In-vitro double-layer blood brain barrier system with controllable opening time

Abstract

The invention relates to the field of biological medicine, and provides an in-vitro double-layer blood-brain barrier system with controllable opening time, and the reversible recovery time of the system is 6-24 hours under the intervention and stimulation of ultrasound and ultrasonic response micro-nano particles. The system can be used for evaluating the permeability of the drug at the specified opening time, is beneficial to better predicting and analyzing the intracerebral delivery efficiency of the drug under the condition of reversibly opening the blood-brain barrier, and provides important support for drug targeted delivery and treatment effect optimization.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to an in vitro double-layer blood-brain barrier system with controllable opening time. Background Art

[0002] Ultrasound combined with microbubble technology can achieve reversible opening of the blood-brain barrier (BBB), which provides an effective method for targeted drug delivery to the central nervous system (CNS). Studies have shown that among brain-targeted delivery drugs, the blood half-life of most bare drugs and drug-loaded nanoparticles injected intravenously is less than 6 hours, and the blood-brain barrier needs to be closed within 24 hours to ensure the safety of the brain environment. Therefore, in order to ensure the effective enrichment of drugs in the brain while minimizing brain damage, the in vivo BBB opening window time needs to be closed within 6-24 hours. However, there is still a lack of an in vitro blood-brain barrier system with controllable opening time in the field, which is used to quantitatively evaluate the drug permeability during the specified opening window of the BBB. Therefore, the present invention provides an in vitro double-layer blood-brain barrier system with controllable opening time, and its reversible recovery time is 6-24 hours. The establishment of this system helps to better predict and analyze the delivery efficiency of drugs in the brain during the safe window period when the BBB can be reversibly opened, providing important support for the targeted delivery of brain drugs and the optimization of therapeutic effects. Summary of the invention

[0003] The present invention provides an in vitro double-layer blood-brain barrier system with controllable opening time, which is used to evaluate the permeability of drugs at a specified opening time, so as to better predict and analyze the efficiency of drug delivery into the brain under the condition of reversible opening of the BBB, and provide important support for targeted drug delivery and optimization of therapeutic effects.

[0004] To achieve the above objectives, the system of the present invention has a reversible recovery time of 6-24 hours under the intervention stimulation of ultrasound combined with ultrasound-responsive micro-nanoparticles.

[0005] The double-layer blood-brain barrier is characterized in that it is composed of an upper layer of cells, a middle layer of supporting layer and a lower layer of cells, wherein the upper layer of cells are endothelial cells, the middle layer of supporting layer is a porous adhesion plate, the lower layer of cells are pericytes or astrocytes, and the upper layer of cells are co-incubated with droplets.

[0006] The endothelial cells are characterized in that they are one or more of mouse brain microvascular endothelial cells or human brain microvascular endothelial cells.

[0007] Preferably, the brain microvascular endothelial cells are one or more of the bEnd.3 cell line derived from mouse brain microvascular endothelial cells; the hCMEC / D3 cell line and the hCMEC / D3-RFP cell line derived from human brain microvascular endothelial cells; and the hBMEC cell line derived from primary cultured human brain microvascular endothelial cells.

[0008] More preferably, the brain microvascular endothelial cells are one or more of a bEnd.3 cell line derived from mouse brain microvascular endothelial cells and a hBMEC cell line derived from primary cultured human brain microvascular endothelial cells.

[0009] The porous adhesion plate is characterized in that the pore size of the porous filter membrane is smaller than the diameter of the cells to be cultured. The lower layer cells are characterized in that they are one or more of mouse pericytes, mouse astrocytes, human pericytes or human astrocytes.

[0010] Preferably, the brain astrocytes are primary cultured astrocytes isolated and cultured from brain tissue of newborn or adult animals (such as mice and humans); derived from mouse astrocytoma SVGp12 cell line; derived from one or more of human astrocyte H4 cell line and HAPI cell line.

[0011] More preferably, the brain astrocytes are one or more of astrocytes derived from mouse C8-D1A cell line and astrocytes derived from human HAPI cell line.

[0012] The ultrasound-responsive micro-nanoparticles are nanodroplets, characterized in that they consist of a shell layer and a liquid encapsulated by the shell layer.

[0013] Preferably, the shell layer is composed of lipid material; Preferably, the liquid consists of perfluorocarbon.

[0014] The ultrasonic intervention stimulation is characterized in that the intervention stimulation parameters are: the ultrasonic power selection range is 50-80 W, the ultrasonic frequency selection range is 1-3 MHz, the ultrasonic time selection range is 1-3 min, and the duty cycle selection range is 20%-30%.

[0015] Preferably, the intervention stimulation parameters are: ultrasound power selection range is 60-70 W, ultrasound frequency selection range is 1-2 MHz, ultrasound time selection range is between 1-2 min, and duty cycle selection range is 20%-25%.

[0016] The reversible recovery is characterized in that the absolute difference in TEER value before and after stimulation is 10-50 Ω·cm 2 , P app The relative difference in values ​​is 10-50 times.

[0017] Preferably, the absolute difference in TEER value before and after stimulation is 20-40Ω·cm 2 , P app The relative difference in value is 20-40 times The method for constructing the system is characterized in that the method comprises the following steps: a) constructing an in vitro double-layer blood-brain barrier model, wherein the model is a double-layer blood-brain barrier model with a TEER value of 200-400 Ω·cm 2 , and its Papp value is 0.1x10 4 -3x10 4 cm / s; Preferably, the TEER value is 300-400 Ω·cm 2 ; Preferably, the P app The value is 0.1x10 4 -0.3x10 4 cm / s.

[0018] b) Add ultrasound-responsive micro-nanoparticles to the endothelial cell side of the blood-brain barrier and maintain their concentration at 1*10 8 -1*10 9 / ml, incubate for 5-10 min; Preferably, the concentration is 1*10 8 -0.5*10 9 / ml; Preferably, the incubation time is 5-6 min.

[0019] c) After the ultrasound-responsive micro-nanoparticles are in uniform contact with the endothelial cells in the blood-brain barrier to partially simulate the in vivo environment, ultrasound intervention stimulation is performed.

[0020] The present invention adopts the above technical solution to achieve the following beneficial effects: The present invention adopts the above method to achieve reversible opening of the in vitro blood-brain barrier model, and provides a reliable measurement tool for quantitatively analyzing the efficiency of multiple drugs crossing the blood-brain barrier during the opening of the blood-brain barrier.

[0021] The ultrasound-responsive micro-nanoparticle concentration and ultrasound stimulation parameters provided by the present invention can open the tight junctions between barrier cells without excessively damaging the barrier cells (barrier layer shedding or barrier cell death), thereby providing a safe and reliable basis for the application technology of central nervous system drug delivery.

[0022] The invention provides the construction of initial TEER value and P app The value can not only achieve the goal of constructing a mature in vitro blood-brain barrier model, but also ensure that the blood-brain barrier model can be reversibly opened under the ultrasound stimulation parameters provided by the present invention.

[0023] The range endpoints and any values ​​disclosed in this article are not limited to the exact range or value, and these ranges or values ​​should be understood to include close to these ranges or values. For numerical ranges, each range endpoint value, each range endpoint value and a single point value, and a single point value can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article. Herein, in the absence of special instructions, data ranges include endpoints. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a schematic diagram of the in vitro double-layer blood-brain barrier system with controllable opening time according to the present invention. DETAILED DESCRIPTION

[0025] The specific implementation of the present invention is described in detail below. It should be understood that the specific implementation described here is only used to illustrate and explain the present invention, and is not used to limit the present invention.

[0026] Unless otherwise defined, all scientific and technical terms used in the present invention have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention relates. Example

[0027] 1. Construct an in vitro double-layer blood-brain barrier model. First, invert the model and lay mouse astrocyte cell line C8-D1A on the bottom layer of the model at a seeding concentration of 4x10 4 After the astrocytes were attached to the model, the model was placed upright and mouse brain endothelial cell line bEnd.3 was spread on the top of the model at a seeding concentration of 2x10 4 After 4 days of contact co-culture, the TEER value was measured using a resistance meter and the TEER value was 400 Ω·cm 2 ; Detection of P using fluorescein yellow penetration method app value, get P app The value is 0.1x10 4 cm / s.

[0028] 2. Add the nanodroplets from the upper layer to the endothelial cell side of the blood-brain barrier and maintain the concentration at 1*10 8 / ml, incubate for 5 min; 3. The nanodroplets were brought into uniform contact with the endothelial cells by positive gravity, followed by ultrasonic stimulation. The ultrasonic parameters were as follows: ultrasonic power of 60 W, ultrasonic frequency of 1 MHz, ultrasonic time of 1 min, and duty cycle of 20%, thereby constructing an in vitro double-layer blood-brain barrier system with controllable opening time.

[0029] 4. Then, the open blood-brain barrier system was used to test the application of gene drug Cy5-siRNA in the open blood-brain barrier: Cy5-siRNA was first added from the endothelial cell side, and its concentration was maintained at 200nM. After standing for 3 hours, 100μL of the solution on the astrocyte side was taken. The sample fluorescence was further measured by an ELISA instrument (Ex = 645 nm, Em = 670 nm) to measure the transport rate (%) of the Cy5-siRNA gene drug.

[0030] 5. Subsequent long-term measurement of TEER and P app The value was used to monitor the reversible recovery time of the double-layer blood-brain barrier in vitro.

[0031] 6. The in vitro double-layer blood-brain barrier system with controllable opening time formed by the above method is denoted as I1, and its TEER value and P app The values, reversible recovery time and drug transport rate (%) are shown in Table 1. Example

[0032] 1. Construct an in vitro double-layer blood-brain barrier model. First, invert the model and place human astrocyte HAPI cells on the lower layer of the model at a seeding concentration of 4x10 4 / well, and let it stand for 5 hours. After the astrocytes are attached to the model, the model is placed upright and human brain endothelial cell line hBMEC is inoculated on the upper layer of the model at a concentration of 2x10 4 After 4 days of contact co-culture, the TEER value was measured using a resistance meter and the TEER value was 350 Ω·cm 2 ; Detection of P using fluorescein yellow penetration method app value, get P app The value is 2x10 4 cm / s.

[0033] 2. Add the nanodroplets from the upper layer to the endothelial cell side of the blood-brain barrier and maintain the concentration at 0.5*10 9 / ml, incubate for 6 min; 3. The nanodroplets were brought into uniform contact with the endothelial cells by positive gravity, followed by ultrasonic stimulation. The ultrasonic parameters were as follows: ultrasonic power of 70 W, ultrasonic frequency of 2 MHz, ultrasonic time of 2 min, and duty cycle of 25%, thereby constructing an in vitro double-layer blood-brain barrier system with controllable opening time.

[0034] 4. Then, an open blood-brain barrier system was used to test the application of fluorescently labeled antibody-drug conjugates (Alexa Fluor 647-anti-HER2 antibody and doxorubicin conjugate) in the open blood-brain barrier: First, Alexa Fluor 647-anti-HER2 antibody and doxorubicin conjugate were added from the endothelial cell side to maintain its concentration at 200 μM. After standing for 3 hours, 100 μL of the solution on the astrocyte side was taken, and the sample fluorescence was further measured by an ELISA reader (Ex = 650 nm, Em = 668 nm) to measure the transport rate (%) of Alexa Fluor 647-anti-HER2 antibody and doxorubicin conjugate.

[0035] 5. Subsequent long-term measurement of TEER and P app The value can be used to monitor the reversible recovery time of the in vitro double-layer blood-brain barrier system.

[0036] 6. The in vitro double-layer blood-brain barrier system with controllable opening time formed by the above method is denoted as I2, and its TEER value and P app The values, reversible recovery time and drug transport rate (%) are shown in Table 1. Example

[0037] 1. Construct an in vitro double-layer blood-brain barrier model. First, invert the model and lay mouse astrocyte cell line C8-D1A on the bottom layer of the model at a seeding concentration of 4x10 4 After the astrocytes were attached to the model, the model was placed upright and mouse brain endothelial cell line bEnd.3 was spread on the top of the model at a seeding concentration of 2x10 4 After 4 days of contact co-culture, the TEER value was measured using a resistance meter and the TEER value was 280 Ω·cm 2 ; Detection of P using fluorescein yellow penetration method app value, get P app The value is 8x10 4 cm / s.

[0038] 2. Add the nanodroplets from the upper layer to the endothelial cell side of the blood-brain barrier and maintain the concentration at 1*10 9 / ml, incubate for 10 min; 3. The nanodroplets were brought into uniform contact with the endothelial cells by positive gravity, followed by ultrasonic stimulation. The ultrasonic parameters were as follows: ultrasonic power of 50 W, ultrasonic frequency of 3 MHz, ultrasonic time of 3 min, and duty cycle of 30%, thereby constructing an in vitro double-layer blood-brain barrier system with controllable opening time.

[0039] 4. Then, the open blood-brain barrier system was used to test the application of fluorescein-labeled chemical drug doxorubicin (Fluorescein-Doxorubicin) in the open blood-brain barrier: First, Fluorescein-Doxorubicin was added from the endothelial cell side to maintain its concentration at 100 μM. After standing for 3 hours, 100 μL of the solution from the astrocyte side was taken and TEER and P were measured again. app The value was used to monitor the degree of opening of the double-layer blood-brain barrier system in vitro. Furthermore, the sample fluorescence was measured by an ELISA instrument (Ex = 490nm, Em = 520nm) to measure the chemical drug transport rate (%) of Fluorescein-Doxorubicin.

[0040] 5. Subsequent long-term measurement of TEER and P app The value can be used to monitor the reversible recovery time of the in vitro double-layer blood-brain barrier system.

[0041] 6. The in vitro double-layer blood-brain barrier system with controllable opening time formed by the above method is denoted as I3, and its TEER value and P app The values, reversible recovery time and drug transport rate (%) are shown in Table 1. Comparative Example 1 The operation was carried out according to the method of Example 1, except that the initial TEER value was 100 Ω·cm 2 and the initial Papp value is 1x10 6 cm / s, and the final in vitro double-layer blood-brain barrier system with controllable opening time was recorded as D1.

[0042] TEER value and P value during opening of an in vitro double-layer blood-brain barrier system with controllable opening time app The values, reversible recovery time and drug transport rate (%) are shown in Table 1. Comparative Example 2 The operation was carried out according to the method of Example 2, except that the concentration of nanodroplets was 0.5*10 8 / ml, incubated for 3 minutes, and finally obtained an in vitro double-layer blood-brain barrier system with controllable opening time, which was recorded as D2.

[0043] TEER value and P value during opening of an in vitro double-layer blood-brain barrier system with controllable opening time app The values, reversible recovery time and drug transport rate (%) are shown in Table 1. Comparative Example 3 The operation was carried out according to the method of Example 3, except that the ultrasonic parameters were: ultrasonic power of 90 W, ultrasonic frequency of 4 MHz, ultrasonic time of 4 min, and duty cycle of 50%. Finally, an in vitro double-layer blood-brain barrier system with controllable opening time was obtained, which was recorded as D3.

[0044] TEER value and P value during opening of an in vitro double-layer blood-brain barrier system with controllable opening time app The values, reversible recovery time and drug transport rate (%) are shown in Table 1. Table 1 Note: In Table 1, TEER value, P app Values ​​and drug transport rates (%) are approximate.

[0045] As can be seen from Table 1, compared with the prior art, the in vitro double-layer blood-brain barrier system with controllable opening time constructed by the present invention has higher safety, reversible recovery function within 6-24 hours and reasonable drug penetration efficiency. The present invention provides technical support for the in vitro evaluation of drug blood-brain barrier permeability and provides a reliable basis for the clinical development of opening the blood-brain barrier and delivering drugs in vivo. It should be noted that, in this article, the terms "comprise", "include" or any other variant thereof are intended to cover non-exclusive inclusion, so that the process, method, article or device including a series of elements includes not only those elements, but also includes other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise one..." do not exclude the presence of other identical elements in the process, method, article or device including the element. In addition, it should be pointed out that the scope of the methods and devices in the embodiments of the present application is not limited to performing functions in the order shown or discussed, and may also include performing functions in a substantially simultaneous manner or in reverse order according to the functions involved, for example, the described method may be performed in an order different from that described, and various steps may also be added, omitted, or combined. In addition, the features described with reference to certain examples may be combined in other examples.

[0046] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An in vitro double-layer blood-brain barrier system with controllable opening time, characterized in that: The reversible recovery time of the system is 6-24 hours under the intervention stimulation of ultrasound combined with ultrasound-responsive micro-nanoparticles.

2. The in vitro double-layer blood-brain barrier according to claim 1, characterized in that: It consists of an upper layer of cells, a middle layer of supporting layer and a lower layer of cells, wherein the upper layer of cells are endothelial cells, the middle layer of supporting layer is a porous adhesion plate, the lower layer of cells are pericytes or astrocytes, and the upper layer of cells are co-incubated with ultrasound-responsive micro-nanoparticles.

3. The endothelial cell according to claim 2, characterized in that The cell is a kind of mouse-derived brain microvascular endothelial cell or human-derived brain microvascular endothelial cell.

4. The adhesive plate with holes according to claim 2, characterized in that: The pore size of the porous filter membrane is smaller than the diameter of the cells to be cultured.

5. The lower layer cell according to claim 2, characterized in that: It is a type of mouse pericyte, mouse astrocyte, human pericyte or human astrocyte.

6. According to claim 1, the ultrasound-responsive micro-nano particles are nano-droplets, characterized in that: The nano droplet consists of a shell layer and a liquid enclosed by the shell layer.

7. The ultrasound intervention stimulation according to claim 1, characterized in that: The intervention stimulation parameters were as follows: ultrasound power selection range was 50-80 W, ultrasound frequency selection range was 1-3 MHz, ultrasound time selection range was 1-3 min, and duty cycle selection range was 20%-30%.

8. The reversible recovery according to claim 1, characterized in that: The absolute difference in TEER value before and after stimulation is 10-50 Ω·cm 2 , P app The relative difference in values ​​is 10-50 times.

9. The in vitro double-layer blood-brain barrier system with controllable opening time according to claim 1, characterized in that: The construction method includes the following steps: a) Construct an in vitro double-layer blood-brain barrier model with a TEER value of 200-400 Ω·cm 2 , and its Papp value is 0.1x10 4 -3x10 4 cm / s; b) Add ultrasound-responsive micro-nanoparticles to the endothelial cell side of the blood-brain barrier and maintain their concentration at 1*10 8 -1*10 9 / ml, incubate for 5-10 min; c) After the ultrasound-responsive micro-nanoparticles are in uniform contact with the endothelial cells in the blood-brain barrier to partially simulate the in vivo environment, ultrasound intervention stimulation is performed.

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