CN102138889A - Targeted drug-bearing ultrasonic microbubble and preparation method thereof - Google Patents
Targeted drug-bearing ultrasonic microbubble and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a targeted drug-bearing ultrasonic microbubble comprising a lipide dimolecular layer outer shell, targeted polypeptide fixed at the outer side of the lipide dimolecular layer outer shell, a biological inert gas wrapped in the lipide dimolecular layer outer shell and medicament granules dispersed in the lipide dimolecular layer outer shell. The targeted polypeptide is a polypeptide or protein derivative containing an amino acid sequence CGNKRTRGC. By connecting the polypeptide or protein derivative containing a tumor targeted peptide sequence outside the lipide dimolecular layer outer shell, the obtained targeted drug-bearing ultrasonic microbubble can target the lymph vessels and the tumor cells of a tumor, can detect and diagnose the generation, the development and the curative effect on the tumor in real time through high-frequency ultrasonic imaging and can crush the microbubble through low-frequency ultrasound to release the medicament granules so as to achieve the aim of controllably and targetedly releasing the medicament, thereby having extremely important meanings to the prevention, diagnose and treatment of the tumor. In addition, the invention also relates to a preparation method of the targeted drug-bearing ultrasonic microbubble.
Description
[technical field]
The present invention relates to ultrasonic microbubble developing technique field, relate in particular to a kind of target drug-carrying ultrasonic microbubble and preparation method thereof.
[background technology]
Nineteen sixty-eight Gramiak reported first can strengthen the minute bubbles of development, i.e. ultrasound microbubble contrast agent (UCA).The frontier of noinvasive ultrasonic diagnosis and treatment has been started in the appearance of UCA.Along with to the deepening continuously of its research, it is found that ultrasound microbubble contrast agent is not only a kind of good ultrasonoscopy contrast medium, and be a kind of important drug delivery vehicle.It is increasingly extensive in the research of medical domain that ultrasound microbubble contrast agent is carried the treatment of gene or drug targeting.The ultrasound microbubble contrast agent targeted therapy comprises the treatment of ultrasonic mediation medicine carrying microvesicle and the treatment of ultrasonic mediation target drug-carrying microvesicle.
The associated methods of microvesicle and medicine has: (1) directly is attached on the microvesicle shell, is attached on the lipid microbubble surface of positively charged easily as the medicine of electronegative group; (2) parcel is gone in the microvesicle shell, and the composition albumin of formation ultrasound microbubble contrast agent shell, phospholipid polysaccharide etc. all are good carriers of medicine, medicine can be adsorbed, integrate or wrap up wherein; (3) parcel is gone into microvesicle inside; this is a kind of more stable combination; high and the complete release of energy under ultrasonication of drug encapsulation rate; can increase the drug level of target tissue; place in the microvesicle by the medicine that will have big side effect, can reach the protection medicine in the local release of target organ, delay medicine release, reduce administration number of times, reduce dosage and increase the purpose of curative effect.
Although ultrasonic mediation target drug-carrying microvesicle has safety, efficient, advantage such as controllability is strong concurrently, tentatively shown wide application prospect, but traditional ultrasonic mediation target drug-carrying microvesicle targeting is not strong, for strengthening its targeting, need further transform it.
[summary of the invention]
Based on this, be necessary to provide a kind of targeting target drug-carrying ultrasonic microbubble preferably.
A kind of target drug-carrying ultrasonic microbubble, comprise the lipid bilayer shell, be fixed on the lipid bilayer shell outside target polypeptide, be wrapped in the biologically inert gas of lipid bilayer enclosure and be scattered in drug particles in the lipid bilayer shell, target polypeptide is polypeptide or the protein derivatives that comprises aminoacid sequence CGNKRTRGC.
Preferably, the lipid bilayer shell comprises each component of following mass fraction:
1,7 parts of 2-distearyl acyl groups-sn-glyceryl-3-phosphatidylcholine,
1,2,000 0.5 parts of 2-distearyl acyl groups-sn-glyceryl-3-phosphatidylcholine-macrogol,
1,0.5 part of 2-distearyl acyl group-sn-glyceryl-3-phosphatidylcholine-macrogol 2000-biotin;
Drug particles is a paclitaxel, and 1, the mass ratio of 2-distearyl acyl group-sn-glyceryl-3-phosphatidylcholine and paclitaxel is 7-8: 2-3.
Preferably, also comprise Avidin, described target polypeptide is connected with biotin, and target polypeptide is by biotin-avidin-biotin bridge and 1, and 2-distearyl acyl group-sn-glyceryl-3-phosphatidylcholine-macrogol 2000-biotin connects.
Preferably, also comprise Streptavidin, described target polypeptide is connected with biotin, and target polypeptide is by biotin-Streptavidin-biotin bridge and 1, and 2-distearyl acyl group-sn-glyceryl-3-phosphatidylcholine-macrogol 2000-biotin connects.
Preferably, the particle diameter of target drug-carrying ultrasonic microbubble is 0.5～8 μ m.
Preferably, biologically inert gas is at least a in perfluoropropane, perfluorinated butane and the sulfur hexafluoride.
Preferably, the lipid bilayer shell is phospholipid or phospholipid derivant.
Preferably; phospholipid or phospholipid derivant are 1; 2-two palmityls-sn-glyceryl-3-phosphatidic acid glyceryl-sodium salt, 1,2-two palmityls-sn-glyceryl-3-phosphatidic acid-sodium salt and 1, at least a in 2-two palmityls-sn-glyceryl-3-phosphatidylcholine.
Preferably, drug particles is at least a in Docetaxel, amycin, camptothecine, 5-fluorouracil, cytosine arabinoside, methotrexate, indometacin, Pu Luobipuluofen, ketone ibuprofen, piroxicam and the diclofenac, and perhaps drug particles is activated protein, polypeptide, vaccine or the gene that is used for the treatment of breast carcinoma.
In order to increase the targeting of medicine carrying ultrasonic microbubble, outside the lipid bilayer shell, connect polypeptide or the protein derivatives that contains the cancer target peptide sequence, the target drug-carrying ultrasonic microbubble that obtains can target tumor lymphatic vessel and tumor cell thereof, can generation, development and the curative effect of tumor be detected in real time and diagnose by the high frequency ultrasound imaging again, also can smash microvesicle and discharge drug particles by low frequency ultrasound, reach the controllably purpose of targeting release medicine, thereby prevention, diagnosis and the treatment of tumor all had extremely important meaning.
In addition, also be necessary to provide a kind of targeting preparation method of target drug-carrying ultrasonic microbubble preferably.
A kind of preparation method of target drug-carrying ultrasonic microbubble comprises the steps:
Step 1: press 7/4～3 part of mass fraction proportioning paclitaxel, 1,7 parts of 2-distearyl acyl groups-sn-glyceryl-3-phosphatidylcholine, 1,2,000 0.5 parts of 2-distearyl acyl groups-sn-glyceryl-3-phosphatidylcholine-macrogol, 1,0.5 part of 2-distearyl acyl group-sn-glyceryl-3-phosphatidylcholine-macrogol 2000-biotin is dissolved in preparation medicine phospholipid suspension in the solvent;
Step 2: with medicine phospholipid suspension mixing, at exsiccant N
2The stream effect removes to desolvate down and makes phospholipid form the layer of even thin film on chamber wall, vacuum drying 1～3 hour;
Step 3: in containing the container of dry phospholipid membrane, add the Tris buffer solution of the pH=7.4 that handles through the degassing, must prepare the medicine carrying phospholipid solution, comprise the glycerol of 10% volume and the propylene glycol of 10% volume in the Tris buffer solution;
Step 4: heating medicine carrying phospholipid solution is to more than the phase transition temperature, and the ultrasonic concussion of water-bath disperses thoroughly until transparent phospholipid solution;
Step 5: transparent medicine carrying phospholipid solution is divided in the device cillin bottle, and the air displacement in the cillin bottle is become biologically inert gas, shake 30～60 seconds preparation medicine carrying ultrasonic microbubbles;
Step 6: centrifugal floating method is cleaned the medicine carrying ultrasonic microbubble 3～4 times, to remove the phospholipid that does not form microvesicle;
Step 7: in the medicine carrying ultrasonic microbubble that cleaned, add Avidin, shake gently to hatch under the room temperature after 15 minutes and clean 3-4 time, remove not coupled Avidin with centrifugal floating method;
Step 8: in the medicine carrying ultrasonic microbubble that step 7 obtains, add biotinylated target polypeptide, shake gently also and hatch more than 10 minutes under the room temperature, remove unconjugated biotinylated target polypeptide 3～4 times with centrifugal floating method cleaning, obtain the target drug-carrying ultrasonic microbubble.
Preferably, in the step 4, the concussion frequency is 4000～5000 times/minute, and shock range is 10～40 millimeters, and the concussion time is 30～90 seconds.
This preparation method principle is simple, easy and simple to handle, low for equipment requirements, can wide popularization and application.
[description of drawings]
Fig. 1 is the target drug-carrying ultrasonic microbubble ideograph of an embodiment;
Fig. 2 is the target drug-carrying ultrasonic microbubble fluorogram that combines target polypeptide;
Fig. 3 A is that non-target drug-carrying ultrasonic microbubble combines sketch map with breast carcinoma MDA-MB-231 cell;
Fig. 3 B is target drug-carrying ultrasonic microbubble and the bonded Electronic Speculum figure of mammary gland MDA-MB-231 cell-specific;
Fig. 4 is the breast carcinoma MDA-MB-231 cell imaging comparison diagram that is added with target drug-carrying ultrasonic microbubble and non-target drug-carrying ultrasonic microbubble;
Fig. 5 is target drug-carrying ultrasonic microbubble killing activity sketch map to breast carcinoma MDA-MB-231 cell under ultrasound condition.
[specific embodiment]
Mainly reaching specific embodiment below in conjunction with the accompanying drawings is described in further detail target drug-carrying ultrasonic microbubble and preparation method thereof.
The target drug-carrying ultrasonic microbubble of one embodiment comprises: the lipid bilayer shell, be fixed on the lipid bilayer shell outside target polypeptide, be wrapped in the biologically inert gas of lipid bilayer enclosure and be scattered in drug particles in the lipid bilayer shell.
Target polypeptide is for comprising the polypeptide or the protein derivatives of aminoacid sequence CGNKRTRGC (LyP-1 polypeptide).Be connected with biotin on the target polypeptide, thereby can connect by biotin-avidin-biotin bridge or biotin-Streptavidin-biotin bridging between target polypeptide and the lipid bilayer shell.
The lipid bilayer shell of present embodiment is phospholipid or phospholipid derivative; as 1; 2-two palmityls-sn-glyceryl-3-phosphatidic acid glyceryl-sodium salt (DPPG), 1; 2-distearyl acyl group-sn-glyceryl-3-phosphatidylcholine (DSPC), 1; 2-two palmityls-sn-glyceryl-3-phosphatidic acid-sodium salt (DPPA), 1,2-two palmityls-sn-glyceryl-3-phosphatidylcholine (DPPC) etc.Accordingly, can be on phospholipid or the phospholipid derivant connecting Polyethylene Glycol (PEG), biotin equimolecular, so that further be connected with other polypeptide, albumen etc.
Be to strengthen the stability of target drug-carrying ultrasonic microbubble, the lipid bilayer shell of present embodiment is enclosed with biologically inert gas, can be in perfluoropropane, perfluorinated butane and the sulfur hexafluoride at least a.
The drug particles of present embodiment can be paclitaxel, Docetaxel, amycin, camptothecine, 5-fluorouracil, cytosine arabinoside, methotrexate, antiinflammatory anodynin such as indometacin, Pu Luobipuluofen, ketone ibuprofen, piroxicam, diclofenac etc., and the soluble agents that can be used for treating breast carcinoma, as: activated protein, polypeptide, vaccine and gene.
In order to increase the targeting of medicine carrying ultrasonic microbubble, outside the lipid bilayer shell, connect polypeptide or the protein derivatives that contains the cancer target peptide sequence, the target drug-carrying ultrasonic microbubble that obtains can target tumor lymphatic vessel and tumor cell thereof, can generation, development and the curative effect of tumor be detected in real time and diagnose by the high frequency ultrasound imaging again, also can smash microvesicle and discharge drug particles by low frequency ultrasound, reach the controllably purpose of targeting release medicine, thereby prevention, diagnosis and the treatment of tumor all had extremely important meaning.
Below be the specific embodiment part:
As shown in Figure 1, the target drug-carrying ultrasonic microbubble of present embodiment is made of class lipid bilayer film wrapped biologically inert gas, and paclitaxel is encapsulated in the bilayer, and the LyP-1 polypeptide is fastened on the bilayer outside.Class lipid bilayer thin film and paclitaxel make by following mass fraction proportioning:
1,2-distearyl acyl group-sn-glyceryl-3-phosphatidylcholine (DSPC): 7 parts
1,2-distearyl acyl group-sn-glyceryl-3-phosphatidylcholine-macrogol 2000 (DSPC-PEG): 0.5 part
1,2-distearyl acyl group-sn-glyceryl-3-phosphatidylcholine-macrogol 2000-biotin (DSPC-PEG-Biotin): 0.5 part
Paclitaxel: 7/4～3 part.
Target polypeptide LyP-1 is by biotin-avidin-biotin bridge and 1, and 2-distearyl acyl group-sn-glyceryl-3-phosphatidylcholine-macrogol 2000-biotin connects.
The particle diameter of the target drug-carrying ultrasonic microbubble of present embodiment can be controlled between 0.5～8 μ m.
The preparation process of this target drug-carrying ultrasonic microbubble, can carry out as follows:
Step S1: press 7/4～3 part of mass fraction proportioning paclitaxel, 1,7 parts of 2-distearyl acyl groups-sn-glyceryl-3-phosphatidylcholine, 1,2,000 0.5 parts of 2-distearyl acyl groups-sn-glyceryl-3-phosphatidylcholine-macrogol, 1,0.5 part of 2-distearyl acyl group-sn-glyceryl-3-phosphatidylcholine-macrogol 2000-biotin is dissolved in the 5mL chloroform, preparation medicine phospholipid suspension;
Step S2: on turbine mixer with medicine phospholipid suspension mixing, at exsiccant N
2Remove chloroform under the stream effect and make phospholipid on chamber wall, form the layer of even thin film, vacuum drying 1～3 hour;
Step S3: in containing the container of dry phospholipid membrane, add the Tris buffer solution of the pH=7.4 that handles through the degassing, must prepare the medicine carrying phospholipid solution, comprise the glycerol of 10% volume and the propylene glycol of 10% volume in the Tris buffer solution;
Step S4: heating medicine carrying phospholipid solution is to more than the phase transition temperature (55～60 ℃), the ultrasonic concussion of water-bath disperses thoroughly until transparent the phospholipid solution of milk shape, wherein shaking frequency is 4000～5000 times/minute, and shock range is 10～40 millimeters, and the concussion time is 30～90 seconds;
Step S5: every milliliter one of transparent medicine carrying phospholipid solution is packed in the 2mL cillin bottle, and the air displacement in the cillin bottle is become perfluoropropane, machinery concussion preparation in 30～60 seconds medicine carrying ultrasonic microbubble;
Step S6: clean the medicine carrying ultrasonic microbubble 3～4 times with the centrifugal floating method of PBS buffer, to remove the phospholipid that does not form microvesicle;
Step S7: in the medicine carrying ultrasonic microbubble that cleaned, add Avidin, shake gently to hatch under the room temperature after 15 minutes and clean 3-4 time, remove not coupled Avidin with centrifugal floating method;
Step S8: the biotinylated target polypeptide of adding in the medicine carrying ultrasonic microbubble that step S7 obtains (biotin-LyP-1), shake gently also and hatch more than 10 minutes under the room temperature, remove unconjugated biotinylated target polypeptide 3～4 times with centrifugal floating method cleaning, obtain the target drug-carrying ultrasonic microbubble.
Below be target drug-carrying ultrasonic microbubble performance detection, study portion to making:
(1) the successful Rhizoma Nelumbinis of checking LyP-1 polypeptide and medicine carrying ultrasonic microbubble connects:
With Fluorescein isothiocyanate (fluorescein isothiocyanate, FITC) biotin-LyP-1 polypeptide is carried out fluorescent labeling, and the biotin behind the labelling-LyP-1 polypeptide and the medicine carrying ultrasonic microbubble that is connected with Avidin hatched, after the centrifugal floating method washing of PBS 3 times, place under the fluorescence microscope and observe, the result can successfully detect green fluorescence among the figure as shown in Figure 2, proof LyP-1 polypeptide and medicine carrying ultrasonic microbubble success Rhizoma Nelumbinis connect, and make LyP-1 target drug-carrying ultrasonic microbubble.
(2) entrapment efficiency of optimization target drug-carrying ultrasonic microbubble:
Add 0.75 respectively, 1.5,3,4.5mg taxol drug, prepare the LyP-1 targeted ultrasound microvesicle that carries the variable concentrations paclitaxel, measure the particle size distribution of respectively organizing microvesicle and the concentration that forms microvesicle, pass through the medicine carrying ultrasonic microbubble of chloroform cracking equivalent again by particle diameter/concentration analyzer, respectively organize the medicament contg of contained paclitaxel with the ultraviolet spectrophotometer assay determination, draw best medicine carrying ultrasonic microbubble parameter.The result is as shown in table 1:
Table 1
| Medicine addition (mg) | Microbubble concentration (10 8MBs/ml) | Envelop rate (%) |
| 0.75 | 5.95+0.79 | 62.92+2.38 |
| 1.5 | 4.99+0.43 | 54.52+2.19 |
| 3.0 | 4.14+0.29 | 50.39+1.68 |
| 4.5 | 3.51+0.36 | 42.98+1.47 |
Table 1 result shows the increase along with the drug particles addition, and the drug loading of medicine carrying ultrasonic microbubble also increases thereupon, but drug particles descends to some extent with the bubble rate that becomes of liposome.
(3) the target drug-carrying ultrasonic microbubble is to the specificity of tumor cell: adopt the LyP-1 targeted ultrasound microvesicle of medicine carrying to verify its adhesion and specific bond ability to breast cancer cell.
At first, prepare LyP-1 target drug-carrying ultrasonic microbubble according to the method described above, use 1 * 10
8Microvesicle/ml concn is added to the MDA-MB-231 breast cancer cell that adhere-wall culture is spent the night, adopt non-target drug-carrying ultrasonic microbubble simultaneously in contrast, after target drug-carrying ultrasonic microbubble and non-target drug-carrying ultrasonic microbubble hatched 5 minutes with breast carcinoma MDA-MB-231 cell respectively, with the centrifugal floating washing of PBS 3-4 time, examine under a microscope adhesiveness and the cell-specific of LyP-1 target drug-carrying ultrasonic microbubble to breast cancer cell.The result is shown in Fig. 3 A and Fig. 3 B, and the result shows that the target drug-carrying ultrasonic microbubble can combine and be attached on its cell surface with breast carcinoma MDA-MB-231 cell, but not the target drug-carrying ultrasonic microbubble then can not combine with breast carcinoma MDA-MB-231 cell.
(4) study the ultra sonic imaging characteristic of medicine carrying targeted microbubble to breast carcinoma:
The MDA-MB-231 cell attachment is grown on the coverslip, adopt LyP-1 target drug-carrying ultrasonic microbubble and cell to hatch, LyP-1 target drug-carrying ultrasonic microbubble is bonded to cell surface, with the non-target drug-carrying ultrasonic microbubble with the LyP-1 polypeptide not is contrast, two groups are carried out the B-mode ultra sonic imaging with ultrasonic probe and analyze, the ultra sonic imaging characteristic that compares target drug-carrying ultrasonic microbubble and non-target drug-carrying ultrasonic microbubble, the result as shown in Figure 4, the result shows that the LyP-1 target drug-carrying ultrasonic microbubble that is incorporated into breast carcinoma MDA-MB-231 cell can detect tangible ultrasonic signal under the B-mode condition.
(5) study the killing activity of target drug-carrying ultrasonic microbubble to breast carcinoma:
With 1 * 10
8The medicine carrying ultrasonic microbubble of microvesicle/ml concn is added to the breast carcinoma MDA-MB-231 cell that adhere-wall culture is spent the night, experiment grouping: complete untreated fish group, medicine carrying ultrasonic microbubble processed group not, non-target drug-carrying ultrasonic microbubble processed group, target drug-carrying ultrasonic microbubble processed group is carried out ultrasonic respectively and supersound process not to above four processed group.Analyze the target drug-carrying microvesicle under ultrasound condition to the mammary tumor cells specific killing activity.The result as shown in Figure 5, the result shows that the more non-target drug-carrying ultrasonic microbubble of target drug-carrying ultrasonic microbubble has stronger killing activity, embodies better breast cancer treatment effect.
Present embodiment adopts and makes target polypeptide at the bonded LyP-1 polypeptide of breast cancer tumour cell-specific, and this polypeptide has characteristics such as high specific, high selectivity and molecular weight are little, can be specifically in conjunction with tumor.The target drug-carrying ultrasonic microbubble that present embodiment makes is a kind of diagnosing tumor and treatment dual-functional ultrasonic contrast agent of having concurrently, by ultra sonic imaging can determine tumor in vivo particular location and generation, the state of development of tumor made diagnosis; Utilize the ultrasonic cavitation effect can make microbubble ruptures, discharge medicine, utilize acoustic horn effect to strengthen local blood capillary and permeability of cell membrane, increase targeting moiety blood drug level, reduce dosage and strengthen the effect of target administration thereby reach.
The above embodiment has only expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but can not therefore be interpreted as the restriction to claim of the present invention.Should be pointed out that for the person of ordinary skill of the art without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (11)
1. target drug-carrying ultrasonic microbubble, it is characterized in that, comprise the lipid bilayer shell, be fixed on the described lipid bilayer shell outside target polypeptide, be wrapped in the biologically inert gas of described lipid bilayer enclosure and be scattered in drug particles in the described lipid bilayer shell, described target polypeptide is polypeptide or the protein derivatives that comprises aminoacid sequence CGNKRTRGC.
2. target drug-carrying ultrasonic microbubble as claimed in claim 1 is characterized in that, described lipid bilayer shell comprises each component of following mass fraction:
1,7 parts of 2-distearyl acyl groups-sn-glyceryl-3-phosphatidylcholine,
1,2,000 0.5 parts of 2-distearyl acyl groups-sn-glyceryl-3-phosphatidylcholine-macrogol,
1,0.5 part of 2-distearyl acyl group-sn-glyceryl-3-phosphatidylcholine-macrogol 2000-biotin;
Described drug particles is a paclitaxel, and described 1, and the mass ratio of 2-distearyl acyl group-sn-glyceryl-3-phosphatidylcholine and described paclitaxel is 7～8: 2～3.
3. target drug-carrying ultrasonic microbubble as claimed in claim 2; it is characterized in that; also comprise Avidin; described target polypeptide is connected with biotin; described target polypeptide by biotin-avidin-biotin bridge with described 1,2-distearyl acyl group-sn-glyceryl-3-phosphatidylcholine-macrogol 2000-biotin connection.
4. target drug-carrying ultrasonic microbubble as claimed in claim 2; it is characterized in that; also comprise Streptavidin; described target polypeptide is connected with biotin; described target polypeptide by biotin-Streptavidin-biotin bridge with described 1,2-distearyl acyl group-sn-glyceryl-3-phosphatidylcholine-macrogol 2000-biotin connection.
5. target drug-carrying ultrasonic microbubble as claimed in claim 1 or 2 is characterized in that, the particle diameter of described target drug-carrying ultrasonic microbubble is 0.5～8 μ m.
6. target drug-carrying ultrasonic microbubble as claimed in claim 1 or 2 is characterized in that, described biologically inert gas is at least a in perfluoropropane, perfluorinated butane and the sulfur hexafluoride.
7. target drug-carrying ultrasonic microbubble as claimed in claim 1 is characterized in that, described lipid bilayer shell is phospholipid or phospholipid derivant.
8. target drug-carrying ultrasonic microbubble as claimed in claim 7; it is characterized in that; described phospholipid or phospholipid derivant are 1; 2-two palmityls-sn-glyceryl-3-phosphatidic acid glyceryl-sodium salt, 1; 2-two palmityls-sn-glyceryl-3-phosphatidic acid-sodium salt and 1, at least a in 2-two palmityls-sn-glyceryl-3-phosphatidylcholine.
9. target drug-carrying ultrasonic microbubble as claimed in claim 1, it is characterized in that, described drug particles is at least a in Docetaxel, amycin, camptothecine, 5-fluorouracil, cytosine arabinoside, methotrexate, indometacin, Pu Luobipuluofen, ketone ibuprofen, piroxicam and the diclofenac, and perhaps described drug particles is activated protein, polypeptide, vaccine or the gene that is used for the treatment of breast carcinoma.
10. the preparation method of a target drug-carrying ultrasonic microbubble as claimed in claim 3 is characterized in that, comprises the steps:
Step 1: press 7/4～3 part of mass fraction proportioning paclitaxel, 1,7 parts of 2-distearyl acyl groups-sn-glyceryl-3-phosphatidylcholine, 1,2,000 0.5 parts of 2-distearyl acyl groups-sn-glyceryl-3-phosphatidylcholine-macrogol, 1,0.5 part of 2-distearyl acyl group-sn-glyceryl-3-phosphatidylcholine-macrogol 2000-biotin is dissolved in preparation medicine phospholipid suspension in the solvent;
Step 2:, under the effect of exsiccant N2 stream, make phospholipid on chamber wall, form the layer of even thin film, vacuum drying 1～3 hour except that desolvating with described medicine phospholipid suspension mixing;
Step 3: in containing the container of dry phospholipid membrane, add the Tris buffer solution of the pH=7.4 that handles through the degassing, must prepare the medicine carrying phospholipid solution, comprise the glycerol of 10% volume and the propylene glycol of 10% volume in the described Tris buffer solution;
Step 4: heat described medicine carrying phospholipid solution to more than the phase transition temperature, the ultrasonic concussion of water-bath disperses thoroughly until transparent phospholipid solution;
Step 5: transparent medicine carrying phospholipid solution is divided in the device cillin bottle, and the air displacement in the cillin bottle is become biologically inert gas, shake 30～60 seconds preparation medicine carrying ultrasonic microbubbles;
Step 6: centrifugal floating method is cleaned the medicine carrying ultrasonic microbubble 3～4 times, to remove the phospholipid that does not form microvesicle;
Step 7: in the medicine carrying ultrasonic microbubble that cleaned, add Avidin, shake gently to hatch under the room temperature after 15 minutes and clean 3-4 time, remove not coupled Avidin with centrifugal floating method;
Step 8: in the medicine carrying ultrasonic microbubble that step 7 obtains, add biotinylated target polypeptide, shake gently also and hatch more than 10 minutes under the room temperature, remove unconjugated biotinylated target polypeptide 3～4 times with centrifugal floating method cleaning, obtain described target drug-carrying ultrasonic microbubble.
11. the preparation method of target drug-carrying microvesicle as claimed in claim 10 is characterized in that, in the described step 4, the concussion frequency is 4000～5000 times/minute, and shock range is 10～40 millimeters, and the concussion time is 30～90 seconds.
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