A kind of targeted developing microvesicle and preparation method thereof for tumour ultrasonic therapyTechnical field
The invention belongs to biomedical materials fields, and in particular to a kind of targeted developing microvesicle for tumour ultrasonic therapyAnd preparation method thereof.
Background technique
Malignant tumour is always to perplex the problem in the world, and increased incidence and mortality is that modern medicine faces year by yearA major challenge.Tumor tissues are made of two big Major Systems ingredients: tumor vessel and tumour cell.The growth of tumour cell andTransfer relies on the formation of new vessels, and new vessels are not only that the growth of tumour cell provides nutriment and oxygen, while also havingEffect transports its metabolite, and nutrition supply and the substance row of tumour cell are unable to satisfy if the not formation of new vesselsIt lets out, then the seriously growth and transfer of limitation tumour cell.Tumor vasculature targeting has been considered as treatment solid tumor at presentImportant means mainly includes intravascular intervention and anti-angiogenic drugs two ways.Intravascular intervention, which is that one kind is invasive, to be controlledIt treats, big, the medium vessels in main occlusion of bone tumors, offer limited effectiveness, and recurrence rate is higher；Equally, anti-angiogenic medicaments treatment is facedBed application effect is not good enough, and overall survival is without significantly improving, and, anti-angiogenic medicaments the same with other anti-tumor drugsThere are drug resistance phenomenon and toxic side effects.
Ultrasonic cavitation is increasingly taken seriously as a kind of noninvasive, safe and effective non-operative treatment.Ultrasonic cavitation have withLower several respects advantage: preferred, microvesicle is a kind of good pharmaceutical carrier, is easy in conjunction with drug and gene；Secondly, microvesicle conductA kind of highly effective cavitation nucleus is easy induced ultrasonic cavitation and occurs, and cavitation occurs to can produce " acoustic aperture effect in tumor vessel inner wallAnswer ", cause permeability of cell membrane to increase, is conducive to drug absorption；Finally, cavitation nucleus is close when blood vessel is perfused by high concentration microvesicleDegree increases significantly, and can cause strong cavitation effect, and mechanical injuries vascular endothelial cell makes basilar memebrane exposure, to startIntrinsic coagulation system leads to tumor microcirculation embolism, blocks tumor feeding.
Patent CN200610095075.8 discloses a kind of therapeutic type ultrasonic microbubble for tumour ultrasonic therapy, it includesThe acoustic contrast agent microvesicle and coagulation factor of core in gassiness body are adsorbed in acoustic contrast agent microbubble surface by coagulation factor, or byCoagulation factor is adsorbed in microbubble surface and is wrapped in the ultrasonic microbubble for constituting in it and combining coagulation factor.The therapeutic type ultrasonic microbubbleUnder treatment ultrasonication appropriate, local organization microcirculation thrombosis can be promoted, can be used for the disease treatments such as tumour.It shouldTherapeutic type ultrasonic microbubble is made in the ultrasonic energy (high intensity focused ultrasound, pulsed focus ultrasound or plane ultrasonic) of diversified formsUnder, controlled in a manner of local microcirculation thrombosis etc. by enhancing ultrasonic cavitation ablated tumor, ultrasound thermal ablation tumour or promotingTreat tumour.Although the method can promote local organization microcirculation thrombosis, it does not have selectivity, in occlusion of bone tumors hairWhile thin blood vessel also can embolism live healthy blood vessel, cause the damage of patient health tissue.Also, the simple mode by perfusionIncrease the concentration of capillary internal coagulant, embolism speed is slower.In addition, simple thrombus can only embolism within a short period of timeFirmly tumour capillary, thrombus gradually can be degraded and be metabolized, thin to tumour without the cooperation of other anti-tumor drugsCellular damage is limited.
Patent CN201110074404.1 discloses a kind of targeted drug-bearing ultrasonic microbubble, including lipid bilayer shell,The biologically inert gas for being fixed on the target polypeptide of lipid bilayer outer side of shell, being wrapped in lipid bilayer interior of shellBody and the drug granule being scattered in lipid bilayer shell, target polypeptide are to include amino acid sequence CGNKRTRGC'sPolypeptide or protein derivatives.Spread out by connecting more peptide or proteins containing cancer target peptide sequence outside lipid bilayer shellBiology, obtained targeted drug-bearing ultrasonic microbubble can with the lymphatic vessel and its tumour cell of target tumor, then by high frequency ultrasound atAs can generation, development and curative effect to tumour be measured in real time and diagnose, can also be smashed by low frequency ultrasound microvesicle releaseDrug granule achievees the purpose that controllably Targeting delivery drug, to have to the prevention of tumour, diagnosis and treatment extremely heavyThe meaning wanted.Moreover, it relates to a kind of preparation method of targeted drug-bearing ultrasonic microbubble.The method can be to the leaching of tumourHand shaft and its tumour cell carry out Targeting delivery drug, but in the case where not cutting off tumor blood supply to lymphangiogenesisAnd its tumour cell carries out Targeting delivery drug, effect is limited.
The present invention passes through the target polypeptide or protein derivatives of lipid bilayer outer side of shell, passes through specific adsorption tumourThe specific expressed vascular endothelial growth factor of vascular endothelial cell (Vascular Endothelial Growth Factor,VEGF) receptor and specific adsorption tumor vessel, and in ultrasonication next part cavitation, coagulation factor is discharged, hair is quickly formedThin blood vessel embolism, tumor vessel can not be concentrated without specific adsorption and coagulation factor by solving patent CN200610095075.8The slow disadvantage of blood vessel embolism caused by cavitation.In addition, drug granule has been discharged into tumour in microvesicle cavitation processesIn blood vessel embolism substance, make drug slow release, while physics embolism, can drug therapy tumour, solve patentThe problem that CN201110074404.1 can only rely on drug therapy tumor effect poor merely.The present invention is to the diagnosis of tumour and controlsTreatment has extremely important meaning.
Summary of the invention
For the above-mentioned prior art, the purpose of the present invention is to provide a kind of targeted developing for tumour ultrasonic therapy is micro-Bubble, the targeted developing microvesicle can not only be developed with high frequency ultrasound, carry out detection and diagnosis to tumour, and can be to tumour capillaryBlood vessel carries out the quick embolism of specificity, and can carry out specific drug treatment to tumor tissues.The invention further relates to a kind of useIn the preparation method of the targeted developing microvesicle of tumour ultrasonic therapy.
To achieve the above object, the present invention uses following scheme:
A kind of targeted developing microvesicle for tumour ultrasonic therapy, which is characterized in that including lipid bilayer shell, be fixed onThe target polypeptide or protein derivatives of outer side of shell wrap up biologically inert gas inside the housing and are scattered in shellCoagulation factor and drug granule.The target polypeptide or protein derivatives are can be with vascular endothelial growth factor receptor specificityIn conjunction with polypeptide or protein derivatives.
The lipid bilayer shell is phosphatide or phospholipid derivative, and the phosphatide or phospholipid derivative are 1,2-, bis- palm fibrePalmitic acid docosahexaenoyl-sn-glycero -3- phosphatidic acid glyceryl-sodium salt DPPG, 1,2- distearyl acyl group-sn- glyceryl -3- phosphatidyl gallbladderAlkali DSPC, 1,2-, bis- palmityl-sn- glyceryl -3- phosphatidic acid-sodium salt DPPA or 1, bis- palmityl-sn- glyceryl of 2- -One of 3- phosphatidyl choline DPPC or a variety of.The phosphatide or phospholipid derivative for wherein having 5-10% are by polyethylene glycol-biologyElement, one of polyethylene glycol a variety of are modified.
The target polypeptide or protein derivatives pass through streptavidin and modify phosphorus by polyethylene glycol-biotinRouge or phospholipid derivative are connected.
The partial size of the targeted developing microvesicle is 1-5 μm.
The biologically inert gas is one of perfluoropropane, perfluorinated butane and sulfur hexafluoride or a variety of.
The coagulation factor is factor, fibrinogen and protamine sulfate, in microvesicle suspension concentration pointIt is not 10-100mg/ml, 20-200mg/ml and 0.1-10mg/ml, mass ratio 1-2:2-1:0.01-1.
The drug granule be Docetaxel, adriamycin, camptothecine, 5 FU 5 fluorouracil, cytarabine, amethopterin,One of indocin, Pu Luobipu ibuprofen, Ketoprofen, feldene, diclofenac, activated protein, polypeptide, gene orIt is a variety of, it is 0.1-1.5mg/ml in microvesicle suspension concentration.
The target polypeptide or protein derivatives be by biotin modification, and include QKRKRKKSRYKS,The polypeptide or protein derivatives of QKRKRKKSRKKH, RKRKRKKSRYIVLS or CHVILHPRC amino sequence, target polypeptide or eggWhite derivative: phosphatide or phospholipid derivative the mass ratio of the material are 5-10:100.
The present invention also provides a kind of preparation method of targeted developing microvesicle for tumour ultrasonic therapy, specific steps are as follows:
(1) phosphatide or phospholipid derivative, anticoagulant factor and drug granule are dissolved in solvent and prepare lipid suspensions in proportion,Middle solvent is one of dimethyl sulfoxide, methanol, ethyl alcohol, methylene chloride, chloroform or a variety of.
(2) lipid suspensions are mixed, removes solvent under dry nitrogen stream effect, makes phosphatide on the wallOne layer of uniform film is formed, is dried in vacuo 3-5 hours.
(3) the Tris buffer solution that the pH value of degassed processing is 7.4 is added in the container containing dry phospholipid membrane,And more than heated solution to phase transition temperature, it is thoroughly transparent that water bath sonicator concussion is dispersed to phospholipid solution.The Tris bufferingSolution includes the glycerol of 10% volume and the 1,2-PD of 10% volume.The oscillation frequency is 4000-5000 beats/min, shakeSwinging amplitude is 10-40mm, and the concussion time is 60-90s.
(4) it will shake into transparent phospholipid solution separating device cillin bottle, and by the air displacement in cillin bottle at biologyInert gas, concussion 30-60s prepare ultrasonic microbubble.It is centrifuged floating method to clean ultrasonic microbubble 3-4 times, to remove not formed microvesiclePhosphatide.
(5) streptavidin is added in the ultrasonic microbubble after cleaning, gently shakes and is incubated for 15 minutes at room temperatureIt is cleaned 3-4 times with centrifugation floating method afterwards, removes the streptavidin not coupled.
(6) target polypeptide or protein derivatives of biotin modification are added into the ultrasonic microbubble that step 5 obtains, gently shakesMove and be incubated for 10 minutes at room temperature or more, the targeting for cleaning the 3-4 unbonded biotin modification of removing with centrifugation floating method is morePeptide or protein derivative obtains a kind of targeted developing microvesicle for tumour ultrasonic therapy.
The invention has the advantages that:
1. microvesicle of the present invention can specific adsorption tumor vessel inner wall realize to tumour capillary to collect cavitationQuick embolism.And it due to the specific adsorption to tumor vessel inner wall, will not be made while occlusion of bone tumors capillaryAt the embolism of healthy blood vessel.
2. anti-tumor drug can be wrapped in thrombus by microvesicle of the present invention while forming embolism, realize anti-swollenThe slow release of tumor medicine carries out continued treatment to tumour.
Detailed description of the invention:
Fig. 1 is a kind of structural schematic diagram of targeted developing microvesicle for tumour ultrasonic therapy.
Fig. 2 is that a kind of targeted developing microvesicle for tumour ultrasonic therapy illustrates tumour capillary specific adsorptionFigure.
Fig. 3 is targeted developing microvesicle oncotherapy effect assessment.Abscissa is the time, and ordinate is gross tumor volume.As a resultShow that targeted developing microvesicle of the present invention plays good tumor inhibition effect.
Specific embodiment
Technical solution of the present invention is described in further detail with comparative example with reference to embodiments.But the present invention is notIt is restricted by the specific examples.Method therefor is conventional method unless otherwise instructed in embodiment.
Embodiment 1: by 90 parts of 1,2-distearoyl-sn-glycero-3-phosphatidylcholine, 5 parts of 1,2- distearylsBase-sn- glyceryl -3- phosphatidyl choline-polyethylene glycol, 5 parts of 1,2-distearoyl-sn-glycero-3-phosphatidylcholine -Polyethylene glycol-biotin, 250mg factor, 500mg fibrinogen, 25mg protamine sulfate and 4mg DocetaxelIt is dissolved in 1ml chloroform, mixes.Room temperature be passed through it is dry be dried with nitrogen, be dried in vacuo 4h, eliminate chloroform completely.PH value, which is added, is7.4 Tris buffer solution 5ml(volume ratio: Tris: glycerol: 1,2-PD=8:1:1).It is placed in 60 DEG C of water bath sonicator cleaningsWith 4500 beats/min of oscillation frequency in instrument, 25mm shock range shakes 75s, obtains transparent phospholipid solution.Cillin bottle point5 bottles are filled, with air in perfluoropropane displacement cillin bottle, after shaking 45s, 4min is centrifuged with 400g/min, is washed with PBS buffer solution3 this.Streptavidin presses every 1 × 1073 μ g are added in a microvesicle, are incubated at room temperature 15min, and centrifuge washing 3 times.ContainThe target polypeptide by biotin modification of QKRKRKKSRKKH amino sequence presses 1 × 1078 μ g, incubation at room temperature is added in a microvesicle15min is centrifuged 3 times, that is, completes a kind of preparation of targeted developing microvesicle for tumour ultrasonic therapy.
Embodiment 2: each step is equal to embodiment 1, but the difference is that 1,2- distearyl acyl group-sn- glyceryl -3- phosphatidePhatidylcholine is 95 parts, 1,2-distearoyl-sn-glycero-3-phosphatidylcholine-polyethylene glycol is 2.5 parts, 1,2- distearylDocosahexaenoyl-sn-glycero -3- phosphatidyl choline-polyethylene glycol-biotin is 2.5 parts.
Embodiment 3: each step is equal to embodiment 1, but the difference is that 1,2- distearyl acyl group-sn- glyceryl -3- phosphatidePhatidylcholine is 93 parts, 1,2-distearoyl-sn-glycero-3-phosphatidylcholine-polyethylene glycol is 3.5 parts, 1,2- distearylDocosahexaenoyl-sn-glycero -3- phosphatidyl choline-polyethylene glycol-biotin is 3.5 parts.
Embodiment 4: each step is equal to embodiment 1, but the difference is that the amino sequence that target polypeptide includes isRKRKRKKSRYIVLS。
Embodiment 5: each step is equal to embodiment 1, but the difference is that the amino sequence that target polypeptide includes isQKRKRKKSRYKS。
Embodiment 6: each step is equal to embodiment 1, but the difference is that polyenoid Japanese yew additional amount is 0.5mg.
Embodiment 7: each step is equal to embodiment 1, but the difference is that polyenoid Japanese yew additional amount is 6mg.
Comparative example 1: each step is equal to embodiment 1, but the difference is that does not add factor, fibrinogen and sulfuric acid fishProtamine.
Comparative example 2: each step is equal to embodiment 1, but the difference is that does not add Docetaxel.
Comparative example 3: each step is equal to embodiment 1, but the difference is that does not add target polypeptide.
Comparative example 4: each step is equal to embodiment 1, but the difference is that 1,2- distearyl acyl group-sn- glyceryl -3- phosphatidePhatidylcholine is 98 parts, 1,2-distearoyl-sn-glycero-3-phosphatidylcholine-polyethylene glycol is 1 part, 1,2- distearylBase-sn- glyceryl -3- phosphatidyl choline-polyethylene glycol-biotin is 1 part.
Targeted developing microvesicle partial size and concentration mensuration test:
The embodiment 1-7 and comparative example 1-4 targeted developing microvesicle suspension prepared is diluted to 2ml with distilled water, takes 10 μ l microvesiclesSuspension measures particle size in AccuSizer780A particle size analyzer and concentration is as shown in table 1.
By embodiment 1, embodiment 6, embodiment 7 and comparative example 2 in table 1 it is found that with anti-tumor drug additional amount increasingMore, microbubble concentration gradually decreases；By embodiment 1-3, comparative example 1 and comparative example 3 it is found that with microvesicle target polypeptide content increasingAdd, the partial size of microvesicle is gradually increased.
The test of targeted developing microcapsular ultrasound imaging effect:
By MDA-MB-231 cell adherent growth in the targeted developing on coverslip, prepared using embodiment 1-7 and comparative example 1-4Targeted developing microvesicle with cell incubation, is bound to cell surface respectively by microvesicle, carries out B-mode ultrasonic imaging with ultrasonic probeAnalysis, compares the ultrasonic imaging characteristic of targeted developing microvesicle, the results showed that the targeting of embodiment 1-7 and comparative example 1-4 preparation is aobviousShadow microvesicle is able to detect that apparent ultrasonic signal under the conditions of B-mode.
Targeted developing microvesicle adherence test:
By tumor vascular endothelial cell culture to 24 orifice plate bottoms of covering, culture solution is removed, is cleaned cell 3 times with PBS, respectively will100 μ l concentration are 2 × 107The targeted developing microvesicle of embodiment 1-7 and comparative example the 1-4 preparation of a/ml is added in 24 orifice plates,It is incubated at room temperature 5min, comes into full contact with microvesicle with cell surface.Microvesicle suspension is drawn, is cleaned cell 5 times with PBS, in 200 times of lightIt learns 5 picture of random picture under inverted microscope and carries out data analysis.The results are shown in Table 2.
As shown in Table 2, the made microvesicle of embodiment to the adherency number of single tumor vascular endothelial cell all reached 5 withOn, and not up to the present invention claims the adherency of the comparative example 4 of range for the comparative example 3 and target polypeptide additional amount for not adding target polypeptideNumber only has 0.01 and 2.15. to illustrate that targeted developing microvesicle of the present invention imitates tumor vascular endothelial cell with specific adhesionFruit.
The test of targeted developing microvesicle oncotherapy effect assessment
(1) mouse prostate cancer cell RM-1 cell culture
It is removed from liquid nitrogen to recovery Prostate Carcinoma of Mice RM-1 cell strain, is put into rapidly in 37 DEG C of water-baths and thaws rapidly, thenIt is centrifuged, removes supernatant, 1ml is added containing the DMEM of 10% peptide cow's serum, 1% penicillin/streptomycin and accompanies feeding base, piping and druming is mixed, then movedEnter to have added the 25cm of the full culture medium of 3ml2Tissue Culture Flask in, in 37 DEG C, 5%CO2It is cultivated in incubator, passage 1 in every 2 to 3 daysIt is secondary, it is tested when cell is grown in logarithmic growth phase.
(2) mouse tumor model of angiogenesis is established
Logarithmic growth phase RM-1 cell, PBS are washed twice, and are carried out digestion 2-3min with pancreatin, cell are collected after centrifugation, simultaneouslyTwice with PBS eccentric cleaning, living cell counting under Trypan Blue microscope；Cell concentration is adjusted to 2 × 10 with PBS7A/The single cell suspension of ml；Iodine disinfection right side of mice skin, 1ml asepsis injector inoculate, every C57 mouse inoculationThe cell of 0.2ml is in right side of mice subcutaneous abdomen.After inoculated tumour cell, mouse inoculation position is gently stroked with finger, to energyTumor tubercle is enough touched, as at tumor markers, observes the tumor formation situation of mouse.10 days to 14 days or so after inoculation, observeMouse tumor formation rate 100%, when tumour growth is about 0.5cm × 0.8cm, mouse tumor model of angiogenesis, which is established, to be completed.
(3) targeted developing microvesicle oncotherapy effect assessment is tested
Grouping: model mice is divided into 4 groups, every group 10, injects embodiment 1, comparative example 1, comparative example 2 and comparative example 3 respectivelyThe targeted developing microvesicle of preparation.The ultrasound of identical energy and time is cooperated to carry out oncotherapy.The following institute of specific implementation methodShow.
Model mice is anaesthetized before experiment, penta bar of 150 μ l 1% is subcutaneously injected with 1ml asepsis injector for every model mouseThan appropriate sodium solution, dosage is by weight 80mg/kg.The chaeta that tumor locus epidermis is sloughed with depilatory cream, then uses mouseAdhesive tape is fixed on experimental bench, and using Sequoia512 diasonograph, 15L8-S high-frequency linear array is popped one's head in, frequency 7.0MHz,Ultrasonic imaging observation is carried out with CPS imaging technique, each image-forming condition is consistent, and is provided with Ultrasound Instrument work station storage imagingMaterial, M is set as 0.09 when ultrasonic imaging, and M is set as 1.9, gain -27dB, focusing range, time gain compensation when microvesicle smashesEtc. parameters be adjusted to optimum state.
Ultrasonic imaging and treatment method: 300 μ l of tail vein injection contains 1 × 108 targeted developing microvesicle, uses CPS after 30sThe ultrasonic signal of imaging method acquisition 10s tumor locus；Wait 7min (microvesicle recycles 7min in vivo) so as to microvesicle and blood vesselEndothelial cell adhesion combines, and the microvesicle not sticked is removed in cyclic process in vivo；Then 10s tumour portion is acquired againThe ultrasonic signal of position.It the use of MI is that 1.9 (centre frequency 5.0MHz) microbubble disruption pulse trains irradiate tumor locus 3s, withSmash all microvesicles in sound field.It observes the specific ultrasonoscopy of tumor vessel blood circumstance and embolization effect is as shown in table 3.It is treatingThe length and width of continuous 14 days measurement tumours afterwards, gross tumor volume be approximately equal to it is long multiplied by wide square divided by 2, concrete outcome such as Fig. 3It is shown.
As table 3 and Fig. 3 it is found that targeted developing microvesicle described in embodiment 1 not only has good development effect, and can shapeAt the targeting embolism of tumour capillary, and sustainable effectively tumour is inhibited.And targeted developing described in comparative example 1 is micro-Coagulation factor is not added for bubble, cannot cause the embolism of tumour capillary.Microvesicle described in comparative example 3 although joined blood coagulation becauseSon, but since it does not have targeting, can not be assembled in tumor vessel, it is discharged to can not be concentrated to coagulation factor,Also fail to cause the embolism of tumour capillary.Although targeted microbubble described in comparative example 2 also results in the target of tumour capillaryTo embolism, but anti-tumor drug is free of in microvesicle, the Drug inhibition of continuous and effective can not be carried out to tumor locus.
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned realityIt applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not depositedIn contradiction, it is all considered to be the range of specification record.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneouslyLimitations on the scope of the patent of the present invention therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the artFor, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to guarantor of the inventionProtect range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.