CN103100093A - Load small interfering RNA nanoscale lipid microbubble ultrasonic contrast agent and preparation method - Google Patents
Load small interfering RNA nanoscale lipid microbubble ultrasonic contrast agent and preparation method Download PDFInfo
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Abstract
The invention discloses a load small interfering RNA (siRNA) nanoscale lipid microbubble ultrasonic contrast agent and a preparation method. The load siRNA lipid microbubble is formed by preparing DPPC (Dipalmitoyl Phosphatidyl Choline), DSPE (1, 2-distearoyl-sn-glycero-3-phosphoethanolamine) and DPPA (Diphenyl Phosphoryl Azide) into microbubbles (containing octafluoropropane) according to the weight part ratio of 18:1:1 and then assembling together with PEG-PLL (Polyethylene Glycol-Polylysne)-coated siRNA nanomicelle. The load siRNA nanoscale lipid is nanosclae, has an obvious ultrasonic contrast effect, and can generate obvious siRNA cell transfection efficiency under low-frequency ultrasonic irradiation, thereby further hopefully having important research values and application prospects in the fields of ultrasonic diagnosis and gene treatment.
Description
Technical field
The present invention relates to ultrasound molecular iconography and biomedical engineering field, particularly, relate to nanoscale lipid microvesicle ultrasound angiography agent of a kind of load siRNA (siRNA) and preparation method thereof.
Background technology
Ultrasonic contrast is as the revolution for the third time in ultrasonic imaging technique, and particularly diagnosis and the Differential Diagnosis of neoplastic disease provide effective foundation for various diseases.Along with the fast development of ultrasonic imaging technique, find that microvesicle can reduce the threshold value of cavitation effect as the cavitation nucleus of ultrasonic cavitation effect as developing agent on the one hand on the other hand in ultrasonic contrast, promote the targeted delivery of genetic fragment.Studies have shown that in a large number, as the cavitation core, under low frequency high-energy ultrasonic irradiation, can produce cavitation effect with microbubble, discharge huge energy, high temperature, high pressure and injection stream in this process.Under the effect of cavitation effect, the cell membrane around microvesicle was opened by moment, produced the half-life and be the ceasma of 20 to 50 milliseconds, rapid closing then, and this effect is called as " acoustic horn effect ".By the cell membrane in the sound hole that is opened, pericellular genetic fragment is delivered to endochylema passively under the effect of cavitation effect in, realize that the cell of gene is sent.This target gene delivery system of realizing by cavitation effect and acoustic horn effect is called ultrasonic targeted microbubble and destroys (UTMD) technology.At present, being applied to the UTMD technology, to carry out the microvesicle of gene delivery be mainly commercial microbubble contrast agent, as sound Novi etc.
But, commercial microvesicle has as gene delivery vector (particularly tumor target gene delivery vector) and exists obvious deficiency: 1. diameter is excessive, because present acoustic contrast agent on the market is all micron level, can only be confined in vascular system, can't be by around tumor vascular endothelium gap arrival tumor cell; 2. can not the load genetic fragment, because on the market microvesicle is surperficial electronegative microvesicle, and genetic fragment (plasmid DNA, siRNA etc.) surface is all with negative electricity, both effectively combinations.Therefore, present commercial microcapsular ultrasound contrast agent can not be called proper genophore.
For the high-efficiency delivery genetic fragment and realize tumor target gene transmission, be necessary to prepare a kind of novel acoustic contrast agent as genophore, and solve simultaneously technical barrier.1. particle diameter is little, is in nanoscale, can realize by the blood vessel endothelium gap (380～780 nm) of tumor broadening the tumor passive target gathering of contrast agent; 2. can the high-efficient carrier genetic fragment, it is loaded in microbubble contrast agent, realize the synchronized delivery of contrast agent and genetic fragment; 3. have good enhancing ultrasonic development ability, realize the application of ultrasonic contrast under the diagnostic ultrasound condition; 4. have ultrasonic sensitive, excite the cavitation effect of microvesicle under the condition of low frequency high-energy ultrasonic irradiation, realize that the targeting of genetic fragment discharges.
Summary of the invention
The object of the invention is to for present existing acoustic contrast agent as the deficiency that the siRNA carrier exists, a kind of nanoscale lipid microvesicle ultrasound angiography agent (hereinafter referred siRNA nanometer microvesicle) that can load siRNA is provided.
Another object of the present invention is to provide the preparation method of above-mentioned microbubble contrast agent.
The present invention is achieved through the following technical solutions above-mentioned purpose:
The nanoscale lipid microvesicle ultrasound angiography agent of a kind of load siRNA, described contrast agent can high-efficient carrier siRNA, and siRNA is loaded on the surface of whole microbubble structure, forms integral body with the agent of nanoscale lipid microvesicle ultrasound angiography; The diameter of the nanoscale lipid microbubble of described load siRNA is 200～500 nm.
Contrast agent is by the positive and negative charge captivation as mentioned above, and the siRNA nano-micelle of surface band positive electricity is loaded on surperficial electronegative nanoscale lipid microvesicle ultrasound angiography agent surface, forms an overall structure.
The preparation method of the nanoscale lipid microvesicle ultrasound angiography agent of a kind of siRNA of load as mentioned above specifically comprises the steps:
S1. take phospholipid composition dipalmitoyl phosphatidyl choline, DSPE and DPPA as the film material, obtain the agent of electronegative nanoscale lipid microvesicle ultrasound angiography by thin film-aquation legal system is standby;
S2. with Polyethylene Glycol (Polyethyeneglycol, can be abbreviated as PEG) and polylysine (Polylysine, can be abbreviated as PLL) form two block cation copolymers and siRNA and independently fill during as 2 ~ 8:1 take the ratio of N/P ratio (mol ratio of the nitrogen-containing group of copolymer and the phosphorus-containing groups of siRNA), obtain the siRNA nano-micelle of positively charged;
S3. in adding excessive S2 in the electronegative nanoscale lipid microbubble that S1 obtains, the siRNA nano-micelle of positively charged, hatch the nanoscale lipid microvesicle ultrasound angiography agent for preparing load siRNA.
Particularly, thin film described in step S1-aquation legal system is as follows for the step that obtains the agent of electronegative nanoscale lipid microvesicle ultrasound angiography:
S11. with phospholipid composition dipalmitoyl phosphatidyl choline (DPPC), DSPE (DSPE), DPPA (DPPA) is dissolved in chloroform and is placed in culture dish,
S12. treating in fume hood that chloroform volatilizees naturally forms phospholipid membrane, add 37 ~ 60 ℃ of aquations of distilled water after 0.5 ~ 2 hour, lipid soln is transferred in centrifuge tube, utilized Ultrasonic Cell Disruptor to carry out sound and shook 1 ~ 10 minute, pass into simultaneously octafluoropropane gas generation lipid microbubble;
S13. with standing 10 ~ 30 minutes of microvesicle liquid, then with 1000 ~ 2000rpm centrifugal 1 ~ 10 minute, draw lower floor's milky white liquid and be prepared surperficial electronegative nanometer microvesicle.
Preferably, the ratio of weight and number of dipalmitoyl phosphatidyl choline, DSPE and DPPA described in step S1 is 18:1:1 ~ 5.
More preferably, the ratio of weight and number of dipalmitoyl phosphatidyl choline, DSPE and DPPA described in step S1 is 18:1:1.
Preferably, for removing the siRNA micelle that is not attached to the microvesicle surface, incubate described in step S3 and can utilize the supercentrifugal process purified product after abundant, namely used 2000 rpm centrifugal 30 minutes, discard subnatant, add 4 milliliters of distilled waters resuspended, cyclic washing three times is used the resuspended end-product that gets of 1 ml phosphate buffer (pH=7.4) at last.
Further, described in S1, electronegative nanoscale lipid microbubble includes octafluoropropane gas.
Preferably, (the Polyethyeneglycol of Polyethylene Glycol described in step S2, can be abbreviated as PEG) form two block cation copolymers (can write a Chinese character in simplified form into PEG-PLL) with polylysine (Polylysine can be abbreviated as PLL), wherein the molecular weight of Polyethylene Glycol is 500 ~ 3500; The mean molecule quantity of polylysine described in step S2 is 6000 ~ 10000, and concrete synthetic method is as follows: the PEG (mPEG-NH of S21. macromole evocating agent Alpha-Methyl-omega-amino-
2) be that the list of references method is by methoxy poly (ethylene glycol) (mPEG-OH) preparation [Neal JC, Stolnik S, Schacht E, Kenawy ER, Garnett MC, Davis SS, IIIum L. Pharm Sci 1998; 87:1242-1248]; S22. benzyloxycarbonyl group is protected lysine anhydride (Benzyloxycarbonyl-L-Lysine N-carboxylic anhydride, CBLLys-NCA) be according to literature method (Daly HW, Poche D. Tetrahedron Lett 1988,29:5859-5862; Zhang XQ, Li JG, Li W, Zhang AF. Biomacromolecules 2007,8:3557-3567) synthetic by lysine hydrochloride (L-Lysine ﹒ HCl, Chinese traditional Chinese medicines chemical reagent company limited); S23. PEG-b-poly-(benzyloxycarbonyl group-lysine) (mPEG-b-PCBLLys) block copolymer be PEG mPEG-NH with Alpha-Methyl-omega-amino-
2As macromole evocating agent, the ring-opening polymerization of the lysine anhydride by causing the benzyloxycarbonyl group protection is synthetic; Concrete steps are, under argon shield, in the reaction bulb of the drying that is added with magnetic stir bar, PEG 0.5 ~ 2 gram and benzyloxycarbonyl group protection lysine anhydride 6 ~ 10 grams that add Alpha-Methyl-omega-amino-, after the anhydrous dimethyl formamide dissolving, be placed on 33 ~ 37 ℃ of oil bath reactions 2 ~ 4 days; After reaction finishes, with the reaction system ether sedimentation, filter, wash, under room temperature at vacuum drying, the white powder that obtains;
S24. the preparation of diblock copolymer PEG-b-PLL (mPEG-b-PLLys): copolymer PEG-b-in S23 poly-(benzyloxycarbonyl group-lysine) is dissolved in trifluoroacetic acid, under condition of ice bath, add the glacial acetic acid solution of hydrogen bromide; After stirring under room temperature, with excessive ether washing reaction system washing; After solvent evaporated, at vacuum drying, obtain the end-product powder of Lycoperdon polymorphum Vitt under room temperature.
The invention has the beneficial effects as follows:
The particle diameter of the nanoscale lipid microvesicle ultrasound angiography agent of the load siRNA for preparing by this method is little, is in nanoscale, can realize that the tumor passive target of contrast agent assembles by the blood vessel endothelium gap (380～780 nm) of tumor broadening.
The load siRNA nanoscale lipid microvesicle ultrasound angiography agent for preparing by this method can high-efficient carrier siRNA, and it is loaded in microbubble contrast agent, realizes the synchronized delivery of contrast agent and siRNA.When carrying siRNA in vivo, can realize the distribution monitoring of siRNA by the method for ultrasonic contrast.
The agent of described load siRNA nanoscale lipid microvesicle ultrasound angiography has good enhancing ultrasonic development ability, realizes the application of ultrasonic contrast under the diagnostic ultrasound condition; Have ultrasonic sensitive, excite the cavitation effect of microvesicle under the condition of low frequency high-energy ultrasonic irradiation, realize that the targeting of siRNA discharges.Can realize the integrated of the interior video picture of tumor body and treatment.
Description of drawings
Fig. 1. siRNA nanometer microvesicle is done the result that transmission electron microscope detects.
Fig. 2. siRNA nanometer microvesicle is carried out the detection of the detection of ultrasonic development ability and ultrasonic irradiation sensitivity; A and C represent respectively siRNA nanometer microvesicle and the nanometer microvesicle development usefulness under the ultrasonic contrast pattern; B and D represent that respectively siRNA nanometer microvesicle and nanometer microvesicle are through the development effect under the ultrasonic contrast pattern after low frequency ultrasound irradiation.
The siRNA transfection usefulness of Fig. 3 .siRNA nanometer microvesicle cell under low frequency ultrasound irradiation detects; Ultrasonic (+) is the siRNA nanometer microvesicle of fluorescent material Cy3 labelling siRNA; Ultrasonic (-) be the Cy3 labelling siRNA but without ultrasonic irradiation; Contrast is for only to add the siRNA of Cy3 labelling but to process without ultrasonic irradiation.
Fig. 4. laser confocal microscope detects the interior distribution results of cell of siRNA; Ultrasonic (+) is the siRNA nanometer microvesicle of fluorescent material Cy3 labelling siRNA; Ultrasonic (-) be the Cy3 labelling siRNA but without ultrasonic irradiation; Contrast as only adding siRNA but process without ultrasonic irradiation.
The specific embodiment
Further explain the present invention below in conjunction with embodiment, but embodiment does not do any type of restriction to the present invention.
In order to narrate conveniently, the agent of load siRNA nanoscale lipid microvesicle ultrasound angiography is referred to as siRNA nanometer microvesicle; The agent of electronegative nanoscale lipid microvesicle ultrasound angiography is referred to as the nanometer microvesicle; The siRNA nano-micelle of positively charged is called for short the siRNA micelle.
Embodiment 1
S1. surperficial electronegative nanoscale lipid microvesicle ultrasound angiography agent (hereinafter referred nanometer microvesicle) preparation:
Utilize thin film-aquation method to prepare the nanometer microvesicle take phospholipid as raw material.With phospholipid composition dipalmitoyl phosphatidyl choline (DPPC), DSPE (DSPE), DPPA (DPPA) 18:1:1 by weight is dissolved in chloroform and is placed in the culture dish of 9 centimetres of diameters, treats in fume hood that chloroform volatilizees naturally to form phospholipid membrane.Add 37 ℃ of aquations of 4 milliliters of distilled waters after 1 hour, lipid soln is transferred in 50 milliliters of centrifuge tubes, utilize Ultrasonic Cell Disruptor to carry out sound and shook 5 minutes, pass into simultaneously octafluoropropane gas generation lipid microbubble.With standing 10 minutes of microvesicle liquid, then with 1000 rpm centrifugal 5 minutes, draw lower floor's milky white liquid and be prepared surperficial electronegative nanometer microvesicle.
S2. the preparation of the siRNA nano-micelle of surface band positive electricity:
Utilize Polyethylene Glycol (Polyethyeneglycol, can be abbreviated as PEG) and polylysine (Polylysine, can be abbreviated as PLL) form two block cation copolymers (can write a Chinese character in simplified form into PEG-PLL), wherein the mean molecule quantity of PEG and PLL is 2000 and 8000.PEG-PLL and siRNA are carried out proportioning take N/P ratio (mol ratio of the nitrogen-containing group of copolymer and the phosphorus-containing groups of siRNA) ratio as 5:1, both hatched 20 minutes under outdoor, can be prepared into positively charged and the siRNA nano-micelle.
Polyethylene Glycol (Polyethyeneglycol can be abbreviated as PEG) is as follows with the preparation method that polylysine (Polylysine can be abbreviated as PLL) forms two block cation copolymers:
S21. PEG (the mPEG-NH of macromole evocating agent Alpha-Methyl-omega-amino-
2) be that the list of references method is prepared by methoxy poly (ethylene glycol) (mPEG-OH).[Neal JC, Stolnik S, Schacht E, Kenawy ER, Garnett MC, Davis SS, IIIum L. Pharm Sci 1998; 87:1242-1248]; S22. benzyloxycarbonyl group is protected lysine anhydride (Benzyloxycarbonyl-L-Lysine N-carboxylic anhydride, CBLLys-NCA) be according to literature method (Daly HW, Poche D. Tetrahedron Lett 1988,29:5859-5862; Zhang XQ, Li JG, Li W, Zhang AF. Biomacromolecules 2007,8:3557-3567) synthetic by lysine hydrochloride (L-Lysine ﹒ HCl, Chinese traditional Chinese medicines chemical reagent company limited); S23. PEG-b-poly-(benzyloxycarbonyl group-lysine) (mPEG-b-PCBLLys) block copolymer be PEG mPEG-NH with Alpha-Methyl-omega-amino-
2As macromole evocating agent, the ring-opening polymerization of the lysine anhydride by causing the benzyloxycarbonyl group protection is synthetic; Concrete steps are under argon shield, in the reaction bulb of the drying that is added with magnetic stir bar, to add the mPEG-NH of amount of calculation
2(1.0 g, 0.5 mmol) and CBLLys-NCA (8.4 g, 27.5 mmol) after 20 ml anhydrous dimethyl formamide (DMF) dissolvings, are placed on 35 ℃ of oil bath reactions 3 days.After reaction finishes, with the reaction system ether sedimentation, filter, wash, under room temperature at vacuum drying, white powder 7.6 grams (92%) that obtain; S24. diblock copolymer PEG-b-PLL (mPEG-b-PLLys) is that copolymer mPEG-b-PCBLLys (2.0 g) is dissolved in 5 ml trifluoroacetic acids, under 0 ° of C condition, add the glacial acetic acid solution (33%) of 2 ml hydrogen bromides.Stir after 2 hours under room temperature, wash for 4 times with excessive ether washing reaction system.After solvent evaporated, at vacuum drying, obtain powder 1 gram (90%) of Lycoperdon polymorphum Vitt under room temperature.
The preparation of S3.siRNA nanometer microvesicle:
Prepare siRNA nanometer microvesicle by autonomous dress.The positive charge siRNA nano-micelle that adds excessive step S2 in the negative charge nanometer microvesicle of above-mentioned steps S1, incubated at room 20 minutes can be prepared siRNA nanometer microvesicle.For removing the siRNA micelle that is not attached to the microvesicle surface, utilize the supercentrifugal process purified product, namely used 2000 rpm centrifugal 30 minutes, discard subnatant, add 4 milliliters of distilled waters resuspended, cyclic washing three times is used the resuspended end-product that gets of 1 ml phosphate buffer (pH=7.4) at last.
The present invention is based on and utilize the nanometer microvesicle to come load siRNA, gained intermediate product and end product diameter and the surface potential of dynamic light scattering determination microvesicle; Form and structure by transmission electron microscope observation siRNA nanometer microvesicle; Utilize the ultrasonic development ability of the contrast-enhanced ultrasound technique contrast agent detection that exsomatizes and the sensitivity that low frequency ultrasound irradiation produces cavitation effect; At last by siRNA transfection usefulness and the distribution to cell under low frequency ultrasound irradiation of flow cytometry and laser confocal microscope contrast agent detection.
Diameter and the surface potential of embodiment 2 intermediate products and end product:
Utilize dynamic light scattering method to detect its diameter and surface potential separately embodiment 1 gained intermediate product (nanometer microvesicle, siRNA micelle) and end-product (siRNA nanometer microvesicle).Result (result data represents with mean ± standard error) shows that the diameter of nanometer microvesicle, siRNA micelle, siRNA nanometer microvesicle is respectively 436.8 ± 5.7 nm, 66.8 ± 2.8 nm, 476.0 ± 6.1 nm; And three's surface potential is-18.4 ± 0.2 mV; 23.4 ± 1.1 mV, 15.3 ± 2.7 mV.Diameter and surface potential have been verified the polymerization process of preparation siRNA nanometer microvesicle.
The morphosis of embodiment 3 siRNA nanometer microvesicles detects:
For further confirming form and the structure of embodiment 1 preparation gained siRNA nanometer microvesicle, adopt transmission electron microscope to detect, test result is seen Fig. 1.By testing the rounded or similar round of visible siRNA nanometer microvesicle, about 200 ~ 500 nm, diameter Distribution is even greatly for diameter Distribution, has no obvious gathering, the microvesicle surface pitting that as seen surface causes at preparation transmission electron microscope process evacuation.Under high-amplification-factor visible (Fig. 1 upper left corner), contrast agent is similar round, and is rough, and the little siRNA micelle of visible a large amount of diameter 50 ~ 70 nm in surface loads on the microvesicle surface, forms an overall structure.
The detection of the stripped ultrasonic development usefulness of embodiment 4 siRNA nanometer microvesicles and low frequency ultrasound radiation sensitivity:
For proof embodiment 1 preparation gained siRNA nanometer microvesicle has similar ultrasonic development ability and low frequency ultrasound sensitivity to the nanometer microvesicle, the present embodiment specially prepares the basic acoustic properties that agarose model (agarose is at the ultrasonic lower echoless that substantially is) with circular opening comes contrast agent detection, and result as shown in Figure 2.Fig. 2 A and C are presented at respectively in the agarose model, and siRNA nanometer microvesicle and the nanometer microvesicle development situation in the ultrasonic contrast pattern both all shows significantly strong echological picture, and after load siRNA was described, the nanometer microvesicle had good ultrasonic development ability equally.Owing to will realizing the transfection effect of siRNA nanometer microvesicle on cell, must utilize the low frequency ultrasound group to make it produce cavitation effect according to siRNA nanometer microvesicle, therefore designed the cavitation effect situation that the low frequency ultrasound radiation sensitivity tests to verify siRNA nanometer microvesicle.Fig. 2 B and D be demonstration respectively, and siRNA nanometer microvesicle and nanometer microvesicle change echoless into by original strong echo after low frequency ultrasound irradiation.Illustrate that siRNA nanometer microvesicle is similar to the nanometer microvesicle, after low frequency ultrasound irradiation, cavitation effect has occured, microbubble destruction has been verified the ultrasonic sensitive of siRNA nanometer microvesicle.
The siRNA transfection usefulness of embodiment 5 embodiment 1 preparation gained siRNA nanometer microvesicles cell under low frequency ultrasound irradiation detects:
Cover plant tumor cell in 6 orifice plates adds with fluorescent material Cy3(and can send red fluorescence) the siRNA nanometer microvesicle of labelling siRNA, then use low frequency ultrasound irradiation, realize siRNA transfection in tumor cell of Cy3 labelling.Utilize only add siRNA nanometer microvesicle but without the cell of ultrasonic irradiation as experiment contrast, only add the siRNA of Cy3 labelling as negative control.Each group cell is collected with trypsinization and with phosphate buffer washing three times, added and carry out flow cytometry after the phosphate buffer re-suspended cell and detect.Testing result as shown in Figure 3, through after ultrasonic irradiation, the transfection efficiency of siRNA is 50.3 ± 2.5 %, and is 5.0 ± 0.2 % without the experiment contrast group transfection efficiency of ultrasonic irradiation.The siRNA transfection ability of presentation of results siRNA nanometer microvesicle itself is not high, but higher siRNA transfection usefulness is arranged under low frequency ultrasound irradiation, meets the requirement as the siRNA carrier.
Embodiment 6 laser confocal microscopes detect interior distribution of cell of siRNA:
Realize the function of siRNA, must guarantee first that siRNA successfully is transported in cytoplasm, therefore, the present embodiment utilizes laser confocal microscope to detect the gene of embodiment 1 preparation gained siRNA nanometer microvesicle under low frequency ultrasound irradiation and carries effect.On cell cover plant and laser co-focusing ware, add the siRNA nanometer microvesicle with Cy3 labelling siRNA, then carry out ultrasonic irradiation.Simultaneously, only add siRNA nanometer microvesicle and the cell that do not carry out ultrasonic irradiation as the experiment contrast group, and the naked siRNA that only adds the Cy3 labelling is as negative control group.Result as shown in Figure 4, siRNA nanometer microvesicle is after ultrasonic irradiation, visible a large amount of red fluorescence (Cy3), obviously many than the experiment contrast group in cytoplasm, show and have no any red fluorescence in nucleus.This presentation of results siRNA nanometer microvesicle can be delivered to cytoplasm with siRNA effectively after ultrasonic irradiation.
Claims (10)
1. the nanoscale lipid microvesicle ultrasound angiography agent of a load siRNA, is characterized in that, siRNA is loaded on the surface of microbubble structure, and the diameter of the nanoscale lipid microbubble of described load siRNA is 200～500 nm.
2. the preparation method of the nanoscale lipid microvesicle ultrasound angiography agent of a load siRNA, it is characterized in that, described contrast agent is by the positive and negative charge captivation, the siRNA nano-micelle of surface band positive electricity is loaded on surperficial electronegative nanoscale lipid microvesicle ultrasound angiography agent surface, form an overall structure.
3. the preparation method of the nanoscale lipid microvesicle ultrasound angiography agent of a load siRNA, is characterized in that comprising the steps:
S1. take phospholipid composition dipalmitoyl phosphatidyl choline, DSPE and DPPA as the film material, obtain electronegative nanometer microcapsular ultrasound contrast agent by thin film-aquation legal system is standby;
S2. Polyethylene Glycol and polylysine are formed two block cation copolymers and siRNA and independently fill during as 2 ~ 8:1 take the ratio of N/P ratio, obtain the siRNA nano-micelle of positively charged; Described N/P ratio refers to the mol ratio of the phosphorus-containing groups of the nitrogen-containing group of copolymer and siRNA;
S3. in adding excessive S2 in the electronegative nanoscale lipid microbubble that S1 obtains, the siRNA nano-micelle of positively charged, hatch the nanoscale lipid microvesicle ultrasound angiography agent for preparing load siRNA.
4. preparation method according to claim 3, is characterized in that, the ratio of weight and number of dipalmitoyl phosphatidyl choline described in S1, DSPE and DPPA is 18:1:1 ~ 5.
5. preparation method according to claim 4, is characterized in that, the ratio of weight and number of dipalmitoyl phosphatidyl choline described in S1, DSPE and DPPA is 18:1:1.
6. preparation method according to claim 3, is characterized in that, thin film described in S1-aquation method concrete steps are as follows:
S11. with the phospholipid composition dipalmitoyl phosphatidyl choline, DSPE and DPPA are dissolved in chloroform;
S12. add 37 ~ 60 ℃ of aquations of distilled water after 0.5 ~ 2 hour after chloroform volatilizees the formation phospholipid membrane naturally, ultrasonic sound shook 1 ~ 10 minute, passed into simultaneously octafluoropropane gas generation lipid microbubble;
S13. with standing 10 ~ 30 minutes of microvesicle liquid, centrifugal, draw lower floor's milky white liquid and be prepared surperficial electronegative nanoscale lipid microvesicle ultrasound angiography agent.
7. preparation method according to claim 3, is characterized in that, described in S1, electronegative nanoscale lipid microbubble includes octafluoropropane gas.
8. preparation method according to claim 3, is characterized in that, the molecular weight of Polyethylene Glycol described in S2 is 500 ~ 3500; The mean molecule quantity of described polylysine is 6000 ~ 10000.
9. preparation method according to claim 3, is characterized in that, the concrete steps that Polyethylene Glycol described in S2 and polylysine form two block cation copolymers are:
S21. prepared the PEG of macromole evocating agent Alpha-Methyl-omega-amino-by methoxy poly (ethylene glycol);
S22. by the synthetic benzyloxycarbonyl group protection of lysine hydrochloride lysine anhydride;
S23. the preparation of poly-(benzyloxycarbonyl group-lysine) block copolymer of PEG-b-: as macromole evocating agent, the ring-opening polymerization of the lysine anhydride by causing the benzyloxycarbonyl group protection is synthetic with the PEG of Alpha-Methyl-omega-amino-; Concrete steps are under argon shield, in reaction bulb, add PEG 0.5 ~ 2 gram and benzyloxycarbonyl group protection lysine anhydride 6 ~ 10 grams of Alpha-Methyl-omega-amino-, after the anhydrous dimethyl formamide dissolving, are placed on 33 ~ 37 ℃ of oil bath reactions 2 ~ 4 days; After reaction finishes, with the reaction system ether sedimentation, filter, wash, under room temperature at vacuum drying, the white powder that obtains;
S24. the preparation of diblock copolymer PEG-b-PLL: copolymer PEG-b-in S23 poly-(benzyloxycarbonyl group-lysine) is dissolved in trifluoroacetic acid, under condition of ice bath, adds the glacial acetic acid solution of hydrogen bromide; After stirring under room temperature, with excessive ether washing reaction system washing; After solvent evaporated, at vacuum drying, obtain the end-product powder of Lycoperdon polymorphum Vitt under room temperature.
10. preparation method according to claim 3, it is characterized in that, utilize the supercentrifugal process purified product after hatching described in S3, namely used 500 ~ 3000 rpm centrifugal 20 ~ 40 minutes, discard subnatant, add distilled water resuspended, cyclic washing three times is at last with the resuspended end-product that gets of phosphate buffer.
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| CN104629045A (en) * | 2015-01-30 | 2015-05-20 | 中国科学院长春应用化学研究所 | Ring opening polymerization method for preparing epsilon-polylysine |
| CN104629045B (en) * | 2015-01-30 | 2016-11-09 | 中国科学院长春应用化学研究所 | The method of epsilon-polylysine is prepared in a kind of ring-opening polymerisation |
| CN105374266B (en) * | 2015-12-16 | 2018-11-13 | 中山大学附属第三医院 | A kind of imitative body Model for simulating tumour ultrasonic contrast |
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| CN106943603B (en) * | 2017-01-24 | 2020-07-14 | 中山大学 | Preparation method of nanogold shell by taking pH sensitive micelle as template |
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| CN111330025A (en) * | 2020-03-03 | 2020-06-26 | 中山大学附属第三医院 | Bionic microbubble ultrasound contrast agent and preparation method thereof |
| CN111330025B (en) * | 2020-03-03 | 2022-07-26 | 中山大学附属第三医院 | Bionic microbubble ultrasound contrast agent and preparation method thereof |
| CN113694040A (en) * | 2020-05-20 | 2021-11-26 | 中山大学孙逸仙纪念医院 | DPPA-based therapeutic liposome nanoparticle and preparation method and application thereof |
| CN113769118A (en) * | 2021-06-28 | 2021-12-10 | 中山大学附属第三医院（中山大学肝脏病医院） | Platelet membrane bionic ultrasonic microbubble and preparation method and application thereof |
| CN117653754A (en) * | 2023-12-05 | 2024-03-08 | 上海市第六人民医院 | Cationic ultrasonic microbubble contrast agent and preparation method thereof |
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