CN102657612B - GDNF-carrying microbubble preparation and method for making the same - Google Patents
GDNF-carrying microbubble preparation and method for making the same Download PDFInfo
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Abstract
The invention discloses a GDNF-carrying microbubble preparation and a method for making the same. The GDNF-carrying microbubble is composed of a lipid bilayer internally wrapping biological inert gas and biotinylated GDNF connected to the outside of the lipid bilayer. Employing MRI real-time guided low-frequency focused ultrasound combined with BBB opening by the GDNF-carrying microbubble to irradiate a craniocerebral parietal cortex area of a rat (the optimal parameters are set to be: probe frequency 1 Hz, microbubble 0.5 mL, irradiation period 60 s, sound pressure 0.8 MPa, and time-delay 60 s) can promote GDNF to penetrate the BBB and increase the effective drug concentration of GDNF in the central nervous system. The pharmacological research after effective macromolecule breaking through of BBB by low-frequency focused ultrasound combined with target microbubble technique will further increase the advantage of neurotrophic factors in treatment of brain diseases and provide the scientific evidence for GDNF to treat nervous centralis diseases.
Description
Technical field
The present invention relates to a kind of year GDNF microbubble agents and preparation method thereof.
Background technology
Glial cell line-derived neurotrophic factor (glial cell line derived neurotrophic factor, GDNF) be one of the most representative member in neurotrophic factor family, being found in the strain of the B49 of rat neurogliocyte at first, is that molecular weight is the high molecular weight protein of 24kDa.GDNF extensively is present in central nervous system and the ripe cerebral tissue of growth; high level expression is in striatum, thalamus, cortex and Hippocampus; to multiple neuron as: neurogliocyte, serotoninergic neuron and dopaminergic neuron all have and promote growth, protection and repair function, and can regulate norepinephrine energy and Gabanergic approach.GDNF obtained generally acknowledging for the effect of the neurodegenerative diseases such as parkinson disease and Alzheimer, and recent clinical and zoopery shows that GDNF plays an important role in the depression treatment.Research finds that also GDNF is relevant to multiple central nervous system disease, as depression, drug dependence, alzheimer disease etc.
Yet, due to GDNF molecular weight large (24kDa), being difficult to see through blood brain barrier (Blood-Brain Barrier, BBB), its therapeutic effect has been subject to serious restriction.Therefore, how promoting GDNF to see through BBB, increase GDNF in the intrasystem active drug concentration of maincenter god, will be a key scientific problems using GDNF treatment central nervous system disease.
BBB refers to the structure that the brain blood capillary stops Cucumber (being mostly to be harmful to) to enter cerebral circulation blood, this structure can make cerebral tissue be subjected to less even not to be subjected to the infringement of harmful substance in blood circulation, thereby in the maintenance cerebral tissue, environment is basicly stable, plays an important role to keeping central nervous system's normal physiological state.The research in recent years results suggest, blood brain barrier is the cell system of a complexity, it is mainly consisted of and has been kept the specific function of blood brain barrier by tight connection, spider cell, perithelial cells and the structures such as circumvascular microglia and basement membrane of endotheliocyte, endotheliocyte, kept the stable of the interior environment of central nervous system.BBB is the invasion and attack of harmful substance to brain in effective block blood, when ensureing the normal performance of its function, the macromolecular drug that has also stopped 98% above small-molecule drug and 100%, studies show that, only has fat-solubility, the small molecular weight material of low-molecular-weight (200-400Da) could be passed through BBB, this restriction has stopped entering of most curative drugs, make brain cancer, parkinson, alzheimer disease, multiple sclerosis, apoplexy, many central nervous system disease such as traumatic brain injury are difficult to Drug therapy, therefore how to open to therapeutic BBB, it is the focus of Recent study.
The traditional method of Opening Blood Brain Barrier is more, but all fails to obtain promising result.Cause the endotheliocyte shrinkage as inject hyperosmotic solution (as mannitol) by intra-arterial, cause the extensive blood brain barrier junction opened of a few hours; The ethanol of solvent such as high dose or dimethyl sulfoxide (DMSO) alkylates such as alkeran, immune adjuvant and cytokine also are used to open BBB.Infiltration and chemical method all require to implement catheter intubation, and this direct injection is invasive and needs open skull, causes that easily the permeability of non-target area cerebral tissue increases, and may cause that even cerebral tissue destroys, hemorrhage, infection; And the easy disperse of hyperosmotic solution and chemical reagent, cause to inject the BBB that tremulous pulse and branch arrange all open, thereby lack selectivity, limited it in clinical application.
Studies show that in a large number, ultrasonic wave energy is open haemal tissue barrier under certain condition.Taniyama etc. realize successfully that in isolated test the DNA plasmid transfection enters Mus carotid artery tissue, have externally confirmed that ultrasonic irradiation can increase biomembranous permeability.Mesiwala equals to select high-intensity focused ultrasound studies confirm that in 2002 subsequently, and selective opening BBB under certain ultrasound intensity does not cause obvious brain tissue impairment.For reducing ultrasonic energy, researcher finds to have realized with the collaborative ectogenic cavitation nucleus (microvesicle) of focus supersonic in the situation that do not cause the of short duration open rabbit BBB of acute neuronal damage, and then thinks that focus supersonic can realize high selectivity, open BBB non-invasively; And in the situation that microvesicle exists, the required ultrasonic energy of open BBB obviously reduces, and this is conducive to realize ultrasonicly reach selective opening BBB and do not damage the purpose of normal cerebral tissue with more low-energy.Based on the focus supersonic associating effective noinvasive of the microvesicle open BBB in ground, after having the scholar to inject on this basis microvesicle optison, the cerebral tissue with the irradiation of low frequency ultrasound multiple spot is windowed adopts multiple means (light microscopic, Electronic Speculum and MRI), all observes the opening of BBB.Experiment confirm ultrasonic in combination microvesicle can local, reversible and noninvasive open BBB, effectively treat for clinical practice intracranial disease drug provision very positive using value.
In the feasibility study that carries out the open BBB of ultrasonic in combination microvesicle, researcher is not ignored the evaluation of safety.Different research groups confirms that all there is safe thresholding in supersonic synergic microvesicle Opening Blood Brain Barrier, and the ultrasound parameter that just uses due to different researcheres, exposure time, microbubble concentration, observational technique etc. are not quite similar, thereby the conclusion that draws is not the same.Researcher finds to adopt tranmitting frequency 1.5MHz, pulse recurrence frequency 1Hz, instantaneous peak value acoustic pressure amplitude 2～12.7MPa ultrasonic irradiation Medulla Leporis seu Oryctolagi 20s, MRI strengthens video picture and histological examination is found, the damage of cerebral tissue occurs when acoustic pressure reaches 6.3MPa, show as blood vessel wall destruction, hemorrhage, accidental necrosis.Another research group adopts the impulse ultrasound irradiation Medulla Leporis seu Oryctolagi 20s of tranmitting frequency 1.63MHz, pulse recurrence frequency 1Hz, acoustic pressure amplitude 0.7～1.0MPa, after irradiation, whole cerebral tissue is carried out pathological study, find at the rarely seen a few cell apoptosis in ultrasonic irradiation district; And carry out Contrast-enhanced MRI inspection or histopathological examination after 4 weeks at ultrasonic irradiation and all do not find the BBB damage.Conclusion in above multinomial research all unanimously shows, the modern image techniques technology can show the integrity of normal BBB and the regional BBB that is damaged by disease in real time.Simultaneously, not observe cerebral tissue impaired in the several different methods iconography research of opening BBB.
Along with going deep into of the open BBB research of focus supersonic associating microvesicle radiography, there are many scholars to utilize ultrasonic open BBB, the mode of taking targeting to discharge, treatment central nervous system's disease.Researcher enters cerebral tissue with the antibody of focus supersonic Opening Blood Brain Barrier transhipment targeting dopamine receptor, after result is presented at ultrasonic open BBB, and the dopamine receptor on the antibody capable of the brain of transporting identification brain cell membrane.In a word, in the time of the open BBB of the further investigation using ultrasound of ultrasonic medicine-carrying microvesicle associating microvesicle, but microvesicle carries medicine or gene targeted therapy intracranial disease, for the treatment of intracranial disease provides new strategy.But at present, about the open BBB of the guided focused ultrasonic in combination medicine-carrying microvesicle of NMR (Nuclear Magnetic Resonance)-imaging (magnetic resonance imaging, MRI), transmit the research of the various central nervous system disease of GDNF treatment and not yet report.
Summary of the invention
The purpose of this invention is to provide a kind of year GDNF microvesicle and preparation method thereof.
Provided by the present invention year GDNF microvesicle is comprised of the lipid bilayer of internal package biologically inert gas and the biotinylated GDNF that is connected in the described lipid bilayer outside.
Wherein, described lipid bilayer specifically can be comprised of the material of following mass parts: DSPC 4.9-5.1 part, PEG-DSPE 2000 0.9-1.2 parts, PEG-DSPE 2000-biotin 0.9-1.2 part; Described lipid bilayer is connected by biotin-avidin-biotin with biotinylated GDNF.
The mean diameter of described year GDNF microvesicle is 2-3 μ m.
The drug loading of described year GDNF microvesicle is 46.54%-46.62%.
Described biologically inert gas can be sulfur hexafluoride or perfluoropropane.
Prepare the method for described year GDNF microvesicle, comprise the steps:
1) DSPC 4.9-5.1 part, PEG-DSPE 20000.9-1.2 part, PEG-DSPE 2000-biotin 0.9-1.2 part are dissolved in mixing in chloroform, at the N of drying
2Remove chloroform under the stream effect, make phospholipid form the uniform thin film of one deck on chamber wall, vacuum drying is more than 2 hours;
2) adding degassed pH value in containing the container of dry phospholipid membrane is 7.4 Tris buffer solution, obtains phospholipid solution, wherein, contains volume fraction in described Tris buffer solution and be 10% glycerol and volume fraction and be 10% propylene glycol;
3) the described phospholipid solution of heating is to it more than phase transition temperature, and phospholipid solution thoroughly disperseed until transparent with water-bath is ultrasonic, then is distributed in cillin bottle, and the air displacement in bottle is become biologically inert gas, shakes, and obtains microvesicle;
4) centrifugal floating method is cleaned microvesicle 3-4 time, removes the phospholipid that does not form microvesicle; Add avidin in microvesicle after cleaning, hatch under room temperature more than 15 minutes, clean 3-4 time with PBS solution centrifugal floating method, remove the not avidin of coupling; Then add biotinylated GDNF in the microvesicle of coupling avidin, hatch under room temperature more than 10 minutes, clean with floating method afterwards and remove unconjugated biotinylation GDNF 3-4 time, obtain carrying the GDNF microvesicle.
Wherein, described step 2), the concentration of phospholipid solution is 2.8-3.1mg/ml.
Step 4) proportioning of microvesicle described in, avidin, biotinylated GDNF is (0.85-1.1) ml: (0.08-0.12) mg: (0.9-1.2) mg.
A further object of the present invention is to provide the administration suit of a kind of year GDNF microvesicle.
The administration suit of provided by the present invention year GDNF microvesicle comprises of the present invention year GDNF microvesicle and low-frequency focusing ultrasonic equipment; Wherein, the dosage of described year GDNF microvesicle is set as 0.5ml, and the parameter of described low-frequency focusing ultrasonic equipment arranges as follows: frequency probe 1MHz, ultrasonic irradiation time 60s, acoustic pressure 0.8MPa, time delay 60s.
The present invention adopts MRI guiding in real time focus supersonic to unite to be loaded with the open BBB irradiation rat cranium brain parietal cortex of GDNF microvesicle district, promotes GDNF to see through blood brain barrier, increases the active drug concentration of GDNF in the central nervous system.Unite the targeted microbubble technology by low-frequency focusing ultrasonic, carry out the pharmaceutical research after effective macromole is broken through BBB, with further increasing neurotrophic factor in the advantage aspect the treatment disease of brain, provide the scientific basis of GDNF treatment nervus centralis disease.
Description of drawings
Fig. 1 is the granularmetric analysis at different time of self-control microvesicle and sound Novi.
Fig. 2 is that the cerebral tissue of the different microvesicle Opening Blood Brain Barrier brains of focus supersonic associating is seen (left figure) substantially and tangent plane is seen (right figure).
Fig. 3 is the content of EB in the different microvesicle Opening Blood Brain Barrier brain cerebral tissue of focus supersonic associating.
Fig. 4 is the different microvesicle Opening Blood Brain Barrier cerebral tissue HE dyeing of focus supersonic associating.
Fig. 5 is that orthogonal test is respectively organized the EB content in ultrasonic in combination microvesicle Opening Blood Brain Barrier cerebral tissue.
Fig. 6 is that orthogonal test is respectively organized ultrasonic in combination microvesicle Opening Blood Brain Barrier brain tissue slice HE dyeing (400 *).
Fig. 7 is that orthogonal test is respectively organized ultrasonic in combination microvesicle Opening Blood Brain Barrier brain tissue slice TUNEL dyeing (400 *).
Fig. 8 is that orthogonal test is respectively organized focus supersonic and induced the apoptotic effect of blood brain barrier.
Fig. 9 is the Change of Ultrastructure that orthogonal test is respectively organized cerebral tissue endotheliocyte and neurocyte.
Figure 10 is that focus supersonic is induced after blood-brain barrier disruption BSA concentration in ultrasonic tissue.
Figure 11 is that focus supersonic is induced the GDNF concentration in ultrasonic tissue after blood-brain barrier disruption.
Figure 12 is the differentiation of the GDNF PC-12 cell of inducing.A microvesicle group; B, GDNF targeted microbubble group; The C:GDNF protein groups.
Figure 13 is the low-frequency focusing ultrasonic associating microvesicle Opening Blood Brain Barrier of MRI guiding and the Blood Brain Barrier (BBB) opening zone that EB shows.
The specific embodiment
The present invention will be described below by specific embodiment, but the present invention is not limited thereto.
Experimental technique described in following embodiment if no special instructions, is conventional method; Described reagent and biomaterial if no special instructions, all can obtain from commercial channels.
The research that GDNF microbubble contrast agent and Opening Blood Brain Barrier thereof are carried in embodiment 1, preparation
1) the GDNF microvesicle is carried in preparation
By a certain percentage with a certain amount of DSPC (DSPC, 10.64mg), DSPE-PEG2k (PEG2000-DSPE, 2.10mg), (DSPE-PEG2k-Biotin 2.26mg) is dissolved in the 0.5ml chloroform mixing on turbine mixer to DSPE-PEG2k-Biotin.N in drying
2Remove chloroform under the stream effect and make phospholipid form the uniform thin film of one deck on test tube wall, dry more than 2 hours in vacuum drying oven.Then add the Tris buffer solution of the degassed pH7.4 of 5ml in containing the test tube of dry phospholipid membrane: contain glycerol (10% volume fraction) and propylene glycol (10% volume fraction) obtains certain density phospholipid solution.The heating phospholipid solution is to more than its phase transition temperature (55-60 ℃), and with the water-bath ultrasonator, the phospholipid solution of milk shape is thoroughly disperseed until transparent, then be divided in the cillin bottle of the every milliliter of 2ml that packs into, air displacement in bottle is become sulfur hexafluoride or perfluoropropane, then use the rear 4 ℃ of storages of aluminium-plastic cap and rubber stopper sealing standby.
During use, after preparing microvesicle with mechanical concussion method (concussion 45s), clean 3-4 time with PBS solution centrifugal floating method, remove the phospholipid that does not form microvesicle.Add 0.1mg avidin (avidin) in the microvesicle of 1ml, shake gently and hatched under room temperature 20 minutes, continue to clean 3-4 time with PBS solution centrifugal floating method, remove the not avidin avidin of coupling, then sneak in the microvesicle that obtains the biotinylated GDNF of 1mg (PeproTech company, 450-51), shake gently and room temperature under hatched 15 minutes, clean with floating method afterwards and remove unconjugated biotinylation GDNF 3-4 time, obtain the microvesicle of GDNF targeting.
The Performance Detection of carrying the GDNF microvesicle to preparation:
Grain count instrument (Accusizer 780/A, the U.S.) detects the particle diameter that carries the GDNF microvesicle: its particle size distribution is 0.5-8 μ m, and mean diameter is 2.3 μ m.
Carry drug loading and envelop rate in the GDNF microvesicle: be respectively 46.58% and 81.52%.
The effect of Opening Blood Brain Barrier (BBB) under focus supersonic (frequency probe 1MHz, microvesicle amount 1ml, irradiation time 60s, acoustic pressure 0.8MPa) effect compares with homemade blank microvesicle and sound Novi, the results are shown in Figure 1-4.
Fig. 1 for self-control microvesicle and sound Novi (Brocco company, H20080059) the different time particle size analyzer detects figure, wherein A: self-control microvesicle 0h organizes; B: self-control microvesicle 24h; C: the sound 0h of Novi group; D: the sound 24h of Novi group.
Fig. 2 is that the different microvesicle Opening Blood Brain Barrier cerebral tissue of focus supersonic associating are seen and tangent plane sight, wherein A substantially: self-control microvesicle 0h group; B: self-control microvesicle 24h; C: the sound 0h of Novi group; D: the sound 24h of Novi group.
Fig. 3 is EB content, wherein A in each group cerebral tissue: self-control microvesicle 0h group; B: self-control microvesicle 24h; C: the sound 0h of Novi group; D: the sound 24h of Novi group.
Fig. 4 is the different microvesicle Opening Blood Brain Barrier cerebral tissue HE dyeing of focus supersonic associating, wherein A: self-control microvesicle 0h group; B: the sound 0h of Novi group.
Result shows, self-control microvesicle harmony Novi does not have significant difference in the opening of BBB, and the effect of the open BBB of self-control microvesicle is more stable.
Take ultrasonic irradiation time, microvesicle dosage, time delay (microvesicle inject time and acoustic shock interval), supersonic frequency, acoustic pressure as the influence factor, three levels of each selecting factors, adopt orthogonal (seeing Table 1), with azovan blue (EB, Evens blue, Sigma company, E2129) seepage discharge is as the index (EB seepage discharge) of blood brain barrier percent of pass, determine the low-frequency focusing ultrasonic associating targeted microbubble partly open BBB of MRI guiding, increase the optimal parameter of maincenter GDNF level.
Test method: the mensuration of permeability adopts the seepage discharge of EB quantitatively to estimate.Get the rat cerebral tissue after the PBS perfusion, the cerebral tissue weight in wet base that local indigo plant is dyed immerses formamide solution by 10ml/g, extracting 24h in 60 ℃ of constant water bath box, formamide solution presents the different blueness of the depth, cerebral tissue presents the water white transparency shape, then the OD value of the 620nm wavelength quantitative EB in place in Methanamide under the UV300 ultraviolet-visible spectrophotometer, the content of EB in quantitative analysis sample cerebral tissue.Pure formamide solution is made as blank, according to standard curve, calculates EB content (μ g/g).
The results are shown in Table 1.
Factor and the level of the optimal parameter of the low-frequency focusing ultrasonic associating targeted microbubble partly open BBB of table 1MRI guiding
The orthogonal design of the optimal parameter of the low-frequency focusing ultrasonic associating targeted microbubble partly open BBB of table 2MRI guiding reaches respectively organizes the EB seepage discharge
The results are shown in Figure 5-9
Fig. 5 is the EB content in different condition ultrasonic in combination microvesicle Opening Blood Brain Barrier cerebral tissue, as seen organize 6, group 11, group 13 and group 14BBB permeability stronger.
Fig. 6 is different condition ultrasonic in combination microvesicle Opening Blood Brain Barrier brain tissue slice HE dyeing (A: matched group; B: group 6; C: group 11; D: group 13; E: group 14,400 *).
Fig. 7 is different condition ultrasonic in combination microvesicle Opening Blood Brain Barrier brain tissue slice TUNEL dyeing (400 *) (A: matched group; B: group 6; C: group 11; D: group 13; E: group 14,400 *).
Fig. 8 is that focus supersonic is induced the apoptotic effect of blood brain barrier.The apoptotic cell number of each sample is counted (with matched group and the 6th group of comparison * P＜0.01st) in five independent experiments.
Fig. 9 is ultramicroscope Ultrastructural observation result (A: matched group; B: group 6; C: group 11; D: group 13; E: group 14,400 *).In D group and E group, endotheliocyte and neurocyte are not observed variation by heavy damage but organize at A, B, C.
Above result shows: frequency probe 1MHz, microvesicle amount 0.5ml, irradiation time 60s, acoustic pressure 0.8MPa, time-delay 60s is Parameter Conditions preferably, BBB degree of opening large (the EB seepage discharge shows its degree of opening) under this condition, and histology and electron microscopic observation do not have obvious structural damage.
The mensuration of protein content in embodiment 3, brain
The ultrasound condition of this embodiment is the optimal parameter condition that draws in embodiment 2, i.e. frequency probe 1MHz, microvesicle amount 0.5ml, irradiation time 60s, acoustic pressure 0.8MPa, time-delay 60s.
Ultrasonic in combination microvesicle Opening Blood Brain Barrier, after intravenous injection albumen, the comparison of determining the protein quantity and two kinds of albumen transmission means in brain.After detecting the open BBB of ultrasonic in combination microvesicle, protein content with enter the cerebral tissue amount.
(1) because the GDNF price is higher, with the low alternative protein B SA simulation experiment condition of another kind of price, determine to enter the amount in cerebral tissue.
Be divided into two groups: ultrasonic+microvesicle+albumen (A group); Ultrasonic+year albumen microvesicle (B group); Set different dosing dosage: 1) 0 μ g/kg, 1) 100 μ g/kg, 2) 200 μ g/kg; 3) 400 μ g/kg, 4) 800 μ g/kg, 5) 1200 μ g/kg.
Experimental result is seen Figure 10, shows that focus supersonic induces after blood-brain barrier disruption BSA concentration (* p＜0.05) in ultrasonic tissue.
(2) tail vein injection carries the GDNF microvesicle, and dosage is 800 μ g/kg.
Experimental result is seen Figure 11, shows that focus supersonic induces the GDNF concentration (* p＜0.05) in ultrasonic tissue after blood-brain barrier disruption.(the A:GDNF in Figure 11 (injecting again GDNF after the injection microvesicle); B carries the GDNF microvesicle)
Above result shows, 1) in cerebral tissue, detected protein content increases with the increase of intravenous injection protein concentration; 2) two kinds of albumen transfer modes are compared, and carry the albumen microvesicle and can promote that more albumen enters cerebral tissue.
The detection of embodiment 4, GDNF fluorescence microvesicle external activity
Whether influential for detecting in targeted microbubble manufacturing process the GDNF activity, survey its external activity by PC12 (Mus pheochromocytoma cells), the PC-12 cell strain derives from a kind of transplantable Mus pheochromocytoma, and this cell has the profiling reaction of reversible neuron to GDNF.
Experiment grouping: A, microvesicle group; B, GDNF targeted microbubble group; C, the gdnf protein group.
Method: cell suspension in the DMEM culture fluid that contains 5%HS and 5%FBS, is inoculated in that in 12 orifice plates, concentration is 2*10
3Cell/ml.Add the GDNF of 50ng/ml restructuring in C, add the GDNF targeted microbubble that contains 50ng/ml GDNF in B, A adds the microvesicle of same amount.Cultivate observation of cell variation after 7 days.
The results are shown in Figure 12, the PC-12 cell that GDNF induces is in differentiation.
Result shows, the contained GDNF of microvesicle is consistent with GDNF effect in the profiling reaction of inducing the PC-12 cell of restructuring, illustrate the contained GDNF of microvesicle be have bioactive.
The lower Blood Brain Barrier (BBB) opening of embodiment 5, MRI monitoring
MRI scanning device (SIEMENS), Germany adopts clinical criteria 3.0T signaling system.Each pre-irradiation corrective system focal coordinates.Employing T1 scanning phase in pre-irradiation and experiment (TE:16ms, TR:1000ms, bandwidth: 16kHz, matrix: 184 * 256, flip angle: 90 °, times of collection: 4 times, scanning bed thickness: 1mm).With 10% chloral hydrate intraperitoneal injection of anesthesia animal, remove irradiated region Mus hair with depilatory after the anesthesia onset.Lie on the back and be fixed in the MRI supersonic therapeutic system, the surface coils of animal head bottom fixed diameter 7.5cm is with the reduce disturbance signal.According to MRIT1 information adjustment system focus to the target area, pre-irradiation 10 seconds, tail vein injection 0.5ml microvesicle (microvesicle is the microvesicle that carries GDNF herein) (5-8 * 10
8/ ml) (parameter that this example medium and low frequency focuses on is: frequency probe 1MHz, microvesicle amount 0.5ml, irradiation time 60s, acoustic pressure 0.8MPa, time-delay 60s), 15 seconds tail vein injection magnevist (Magnevist) contrast agent (0.2ml/kg) after ultrasonic irradiation are after the T1 scanning phase, tail vein injection EB (100mg/kg) shows the zone of Blood Brain Barrier (BBB) opening.
The results are shown in Figure 13, be the low-frequency focusing ultrasonic associating microvesicle Opening Blood Brain Barrier of MRI guiding and the Blood Brain Barrier (BBB) opening zone of EB demonstration.The cross-section bit image of A:MRI wherein; B:MRI Coronal image strengthens the high signal of contrast agent that develops in brain essence shown in figure A and B arrow, illustrate that local BBB is open, and the MRI contrast agent enters brain essence; C: the brain tissue slice corresponding with figure A seen; D: see with the corresponding brain tissue slice of figure B, figure C and D see that all the cerebral tissue indigo plant corresponding with the MRI image dyes, and show that local blood brain barrier opens, and the dyestuff azovan blue enters cerebral tissue.
Result shows, after ultrasonic in combination microvesicle Opening Blood Brain Barrier to dye zone (Figure 13 C, D) consistent with substantially seeing lower EB indigo plant the lower shown high signal area of contrast agent (Figure 13 A, B) of MRI scanning, illustrates that MRI can monitor Blood Brain Barrier (BBB) opening.
Claims (7)
1. one kind carries the GDNF microvesicle, is comprised of the lipid bilayer of internal package biologically inert gas and the biotinylated GDNF that is connected in the described lipid bilayer outside; Described lipid bilayer is comprised of the material of following mass parts: DSPC 4.9-5.1 part, PEG-DSPE 20000.9-1.2 part, PEG-DSPE 2000-biotin 0.9-1.2 part; Described lipid bilayer is connected by biotin-avidin-biotin with biotinylated GDNF.
2. according to claim 1 year GDNF microvesicle, it is characterized in that: the mean diameter of described year GDNF microvesicle is 2-3 μ m.
3. according to claim 1 and 2 year GDNF microvesicle, it is characterized in that: described biologically inert gas is sulfur hexafluoride or perfluoropropane.
4. prepare the method for described year GDNF microvesicle of any one in claim 1-3, comprise the steps:
1) DSPC 4.9-5.1 part, PEG-DSPE 20000.9-1.2 part, PEG-DSPE 2000-biotin 0.9-1.2 part are dissolved in mixing in chloroform, at the N of drying
2Remove chloroform under the stream effect, make phospholipid form the uniform thin film of one deck on chamber wall, vacuum drying is more than 2 hours;
2) adding degassed pH value in containing the container of dry phospholipid membrane is 7.4 Tris buffer solution, obtains phospholipid solution, wherein, contains volume fraction in described Tris buffer solution and be 10% glycerol and volume fraction and be 10% propylene glycol;
3) the described phospholipid solution of heating is to it more than phase transition temperature, and phospholipid solution thoroughly disperseed until transparent with water-bath is ultrasonic, then is distributed in cillin bottle, and the air displacement in bottle is become biologically inert gas, shakes, and obtains microvesicle;
4) centrifugal floating method is cleaned microvesicle 3-4 time, removes the phospholipid that does not form microvesicle; Add avidin in microvesicle after cleaning, hatched under room temperature 20 minutes, clean 3-4 time with PBS solution centrifugal floating method, remove the not avidin of coupling; Then add biotinylated GDNF in the microvesicle of coupling avidin, hatched under room temperature 15 minutes, clean with floating method afterwards and remove unconjugated biotinylation GDNF 3-4 time, obtain carrying the GDNF microvesicle.
5. method according to claim 4, it is characterized in that: the concentration of phospholipid solution described step 2) is 2.8-3.1mg/ml.
6. according to claim 4 or 5 described methods, is characterized in that: step 4) described in the proportioning of microvesicle, avidin, biotinylated GDNF be (0.85-1.1) ml: (0.08-0.12) mg: (0.9-1.2) mg.
7. administration suit that carries the GDNF microvesicle comprises in claim 1-3 described year GDNF microvesicle of any one and focus supersonic equipment; The dosage of described year GDNF microvesicle is set as 0.5ml, and the parameter of described focus supersonic equipment arranges as follows: frequency probe 1MHz, ultrasonic irradiation time 60s, acoustic pressure 0.8MPa, time delay 60s.
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