CN102772808B - A kind of multi-modality imaging microbubble construction, Preparation method and use - Google Patents
A kind of multi-modality imaging microbubble construction, Preparation method and use Download PDFInfo
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Abstract
The present invention relates to the crossing domain of Chemical Engineering, material and nanometer medicine, specifically a kind of multi-modality imaging probe structure and preparation method thereof.Multi-modality imaging probe of the present invention is microbubble containing core-shell structure, Shell membrane materials are made up of the good macromolecular material of biodegradable, biocompatibility and phospholipid material, the water-soluble quantum dot solution of the different fluorescent characteristicss of center embedding, it is filled with perfluorocarbon alkane gas, the double emulsifying lyophilization inflation method of application prepares size tunable, good dispersion and the stable multi-modal probe easily preserving.Microbubble of the present invention be reticuloendothelial system specificity contrast medium, can not only fluorescence imaging, and enable Enhance ultrasonography and MRI imaging, microbubble multi-modal imaging, have broad application prospects.
Description
Technical field
The present invention relates to the crossing domain of biomedical, material and nanometer medicine is and in particular to a kind of multi-modal targeting becomes
As microbubble structure, Preparation method and use.
Background technology
With the development of Medical Imaging, application in clinic for all kinds of contrast agent is also more and more extensive.Contrast agent can
Increase contrast in tissue, improve the ability of image qualitative positioning, improve rate of correct diagnosis.At present, various imaging techniques all have respectively
From contrast agent, such as the microbubble contrast agent of ultra sonic imaging, for the diodone of CT imaging, for nuclear magnetic resonance
Gd-DTPA and Superparamagnetic Iron Oxide etc..Same patient is to clarify a diagnosis, and usually needs to accept a large amount of, multiple radiographies in a short time
Agent, has not only increased the risk that organism metabolism burden and adverse reactions to contrast medium occur, has also increased medical expense, thus, face
In the urgent need to a kind of contrast agent that can be simultaneously used for derived techniques on bed, i.e. multi-functional contrast agent.Chinese scholars are
Multi-functional contrast agent is carried out with correlational study, ultra sonic imaging is combined and glimmering with nuclear magnetic resonance, radionuclide imaging, fluorescence imaging
United contrast agent is reported photoimaging successively with radionuclide imaging, but can be simultaneously used for ultrasonic, fluorescence and nuclear magnetic resonance
There is not been reported for contrast agent.
Ultra sonic imaging is a kind of conventional methods for clinical diagnosis, with its characteristic such as quick, safe, cheap, portable, in the world
In the range of be widely used.But due to the restriction of of ultrasonic diagnosises method itself, the resolution of ultra sonic imaging and accuracy are remote
Far from the requirement meeting advanced clinical diagnosises.Ultrasonic contrast medium can significantly increase medical ultrasound diagnosis signal, obtains than general
Logical ultra sonic imaging is more rich, more accurately diagnostic message, and the diagnoses and treatment for disease provides more foundations.It is applied to scientific research and face
On bed, most acoustic contrast agents is microvesicle (MBs) contrast medium, and it is by outer layer peplos shell (phospholipid, surfactant, macromolecule
Deng) and internal package gas (air, nitrogen, perfluocarbon, sulfur hexafluoride etc.) composition.
Gd-DTPA (Magnevist) is a kind of nuclear magnetic resonance agent of Clinical practice, good stability, and magnetic property is excellent
Good.But discovered in recent years Gd-DTPA can cause hepatocyte chronic fibrosises, some countries stop using.Can keep
Can be new strategy from smoothly discharging in vivo while Gd-DTPA premium properties, such as again：Make Gd-DTPA molecule and albumen, saccharide
Connect and modified with suitable polymer microsphere parcel etc., obtain preferable progress.
Quantum dot (quantumn dots QDs) is as a kind of semiconductor fluorescence nano-particle, due to its excellent light
Learn characteristic, be a study hotspot of nano biological medical domain in recent years.Typically by second family and the 6th race's element or the 3rd
Race and pentels are constituted.Quantum dot has a very wide absworption peak scope, narrow and symmetrical launch wavelength, larger stoke
This displacement, higher quantum yield and very strong anti-light Bleachability, there is the incomparable optics of traditional organic fluorescent substance
Characteristic.Some famous publications international constantly report quantum dot in biomedical and nanometer field of medicaments applied research in recent years,
Quantum dot application has gradually penetrated into biomedical every field, including cytobiology, molecular biology, protein science
And nano-probe, become a kind of preferable in vitro and in vivo cell/tissue fluorescence imaging diagnostic reagent.How quick, low one-tenth
This acquisition, high yield, the quantum dot microsphere of small toxicity are biomedical applications field problems anxious to be resolved.Different qualities,
Difference in functionality quantum dot microsphere and microbubble have high researching value and application prospect.
Content of the invention
The present invention is directed to different imaging techniques not only need to increase organism metabolism burden using different contrast agent, and radiography
The problem of agent side reaction, provides a kind of multi-modal ultrasonic imaging agent, and to realizing the purpose of " probe is multi-functional ", the present invention is also
The method preparing described microbubble is provided.
For reaching above-mentioned purpose, technical scheme provided by the present invention is such：
A kind of multi-modality imaging microbubble, is core-shell structure, and the Shell membrane materials of described core-shell structure are received by high molecular polymerization
Rice material and phospholipid composition, nuclear material is water-soluble quantum dot and noble gases, and described water-soluble quantum dot is repaiied for mercaptopropionic acid
The cadmium telluride of decorations, particle diameter radius is 3.2-9.0nm.
The method preparing described multi-modality imaging microbubble, comprises the following steps：
Step one, phospholipid and the macromolecule polymeric material containing end carboxyl are dissolved in by 1: 8.75-9.25 mol ratio
In dichloromethane solvent, it is subsequently adding water-soluble quantum dot, sound and vibration 35-45s obtains colostric fluid；Described colostric fluid and concentration are
The poly-vinyl alcohol solution mixing of 3%-5%, and homogeneous dispersion effect in rotating speed is for the homogenizer of 2000-10000r/min
5min, obtains microsphere and redissolves suspension, adds the aqueous isopropanol that concentration is 2%-4%, 2-5h is stirred at room temperature, through centrifugation
Rinsing, collects lower floor microsphere, and will be freeze-dried for the microsphere collected after be filled with noble gases obtain many in lyophilized powder
Modality microbubble.
Further, described noble gases are octafluoropropane.
Further, described shell membrane is provided with the selectively targeted part bonded by chemical covalent coupling method-amide.
Further, described macromolecule polymeric material is carboxyl by the homopolymer after lactic acid, hydroxyacetic acid polymerization for end group
Or copolymer.
Further, described selectively targeted part includes antibody, small molecule complexes or the aptamer with free amine group.
Further, described phospholipid is phosphoglyceride.
Further, also include step 2, take step one multi-modality imaging microbubble lyophilized powder to be obtained in deoxyribonuclease
Dissolve in the MES-TRIS buffer of deoxyribonuclease aqueous solution or PH=6.0, and add coupling activator EDC/NHS,
Using ice bath or shaking table constant-temperature incubation 30～45min, then the MES-TRIS buffer by centrifugation of described PH=6.0 is rinsed three
Secondary, and limit centrifugal rotational speed as 3000r/min, in order to remove unreacted EDC/NHS in microbubble, by gained after centrifugal rinsing
Microbubble be dissolved in deoxyribonuclease deoxyribonuclease aqueous solution or the MES-TRIS buffer of PH=8.0, and
Target ligand is added to mix low temperature incubation 2-4h；Again with described deoxyribonuclease deoxyribonuclease aqueous solution or PH=
8.0 MES-TRIS buffer by centrifugation rinses three times, and the microbubble of collection is multi-modal targeted imaging microbubble.
Described multi-modality imaging microbubble has the purposes for preparing MRI contrast agent.
The present invention successfully prepares embedded quantum dots microbubble with double emulsifying freeze-dryings, and its stable in properties is it is easy to store；
Synthesis technique is simple, and material requested is simple, cheap, takes few, is easy to produce in batches.
Multi-modality imaging microbubble of the present invention, cytotoxicity is little, possesses fluorescence imaging function, can not only increase simultaneously
Strong ultrasonoscopy, on the premise of without existing disclosed MRI image forming material but also can act as MRI contrast agent, in vivo
It is respectively provided with outward imaging results, and outstanding added with the multi-modality imaging microbubble imaging results of targeting antibodies or small molecule complexes in vitro
For notable.
Multi-modality imaging microbubble of the present invention, can tentatively realize the imaging of target tumor.
Different from conventional ultrasound microbubble contrast agent, magnetic resonance Gd-DTPA contrast agent, fluorescence imaging agent, of the present invention
Multi-modality imaging microbubble, circulating half-life is long in vivo, after intravenous administration, is swallowed by reticuloendothelial system cell, thus
Realize the lasting passive target strengthening of the internal organs such as liver, spleen, be easy to rechecking, dynamic monitoring and curative effect evaluation of pathological changes etc..
The multi-modality imaging targeted micro-bubble being coupled target ligand more make the early diagnosiss of tumor, in time treatment become can the phase thing.
Target ligand chemical covalent is sent out and is coupled on shell membrane, enhance the anti-blood flow shear ability of targeted microbubble, so
Ultrasonic distinctive cavitation effect can be utilized in imaging simultaneously, realize the clear positioning of target tumor, packaging medicine targeting is fixed
Position release.
Shell membrane materials are made up of high molecular polymerization nano material and phospholipid although the ultrasonic contrast effect of phospholipid material is obvious
Better than high molecular polymerization nano material, but structural stability is relatively poorer than high molecular polymerization nano material, due to the present invention's
Water-soluble quantum dot is toxicant, for security consideration, therefore adds metastable high molecular polymerization in membrane material
Nano material.But this needs certain ratio to control it should (can not be entirely high based on high molecular polymerization nano material again
Molecule aggregation nano material, such ultrasonic development effect is poor, and lipid amount is many, can affect stablizing of overall shell membrane structure again
Property), in the present patent application, high molecular polymerization nano material and the mol ratio of lipid control in 8.75-9.25: 1.
Brief description：
Fig. 1 Malvern laser analyzer detects the grain-size graph of Multifunctional ultrasound contrast agent；
The images of transmissive electron microscope (80KV × 9800) of the multi-modal microbubble of Fig. 2 and scanning electron microscope image (1.0Kv-D7.9mm
X10.0k) compares figure；
MRI imaging compares figure in Fig. 3 multi-modal microbubble body.Left figure injects the multi-modal non-targeted microbubble 1min of the present invention
SD rat kidney radiography afterwards, right figure injection common microvesicle 1min comparison, middle figure is the 1min comparison of only injection quantum dot solution, figure
The position of middle arrow indication is the kidney position of SD rat；
The external MRI of the multi-modal microbubble of Fig. 4 images compares figure, and in figure MB represents common microvesicle, MBQDSRepresent the present invention
The non-targeted microbubble contrast agent MB being encapsulated with quantum dot being protectedQDSWhat-T represented that the present invention protected is encapsulated with quantum dot
Targeted microbubble contrast medium；
The multi-modal microbubble of Fig. 5 strengthens nude mice lotus knurl acoustic image compares figure, respectively injects contrast agent pre-neoplastic ultra sonic imaging
Figure, inject non-targeted microbubble contrast 1min after ultra sonic imaging figure and injection targeted micro-bubble contrast agent 1min after ultrasonic
Image；
Fig. 6 multi-modal microbubble fluorescence imaging compares figure, fluorescence imaging under left figure small animal living body white light patterns, right figure is
Fluorescence imaging under small animal living body gold-tinted (wavelength 485nm) pattern；
Fig. 7 is multi-modal micro air bubble ultrasonic imaging contrast figure, and left figure is entirely high molecular polymerization nanometer material for Shell membrane materials
Ultrasonoscopy figure during material PLGA, right figure is to form Shell membrane materials during mol ratio 9: 1 of high molecular polymerization nano material and lipid
When ultrasonoscopy figure.
Specific embodiment：
With reference to specific embodiment, the present invention is described in further detail, but present disclosure is not limited to be lifted
Embodiment
High molecular polymerization nano material PLGA used by the following example of the present invention is lactic acid: hydroxyacetic acid=50: 50, molecule
Measure as 12,000 high molecular polymer, the mol ratio 9: 1 of high molecular polymerization nano material and lipid, water-soluble quantum dot is mercapto
The cadmium telluride that base propanoic acid is modified, particle diameter radius is 5.1nm.
The preparation of the multi-modal microbubble of embodiment 1 present invention
Electricity point balance weighs 50mg PLGA (50: 50, MW 12,000), and 0.15mg PE is dissolved in 2ml dichloromethane solvent
In, molten rear addition 100 μ l water-soluble quantum dots entirely, sound and vibration 35-45s obtains colostric fluid (W/O)；By colostric fluid and the poly- second of 6ml 4%
Enolate solution mixes, and acts on 5min in the homogeneous dispersion equipment of 9600r/min, obtains microsphere (W/O/W), adds 5ml
2% aqueous isopropanol, room temperature magnetic stirrer at the uniform velocity stirs 2-5h, so that microsphere surface is solidified, dichloromethane nature volatilization as far as possible.
Aforesaid liquid is distributed in 5ml centrifuge tube, high speed centrifugation (3500rpm, 5min) abandons supernatant.Rejoin double steamings in right amount
Water, is fully mixed, washs, is centrifuged with vortex mixed instrument, abandon supernatant.Washing, centrifugation 5 times altogether.Collect lower floor liquid about 2ml.
The centrifuge tube filling PLGA microsphere is added appropriate distilled water, vortex mixed instrument fully mixes, put freezing in -20 DEG C of refrigerators
30min.Put vacuum lyophilization 48h in -50 DEG C of vacuum freeze driers, be filled with perfluoropropane gas and obtain white powder parcel
The PLGA of quantum dotQDsMicrobubble.Put after weighing in 4 DEG C of refrigerators preserve, standby.
Light Microscopic observation this nanoparticle visible is spherical in shape, uniform in size, form rule, well dispersed；Malvern laser divides
It is (662 ± 20.5) nm that analyzer records nanoparticle particle diameter, narrowly distributing (Fig. 1)；Transmission electron microscope visible CdTe nano-particle is distributed in
In nanoparticle core (Fig. 2 is left).Scanning electron microscope shows that double emulsifying-freeze-dryings have been obtained porous nano grain (Fig. 2 is right).Lyophilizing
4 DEG C of freezer storages placed by powder sample, redissolve after several weeks, and light Microscopic observation form, size and its distribution all significantly do not change,
Fluorescence intensity no changes, and light stability is fine.
Embodiment 2A10-PLGAQDsThe conjugation of targeted micro-bubble
White powder PLGA being obtained in Example 1QDsMicrobubble is dissolved in the no ribose core that concentration is 10 μ g/ μ L
In sour enzyme deoxyribonuclease aqueous solution (Dnase RNase-free water), and (rub at 4: 1 to add 400 μ L EDC/NHS
You compare), it is incubated 45 minutes using constant-temperature incubation shaking table.The microbubble suspension being activated by NHS is through the centrifugation of three buffer
After rinsing (centrifugal rotational speed is 3000r/min), being redissolved in concentration is in 1 μ g/ μ L Dnase RNase-free water, and
Add the A10PSMA aptamer that 50 μ L 3 '-NH2 modifies, low temperature is incubated 2-4h, then with deoxyribonuclease deoxyribose core
Sour enzyme aqueous solution centrifugal rinsing three times, obtains A10 aptamer and microbubble covalent coupling product, and is protected with form of suspension
Deposit.Flow cytometer and fluorescence microscope detect A10PSMA and PLGA that 0.5mg/mL 3 '-NH2 modifiesQDs- COOH microbubble
Coupled product A10-PLGAQDs.
Described three times and multiple centrifugal rinsing refer to plus buffer solution carry out centrifugal treating after go the supernatant, then plus
Buffer solution is centrifuged ..., the number of times being operated with this, to discuss the number of times determining centrifugal rinsing.From target ligand be
Antibody, small molecule complexes or aptamer all can be using the Dnase in MES-TRIS buffer equivalent the present embodiment
RNase-free water, but because aptamer belongs to nucleic acid, from Dnase RNase-free water as buffer meeting
More particularly suitable.
The internal MRI experiment of embodiment 3 multi-modal microbubble of the present invention
SD rat is after 3% pentobarbital sodium (1ml/kg) intraperitoneal injection of anesthesia, common using clinical GE 3.0T superconduction type magnetic
Vibration Meter scans, using the orthogonal circle scanning of the more uniform head of internal magnetic field, SE sequence, T1W1, parameter：TR/TE=824ms/
10ms, visual field FOV=80mm*80mm.It is two rat control methods of 220g using body weight, press 5ml/kg dosage during radiography through big
The multi-modal microbubble PLGA of tail vein injection embodiment 1 preparationQDsWith blank microbubble PLGA, observe liver and nephrography
Time of developing and imaging results (as shown in Figure 3) afterwards are it can be clearly seen that the present invention is protected multi-modal non-targeted microbubble
There is the purposes of preparation MRI contrast agent, the predictable ground present invention is protected multi-modal targeted micro-bubble to should also be as with preparation
The purposes of MRI contrast agent.
The targeting MRI experiment of embodiment 4 multi-modal microbubble of the present invention
Ovarian cancer cell SKOV3 is planted six orifice plates, after numbering, instills the targeted micro-bubble RGD-PLGA having preparedQDs
(2 hole), non-targeted microbubble PLGAQDs(2 hole), the saline solution of blank microbubble PLGA (not having embedded quantum dots) is comparison
Background (2 hole), incubated at room 30min, appropriate normal saline flushing three times, three groups of (each 2 holes) cell dissociations are transferred to 3 EP
Make cell suspension in pipe, scanned using clinical GE 3.0T superconduction type magnetic resonance device, using the more uniform head of internal magnetic field just
Hand over circle scanning, SE sequence, T1W1, parameter：TR/TE=112ms/20ms, visual field FOV=160mm*160mm, are shown in Fig. 4, multi-modal
Imaging targeted micro-bubble is combined with cell-specific identification, compares letter with non-targeted and blank background microbubble cell incubation system
Number by force a lot, both illustrated that antibody was coupled with the success of microbubble and also indicated that this multi-modal microbubble also acted as MRI positive contrast
Agent, the signal effect of wherein blank background microbubble cell incubation system is almost invisible.Because the effect of targeting is so that micro- gas
Steep in ad-hoc location Relatively centralized so that multi-modal targeted imaging microbubble and non-targeted phase comparison signal are a lot of by force, but from this
The present invention is still demonstrated on matter and is protected the multi-modal non-targeted purposes being respectively provided with preparation MRI contrast agent with targeted micro-bubble.
In terms of food inspection, the external identification of forensic identification (as cause of the death identification), medical matters teaching and linked groups, many
Mode images the purposes that targeted micro-bubble is respectively provided with preparation MRI contrast agent.
In the multi-modal microbubble body of embodiment 5, targeted ultrasound strengthens development and fluorography experiment
It is 5 × 10 that the SKOV3 cell RPMI-1640 that phase of taking the logarithm grows is diluted to concentration7/ ml cell suspension, and female
Property BALB/c nude mice back of the body buttocks subcutaneous injection cell suspension 300 μ l, 8, set up lotus knurl model, treat tumor length to 1.0-2.0cm
Carry out imaging experiment.Tail vein random injection targeted micro-bubble RGD-PLGAQDsWith non-targeted microbubble PLGAQDs150-200 μ l,
With the ultrasonic enhancing imaging of Philips iU22 colorful Doppler ultrasound diagnostic apparatus (L12-5 pops one's head in, frequency probe 7-13MHz).Knot
At once, tumor there are no and is remarkably reinforced fruit injection contrast agent, 0.5min after injection, injects targeted micro-bubble RGD-PLGAQDsNaked
Mus tumor perienchyma start occur strengthen, and in injection after 1min about peak, last about 15min (Fig. 5).Use simultaneously
Toy carries out fluorescence imaging in body phosphorimager, injects targeted micro-bubble RGD-PLGAQDsAfterwards 1min about arrive 15min,
Nude mouse tumor perienchyma is sustainable to observe fluorescence, and matched group fluorescence is difficult to observe (Fig. 6) in same time.
Above-described embodiment shows that multi-modality imaging microbubble of the present invention can strengthen tumor ultrasonoscopy and simultaneously real
Existing fluorescence imaging, MRI imaging.Therefore, acoustic contrast agent of the present invention has broad application prospects.
Embodiments of the invention show, multi-modal microbubble of the present invention is mainly the contrast of reticuloendothelial system specificity
Agent, internal stability is high, and circulating half-life is long, enables the lasting passive target strengthening of the internal organs such as liver, spleen, can strengthen super simultaneously
Sound, MRI imaging；After connecting target ligand, there is certain active targeting, the diagnosis to diseases such as tumors, pathological changes dynamic monitoring
And curative effect evaluation has potential using value.
The Shell membrane materials of the present invention select high molecular polymer to mix with lipid, rather than individually by high molecular polymer group
Become, as shown in fig. 7, its ultrasonic development effect of shell membrane being individually made up of high molecular polymer is substantially not as good as disclosed in this invention
Ultrasonic development effect during mol ratio 9: 1 of high molecular polymerization nano material and lipid.
The above is only the preferred embodiment of the present invention it is noted that under the premise without departing from the principles of the invention, also may be used
To make some improvements and modifications, such as using other ligands specifics, other ratios (such as lactic acid: hydroxyacetic acid=75: 25)
High molecular polymer, water-soluble quantum dot particle diameter radius can also for 3.4,5.8,7.6, these improvements and modifications of 8.9nm
Should be regarded as protection scope of the present invention.In addition the reason cadmium telluride that water-soluble quantum dot is modified from mercaptopropionic acid be return study carefully in
Experiment condition limits, and after the present invention there is no method to draw the upper equivalent of other water-soluble quantum dots construction, and cannot realize this
The conclusion of bright multi-modality imaging microbubble equivalent effect, the cadmium telluride particle diameter radius in addition limiting mercaptopropionic acid modification is as 3.2-
9.0nm, is to find through many experiments, its MRI imaging results of cadmium telluride that the base propanoic acid higher than 10nm or less than 2nm is modified,
Basically identical with the imaging results of blank, therefore it is particularly limited to the cadmium telluride that the mercaptopropionic acid that particle diameter is 3.2-9.0nm is modified
Optimized choice as the present invention.
Claims (3)
1. a kind of method preparing multi-modal targeted imaging microbubble is it is characterised in that described multi-modal targeted imaging microbubble
It is coupled with target ligand by multi-modality imaging microbubble and is obtained, described multi-modality imaging microbubble is core-shell structure, described shell core
The Shell membrane materials of structure are made up of high molecular polymerization nano material and phospholipid, and nuclear material is water-soluble quantum dot and noble gases,
The cadmium telluride that described water-soluble quantum dot is modified for mercaptopropionic acid, particle diameter radius is 3.2-9.0nm, described high molecular polymerization nanometer
Material for end group be carboxyl by the copolymer after lactic acid, hydroxyacetic acid polymerization, described multi-modality imaging refers to ultra sonic imaging, glimmering
Photoimaging is imaged with MRI；
Comprise the following steps：
Step one, phospholipid and described high molecular polymerization nano material are dissolved in dichloromethane by 1: 8.75-9.25 mol ratio
In solvent, it is subsequently adding water-soluble quantum dot, sound and vibration 35-45s obtains colostric fluid；Described colostric fluid and concentration are 3%-5%
Poly-vinyl alcohol solution mixing, and in rotating speed is for the homogenizer of 2000-10000r/min homogeneous dispersion effect 5min, obtain micro-
Ball redissolves suspension, adds the aqueous isopropanol that concentration is 2%-4%, 2-5h is stirred at room temperature, through centrifugal rinsing, under collection
Layer microsphere, and will be freeze-dried for the microsphere collected after be filled with noble gases and obtain gas micro- in lyophilized powder multi-modality imaging
Bubble；
Step 2, take step one that multi-modality imaging microbubble lyophilized powder is obtained to dissolve in the MES-TRIS buffer of pH=6.0,
And add coupling activator EDC/NHS, using shaking table constant-temperature incubation 30～45min, then by the MES-TRIS of described pH=6.0
Buffer by centrifugation rinses three times, and limits centrifugal rotational speed as 3000r/min, in order to remove unreacted EDC/NHS in microbubble,
The microbubble of gained after centrifugal rinsing is dissolved into the deoxyribonuclease aqueous solution of deoxyribonuclease or pH=8.0
In MES-TRIS buffer, and target ligand is added to mix low temperature incubation 2-4h；Again with the deoxidation core of described deoxyribonuclease
The MES-TRIS buffer by centrifugation of ribonuclease T. aqueous solution or pH=8.0 rinses three times, and the microbubble of collection is multi-modal target
To imaging microbubble.
2. the method preparing multi-modal targeted imaging microbubble according to claim 1 is it is characterised in that described indifferent gas
Body is octafluoropropane.
3. the method preparing multi-modal targeted imaging microbubble according to claim 1 is it is characterised in that described phospholipid is
Phosphoglyceride.
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