CN102657612B - GDNF-carrying microbubble preparation and method for making the same - Google Patents

Abstract

The invention discloses a GDNF-loaded microbubble and a preparation method thereof. The GDNF-loaded microvesicle is composed of a lipid bilayer that wraps biological inert gas inside and biotinylated GDNF connected to the outside of the lipid bilayer. Using MRI-guided real-time low-frequency focused ultrasound combined with GDNF-loaded microbubbles to open the BBB to irradiate the parietal cortex of the rat brain (the best parameters are set as probe frequency 1MHz, microbubble volume 0.5ml, irradiation time 60s, and sound pressure 0.8MPa , delay 60s), can promote GDNF through the blood-brain barrier, increase the effective drug concentration of GDNF in the central nervous system. Through low-frequency focused ultrasound combined with targeted microbubble technology, pharmacological research on effective macromolecules breaking through the BBB will further increase the advantages of neurotrophic factors in the treatment of brain diseases and provide a scientific basis for GDNF in treating central nervous diseases.

Description

A kind of GDNF-loaded microbubble preparation and preparation method thereof

technical field

The invention relates to a GDNF-loaded microbubble preparation and a preparation method thereof. the

Background technique

Glial cell line derived neurotrophic factor (GDNF) is one of the most representative members of the neurotrophic factor family. It was first discovered in the B49 glial cell line of rats. It is a 24kDa macromolecular protein. GDNF widely exists in the developing central nervous system and mature brain tissue, and is expressed at high levels in the striatum, thalamus, cortex and hippocampus, and has effects on various neurons such as glial cells, serotonergic neurons and dopamine Neurons have growth-promoting, protective and repairing functions, and can also regulate noradrenergic and GABAergic pathways. The role of GDNF in neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease has been recognized. Recent clinical and animal experiments have shown that GDNF plays an important role in the treatment of depression. Studies have also found that GDNF is related to various central nervous system diseases, such as depression, drug dependence, Alzheimer's disease and so on. the

However, due to the large molecular weight of GDNF (24kDa), it is difficult to penetrate the blood-brain barrier (Blood-Brain Barrier, BBB), and its therapeutic effect is severely limited. Therefore, how to promote GDNF to pass through the BBB and increase the effective drug concentration of GDNF in the central nervous system will be a key scientific issue in the application of GDNF to treat central nervous system diseases. the

BBB refers to the structure of brain capillaries that prevent certain substances (mostly harmful) from entering the cerebral blood circulation. This structure can make brain tissue less or even not damaged by harmful substances in the circulating blood, thereby maintaining the environment of the brain tissue. It is basically stable and plays an important role in maintaining the normal physiological state of the central nervous system. Recent research results suggest that the blood-brain barrier is a complex cellular system, which is mainly composed of endothelial cells, tight junctions of endothelial cells, astrocytes, pericytes, microglia around blood vessels, and basement membranes. The special function of the blood-brain barrier is guaranteed, and the stability of the internal environment of the central nervous system is maintained. While effectively blocking harmful substances in the blood from invading the brain and ensuring its normal function, the BBB also blocks more than 98% of small molecule drugs and 100% of macromolecular drugs. Studies have shown that only highly fat-soluble, low molecular weight ( 200-400Da) small molecular weight substances can pass through the BBB, this restriction blocks the entry of most therapeutic drugs, making brain malignancies, Parkinson's disease, Alzheimer's disease, multiple sclerosis, stroke, traumatic Many diseases of the central nervous system, such as brain injury, are difficult to treat with drugs, so how to open the BBB therapeutically has become a research hotspot in recent years. the

There are many traditional methods for opening the blood-brain barrier, but none of them have achieved satisfactory results. For example, intra-arterial infusion of hypertonic solutions (such as mannitol) causes endothelial cell shrinkage, resulting in extensive tight junction opening of the blood-brain barrier for several hours; solvents such as high doses of alcohol or dimethyl sulfoxide (DMSO) alkyls such as Phenylalanine mustard, immune adjuvants, and cytokines are also used to open the BBB. Both infiltration and chemical methods require catheter intubation. This direct injection is invasive and requires an open skull, which can easily cause increased permeability of non-target brain tissue, and may even cause brain tissue destruction, hemorrhage, infection; and high The infiltration solution and chemical reagents are easy to diffuse, causing all the BBBs controlled by the injected arteries and branches to open, so the lack of selectivity limits its clinical application. the

A large number of studies have shown that ultrasound can open the blood tissue barrier under certain conditions. Taniyama et al. successfully transfected DNA plasmids into mouse carotid artery tissue in vitro experiments, and confirmed that ultrasound irradiation can increase the permeability of biofilms in vitro. Subsequently, Mesiwala et al. used high-power focused ultrasound research in 2002 to confirm that selective opening of the BBB under a certain ultrasound intensity did not cause obvious brain tissue damage. In order to reduce the ultrasonic energy, the researchers found that the use of focused ultrasound in conjunction with exogenous cavitation nuclei (microbubbles) achieved short-term opening of the rabbit BBB without causing acute neuronal damage, and further believed that focused ultrasound can achieve high selectivity, The BBB can be opened non-invasively; in the presence of microbubbles, the ultrasonic energy required to open the BBB is significantly reduced, which is conducive to achieving the purpose of selectively opening the BBB with lower energy ultrasound without damaging normal brain tissue. Based on the combination of focused ultrasound and microbubbles to open the BBB effectively and non-invasively, some scholars injected microbubble optison on this basis, then irradiated the brain tissue with low-frequency ultrasound at multiple points, and adopted various methods (light microscope, electron microscope and MRI). Opening of the BBB was observed. Experiments have confirmed that ultrasound combined with microbubbles can open the BBB locally, reversibly and non-invasively, which provides a very positive application value for the clinical application of drugs that effectively treat intracranial diseases. the

While conducting the feasibility study of opening the BBB with ultrasound combined with microbubbles, the researchers did not neglect the evaluation of safety. Different research groups have confirmed that there is a safe threshold for opening the blood-brain barrier with ultrasound and microbubbles, but because different researchers use different ultrasound parameters, irradiation time, microbubble concentration, and observation methods, the conclusions drawn are different. Be consistent. The researchers found that the rabbit brain was irradiated with ultrasound with a transmission frequency of 1.5MHz, a pulse repetition frequency of 1Hz, and an instantaneous peak sound pressure amplitude of 2-12.7MPa for 20s. MRI-enhanced imaging and histological examination revealed that brain tissue appeared when the sound pressure reached 6.3MPa. Injury, manifested as vessel wall destruction, hemorrhage, and occasionally necrosis. Another research team irradiated the rabbit brain with pulsed ultrasound with a transmission frequency of 1.63MHz, a pulse repetition frequency of 1Hz, and a sound pressure amplitude of 0.7-1.0MPa for 20s. After irradiation, the whole brain tissue was observed histopathologically and found that only A small number of cells were apoptotic; and no BBB damage was found in enhanced MRI examination or histopathological examination after 4 weeks of ultrasound irradiation. The conclusions in the above multiple studies have consistently shown that modern imaging techniques can reveal the integrity of the normal BBB and the regional BBB damaged by disease in real time. At the same time, brain tissue damage was not observed in imaging studies that opened the BBB by various methods. the

With the in-depth research on opening the BBB with focused ultrasound combined with microbubble contrast, many scholars use ultrasound to open the BBB and adopt a targeted release method to treat diseases of the central nervous system. The researchers used focused ultrasound to open the blood-brain barrier and transport antibodies targeting dopamine receptors into brain tissue. The results showed that after ultrasound opened the BBB, the antibodies transported into the brain could recognize dopamine receptors on the brain cell membrane. In conclusion, the in-depth study of ultrasonic drug-loaded microbubbles uses ultrasound combined with microbubbles to open the BBB, and at the same time, microbubbles carrying drugs or genes can be targeted for the treatment of intracranial diseases, providing a new strategy for the treatment of intracranial diseases. However, at present, there is no report on the treatment of various central nervous system diseases by using magnetic resonance imaging (MRI)-guided focused ultrasound combined with drug-loaded microbubbles to open the BBB and deliver GDNF. the

Contents of the invention

The purpose of the present invention is to provide a GDNF-loaded microvesicle and a preparation method thereof. the

The GDNF-carrying microbubble provided by the present invention is composed of a lipid bimolecular layer wrapped with biologically inert gas inside and biotinylated GDNF connected to the outside of the lipid bimolecular layer. the

Wherein, the lipid bilayer can be specifically composed of the following parts by mass: 4.9-5.1 parts of distearoylphosphatidylecholine, 0.9-1.2 parts of distearoylphosphatidylethanolamine-polyethylene glycol 2000, Distearoylphosphatidylethanolamine-polyethylene glycol 2000-biotin 0.9-1.2 parts; the lipid bilayer is connected with biotinylated GDNF through biotin-avidin-biotin. the

The average particle diameter of the GDNF-loaded microbubbles is 2-3 μm. the

The drug loading of the GDNF-loaded microbubbles is 46.54%-46.62%. the

The biologically inert gas can be sulfur hexafluoride or perfluoropropane. the

The method for preparing the described GDNF-loaded microvesicles comprises the following steps:

1) Distearoylphosphatidylethanolamine-polyethylene glycol 4.9-5.1 parts, distearoylphosphatidylethanolamine-polyethylene glycol 20000.9-1.2 parts, distearoylphosphatidylethanolamine-polyethylene glycol 2000-biotin 0.9 - Dissolve 1.2 parts in chloroform and mix well, remove the chloroform under the action of dry _N2 flow, so that the phospholipids form a uniform film on the container wall, and vacuum dry for more than 2 hours;

2) Add a degassed Tris buffer solution with a pH value of 7.4 to a container containing a dry phospholipid film to obtain a phospholipid solution, wherein the Tris buffer solution contains 10% glycerol and 10% tris by volume fraction Propylene glycol;

3) Heating the phospholipid solution to above its phase transition temperature, and ultrasonically dispersing the phospholipid solution in a water bath until it becomes transparent, then filling it into vials, replacing the air in the vials with a biologically inert gas, and shaking to obtain microbubbles ;

4) Wash the microbubbles 3-4 times by centrifugal flotation method to remove phospholipids that do not form microbubbles; add avidin to the microbubbles after cleaning, incubate at room temperature for more than 15 minutes, and wash 3-4 times with PBS solution centrifugal flotation method 4 times to remove uncoupled avidin; then add biotinylated GDNF to avidin-coupled microbubbles, incubate at room temperature for more than 10 minutes, and then wash 3-4 times with floating method to remove unbound Biotinylated GDNF to obtain GDNF-loaded microvesicles. the

Wherein, the concentration of the phospholipid solution in the step 2) is 2.8-3.1 mg/ml. the

The proportion of microbubbles, avidin and biotinylated GDNF in step 4) is (0.85-1.1) ml: (0.08-0.12) mg: (0.9-1.2) mg. the

Another object of the present invention is to provide a drug delivery kit loaded with GDNF microbubbles. the

The administration set of GDNF-loaded microbubbles provided by the present invention includes GDNF-loaded microbubbles and low-frequency focused ultrasound equipment of the present invention; wherein, the dosage of the GDNF-loaded microbubbles is set to 0.5ml, and the low-frequency focused The parameters of the ultrasound equipment are set as follows: probe frequency 1MHz, ultrasound irradiation time 60s, sound pressure 0.8MPa, delay time 60s. the

The invention adopts MRI real-time guided focused ultrasound combined with microbubbles loaded with GDNF to open BBB to irradiate the parietal lobe cortex area of the rat brain, so as to promote GDNF to penetrate the blood-brain barrier and increase the effective drug concentration of GDNF in the central nervous system. Through low-frequency focused ultrasound combined with targeted microbubble technology, pharmacological research on effective macromolecules breaking through the BBB will further increase the advantages of neurotrophic factors in the treatment of brain diseases and provide a scientific basis for GDNF in treating central nervous diseases. the

Description of drawings

Figure 1 shows the particle size analysis of self-made microbubbles and Sonovent at different times. the

Figure 2 shows the general view (left image) and cross-sectional view (right image) of the brain tissue of the brain with focused ultrasound combined with different microbubbles to open the blood-brain barrier. the

Figure 3 shows the EB content in the brain tissue of focused ultrasound combined with different microbubbles to open the blood-brain barrier. the

Figure 4 is HE staining of brain tissue with focused ultrasound combined with different microbubbles to open the blood-brain barrier. the

Figure 5 shows the EB content in the brain tissue of each group in the orthogonal test of ultrasound combined with microbubbles to open the blood-brain barrier. the

Figure 6 shows the HE staining (400×) of brain tissue sections in each group of orthogonal test combined with ultrasound and microbubbles to open the blood-brain barrier. the

Fig. 7 is the TUNEL staining (400×) of the brain tissue sections of each group of the orthogonal test combined with ultrasound and microbubbles to open the blood-brain barrier. the

Figure 8 shows the effects of focused ultrasound in each group on the induction of blood-brain barrier cell apoptosis in orthogonal experiments. the

Figure 9 shows the ultrastructural changes of brain tissue endothelial cells and nerve cells in each group of orthogonal experiments. the

Figure 10 shows the concentration of BSA in ultrasound tissue after focused ultrasound-induced blood-brain barrier disruption. the

Figure 11 shows the concentration of GDNF in ultrasound tissue after focused ultrasound-induced blood-brain barrier disruption. the

Figure 12 shows the differentiation of PC-12 cells induced by GDNF. A microvesicle group; B, GDNF-targeted microvesicle group; C: GDNF protein group. the

Figure 13 shows the opening of the blood-brain barrier by MRI-guided low-frequency focused ultrasound combined with microbubbles and the opening area of the blood-brain barrier displayed by EB. the

Detailed ways

The present invention will be described below through specific examples, but the present invention is not limited thereto. the

The experimental methods described in the following examples, unless otherwise specified, are conventional methods; the reagents and biological materials, unless otherwise specified, can be obtained from commercial sources. the

Example 1. Preparation of GDNF-loaded microbubble contrast agent and its research on opening the blood-brain barrier

1) Preparation of GDNF-loaded microbubbles

A certain amount of DSPC (distearoylphosphatidylethanolamine, 10.64mg), DSPE-PEG2k (polyethylene glycol-distearoylphosphatidylethanolamine, 2.10mg), DSPE-PEG2k-Biotin (polyethylene glycol) was added in a certain proportion. Ethylene glycol-distearoylphosphatidylethanolamine-biotin, 2.26 mg) was dissolved in 0.5 ml of chloroform and mixed on a vortex mixer. Remove chloroform under the action of dry _N2 flow to form a uniform film of phospholipid on the test tube wall, and dry it in a vacuum oven for more than 2 hours. Then add the Tris buffer solution of 5ml degassed pH7.4 in the test tube that contains dry phospholipid film: containing glycerol (10% volume fraction) and propylene glycol (10% volume fraction) obtain the phospholipid solution of certain concentration. Heat the phospholipid solution to above its phase transition temperature (55-60°C), and disperse the milky phospholipid solution thoroughly with a water-bath ultrasonic oscillator until it is transparent. Replace it with sulfur hexafluoride or perfluoropropane, seal it with an aluminum-plastic cap and a rubber stopper, and store it at 4°C for later use.

When in use, after the microbubbles are prepared by a mechanical shaking method (shocking for 45s), the microbubbles are washed 3-4 times with a PBS solution centrifugal floating method to remove phospholipids that do not form microbubbles. Add 0.1 mg of avidin to 1 ml of microbubbles, incubate at room temperature for 20 minutes with gentle shaking, continue to wash with PBS solution centrifugal floating method for 3-4 times, remove uncoupled avidin, and then 1 mg of biotinylated GDNF (PeproTech Company, 450-51) was mixed into the obtained microbubbles, shaken gently and incubated at room temperature for 15 minutes, and then washed 3-4 times by flotation to remove unbound biotinylated GDNF, GDNF-targeted microvesicles were obtained. the

Performance testing of the prepared GDNF-loaded microbubbles:

Particle counter (Accusizer 780/A, USA) detected the particle size of GDNF-loaded microbubbles: the particle size distribution was 0.5-8 μm, and the average particle size was 2.3 μm. the

The drug loading and encapsulation efficiency in GDNF-loaded microbubbles: 46.58% and 81.52%, respectively. the

The effect of opening the blood-brain barrier (BBB) was compared between self-made blank microbubbles and Sonovi under the action of focused ultrasound (probe frequency 1MHz, volume of microbubbles 1ml, irradiation time 60s, sound pressure 0.8MPa), the results are shown in Figure 1 -4. the

Figure 1 is the detection diagram of self-made microbubbles and Sonovo (Brocco, H20080059) particle size analyzer at different times, wherein A: self-made microbubble 0h group; B: self-made microbubble 24h; C: Sonovel 0h group; D : Sonovi 24h group. the

Figure 2 shows the general view and cross-sectional view of the brain tissue with focused ultrasound combined with different microbubbles to open the blood-brain barrier, in which A: self-made microbubble 0h group; B: self-made microbubble 24h; C: SonoVue 0h group; D: SonoVide 24h group. the

Figure 3 shows the EB content in the brain tissue of each group, where A: self-made microbubble 0h group; B: self-made microbubble 24h; C: Sonovo 0h group; D: Sonovo 24h group. the

Figure 4 is HE staining of brain tissue with focused ultrasound combined with different microbubbles to open the blood-brain barrier, in which A: self-made microbubble 0h group; B: Sonovo 0h group. the

The results showed that there was no significant difference between self-made microbubbles and Sonovo in the opening of the BBB, and the effect of self-made microbubbles on opening the BBB was more stable. the

Example 2. Determining the optimal parameters of MRI-guided low-frequency focused ultrasound combined with targeted microbubbles to partially open the BBB

Taking ultrasonic irradiation time, microbubble dose, delay time (microbubble injection time and acoustic shock time interval), ultrasonic frequency, and sound pressure as influencing factors, three levels were selected for each factor, and an orthogonal experimental design was adopted (see Table 1 ), taking Evans blue (EB, Evens blue, Sigma company, E2129) exudation as an indicator of the blood-brain barrier passage rate (EB exudation), to determine the partial opening of the BBB by MRI-guided low-frequency focused ultrasound combined with targeted microbubbles , the optimal parameter to increase central GDNF levels. the

Test method: The permeability is measured by the quantitative estimation of the exudation of EB. Take the rat brain tissue perfused with PBS, immerse the partially blue-stained brain tissue in formamide solution at a wet weight of 10ml/g, and extract it in a constant temperature water bath at 60°C for 24 hours. The formamide solution presents different shades of blue. The tissue was colorless and transparent, and then the OD value of EB in formamide was quantified at a wavelength of 620nm under a UV300 ultraviolet-visible spectrophotometer, and the content of EB in the sample brain tissue was quantitatively analyzed. Pure formamide solution was set as blank control, and the EB content (μg/g) was calculated according to the standard curve. the

The results are shown in Table 1. the

Table 1 Factors and levels of optimal parameters for MRI-guided low-frequency focused ultrasound combined with targeted microbubbles to partially open the BBB

Table 2 Orthogonal design of the optimal parameters of MRI-guided low-frequency focused ultrasound combined with targeted microbubbles to partially open the BBB and EB exudation in each group

The results are shown in Figure 5-9

Figure 5 shows the EB content in the brain tissue of the blood-brain barrier opened by ultrasound combined with microbubbles under different conditions. It can be seen that the BBB permeability of group 6, group 11, group 13 and group 14 is stronger. the

Figure 6 shows the HE staining of brain tissue sections under different conditions of ultrasound combined with microbubbles to open the blood-brain barrier (A: control group; B: group 6; C: group 11; D: group 13; E: group 14, 400×). the

Figure 7 is the TUNEL staining (400×) of the brain tissue sections of ultrasound combined with microbubbles to open the blood-brain barrier under different conditions (A: control group; B: group 6; C: group 11; D: group 13; E: group 14, 400× ). the

Figure 8 shows the effect of focused ultrasound on inducing apoptosis of blood-brain barrier cells. The number of apoptotic cells in each sample was counted in five independent experiments (*P<0.01 compared with control group and group 6). the

Fig. 9 is the result of ultrastructural observation by electron microscope (A: control group; B: group 6; C: group 11; D: group 13; E: group 14, 400×). In groups D and E, endothelial cells and nerve cells were severely damaged, but no changes were observed in groups A, B, and C. the

The above results show that the probe frequency is 1MHz, the amount of microbubbles is 0.5ml, the irradiation time is 60s, the sound pressure is 0.8MPa, and the delay time is 60s are good parameter conditions. degree), while histological and electron microscope observations showed no obvious structural damage. the

Embodiment 3, the mensuration of protein content in brain

The ultrasonic conditions of this embodiment are the optimal parameter conditions obtained in Example 2, that is, the probe frequency is 1 MHz, the amount of microbubbles is 0.5 ml, the irradiation time is 60 s, the sound pressure is 0.8 MPa, and the time delay is 60 s. the

Ultrasound combined with microbubbles to open the blood-brain barrier, after intravenous injection of protein, the determination of protein content in the brain and the comparison of the two protein transport methods. Detect the amount of protein and the amount of protein entering the brain tissue after ultrasound combined with microbubbles to open the BBB. the

(1) Due to the high price of GDNF, another low-priced alternative protein BSA was used to simulate the experimental conditions to determine the amount entering the brain tissue. the

Divided into two groups: ultrasound + microbubbles + protein (group A); ultrasound + protein-loaded microbubbles (group B); set different dosages: 1) 0 μg/kg, 1) 100 μg/kg, 2) 200 μg/kg kg; 3) 400 μg/kg, 4) 800 μg/kg, 5) 1200 μg/kg. the

The experimental results are shown in Figure 10, which shows the concentration of BSA in ultrasonic tissue after focused ultrasound-induced blood-brain barrier destruction (*p<0.05). the

(2) GDNF-loaded microbubbles were injected into the tail vein at a dose of 800 μg/kg. the

The experimental results are shown in Figure 11, which shows the concentration of GDNF in ultrasonic tissue after focused ultrasound-induced blood-brain barrier destruction (*p<0.05). (A in Figure 11: GDNF (injection of GDNF after injection of microbubbles); B loaded with GDNF microbubbles)

The above results showed that 1) the protein content detected in the brain tissue increased with the increase of the protein concentration in intravenous injection; 2) compared with the two protein delivery methods, the protein-loaded microbubbles could promote the protein to enter the brain tissue better. the

Embodiment 4, the detection of GDNF fluorescent microbubble activity in vitro

In order to detect whether the activity of GDNF is affected during the production of targeted microbubbles, its in vitro activity was measured by PC12 (mouse pheochromocytoma cells). The PC-12 cell line was derived from a transplantable mouse pheochromocytoma. There is a reversible neuronal phenotype response to GDNF. the

Experimental groups: A, microbubble group; B, GDNF-targeted microbubble group; C, GDNF protein group. the

Method: Suspend the cells in DMEM medium containing 5% HS and 5% FBS, and inoculate them in a 12-well plate at a concentration of 2*10 ³ cell/ml. 50ng/ml recombinant GDNF was added to C, GDNF-targeted microbubbles containing 50ng/ml GDNF were added to B, and the same amount of microbubbles was added to A. Cell changes were observed after 7 days of culture.

The results are shown in Figure 12, GDNF-induced PC-12 cells are differentiating. the

The results showed that the GDNF contained in the microvesicles and the recombinant GDNF had the same effect on inducing the phenotype response of PC-12 cells, which indicated that the GDNF contained in the microvesicles had biological activity. the

Example 5, Opening of the blood-brain barrier under MRI monitoring

MRI scanner (SIEMENS), Germany adopts clinical standard 3.0T signal system. Correct the focal point coordinates of the system before each irradiation. T1 phase scanning (TE: 16ms, TR: 1000ms, bandwidth: 16kHz, matrix: 184×256, flip angle: 90°, acquisition times: 4 times, scanning layer thickness: 1mm) was used before irradiation and in the experiment. The animals were anesthetized by intraperitoneal injection of 10% chloral hydrate, and the rat hair in the irradiated area was removed with depilatory agent after the anesthesia took effect. Supine fixed in the MRI ultrasound therapy system, the surface coil with a diameter of 7.5cm was fixed under the head of the animal to reduce interference signals. Adjust the focus of the system to the target area according to the MRIT1 information, and 10 seconds before irradiation, inject 0.5ml of microbubbles (here the microbubbles are GDNF-loaded microbubbles) (5-8×10 ⁸ /ml) into the tail vein (in this example, low-frequency focusing The parameters are: probe frequency 1MHz, microbubble volume 0.5ml, irradiation time 60s, sound pressure 0.8MPa, delay 60s), and inject Magnevist contrast agent (0.2ml/kg) into tail vein 15 seconds after ultrasound irradiation, After the T1 phase scan, EB (100 mg/kg) was injected into the tail vein to reveal the open area of the blood-brain barrier.

The results are shown in Figure 13, which shows the opening of the blood-brain barrier by MRI-guided low-frequency focused ultrasound combined with microbubbles and the open area of the blood-brain barrier displayed by EB. Among them, A: MRI transverse image; B: MRI coronal image, the contrast agent hyperintensities in the brain parenchyma shown by the arrows in Figures A and B indicate that the local BBB is open, and the MRI contrast agent enters the brain parenchyma; C: the same as Figure A Corresponding view of brain tissue section; D: view of brain tissue section corresponding to Figure B, both Figures C and D show blue staining of brain tissue corresponding to the MRI image, indicating that the local blood-brain barrier is opened and the dye Evans blue enters brain tissue. the

The results showed that after ultrasound combined with microbubbles to open the blood-brain barrier, the high-intensity area of contrast agent (Fig. 13A, B) displayed on MRI scanning was consistent with the blue-stained area of EB in gross view (Fig. 13C, D), indicating that MRI can monitor the blood-brain barrier. The brain barrier opens. the

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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