, size down and distributed and all changes less than tangible, and fluorescence intensity does not have change, and light stability is fine.
Embodiment 2A10-PLGA
QDsThe coupling experiment of targeted micro-bubble
Get the white powder PLGA that makes among the embodiment 1
QDsMicrobubble is dissolved in the deoxyribonuclease DNase aqueous solution that concentration is 10 μ g/ μ L (Dnase RNase-free water), and adds 400 μ L EDC/NHS (4: 1 mol ratios), adopts constant temperature to hatch shaking table and hatches 45 minutes.After of the centrifugal rinsing (centrifugal rotational speed be 3000r/min) of the activatory microbubble suspension of NHS through three buffer; Being dissolved into concentration again is among the 1 μ g/ μ L Dnase RNase-free water; And adding the A10PSMA aptamer that 50 μ L, 3 '-NH2 modifies, low temperature is hatched 2-4h, the centrifugal rinsing of reuse deoxyribonuclease DNase aqueous solution three times; Obtain adaptive son of A10 and microbubble covalent coupling product, and preserve with form of suspension.Flow cytometer and fluorescence microscope detect A10PSMA and the PLGA that 0.5mg/mL 3 '-NH2 modifies
QDs-COOH microbubble coupled product A10-PLGA
QDs
Said three times and repeatedly centrifugal rinsing are meant that dissolving is carried out going the supernatant after the centrifugal treating with buffer, and dissolving is carried out centrifugal with buffer again ...,, come the number of times of the fixed centrifugal rinsing of opinion with the number of times of this operation.The target ligand of selecting for use is that antibody, micromolecule complex or adaptive son all can adopt the MES-TRIS buffer to be equal to the Dnase RNase-free water in the replacement present embodiment; But, select for use the Dnase RNase-free water can be more suitable as buffer because adaptive son belongs to nucleic acid.
MRI development experiment in the body of embodiment 3 multi-modal microbubbles according to the invention
The SD rat is used clinical GE 3.0T superconduct magnetic resonance device scanning behind 3% pentobarbital sodium (1ml/kg) intraperitoneal injection of anesthesia, use internal magnetic field head quadrature circle scanning uniformly; The SE sequence; T1W1, parameter: TR/TE=824ms/10ms, visual field FOV=80mm*80mm.Adopting body weight is two rat contrast methods of 220g, presses the multi-modal microbubble PLGA of 5ml/kg dosage through rat tail vein injection embodiment 1 preparation during radiography
QDsWith blank microbubble PLGA; Time of developing and imaging results (as shown in Figure 3) behind observation liver and the nephrography; Can find out obviously that the present invention protects multi-modal non-targeted micro-bubble to have the purposes of preparation MRI contrast medium, the present invention of predictable ground protects multi-modal targeted micro-bubble also should have the purposes of preparation MRI contrast medium.
The external targeting MRI of embodiment 4 multi-modal microbubbles according to the invention develops and tests
Ovarian cancer cell SKOV3 kind is planted six orifice plates, splash into the targeted micro-bubble RGD-PLGA that has prepared after the numbering
QDs(2 hole), non-targeted micro-bubble PLGA
QDs(2 hole), the saline solution of blank microbubble PLGA (not having embedded quantum dots) are control background (2 hole), incubated at room 30min; Normal saline flushing is three times in right amount, three groups of (each 2 hole) cell dissociations is transferred in 3 EP pipes processes cell suspension, uses clinical GE 3.0T superconduct magnetic resonance device scanning; Use internal magnetic field head quadrature circle scanning uniformly, SE sequence, T1W1; Parameter: TR/TE=112ms/20ms, visual field FOV=160mm*160mm sees Fig. 4; Multi-modal video picture targeted micro-bubble combines with cell-specific identification; A lot of by force with non-targeting and blank background microbubble cell incubation system comparison signal, both explained to show also antibody and successfully connecting of microbubble that this multi-modal microbubble also can be used as the positive contrast medium of MRI that wherein the signal effect of blank background microbubble cell incubation system was visible hardly.Because the effect of targeting; Make microbubble concentrated relatively at ad-hoc location; Make that multi-modal targeted imaging microbubble and non-targeting phase comparison signal are a lot of by force, but from having proved still that in essence the present invention protects multi-modal non-targeting and targeted micro-bubble all to have the purposes of preparation MRI contrast medium.
Aspect the external evaluation of food inspection, legal medical expert's evaluation (identifying like the cause of the death), medical matters teaching and linked groups, multi-modal video picture targeted micro-bubble all has the purposes of preparation MRI contrast medium.
Ultrasonic enhancing development of targeting and fluorography experiment in the embodiment 5 multi-modal microbubble bodies
It is 5 * 10 that the SKOV3 cell of phase growth of taking the logarithm uses RPMI-1640 to be diluted to concentration
7/ ml cell suspension, with female BALB/c nude mice back of the body buttocks subcutaneous injection cell suspension 300 μ l, 8, set up lotus tumor model, treat that tumor grows to 1.0-2.0cm and carry out the video picture experiment.The tail vein is injected targeted micro-bubble RGD-PLGA at random
QDsWith non-targeted micro-bubble PLGA
QDs150-200 μ l is with the ultrasonic enhancing video picture of Philips iU22 colorful Doppler ultrasound diagnostic apparatus (L12-5 probe, frequency probe 7-13MHz).As a result injection of contrast medium at once, tumor is not seen has obvious enhancing, the 0.5min in injection back, injection targeted micro-bubble RGD-PLGA
QDsNude mice tumor perienchyma begins to occur strengthening, and in about the 1min of injection back, peaks, and continues about 15min (Fig. 5).Carry out fluorescence imaging with toy at body fluorescence imaging appearance simultaneously, injection targeted micro-bubble RGD-PLGA
QDsArrive 15min about back 1min, the sustainable fluorescence of observing of nude mice tumor perienchyma, and matched group fluorescence is difficult for observing (Fig. 6) in the identical time.
The foregoing description shows that multi-modality imaging microbubble according to the invention can strengthen the tumor ultrasonoscopy and realize fluorescence imaging, MRI imaging simultaneously.Therefore, acoustic contrast agent according to the invention has broad application prospects.
Embodiments of the invention show; Multi-modal microbubble according to the invention is mainly reticuloendothelial system specificity contrast medium, and the body internal stability is high, and circulating half-life is long; The lasting passive target reinforcement of internal organs such as liver, spleen can be realized, ultrasonic, MRI video picture can be strengthened simultaneously; Have certain active targeting property after connecting target ligand, to the diagnosis of diseases such as tumor, pathological changes dynamic monitoring and curative effect assessment have potential using value.
Shell membrane material of the present invention selects high molecular polymer to mix with lipid; Rather than form by high molecular polymer separately; Obvious 9: 1 o'clock the ultrasonic development effect of mol ratio of its ultrasonic development effect of shell membrane as shown in Figure 7, as to form by high molecular polymer separately not as good as disclosed high molecular polymerization nano material of the present invention and lipid.
It below only is preferred implementation of the present invention; Should be understood that; Under the prerequisite that does not break away from the principle of the invention; Can also make some improvement and retouching, as adopt other ligands specifics, (lactic acid for example: high molecular polymer hydroxyacetic acid=75: 25), water-soluble quantum dot particle diameter radius can also be for 3.4,5.8,7.6, these improvement of 8.9nm and retouching also should be regarded as protection scope of the present invention for other ratios.To select the reason of the cadmium telluride that mercaptopropionic acid modifies for use be to return to study carefully in experiment condition to limit to water-soluble quantum dot in addition; After the present invention can't draw still that other water-soluble quantum dots are textural and is equal to replacement; And can't realize the conclusion of the equal effect of multi-modality imaging microbubble of the present invention; Limiting the cadmium telluride particle diameter radius that mercaptopropionic acid modifies in addition is 3.2-9.0nm, is to find through experiment repeatedly, is higher than 10nm or is lower than its MRI imaging results of cadmium telluride that the basic propanoic acid of 2nm is modified; With the imaging results basically identical of blank, so the cadmium telluride that special defined particle size is the mercaptopropionic acid of 3.2-9.0nm to be modified is as optimized choice of the present invention.
Claims (9)
1. multi-modality imaging microbubble; Be core-shell structure; It is characterized in that the shell membrane material of said core-shell structure is made up of high molecular polymerization nano material and phospholipid, nuclear material is water-soluble quantum dot and noble gas; Said water-soluble quantum dot is the cadmium telluride that mercaptopropionic acid is modified, and the particle diameter radius is 3.2-9.0nm.
2. multi-modality imaging microbubble according to claim 1 is characterized in that, said shell membrane is provided with the selectively targeted part that connects through chemical e-amido link.
3. multi-modality imaging microbubble according to claim 1 is characterized in that, said macromolecule polymeric material be end group be carboxyl by homopolymer or copolymer after lactic acid, the hydroxyacetic acid polymerization.
4. multi-modality imaging microbubble according to claim 2 is characterized in that, said selectively targeted part comprises antibody, micromolecule complex or the adaptive son with free amine group.
5. prepare the method for the said multi-modality imaging microbubble of claim 1, it is characterized in that, may further comprise the steps:
Step 1, with phospholipid with contain hold carboxyl macromolecule polymeric material by 1: the mol ratio of 8.75-9.25 is dissolved in the dichloromethane solvent, adds water-soluble quantum dot then, and the 35-45s that shakes obtains colostric fluid; With said colostric fluid and concentration is that the poly-vinyl alcohol solution of 3%-5% mixes; And be homogeneous dispersion effect 5min in the homogenizer of 2000-10000r/min at rotating speed, obtain microsphere and redissolve suspension, add the aqueous isopropanol that concentration is 2%-4% again; At room temperature stir 2-5h; Through centrifugal rinsing, collect lower floor's microsphere, and the microsphere of collecting is charged into noble gas promptly obtain being lyophilized powder multi-modality imaging microbubble after lyophilization.
6. the method for preparing of multi-modality imaging microbubble according to claim 5 is characterized in that, said noble gas is an octafluoropropane.
7. the method for preparing of multi-modality imaging microbubble according to claim 5 is characterized in that, said phospholipid is phosphoglyceride.
8. the method for preparing the multi-modality imaging microbubble as claimed in claim 5; It is characterized in that; Also comprise step 2, get step 1 and make multi-modality imaging microbubble lyophilized powder and in the MES-TRIS of deoxyribonuclease DNase aqueous solution or PH=6.0 buffer, dissolve; And add coupling activator EDC/NHS, adopt ice bath or shaking table constant temperature to hatch 30～45min, then with the centrifugal rinsing of the MES-TRIS buffer of said PH=6.0 three times; And the qualification centrifugal rotational speed is 3000r/min; In order to removing unreacted EDC/NHS in the microbubble, the microbubble of gained after the centrifugal rinsing is dissolved in the MES-TRIS buffer of deoxyribonuclease DNase aqueous solution or PH=8.0, and adds target ligand mixing low temperature and hatch 2-4h; The centrifugal rinsing of the MES-TRIS buffer of reuse said deoxyribonuclease DNase aqueous solution or PH=8.0 three times, the microbubble of collection is multi-modal targeted imaging microbubble.
9. claim 1 or 2 described multi-modality imaging microbubbles have the purposes that is used to prepare the MRI contrast medium.
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| CN103977433B (en) * | 2014-05-14 | 2017-01-11 | 上海中医药大学附属岳阳中西医结合医院 | An ultrasonic prostate cancer diagnosis targeting reagent and a preparing method thereof |
| CN104622848A (en) * | 2015-02-13 | 2015-05-20 | 西安交通大学 | Plasma activation encapsulated micro-bubbles |
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