Preparation method and application of a bionic brain-targeted cyclosporine A nanocrystal drug

Abstract

The present invention discloses a preparation method and application of a biomimetic brain-targeted cyclosporine A (CsA) nanoparticle crystal drug. The composition of the biomimetic brain-targeted CsA nanocrystal drug is CsA and platelet membrane (M- PLT ), and M- PLT is wrapped on the surface of CsA nanocrystals by the polycarbonate membrane extrusion method (CsA@M- PLT ), improving the stability of CsA nanocrystals and the targeting property to the brain injury site. The drug loading of CsA in CsA@M- PLT is 49.17%. After being taken up by neurons, CsA@M- PLT can restore the mitochondrial membrane potential of neurons, reduce the release of mitochondrial ROS, and inhibit the apoptosis of neuron cells; CsA@M- PLT can reduce the damage of the blood-brain barrier, reduce brain edema, and treat secondary brain injury efficiently through multiple pathways.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceuticals, and particularly to a novel and highly efficient brain-targeted drug delivery system for treating secondary brain injury. Background Art

[0002] Traumatic brain injury (TBI) is a common traumatic disease. According to epidemiological data from different countries and different periods, there are approximately 55 million new TBI cases globally every year. Secondary brain injury is tissue cell damage caused by a series of biochemical changes triggered by traumatic brain injury, which can further exacerbate brain injury, leading to epilepsy, cognitive dysfunction, permanent neurological damage, emotional disorders, personality changes, mental abnormalities, etc. It is one of the important reasons for the high mortality and poor prognosis of TBI patients. At present, although a large number of experimental studies are dedicated to the neuroprotection of secondary brain injury, due to the fact that neuroprotective agents can only block a certain injury mechanism after trauma and have large toxic and side effects, most of their clinical studies have ended in failure. Therefore, developing a neuroprotective agent that can treat secondary brain injury through multiple pathways has important clinical value.

[0003] Previous studies have shown that neuroinflammation is the most important mechanism for exacerbating secondary brain injury. The over-activated inflammatory response will cause changes in the permeability of the blood-brain barrier, resulting in cerebral edema, secondary cerebral perfusion insufficiency, etc., and ultimately leading to neuronal apoptosis. Cyclosporine A (CsA) can inhibit the abnormal opening of the mitochondrial permeability transition pore, limit the influx of Ca 2＋ ions, maintain the ion homeostasis and membrane potential in mitochondria, thereby maintaining the energy supply in the damaged area and playing a protective role on neurons. Previous studies have shown that CsA can also promote the transformation of M1 microglia into M2 microglia, thereby reducing neuroinflammation. In summary, using cyclosporine A (CsA) can play a role in treating secondary brain injury from two aspects: reducing apoptosis of damaged neurons and inhibiting neuroinflammation. However, CsA has poor solubility, and after entering the blood circulation, CsA is mainly distributed in tissues with a high fat content, such as fat, liver, adrenal gland, and pancreas, etc. It rarely enters the central nervous system, and the plasma protein binding rate of CsA is relatively high. If a high concentration of CsA is to be achieved in the brain, a large dosage needs to be increased, but high-dose CsA will cause systemic immunosuppression and endanger the life of patients instead. Therefore, how to efficiently deliver CsA to the brain injury tissue is the key to using CsA to treat secondary brain injury.

[0004] Nanocrystals (NC) have the advantages of not requiring carrier materials, being easy to industrialize, and having diverse dosage forms. Cyclosporine A (CsA) has poor water solubility. Under suitable conditions, preparing it into nanocrystalline drugs can significantly improve the solubility of CsA. Moreover, compared with traditional nanocarrier-based drug delivery systems, nanocrystalline drugs have extremely high drug-loading capacities. However, nanocrystalline drugs are prone to Oswald ripening phenomena such as aggregation and crystal growth, resulting in an increase in particle size and drug precipitation. Therefore, a large amount of surfactant needs to be added during the preparation of nanocrystalline drugs to increase their stability, but the large addition of surfactants will significantly increase the toxicity of nanocrystalline drugs. Research has found that coating a cell membrane on the surface of nanocrystalline drugs can significantly increase their stability and does not require the addition of surfactants during the preparation process. Previous studies have shown that coating nanoparticles with platelet membranes can not only prolong the circulation time of nanoparticles in the blood but also have strong targeting to damaged sites. Summary of the Invention

[0005] The object of the present invention is to provide a preparation method and application of a biomimetic brain-targeted cyclosporine A nanocrystalline drug. By wrapping platelet membranes on the surface of CsA nanocrystals, a biomimetic brain-targeted cyclosporine A nanocrystalline drug CsA-NC@M is prepared. -PLT to improve the stability of CsA nanocrystals and their targeting to damaged brain tissues and to be used for the treatment of secondary brain injury. CsA-NC@M -PLT can target and accumulate in damaged brain tissues through the platelet membranes on its surface, release CsA, restore the mitochondrial membrane potential, inhibit the release of ROS in the cytoplasm, thereby reducing nerve cell damage, repairing the damaged blood-brain barrier, and improving the treatment effect of secondary brain injury.

[0006] The technical solution of the present invention is: a preparation method of a biomimetic brain-targeted cyclosporine A (CsA) nanocrystalline drug. The composition of the biomimetic brain-targeted cyclosporine A nanocrystalline drug is CsA and platelet membrane M- PLT , and its characteristic is that M- PLT is used as the stabilizer of CsA nanocrystals, and M- PLT is wrapped on the surface of CsA nanocrystals through the polycarbonate membrane extrusion method to form CsA@M- PLT . The M- PLT on the surface of CsA@M- PLT has a high affinity for damaged vascular endothelium, so that CsA@M- PLT can accumulate in the brain injury site, improving the stability of CsA nanocrystals and their targeting to the brain injury site; the particle size of CsA@M- PLT is 178.7 nm, the zeta potential is -23.2 mV, and the CsA drug-loading capacity is 49.17%.

[0007] The present invention first examines the targeting ability of CsA@M- to damaged brain tissues through an in vivo imager; secondly, it uses a flow cytometer to examine the effects of CsA@M- on the opening of the mitochondrial permeability transition pore and the content of mitochondrial ROS after being taken up by neurons; finally, it examines the protective effects of CsA@M- on neurons and the blood-brain barrier at the animal level. It clarifies the effects and mechanisms of CsA@M- in treating secondary brain injury through multiple pathways. PLT The present invention first examines the targeting ability of CsA@M- to damaged brain tissues through an in vivo imager; secondly, it uses a flow cytometer to examine the effects of CsA@M- on the opening of the mitochondrial permeability transition pore and the content of mitochondrial ROS after being taken up by neurons; finally, it examines the protective effects of CsA@M- on neurons and the blood-brain barrier at the animal level. It clarifies the effects and mechanisms of CsA@M- in treating secondary brain injury through multiple pathways. PLT The present invention first examines the targeting ability of CsA@M- to damaged brain tissues through an in vivo imager; secondly, it uses a flow cytometer to examine the effects of CsA@M- on the opening of the mitochondrial permeability transition pore and the content of mitochondrial ROS after being taken up by neurons; finally, it examines the protective effects of CsA@M- on neurons and the blood-brain barrier at the animal level. It clarifies the effects and mechanisms of CsA@M- in treating secondary brain injury through multiple pathways. PLT The present invention first examines the targeting ability of CsA@M- to damaged brain tissues through an in vivo imager; secondly, it uses a flow cytometer to examine the effects of CsA@M- on the opening of the mitochondrial permeability transition pore and the content of mitochondrial ROS after being taken up by neurons; finally, it examines the protective effects of CsA@M- on neurons and the blood-brain barrier at the animal level. It clarifies the effects and mechanisms of CsA@M- in treating secondary brain injury through multiple pathways. PLT The present invention first examines the targeting ability of CsA@M- to damaged brain tissues through an in vivo imager; secondly, it uses a flow cytometer to examine the effects of CsA@M- on the opening of the mitochondrial permeability transition pore and the content of mitochondrial ROS after being taken up by neurons; finally, it examines the protective effects of CsA@M- on neurons and the blood-brain barrier at the animal level. It clarifies the effects and mechanisms of CsA@M- in treating secondary brain injury through multiple pathways.

[0008] Innovation of the present invention: ① Developed a new method to improve the stability of CsA nanocrystals. By wrapping the platelet membrane on the surface of CsA nanocrystals, it not only reduces the surfactants used in large amounts when preparing nanocrystal drugs by traditional methods but also improves the stability of CsA nanocrystals. ② Constructed a drug delivery system that can target and accumulate in damaged brain tissues and effectively treat secondary brain injury. Brief Description of the Drawings

[0009] Figure 1 CsA-NC@M -PLT Characterization of (A) particle size distribution and morphology of CsA-NC; (B) particle size distribution and morphology of CsA-NC@M; (C) Zeta potential distribution of CsA-NC; (D) Zeta potential distribution of CsA-NC@M; -PLT Characterization of (A) particle size distribution and morphology of CsA-NC; (B) particle size distribution and morphology of CsA-NC@M; (C) Zeta potential distribution of CsA-NC; (D) Zeta potential distribution of CsA-NC@M; -PLT Characterization of (A) particle size distribution and morphology of CsA-NC; (B) particle size distribution and morphology of CsA-NC@M; (C) Zeta potential distribution of CsA-NC; (D) Zeta potential distribution of CsA-NC@M;

[0010] Figure 2 Detection of the uptake of CsA-NC@M by scratched N2a cells by flow cytometry; -PLT Detection of the uptake of CsA-NC@M by scratched N2a cells by flow cytometry;

[0011] Figure 3 Detection of the effect of CsA-NC@M on the mitochondrial membrane potential of scratched N2a cells by flow cytometry; (A) Typical flow cytometry pictures of green fluorescence intensity in scratched N2a cells; (B) Typical flow cytometry pictures of red fluorescence intensity in scratched N2a cells; (C) Ratio of red fluorescence intensity to green fluorescence intensity in scratched N2a cells; n = 3, ±SD; -PLT Detection of the effect of CsA-NC@M on the mitochondrial membrane potential of scratched N2a cells by flow cytometry; (A) Typical flow cytometry pictures of green fluorescence intensity in scratched N2a cells; (B) Typical flow cytometry pictures of red fluorescence intensity in scratched N2a cells; (C) Ratio of red fluorescence intensity to green fluorescence intensity in scratched N2a cells; n = 3, ±SD; ** P <0.01;

[0012] Figure 4 Detection of the effect of CsA-NC@M- on the ROS content of scratched N2a cells by flow cytometry; PLT Detection of the effect of CsA-NC@M- on the ROS content of scratched N2a cells by flow cytometry;

[0013] Figure 5 CsA-NC@M -PLTTargeting of damaged brain tissue in mice with mild secondary brain injury model; (A) CsA-NC@M -PLT Distribution in tissues such as brain, heart, liver, spleen, lung and kidney; (B) CsA-NC@M -PLT Distribution in the brain; (C) CsA-NC@M -PLT Distribution in brain tissue sections;

[0014] Figure 6 Evans blue staining experiment to investigate CsA-NC@M -PLT Repair effect on blood-brain barrier in mice with mild secondary brain injury model; (A) Penetration of Evans blue in brain tissue of mice with mild secondary brain injury model after intravenous injection of Evans blue; (B) Observation of Evans blue penetration in brain tissue of mice with mild secondary brain injury model by laser confocal microscopy;

[0015] Figure 7 CsA-NC@M -PLT Effect of CsA-NC@M on water content in brain tissue of mice with mild secondary brain injury model, n = 3, ±SD; ** P <0.01;

[0016] Figure 8 CsA-NC@M -PLT Effect of CsA-NC@M on brain tissue morphology and neurons in mice with mild secondary brain injury model; (A) H&E staining experiment to investigate CsA-NC@M -PLT Effect of CsA-NC@M on brain tissue morphology and neuron morphology in mice with mild secondary brain injury model; (B) Nissl staining to investigate CsA-NC@M -PLT Effect of CsA-NC@M on neuron density in brain tissue of mice with mild secondary brain injury model;

[0017] Figure 9 CsA-NC@M -PLT Effect of CsA-NC@M on ROS content in brain tissue of mice with mild secondary brain injury model. Specific implementation manners

[0018] 1 Research methods

[0019] 1.1 Preparation of platelet membrane-coated cyclosporine A nanocrystalline drug

[0020] Accurately weigh 12.5 mg of CsA, dissolve it in 100 μL of a mixed solvent (polyoxyethylene castor oil: ethanol = 65:35). After mixing well by ultrasonic bath, add it to 2 mL of distilled water, vortex for 30 s, filter through a 0.45 μm microporous membrane to obtain cyclosporine A nanocrystals (CsA-NC). After preparing CsA-NC, add 2.5 mL of platelet membrane solution (containing 2.5 mg of platelet membrane), mix well, probe ultrasound (5 min, 100 W), incubate at room temperature for 30 min, and use a liposome extruder to pass it through polycarbonate membranes with pore sizes of 0.8 μm, 0.4 μm, and 0.2 μm successively, and extrude repeatedly 11 times. Then transfer it to an ultrafiltration centrifugal tube, add 8 mL of pure water, centrifuge (3000 g, 10 min, 25 °C), repeat twice to remove polyoxyethylene castor oil, ethanol, and free CsA, thus obtaining platelet membrane-coated cyclosporine A nanocrystal drug CsA-NC@M -PLT 。

[0021] 1.2 CsA-NC@M -PLT Characterization

[0022] Absorb 1 mL of the CsA-NC@M -PLT solution, and use a laser particle size analyzer to measure the particle size, PDI, and Zeta potential of CsA-NC@M -PLT 。

[0023] Use high performance liquid chromatography to determine the drug loading of CsA in CsA-NC@M -PLT 。CsA high performance liquid chromatography detection conditions: Chromatographic column: C 18 column (4.6 mm × 250 mm, 5 µm, Dikma); Mobile phase: acetonitrile: aqueous phase = 90:10 (V / V); Flow rate: 1.0 mL / min; Column temperature: 55 °C; Injection volume: 20 μL; Detection wavelength: 210 nm.

[0024] Mix 9 mL of chromatographic grade acetonitrile with 1 mL of pure water as a diluent. Accurately weigh 10 mg of CsA, add it to the diluent, and prepare CsA standard solutions with concentrations of 5, 2.5, 1.25, 0.5, 0.25, 0.1, 0.05, and 0.01 mg / mL. Take 200 μL of the above CsA solutions with different concentrations, transfer them to an inner insert tube, then place the inner insert tube into an injection vial, inject and detect according to the above chromatographic conditions. After recording the peak time and peak area, use the peak area as the ordinate and the CsA concentration as the abscissa to plot a working curve and calculate the regression equation of the working curve.

[0025] After freeze-drying CsA-NC@M -PLT accurately weigh 1 mg of CsA-NC@M -PLT, add 200 μL of distilled water and 800 μL of chromatographic grade acetonitrile, vortex and mix well, centrifuge (14000 g, 25 °C, 10 min), take the supernatant, repeat twice, transfer 200 μL of the sample solution to an inner cannula, then place the inner cannula into an injection vial, inject and detect according to the above chromatographic conditions, and calculate the drug loading of CsA in CsA-NC@M according to the working curve regression equation. -PLT The drug loading of CsA in

[0026] 1.3 Observation of CsA-NC@M by transmission electron microscopy -PLT Morphology and platelet membrane coating

[0027] Take a small amount of CsA-NC and CsA-NC@M solutions respectively -PLT , drop them on a copper mesh. After 5 min, blot dry with filter paper, add a drop of 1% phosphotungstic acid, after staining for 2 min, blot dry with filter paper, and observe the morphology of CsA-NC@M by transmission electron microscopy (TEM) -PLT to observe whether the platelet membrane is coated on the surface of CsA-NC.

[0028] 1.4 Observation of the uptake of CsA-NC@M by scratched N2a cells by flow cytometry -PLT

[0029] Take N2a cells in the logarithmic growth phase, wash them with 5 mL of PBS buffer to remove the residual old medium, then add 2 mL of trypsin solution (0.25%) to digest the adherent cells. After about 2 min, add 5 mL of complete DMEM medium to terminate the digestion, and repeatedly pipette the adherent cells with a Pasteur pipette to make the cells evenly dispersed in the culture medium. Then transfer the cell suspension to a centrifuge tube, centrifuge (1000 g, 25 °C, 3 min), discard the supernatant, resuspend the cell pellet with complete medium, take a small amount of the cell suspension to count with a cell counting chamber, and then dilute the cell concentration to 1×10 6 / mL with complete DMEM medium. Add 2 mL of the cell suspension to each well of a 6-well plate. After culturing for 24 h, use a pipette tip to make evenly distributed scratches in the horizontal and vertical directions in the 6-well plate to construct an injured cell model. After 3 h, add CsA-NC@M -PLT-DiO (using CsA-NC@M -RBC-DiO as a control). After incubating for 1 h and 4 h respectively, add PBS buffer, wash to remove the residual medium, collect the cells, add 500 μL of PBS buffer to resuspend the cell pellet, and detect the uptake of CsA-NC@M by N2a cells with a flow cytometer -PLT .

[0030] 1.5 Effect of CsA-NC@M on the mitochondrial membrane potential of scratched N2a cells -PLT

[0031] Take N2a cells in the logarithmic growth phase, wash them with 5 mL of PBS buffer to remove the residual old medium, then add 2 mL of trypsin solution (0.25%) to digest the adherent cells. After about 2 minutes, add 5 mL of complete DMEM medium to terminate the digestion, and repeatedly pipette the adherent cells with a Pasteur pipette to evenly disperse the cells in the culture medium. Then transfer the cell suspension to a centrifuge tube, centrifuge (1000 g, 25 °C, 3 min), discard the supernatant, resuspend the cell pellet with complete medium, take a small amount of cell suspension to count with a cell counting chamber, and then dilute the cell concentration to 1×10 6 / mL with complete DMEM medium. Add 2 mL of cell suspension to each well of a 6-well plate. After culturing for 24 h, use a pipette tip to create evenly distributed scratches in the horizontal and vertical directions in the 6-well plate to construct an injured cell model. After 3 h, add CsA-NC@M -PLT (using CsA-NC@M -RBC as a control). After incubation for 4 h, take out the 6-well plate, pour out the medium, add 2 mL of high-glucose DMEM medium, gently shake it and then pour it out, and repeat twice. Add 1 mL of JC-1 staining working solution to each well and incubate at 37 °C for 20 min. Then pour out the staining working solution, add 1 mL of JC-1 staining buffer to each well, gently shake it and then pour it out, and repeat twice. Add 1 mL of PBS solution to each well, collect the cells, and add 500 μL of PBS buffer to resuspend the cell pellet. Use a flow cytometer to detect the intensities of red fluorescence (excitation wavelength 585 nm, emission wavelength 590 nm) and green fluorescence (excitation wavelength 515 nm, emission wavelength 529 nm) in the cells.

[0032] 1.6 CsA-NC@M -PLT Effect of CsA-NC@M on the ROS content in scratched N2a cells

[0033] Take N2a cells in the logarithmic growth phase, wash them with 5 mL of PBS buffer to remove the residual old medium, then add 2 mL of trypsin solution (0.25%) to digest the adherent cells. After about 2 minutes, add 5 mL of complete DMEM medium to terminate the digestion, and repeatedly pipette the adherent cells with a Pasteur pipette to evenly disperse the cells in the culture medium. Then transfer the cell suspension to a centrifuge tube, centrifuge (1000 g, 25 °C, 3 min), discard the supernatant, resuspend the cell pellet with complete medium, take a small amount of cell suspension to count with a cell counting chamber, and then dilute the cell concentration to 1×10 6 / mL with complete DMEM medium. Add 2 mL of cell suspension to each well of a 6-well plate. After culturing for 24 h, use a pipette tip to create evenly distributed scratches in the horizontal and vertical directions in the 6-well plate to construct an injured cell model. After 3 h, add CsA-NC@M -PLT(using CsA-NC@M -RBC as a control). After incubation for 4 h, take out the 6-well plate, pour out the culture medium, add 2 mL of high-glucose DMEM medium, gently shake and then pour out, repeat twice. Subsequently, add 1 mL of DCFH-DA staining working solution to each well, incubate at 37 °C for 20 min. Then pour out the staining working solution, add 2 mL of high-glucose DMEM medium to each well, gently shake and then pour out, repeat three times to remove the DCFH-DA that has not entered the cells. Collect the cells, add 500 μL of PBS buffer to resuspend the cell pellet. Detect the ROS content in N2a cells by flow cytometry.

[0034] 1.7 Establishment of a mouse model of mild secondary brain injury

[0035] After weighing the mice, inject 3.6% chloral hydrate intraperitoneally at a dose of 10 μL / g. After the mice are anesthetized, place them prone on a foam board, disinfect the skin of the head and neck with alcohol, longitudinally cut about 2 cm of the scalp in the middle, expose the left parietal bone, peel off the periosteum with forceps, drill a bone window with a diameter of about 3 mm in the middle of the left parietal bone, and use the modified Feeney's free fall method to strike the left brain with a 30 g weight freely falling from a height of 10 cm to cause mild brain injury. If bleeding occurs, immediately stop the bleeding with a cotton swab, then drop a drop of penicillin-streptomycin mixed solution around the bone window, seal the bone window with bone wax, suture the scalp, and place it on a warming blanket to recover to obtain a mouse model of mild secondary brain injury.

[0036] 1.8 Targeting of CsA-NC@M -PLT to mild secondary brain injury tissue

[0037] Take the mice with the established mild secondary brain injury model above, and inject CsA-NC@M -PLT-DiO (using CsA-NC@M -RBC-DiO as a control) via the tail vein. After 4 h, 12 h, and 24 h of injection, sacrifice the mice, obtain brain, heart, liver, spleen, lung, and kidney tissues, and observe the distribution of CsA-NC@M -PLT-DiO in the brain, heart, liver, spleen, lung, and kidney tissues of the mice with mild secondary brain injury model by in vivo imaging. Cut the brain tissue into 5 slices evenly, and observe the distribution of CsA-NC@M -PLT-DiO in the brain by in vivo imaging. Fix the brain tissue in 4% paraformaldehyde solution, after 24 h, make paraffin sections, stain with DAPI, and observe the distribution of CsA-NC@M -PLT-DiO in the brain by laser confocal microscopy.

[0038] 1.9 Repair effect of CsA-NC@M -PLT on the blood-brain barrier of mice with mild secondary brain injury model

[0039] Take the mice with the constructed mild secondary brain injury model, and divide the mice into a model group treated with normal saline, a sham operation group, a free CsA group (3 mg / kg), a CsA-NC@M -PLT group (the dose of CsA is 3 mg / kg), and a CsA-NC@M -RBC group (the dose of CsA is 3 mg / kg), a total of five groups. Immediately after model construction, administer the drug by tail vein injection, once every 24 h, for a total of 3 times. On the 3rd day after drug administration, inject 0.2 mL of 2% Evans blue solution (Evans blue, EB) by tail vein. After 3 h, anesthetize the mice by intraperitoneal injection of 0.2 mL of 3.6% chloral hydrate, fix them, cut open the abdominal cavity and thoracic cavity to expose the heart, perfuse 30 mL of normal saline and 20 mL of 4% paraformaldehyde solution into the left ventricle with a venous infusion needle, then completely remove the whole brain, and observe the distribution of EB in the brain. Cut the brain tissue into 5 slices evenly to observe the penetration of EB in the brain. Fix the brain tissue in 4% paraformaldehyde, and after 24 h, make paraffin sections. After DAPI staining, use a laser confocal microscope to observe the penetration of EB in the brain.

[0040] 1.10 CsA-NC@M -PLT Effect on brain water content in mice with mild secondary brain injury model

[0041] On the 3rd day after drug administration, sacrifice the mice, take out the whole brain, weigh the wet weight, then place the brain tissue in a vacuum drying oven and dry it for 48 h to complete dehydration, take it out and weigh the dry weight, and calculate the brain water content according to the formula: water content = (wet weight - dry weight) / wet weight × 100%.

[0042] 1.11 CsA-NC@M -PLT Effect on brain tissue morphology and neurons in mice with mild secondary brain injury model

[0043] On the 7th day after drug administration, sacrifice the mice, take out the whole brain, fix the brain tissue in 4% paraformaldehyde, and after 24 h, make paraffin sections. After sectioning, perform H&E and Nissl staining to observe the effect of CsA-NC@M -PLT on brain tissue morphology and neurons in model mice.

[0044] 1.12 CsA-NC@M -PLT Effect on the content of ROS in brain tissue of mice with mild secondary brain injury model

[0045] On the 3rd day after drug administration, the mice were sacrificed, and the whole brain was removed and frozen-sectioned. The DCFH-DA dye was diluted with serum-free DMEM medium at a ratio of 1:1000, and the frozen sections were immersed in the DCFH-DA staining solution and incubated at 37 °C for 60 min in the dark. The sections were placed in PBS buffer and washed 3 times on a shaker for 7 min each time. After the frozen sections were dried by centrifugation, they were immersed in the DAPI staining solution and incubated at room temperature for 10 min, and then washed with PBS buffer in the same way. After the frozen sections were dried by centrifugation, they were sealed with an anti-fluorescence quenching agent, and finally observed by laser confocal microscopy for the effect of CsA-NC@M -PLT on ROS in the brain tissue of model mice.

[0046] 2 Experimental results

[0047] 2.1 Particle size and morphology of CsA-NC@M -PLT As shown in

[0048] Figure 1 -PLT -PLT the average particle size of CsA-NC@M was 178.7 nm, and the Zeta potential was -23.2 mV. The morphology of CsA-NC@M was spherical, with an obvious core-shell structure, preliminarily proving that the platelet membrane was successfully wrapped on the surface of CsA-NC.

[0049] Taking the CsA concentration as the abscissa (X) and the peak area as the ordinate (Y), a linear regression was performed, and the regression equation of the working curve was obtained as Y = 44.85X + 0.1344, R 2 = 0.9997. Within the set concentration range, the regression equation had a good linear relationship and could be used for the determination of the drug loading of CsA. After detection, the drug loading of CsA in CsA-NC@M -PLT was 49.17%.

[0050] 2.2 Uptake of CsA-NC@M -PLT by scratched N2a cells

[0051] The results of flow cytometry showed that CsA-NC@M -PLT could be taken up by scratched N2a cells in a time-dependent manner, and compared with CsA-NC@M -RBC -PLT Figure 2 the accumulation of CsA-NC@M in N2a cells was significantly increased, as shown in

[0052] -PLT The above results indicate that wrapping CsA-NC with the platelet membrane can improve the targeting of CsA-NC to scratched N2a cells. ​​​​​​

[0053] JC-1 is an ideal fluorescent probe widely used for detecting mitochondrial membrane potential. When the mitochondrial membrane potential is high, JC-1 aggregates in the mitochondrial matrix to form polymers, which can produce red fluorescence; when the mitochondrial membrane potential is low, JC-1 cannot aggregate in the mitochondrial matrix, and at this time JC-1 is in monomer form and can produce green fluorescence. Usually, normal cells have a high mitochondrial membrane potential and emit red fluorescence, while damaged cells have a decreased mitochondrial membrane potential and enhanced green fluorescence. By measuring the intensity ratio of red fluorescence and green fluorescence in cells, the change of mitochondrial membrane potential in cells can be reflected. Flow cytometry detection results show that compared with normal cells, the ratio of red fluorescence intensity / green fluorescence intensity in scratched N2a cells decreased significantly, indicating that the mitochondrial membrane potential of scratched N2a cells decreased. CsA-NC@M -PLT After treatment, the ratio of red fluorescence intensity / green fluorescence intensity in scratched N2a cells increased significantly, and the effect was significantly better than that of free CsA and CsA-NC@M -RBC treatment groups, such as Figure 3 shown. The above results indicate that CsA-NC@M -PLT can restore the mitochondrial membrane potential of scratched N2a cells.

[0054] 2.4 Effect of CsA-NC@M -PLT on the ROS content in scratched N2a cells

[0055] To observe the effect of CsA-NC@M -PLT on the ROS content in scratched N2a cells, cells were treated with DCFH-DA. ROS in the cells breaks the chemical bond between DCFH-DA, releasing DCFH to produce green fluorescence, thereby reflecting the level of ROS in the cells. Flow cytometry detection results show that the green fluorescence intensity in scratched N2a cells is significantly higher than that in other groups, indicating that scratched N2a cells produce a large amount of ROS. CsA-NC@M -PLT After treatment, the green fluorescence intensity in scratched N2a cells decreased significantly, and the effect was significantly better than that of free CsA and CsA-NC@M -RBC treatment groups, such as Figure 4 shown. The above results indicate that CsA-NC@M -PLT can effectively inhibit the generation of ROS in scratched N2a cells.

[0056] 2.5 Targeting of CsA-NC@M -PLT to the damaged brain tissue of mice with mild secondary brain injury model

[0057] In vivo imaging results show that CsA-NC@M -PLTIt can be distributed in the brain tissue of mice with a mild secondary brain injury model. Moreover, after the same administration time, CsA-NC@M -PLT showed significantly more distribution in the brain than CsA-NC@M -RBC . After the brain tissue was evenly cut into 5 slices, it was observed that CsA-NC@M -PLT showed significantly more distribution in the brain injury area than in the normal brain tissue area. The results of laser confocal microscopy showed that CsA-NC@M -PLT showed significantly more distribution in the brain than CsA-NC@M -RBC , and CsA-NC@M -PLT showed significantly more distribution in the brain injury area than in the normal brain tissue area, as Figure 5 shown. The above results indicate that CsA-NC@M -PLT has good targeting to damaged brain tissue.

[0058] 2.6 Repair effect of CsA-NC@M -PLT on the blood-brain barrier of mice with a mild secondary brain injury model

[0059] Evans blue (EB) belongs to an azo dye preparation and is often used as a tracer to detect whether the blood-brain barrier (BBB) is open. When the blood-brain barrier is intact, EB cannot cross the blood-brain barrier and has no distribution in the brain tissue. When the BBB is abnormal, Evans blue can enter the brain tissue and stain it. On the 3rd day after administration to TBI model mice, Evans blue was injected into the tail vein. The results showed that there was no obvious Evans blue penetration in the brain tissue of the sham operation group, while obvious Evans blue distribution could be observed in the brain injury site of the mild secondary brain injury model mice treated with physiological saline. After the brain tissue was evenly cut into 5 slices, obvious infiltration of Evans blue in the brain could be observed, and a large amount of Evans blue penetration could also be observed in the brain in paraffin sections. The infiltration of Evans blue in the CsA-NC@M -PLT administration group was significantly less, and the effect was better than that of free CsA and CsA-NC@M -RBC , as Figure 6 shown. The above results indicate that CsA-NC@M -PLT can repair the blood-brain barrier to a certain extent and protect the brain tissue.

[0060] 2.7 Effect of CsA-NC@M -PLT on the water content of the brain tissue of mice with a mild secondary brain injury model

[0061] The water content of the brain reflects the degree of brain edema to a certain extent. After TBI occurs, the blood-brain barrier is damaged, and severe brain edema usually occurs. The experimental results showed that the water content of the brain in the model group mice treated with physiological saline increased significantly compared with that in the sham operation group, indicating that obvious brain edema occurred in the model mice; while CsA-NC@M -PLTAfter treatment, it can significantly reduce the brain water content, almost equal to that of the sham operation group, and the effect is better than that of free CsA and CsA-NC@M -RBC , such as Figure 7 shown. The above research results indicate that after CsA-NC@M -PLT repairs the blood-brain barrier, it can effectively inhibit the occurrence of brain edema.

[0062] 2.8 Effects of CsA-NC@M -PLT on the brain tissue morphology and neurons of mice with mild secondary brain injury model

[0063] The results of H&E staining showed that in the model mice treated with normal saline, after 7 days, a large area of necrosis appeared in the brain injury area, neuronal chromatin agglutination, nuclear pyknosis. After CsA-NC@M -PLT treatment, the necrotic area was significantly reduced, and the neuronal chromatin was in a trilobed shape, with no obvious difference from normal neurons. Through Nissl staining, it can be seen that compared with the model mice treated with normal saline, the CsA-NC@M -PLT treatment group could significantly increase the neuronal density in the brain injury area, as Figure 8 shown. The above results indicate that CsA-NC@M -PLT can effectively inhibit neuronal damage and play a protective role in the damaged brain tissue.

[0064] 2.9 Effects of CsA-NC@M- PLT on ROS in the damaged brain tissue of mice with mild secondary brain injury model

[0065] The results of DCFH-DA staining showed that compared with the sham operation group, in the brain tissue of the model group mice treated with normal saline, more ROS were produced. The ROS content in the CsA-NC@M -PLT group was significantly less than that in the free CSA group and the drug group wrapped with erythrocyte membrane, and was similar to that of the sham operation group. The results indicate that CsA-NC@M -PLT can reduce the level of ROS in the brain injury area, and the effect is better than that of free CsA and CsA-NC@M -RBC , such as Figure 9 shown.

[0066] 3 Conclusion:

[0067] The cyclosporine A nanocrystal drug CsA-NC@M-PLT wrapped with platelet membrane has good targeting to the damaged brain tissue, can reduce the ROS content in the damaged brain tissue, repair the blood-brain barrier to a certain extent, reduce brain edema, reduce neuronal apoptosis, and significantly improve the therapeutic effect of CsA on secondary brain injury, showing certain application prospects.

Claims

1. A method for preparing a bionic brain-targeted cyclosporin A nanocrystal drug, characterized in that: Platelet membrane M- PLT As a stabilizer, the platelet membrane M- PLT Wrapped on the surface of cyclosporin nanocrystals CsA-NC, CsA-NC@M- PLT Surface M- PLT It has high affinity with damaged vascular endothelium, making CsA-NC@M- PLT It can accumulate in the damaged part of the brain, thus improving the stability of CsA-NC and its targeting to the damaged part of the brain; CsA-NC@M- PLT The particle size was 178.7 nm, the zeta potential was -23.2 mV, and the drug loading was 49.17%; The specific preparation method is as follows: 12.5 mg CsA is accurately weighed, dissolved in 100 μL of mixed solvent, mixed by water bath ultrasound, added to 2 mL of distilled water, vortexed for 30 s, and filtered through a 0.45 μm microporous filter membrane to obtain CsA-NC; after obtaining CsA-NC, 2.5 mL of platelet membrane solution is added, mixed, and probe ultrasound is used for 5 min, 100 W, incubated at room temperature for 30 min, and a liposome extruder is used to pass it through 0.8 μm, 0.4 μm, and 0.2 μm polycarbonate membranes in sequence. After repeated extrusion 11 times, it is transferred to an ultrafiltration centrifuge tube, 8 mL of pure water is added, and centrifugation is performed at 3000 g, 10 min, and 25°C. Repeat twice to remove polyoxyethylene castor oil, ethanol, and free CsA to obtain CsA-NC@M. -PLT ; The mixed solvent is a mixture of polyoxyethylene castor oil and ethanol, wherein the volume ratio of polyoxyethylene castor oil to ethanol is 65:

35.

2. The method for preparing the bionic brain-targeted cyclosporin A nanocrystal drug according to claim 1, characterized in that: The platelet membrane solution contains 2.5 mg of platelet membranes.

3. CsA-NC@M obtained by the method for preparing a bionic brain-targeted cyclosporine A nanocrystal drug according to any one of claims 1 to 2 -PLT Application in the preparation of drugs for treating secondary brain injury diseases.

Images