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Method for evaluating blood-brain barrier permeability of nano-drug

A nano-drug and blood-brain barrier technology, applied in biochemical equipment and methods, drug screening, compound screening, etc., can solve the problems of culture medium spectral interference, complex components, and high detection costs

Inactive Publication Date: 2018-08-10
ACADEMY OF MILITARY MEDICAL SCI
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  • Abstract
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Problems solved by technology

[0007] (1) In the classic transwell in vitro blood-brain barrier model method, it is necessary to accurately test the drug concentration in the lower pool to calculate the permeability of the drug, but the high-concentration culture solution in the lower pool is difficult to detect by liquid chromatography. Several methods can be used The detection methods also have their own defects. For example, if the drug itself has fluorescence or can absorb fluorescence, it can be considered to use a fluorescence spectrophotometer or a UV-visible spectrophotometer for detection, but the culture medium in the lower tank is prone to spectral interference, which seriously reduces the The detection limit of the method has a large error; and for example, if the common high-sensitivity radioactive detection method is used, the drug to be tested needs to be modified (such as radioactive isotope labeling, etc.) before detection, which makes the detection cost too high, and the above The method is suitable for the detection of drug molecules dissolved and dispersed in the system in an ionic state, but not suitable for the evaluation of blood-brain barrier permeability of centrally-targeted nano-drugs in the state of solid particles
[0008] (2) In the zebrafish model method, the operation of injecting drugs into the heart is cumbersome and difficult, and it is difficult to distinguish whether the fluorescence observed in the brain is from the outer layer of the brain or the inner cortex of the brain, so it is impossible to accurately evaluate the blood flow of drugs. brain barrier permeability
[0009] (3) In the rat model method, the components of rat brain tissue fluid, cerebrospinal fluid, and brain homogenate are complex, with large interference, and it is difficult to qualitatively or quantitatively determine the drug content in them

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Examples

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Embodiment 1

[0121] Example 1 Evaluation of the blood-brain barrier permeability of 5nm, 100nm and 360nm silicon nanomedicine (1) Isolation of SD rat brain microvascular endothelial cells and establishment of transwell in vitro blood-brain barrier model:

[0122] Separate the cerebral cortex of 2-week-old SD rats, add 4 mL of D-Hank's solution containing 0.1% (W / W) type II collagenase (containing 120 μL DNase I), place it in a water bath at 37 ° C for 1.5 hours, and then dissolve it at 1000 r Centrifuge at room temperature for 8 minutes at a speed of / min, discard the supernatant; add 20% (W / W) BSA to resuspend and centrifuge (1000g, 20 minutes, 4°C), remove the middle layer tissue and large blood vessels, and keep the bottom sediment Add 2 mL of D-Hank's solution containing 0.1% (W / W) collagenase / dispase (containing 40 μL DNaseI), digest for 1 hour, then centrifuge at 1000 r / min for 8 minutes at room temperature, and discard the supernatant , and purified by density gradient centrifugat...

Embodiment 2

[0135] Example 2 Evaluation of the blood-brain barrier permeability of the nano-medicine modified by the outer layer

[0136] The preparation method of the 100nm silicon nanomedicine modified by transferrin is as follows: Weigh 40mg of 100nm silicon nanomedicine and ultrasonically disperse it in 10mL toluene to form a suspension; inject the suspension into a round bottom flask, and then add the suspension to the suspension Add 150μL (3-oxiranylmethoxypropyl)trimethoxysilane to the solution, and react at 60°C under nitrogen protection for 5 hours; after the reaction, wash the sample with absolute ethanol and centrifuge three times, and the nano (3-oxiranylmethoxypropyl)trimethoxysilane molecules have been bonded to the surface of the drug particles. The obtained sample was dispersed in 5 mL of PBS buffer, and 450 μL of transferrin was added to it, and reacted in the dark for 30 minutes; after the reaction was completed, the sample was washed with deionized water and centrifug...

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Abstract

The invention belongs to the field of detection of nano-drugs and in particular relates to a method for evaluating blood-brain barrier permeability of the nano-drug. The method comprises the followingsteps: (1) establishing a transwell in-vitro blood-brain barrier model; (2) adding suspension liquid containing a fluorescence nano-drug and / or a fluorescence dyed nano-drug into an upper tank of themodel established in step (1) and standing; (3) after finishing standing, taking out a membrane in a small chamber; carrying out fluorescence dyeing on cells on the membrane to obtain a sample to bedetected, wherein a dyed fluorescence color can be distinguished from a fluorescence color in step (2); (4) carrying out layer-by-layer scanning and photographing on the sample to be detected in step(3) along the thickness direction by adopting a laser co-focusing microscope; (5) superimposing pictures obtained by step (4) according to a scanning or photographing sequence and then taking a lateral view; evaluating the blood-brain barrier permeability of the nano-drug through observing the lateral view. The method provided by the invention can be used for accurately and rapidly evaluating theblood-brain barrier permeability of the nano-drug in vitro.

Description

technical field [0001] The invention belongs to the field of nano-medicine detection, and in particular relates to a method for evaluating the blood-brain barrier permeability of nano-medicine. Background technique [0002] The blood-brain barrier is a physiological barrier formed by the walls of brain capillaries and glial cells. Except for certain amino acids, hexoses, and neuropeptides that can penetrate the blood-brain barrier through special carriers, the blood-brain barrier can prevent about 95% of small molecules and about 100% of large molecules from entering the brain. Therefore, the blood-brain barrier It plays an important role in maintaining the dynamic stability of the central nervous system. But at the same time, it is difficult for most drugs to penetrate the blood-brain barrier and enter the central system to take effect, which makes it more difficult for the treatment of brain diseases such as the nervous system. Based on this situation, for drugs for the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/02C12N5/079C12N5/071
CPCC12N5/0622C12N5/069C12N2503/02G01N33/5008G01N2500/10
Inventor 王永安孟凡荣杨军范丽雪王甲朋李万华隋昕骆媛高翔
Owner ACADEMY OF MILITARY MEDICAL SCI
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