System for opening in-vitro double-layer blood brain barrier by adopting ultrasonic response micro-nano particles

Abstract

The invention relates to the field of biological medicine, and provides a system for opening an in-vitro double-layer blood brain barrier by adopting ultrasonic response micro-nano particles. The system comprises the following modules: (1) an ultrasonic response micro-nano particle incubation module; (2) an ultrasonic intervention module; according to the system, an open blood brain barrier model is formed by a method for opening an in-vitro double-layer blood brain barrier through ultrasonic response micro-nano particles. The model can be used for quantitatively analyzing the blood brain barrier crossing efficiency of various drugs, is beneficial to screening and optimizing drug candidate compounds suitable for ultrasonic enhanced administration, and provides a basis for clinical transformation application.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a system for opening an in vitro double-layer blood-brain barrier by using ultrasound-responsive micro-nano particles. Background Art

[0002] Focused ultrasound combined with micro-nanoparticles to open the blood-brain barrier (BBB) ​​is an emerging brain drug delivery technology. The blood-brain barrier protects the brain from harmful substances in the blood. Focused ultrasound combined with micro-nanoparticle technology has been shown to partially open the blood-brain barrier in vivo and achieve drug delivery in the brain. However, since it is impossible to quantitatively evaluate the penetration efficiency of drugs in the brain in vivo, researchers need to use an in vitro blood-brain barrier model to simulate the in vivo environment for quantitative evaluation of drugs. However, the relevant technologies in the field are currently limited to in vitro quantitative analysis of the permeability of drugs in the brain after ultrasound opens the blood-brain barrier: the blood-brain barrier after opening lacks safety, comprehensiveness and stability, which leads to doubts about the authenticity and rationality of the measurement results, and the results are difficult to repeat. The reason is that the existing technology cannot achieve uniform and comprehensive contact between micro-nanoparticles and the blood-brain barrier model. The present invention aims at this limitation and proposes a system for opening the double-layer blood-brain barrier in vitro using ultrasound-responsive micro-nanoparticles. The system can open the blood-brain barrier model safely (the barrier exists intact and cells are not dead), comprehensively (acting on the entire range of the barrier) and stably (ensuring effective opening every time), providing an effective means to evaluate the ability of ultrasound-enhanced drugs to penetrate the blood-brain barrier, helping to screen and optimize drug candidate compounds suitable for ultrasound-enhanced drug delivery technology, and providing a basis for clinical translational applications. Summary of the invention

[0003] The present invention provides a system for opening a double-layer blood-brain barrier in vitro using ultrasound-responsive micro-nanoparticles, providing an effective way to quantitatively analyze the permeability efficiency of different drugs in the blood-brain barrier.

[0004] To achieve the above objectives, the system of the present invention, under the stimulation of ultrasound intervention, combined with ultrasound-responsive micro-nanoparticles, can achieve safe (the barrier exists intact and there is no cell death), comprehensive (acting on the entire range of the barrier) and stable (ensuring effective opening every time) opening of the blood-brain barrier in vitro.

[0005] A method for opening a double-layer blood-brain barrier in vitro using ultrasound-responsive micro-nanoparticles, characterized in that the method comprises the following steps: (1) For the in vitro double-layer blood-brain barrier model, ultrasound-responsive micro-nanoparticles were added to the endothelial cell side of the blood-brain barrier, and the concentration was maintained at A. After filling with complete culture medium, a sealing film was installed, and the model was inverted 180° and incubated for 3-20 minutes to allow the ultrasound-responsive micro-nanoparticles to be in uniform contact with the endothelial cells in the blood-brain barrier; Preferably, the incubation time is 5-10 min.

[0006] (2) After the ultrasound-responsive micro-nanoparticles are in uniform contact with the endothelial cells in the blood-brain barrier, ultrasound intervention stimulation is performed to stably open the blood-brain barrier. The TEER value of the opened blood-brain barrier is 100-200 Ω·cm 2 , its P app The value is 4*10 4 -1*10 5 cm / s.

[0007] Preferably, the TEER value is 150-180 Ω·cm 2 .

[0008] Preferably, the P app The value is 5*10 4 -1*10 5 cm / s.

[0009] The in vitro double-layer blood-brain barrier model is characterized in that the upper layer is brain microvascular endothelial cells, the lower layer is brain astrocytes, and the TEER value is detected using a resistor meter. app The value was detected by the fluorescein yellow permeation method, and the TEER value was 200-400Ω·cm 2 , its P app The value is 0.1*10 4 -3*10 4 cm / s.

[0010] Preferably, the TEER value is 300-400 Ω·cm 2 .

[0011] Preferably, the P app The value is 0.1*10 4 -0.3*10 4 cm / s.

[0012] The double-layer blood-brain barrier model of the present invention is characterized in that the upper layer is brain microvascular endothelial cells, the lower layer is brain astrocytes, and the TEER value is detected using a resistor meter. app The values ​​were detected using the fluorescein yellow permeabilization method.

[0013] Preferably, the brain microvascular endothelial cells are bEnd.3 cell line, bEnd.5 cell line, bEnd.1 cell line derived from mouse brain microvascular endothelial cells; hCMEC / D3 cell line, hCMEC / D3-RFP cell line derived from human brain microvascular endothelial cells; hBMEC cell line derived from primary cultured human brain microvascular endothelial cells; rBMEC cell line derived from primary cultured rat brain microvascular endothelial cells; pBMEC cell line derived from primary cultured pig brain microvascular endothelial cells; rbBMEC cell line derived from primary cultured rabbit brain microvascular endothelial cells; or one or more of nhPBMEC cell line derived from primary cultured non-human primate (such as marmoset, macaque) brain microvascular endothelial cells.

[0014] 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.

[0015] Preferably, the brain astrocytes are primary cultured astrocytes isolated and cultured from brain tissue of newborn or adult animals (such as mice, rats, humans); derived from rat glioma C6 cell line; derived from mouse astrocyte tumor SVGp12 cell line; derived from human astrocyte H4 cell line, HAPI cell line; derived from rat astrocyte tumor RBA-1 cell line; derived from non-human primate (such as marmoset, macaque) astrocyte primary cultured cell nhPAst cell line or more.

[0016] 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.

[0017] The ultrasound-responsive micro-nanoparticles of the present invention are characterized in that they can realize micro-nanoparticle oscillation, contraction and explosion under ultrasound stimulation, and specifically select gas-carrying lipid microparticles or nanodroplets. The gas-carrying lipid microparticles are composed of a lipid shell layer and a body encapsulated by the shell layer, and the nanodroplets are composed of a shell layer and a liquid encapsulated by the shell layer.

[0018] The concentration A of the present invention, for the lipid particles with gas, is 1*10 6 -1*10 9 / ml.

[0019] Preferably, the concentration of the gas-carrying lipid particles is 1*10 8 -1*10 9 / ml; The uniform contact of the present invention is characterized in that, for the gas-carrying lipid particles, an inverted buoyancy operation is used to achieve it.

[0020] The ultrasonic stimulation described in the present invention is characterized in that its ultrasonic parameters are: acoustic power of 50-250 W, ultrasonic frequency of 0.3-5 MHZ, ultrasonic time of 1-10 min, and duty cycle of 20%-60%.

[0021] Preferably, the ultrasonic parameters are: acoustic power 100W-200W, ultrasonic frequency 3-5 MHZ, ultrasonic time 5min-8min, duty cycle 20%-30%.

[0022] The system of the present invention can be used to quantitatively analyze the efficiency of multiple drugs crossing the blood-brain barrier.

[0023] The present invention adopts the above technical solution to achieve the following beneficial effects: 1. The present invention adopts the above system to achieve safe (the barrier exists intact and cells are not dead), comprehensive (acting on the entire range of the barrier) and stable (ensuring effective opening every time) opening of the blood-brain barrier in vitro.

[0024] 2. The blood-brain barrier model obtained by selecting the ultrasound-responsive micro-nanoparticle concentration and ultrasound stimulation parameters of the present invention can avoid excessive damage to barrier cells (barrier cells do not fall off), open the blood-brain barrier more safely, and provide a reliable basis for central nervous system drug delivery.

[0025] 3. Initial TEER value and P of the blood-brain barrier constructed by the present invention app The value can not only achieve the goal of building a mature blood-brain barrier, but also ensure that the in vitro model can be opened safely, comprehensively and stably under the ultrasound stimulation parameters preferred by the present invention.

[0026] The endpoints and any values ​​of the ranges 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

[0027] Figure 1 Schematic diagram of the system for opening the double-layer blood-brain barrier in vitro using ultrasound-responsive micro-nanoparticles according to the present invention. DETAILED DESCRIPTION

[0028] The specific implementation of the present invention is described in detail below. It should be understood that the specific implementation described herein is only used to illustrate and explain the present invention, and is not used to limit the present invention.

[0029] 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 1

[0030] 1. Construct a 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 4*10 4 / well, let it stand for 5 hours, and when the astrocytes are close to the model, the model is placed upright, and mouse brain endothelial cell line bEnd.3 is spread on the top of the model, with an inoculation concentration of 2*10 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 0.1*10 4 cm / s.

[0031] 2. Add the air-carrying lipid microparticles to the endothelial cell side of the blood-brain barrier and maintain the concentration at 1*10 9 / ml, fill with complete culture medium, install sealing film, invert and incubate for 3-20 min; incubate for 5 min.

[0032] 3. The inverted buoyancy effect was used to achieve uniform contact between the gas-carrying lipid particles and the endothelial cells to partially simulate the in vivo environment, and then ultrasonic stimulation was performed; the ultrasonic parameters were acoustic power of 150 W, ultrasonic frequency of 4 MHZ, ultrasonic time of 5 min, and duty cycle of 20%, thereby completing the construction of an open blood-brain barrier system.

[0033] 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 to maintain its concentration at 200 nM. 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 opening degree of the double-layer blood-brain barrier. Finally, the fluorescence of the sample was measured by an ELISA reader (Ex = 645 nm, Em = 670 nm) to measure the transport rate (%) of the Cy5-siRNA gene drug.

[0034] 5. The system formed by the above method is denoted as I1, and its TEER value and P app The values ​​and drug transport rates (%) are shown in Table 1. Example 2

[0035] 1. Construct a double-layer blood-brain barrier model. First, invert the model and place human astrocyte HAPI cells on the bottom layer of the model at a seeding concentration of 4*10 4 / well, let it stand for 5 hours, and when the astrocytes are close 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 2*10 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 0.2*10 4 cm / s.

[0036] 2. Add the air-carrying lipid microparticles to the endothelial cell side of the blood-brain barrier and maintain the concentration at 1*10 8 / ml, incubate for 20 min.

[0037] 3. The inverted buoyancy effect was used to achieve uniform contact between the gas-carrying lipid particles and the endothelial cells to partially simulate the in vivo environment, and then ultrasonic stimulation was performed; the ultrasonic parameters were acoustic power of 180 W, ultrasonic frequency of 5 MHZ, ultrasonic time of 6 min, and duty cycle of 30%, thereby completing the construction of an open blood-brain barrier system.

[0038] 4. Then, the 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, maintaining its concentration at 200 μM. After standing for 3 hours, 100 μL of the solution on the astrocyte side was taken and TEER and P were measured again. app The value was used to monitor the opening degree of the double-layer blood-brain barrier. Finally, the sample fluorescence was measured by an ELISA reader (Ex = 650 nm, Em = 668 nm) to measure the transport rate (%) of the Alexa Fluor 647-anti-HER2 antibody and doxorubicin conjugate.

[0039] 5. The system formed by the above method is denoted as I2, and its TEER value and P app The values ​​and drug transport rates (%) are shown in Table 1. Example 3

[0040] 1. Construct a double-layer blood-brain barrier model. First, invert the model and lay mouse astrocyte cell line C8-D1A on the lower layer of the model at a seeding concentration of 4*10 4After 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 2*10 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 2*10 4 cm / s.

[0041] 2. Add the gas-carrying lipid particles to the endothelial cell side of the blood-brain barrier and maintain the concentration at 1*10 6 / ml, incubate for 15 min.

[0042] 3. The nanodroplets were uniformly contacted with the endothelial cells by positive gravity to partially simulate the in vivo environment, and then ultrasonic stimulation was performed; the ultrasonic parameters were acoustic power of 50 W, ultrasonic frequency of 2 MHZ, ultrasonic time of 3 min, and duty cycle of 50%, thereby completing the construction of an open blood-brain barrier system.

[0043] 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 opening degree of the double-layer blood-brain barrier. Finally, the sample fluorescence was measured by an ELISA reader (Ex = 490 nm, Em = 520 nm) to measure the chemical drug transport rate (%) of Fluorescein-Doxorubicin.

[0044] 5. The system formed by the above method is denoted as I3, and its TEER value and P app The values ​​and drug transport rates (%) are shown in Table 1.

[0045] Comparative Example 1 The operation was carried out according to the method of Example 1, except that the gas-carrying lipid particles were in non-uniform contact with the blood-brain barrier model through the normal effect, and the final system was recorded as D1.

[0046] System TEER value and P app The values ​​and drug transport rates (%) are shown in Table 1.

[0047] Comparative Example 2 The operation was carried out according to the method of Example 2, except that the gas-carrying lipid particles were in non-uniform contact with the blood-brain barrier model through the normal effect, and the final system was recorded as D2.

[0048] System TEER value and P app The values ​​and drug transport rates (%) are shown in Table 1.

[0049] Comparative Example 3 The operation was carried out according to the method of Example 3, except that the gas-carrying lipid particles were in non-uniform contact with the blood-brain barrier model through the upright effect, and the final system was recorded as D3.

[0050] System TEER value and P app The values ​​and drug transport rates (%) are shown in Table 1.

[0051] Table 1 serial number <![CDATA[TEER value (Ω·cm 2 )]]> <![CDATA[P app Value (cm / s)]]> Drug penetration rate (%) I1 150 <![CDATA[9*10 4 ]]> 83 I2 180 <![CDATA[5*10 4 ]]> 78 I3 200 <![CDATA[4*10 4 ]]> 65 D1 380 <![CDATA[0.15*10 4 ]]> 1 D2 340 <![CDATA[1*10 4 ]]> 10 D3 250 <![CDATA[3*10 4 ]]> 5 Note: In Table 1, TEER value, P app Values ​​and drug transport rates (%) are approximate.

[0052] It can be seen from Table 1 that compared with the prior art, the system constructed by the present invention has higher safety, higher blood-brain barrier opening efficiency and 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.

[0053] 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.

[0054] 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. A method for opening the double-layer blood-brain barrier in vitro using ultrasound-responsive micro-nanoparticles, characterized in that: The method comprises the following steps: (1) For the in vitro double-layer blood-brain barrier model, ultrasound-responsive micro-nanoparticles were added to the endothelial cell side of the blood-brain barrier, and the concentration was maintained at A. After filling with complete culture medium, a sealing film was installed, and the model was inverted 180° and incubated for 3-20 minutes to allow the ultrasound-responsive micro-nanoparticles to evenly contact the endothelial cells in the blood-brain barrier; (2) After the ultrasound-responsive micro-nanoparticles are in uniform contact with the endothelial cells in the blood-brain barrier, ultrasound intervention stimulation is performed to stably open the blood-brain barrier. The TEER value of the opened blood-brain barrier is 100-200 Ω·cm 2 , its P app The value is 4*10 4 -1*10 5 cm / s.

2. The in vitro double-layer blood-brain barrier model according to claim 1, characterized in that: The upper layer is the brain microvascular endothelial cells, and the lower layer is the brain astrocytes. The TEER value is detected using a resistor meter. app The value was detected by the fluorescein yellow permeation method, and the TEER value was 200-400 Ω·cm 2 , its P app The value is 0.1*10 4 -3*10 4 cm / s.

3. The ultrasound-responsive micro-nano particles according to claim 1, characterized in that: Ultrasonic stimulation can cause the micro-nanoparticles to oscillate, shrink and explode. The micro-nanoparticles are selected from gas-carrying lipid microparticles, which are composed of a lipid shell layer and a gas encapsulated by the shell layer.

4. The concentration A according to claim 1, characterized in that: For gas-carrying lipid particles, the concentration A is 1*10 6 -1*10 9 / ml.

5. The ultrasound intervention stimulation according to claim 1, characterized in that: The ultrasonic parameters include acoustic power of 50-250 W, ultrasonic frequency of 0.3-5 MHZ, ultrasonic time of 1-10 min, and duty cycle of 20%-60%.

6. A system for opening the double-layer blood-brain barrier in vitro using ultrasound-responsive micro-nanoparticles, characterized in that: Includes the following modules: (1) An ultrasound-responsive micro-nanoparticle incubation module, used to achieve uniform contact between ultrasound-responsive micro-nanoparticles and the blood-brain barrier endothelial cell layer; (2) Ultrasound intervention module, used to achieve stable opening of the blood-brain barrier under ultrasound intervention stimulation.

7. The system according to claim 6, characterized in that: It can be used to quantitatively analyze the efficiency of multiple drugs crossing the blood-brain barrier.

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