Application of Bestrophin 1 as ischemic stroke subacute stage treatment target

Abstract

The invention relates to an application of Bestrophin 1 as a treatment target in an ischemic stroke subacute stage, and belongs to the technical field of biological medicines. The research finds that the expression of an ischemic stroke model animal in subacute stage astrocyte Bestrophin 1 (BEST1) is obviously increased, the motor function recovery is hindered, and the motor function defect can be improved by down-regulating the BEST1, so that the BEST1 can be used as an important target for developing the ischemic stroke subacute stage medicine. On the other hand, the invention further designs a long-circulating nano-carrier of siRNA (siBest1) entrapping Bestphin 1, and the long-circulating nano-carrier is used for intravenous injection, can deliver siBest1 to a brain injury part of an ischemic stroke model animal and effectively knock down the expression of astrocyte BEST1, and can achieve the effect of improving motor function defects for a long time through short treatment 5-11 days after stroke.

Description

Technical Field

[0001] The invention relates to application of Bestrophin 1 (BEST1) as a therapeutic target for subacute phase of ischemic stroke, and belongs to the technical field of biomedicine. Background Art

[0002] Stroke is a cerebrovascular disease caused by impaired cerebral blood circulation, of which ischemic stroke accounts for about 80%. In recent years, drug thrombolysis and intravascular therapy have made great progress in the treatment of acute ischemic stroke, further highlighting the medical and social problems caused by the high disability rate. According to statistics, about 75% of the 7 million stroke survivors are left with disabilities and lose their ability to work and live to varying degrees. After the acute phase of ischemic stroke, there is a subacute phase that is extremely critical for the repair of neurological function, which starts from a few days and lasts for weeks or even months. However, in clinical practice, except for rehabilitation exercises, there is currently a lack of clear targets and effective drugs for the treatment of subacute ischemic stroke.

[0003] Bestrophins are calcium-activated chloride channels (CACCs) located on the plasma membrane and are mainly expressed in retinal pigment epithelial cells. Among the members of the Bestrophins family, Bestrophin 1 (BEST1) encodes CACCs that play an important role in synaptic transmission. Studies have shown that BEST1 mRNA is widely distributed in the brain, especially in the olfactory bulb, hippocampus and cerebellum, and is significantly expressed in neurons and astrocytes. BEST1 ion channels can permeate Cl - and HCO3 - In addition to anions, the most important characteristic of BEST1 in the central nervous system is its high permeability to two neurotransmitters, glutamate and γ-aminobutyric acid. Especially in pathological conditions, the release of neurotransmitters mediated by BEST1 is of great significance. However, in the subacute stage of ischemic stroke, there are no reports on the value of BEST1 in the recovery of ischemic stroke neurological function and whether it can be used as a therapeutic target.

[0004] At present, for potential therapeutic targets such as BEST1 that do not have specific small molecule inhibitors, small interfering RNA (siRNA) can be used to downregulate the expression of specific genes. This RNA interference (RNAi) technology has opened up a new way of thinking for the treatment of many major diseases and the identification and confirmation of effective therapeutic targets, and has a very broad application prospect. However, siRNA has a large molecular weight and a large amount of negative charge on the surface, which leads to its extremely low ability to transport across biological membranes. In addition, siRNA is easily degraded by nucleases and has poor stability. Its in vivo and in vitro delivery usually requires the help of a carrier. Existing siRNA delivery vectors are divided into viral vectors and non-viral vectors: although viral vectors such as retrovirus, lentivirus and adenovirus have good infection efficiency, their clinical application is limited due to their strong immunogenicity and great biosafety risks; non-viral vectors such as cationic liposomes and even lipid or polymer nanoparticles currently used in clinical practice are safer than viral vectors, but have low transfection efficiency, and are rapidly cleared from the blood after traditional intravenous administration, which is not conducive to the enrichment of siRNA in the target site, lack of target site selectivity, and limited transmembrane transport capacity, making it difficult to cross the blood-brain barrier. Therefore, in the relevant research on the application of BEST1-specific small interfering RNA (siBest1) in the treatment of ischemic stroke, it is very necessary to construct a safe and effective nanocarrier system to efficiently deliver siBest1 to the ischemic injury site of the brain. At present, there is no report on self-assembled nanoparticles for targeted delivery of siBest1 into the brain. Summary of the invention

[0005] The present invention aims at the clinical problems of narrow therapeutic window of ischemic stroke and lack of subacute therapeutic drugs with clear targets, and provides a target BEST1 for subacute treatment of ischemic stroke; the present invention finds that downregulating or knocking down the expression of BEST1 in astrocytes can increase the excitability of neurons in the peri-infarct area and promote neural network remodeling of the sensorimotor cortex, thereby improving motor function.

[0006] The technical solution adopted by the present invention to solve the technical problem is:

[0007] In a first aspect, the present invention seeks to protect the use of Bestrophin 1 (BEST1) as a therapeutic target for subacute ischemic stroke in the preparation of a drug for the treatment of subacute ischemic stroke.

[0008] Wherein, the Bestrophin 1 is mammalian Bestrophin 1.

[0009] Preferably, the mammalian Bestrophin 1 is mouse Bestrophin 1 or human Bestrophin 1.

[0010] More preferably, the mouse Bestrophin 1 gene sequence is shown in https: / / www.ncbi.nlm.nih.gov / nuccore / NC_000085.7?from=9962536&to=9978997&report=genban k&strand=true, and the protein amino acid sequence is shown in https: / / www.ncbi.nlm.nih.gov / protein / NP_036043.2, specifically:, as shown in SEQ ID No: 1.

[0011] More preferably, the human Bestrophin 1 gene sequence is shown in https: / / www.ncbi.nlm.nih.gov / nuccore / NG_009033.1?from=5468&to=19578&report=genbank, and the protein amino acid sequence is shown in https: / / www.ncbi.nlm.nih.gov / protein / NP_001132915.1, specifically: as shown in SEQ ID No: 2.

[0012] In specific embodiments, the medicament is for improving motor function.

[0013] In a second aspect, the present invention seeks to protect the use of a drug for downregulating or knocking out BEST1 in the preparation of a drug for treating subacute ischemic stroke.

[0014] In a specific embodiment, the medicament is used to promote recovery of motor function after stroke.

[0015] In a specific embodiment, the active ingredient in the drug for downregulating or knocking out BEST1 is siRNA of Bestrophin 1 (siBest1) or a small molecule compound that can specifically inhibit BEST1.

[0016] In a more specific embodiment, the nucleic acid sequence of the siBest1 is: UUGCCAACUUGUCAAUGAATT. The siBest1 has a good interference effect on both mouse and human Bestrophin 1.

[0017] In a specific embodiment, the drug is a composite nanoformulation named SNP-siBest1, which has polylactic-co-glycolic acid (PLGA) as the main core, encapsulating amphiphilic cationic materials polyethyleneimine-epoxydodecane (PEI-C12) and BEST1, and Tween 80 is distributed on the surface of the core, with a particle size of 110 nm.

[0018] In a specific embodiment, the mass ratio of the PLGA to the PEI-C12 is 3 to 5:1, such as 3:1, 4:1, 5:1, etc.

[0019] In a specific embodiment, the mass ratio of the PEI-C12 to the Bestrophin 1 siRNA is 20-30:1, preferably 20-27:1, 23-27:1, 24-30:1, etc., specifically 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 30:1, etc., and can be adjusted according to actual conditions.

[0020] In a specific embodiment, the concentration of Tween 80 in the aqueous phase is 1-5 mg / mL, preferably 1-3 mg / mL, 2-4 mg / mL, 3-5 mg / mL, more specifically 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, etc.

[0021] In a specific embodiment, the preparation method of the composite nanoformulation is as follows: the organic phase is added dropwise to the aqueous phase under vigorous stirring to obtain nanoparticles.

[0022] In a more specific embodiment, the preparation method of the composite nanoformulation is as follows:

[0023] (1) PLGA and PEI-C12 were added to N,N-dimethylformamide (DMF), stirred evenly, and Bestrophin 1 siRNA dissolved in DEPC water was added, and stirred to obtain an organic phase;

[0024] (2) adding Tween 80 to water and stirring to obtain an aqueous phase;

[0025] (3) The organic phase was added dropwise into the aqueous phase and stirred vigorously to obtain nanoparticles.

[0026] In a third aspect, the present invention protects a drug, which comprises a pharmaceutically acceptable carrier and an effective amount of an active ingredient, wherein the active ingredient is Bestrophin 1 siRNA.

[0027] In a specific embodiment, the nucleic acid sequence of the Bestrophin 1 siRNA is: UUGCCAACUUGUCAAUGAATT. The siBest1 has a good interference effect on both mouse and human Bestrophin 1, and can be applied to both mice and humans.

[0028] In a specific embodiment, the drug is a composite nanoformulation, which has polylactic-co-glycolic acid (PLGA) as the main core, encapsulating the amphiphilic cationic material polyethyleneimine-epoxydodecane (PEI-C12) and Bestrophin 1 siRNA, and Tween 80 is distributed on the surface of the core.

[0029] In a specific embodiment, the mass ratio of the PLGA to the PEI-C12 is 4:1, the mass ratio of the PEI-C12 to the Bestrophin 1 siRNA is 25:1, and the concentration of Tween 80 in the aqueous phase is 2 mg / mL.

[0030] In a specific embodiment, the preparation steps of the composite nanoformulation are as follows:

[0031] (1) PLGA and PEI-C12 were added to N,N-dimethylformamide (DMF), stirred evenly, and Bestrophin 1 siRNA dissolved in DEPC water was added, and stirred to obtain an organic phase;

[0032] (2) adding Tween 80 to water and stirring to obtain an aqueous phase;

[0033] (3) The organic phase was added dropwise into the aqueous phase and stirred vigorously to obtain nanoparticles.

[0034] In order to safely and efficiently deliver siBest1 into the brain, the present invention synthesizes a cationic lipid-like amphiphilic polymer (PEI-C12) composed of low molecular weight polyethyleneimine (polyetherimide, PEI) and 1,2-epoxydodecane (1,2-epoxydodecane, O-C12). PEI-C12 can effectively compress siBest1 through electrostatic interaction to form a nanoscale complex, which can then be actively encapsulated in PLGA nanoparticles with a Tween 80 shell to self-assemble into composite nanoparticles encapsulating siBest1. The nanoparticles designed by the present invention can well protect the siBest1 encapsulated therein from nuclease degradation. In addition, the Tween 80 on the surface of the nanoparticles increases its surface hydrophilicity, making the particles less likely to be engulfed by phagocytes, prolonging the circulation time of the nanoparticles in the body, and making the nanoparticles and the siBest1 encapsulated therein easier to reach the site of action in the body. At the same time, Tween 80 can bind to endogenous apolipoproteins, thereby recognizing the low-density lipoprotein receptor-related protein-1 (LRP-1) highly expressed on the blood-brain barrier, and promoting the nanoparticles to penetrate the blood-brain barrier through LRP-1-mediated transmembrane transport. Cationic polymer PEI has a strong proton buffering capacity, which can promote the lysosomal escape of siBest1, thereby helping to achieve an efficient gene silencing effect. Since the toxicity of PEI depends on its molecular weight and increases with the increase of molecular weight, the present invention selects low-toxic PEI with a molecular weight of 800Da when preparing nanoparticles. In addition, PLGA and Tween 80 used in the present invention are both approved pharmaceutical excipients.

[0035] This application has at least one of the following technical effects or advantages:

[0036] (1) The study found that after ischemic brain injury, astrocytes are activated in large numbers, inducing BEST1 to maintain a high level of expression for a long time starting from the 7th day after cerebral ischemia, mediating the massive release of extrasynaptic γ-aminobutyric acid, forming abnormal tension inhibition on neurons, thereby affecting the excitability of neurons in the subacute peri-infarct area and the remodeling of the neural network in the sensorimotor cortex, making it difficult to recover the damaged neurological function. By specifically knocking down the expression of BEST1 in astrocytes through siBest1, a therapeutic target is provided for the treatment of subacute ischemic stroke.

[0037] (2) Prepare a long-circulating nanoformulation for brain-targeted delivery of siBest1, which is administered by intravenous injection after cerebral ischemia. It can enrich and effectively knock down the expression of BEST1 in astrocytes in the brain injury area, thereby significantly improving motor function defects after cerebral ischemia and achieving long-term therapeutic effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The synthesis and characterization of the amphiphilic cationic material PEI-C12 of Example 1 of the present application, wherein:

[0039] Figure 1 A is a schematic diagram of PEI-C12 synthesis.

[0040] Figure 1 B is the FT-IR spectra of O-C12, PEI and PEI-C12.

[0041] Figure 1 C is agarose gel electrophoresis showing the interaction between PEI-C12 and siBest1.

[0042] Figure 2 The synthetic diagram and characterization of SNP-siBest1 of Example 1 of the present application, wherein:

[0043] Figure 2 A is a schematic diagram of PEI-C12 synthesis.

[0044] Figure 2 B is the morphology of composite nanoparticles under electron microscope. Scale bar: 200 nm.

[0045] Figure 2 C is that the average particle size of SNP-siBest1 is about 110nm, the polydispersity coefficient is less than 0.2, the zeta potential is about -3.09mV, the particle size is uniform, and it has good dispersibility. The encapsulation efficiency is 91.4%, and the drug loading is 0.42%.

[0046] Figure 2 D is the serum stability of SNP-encapsulated siBest1.

[0047] Figure 2 E is the biocompatibility evaluation of SNP-siBest1. Data are mean ± SEM, n = 3.

[0048] Figure 3 The SNP-siBest1-Cy5 of Example 2 of the present application can be taken up by astrocytes and can knock down astrocyte BEST1, wherein,

[0049] Figure 3 A is a representative immunofluorescence image. GFAP, Cy5 and Hoechst33258 are staining markers, and the dose of SNP-siBest1 is 100 nM. Scale bar: 20 μm.

[0050] Figure 3B: The cell membrane is labeled with the fluorescent dye Dio, and the cell nucleus is stained with Hoechst33258. Dio can well outline the cell morphology and facilitate the observation of the distribution of particles in the cytoplasm. Scale bar: 20μm.

[0051] Figure 3 C is Western blot detection of BEST1 expression in astrocytes cultured in vitro treated with different concentrations of SNP-siBest1 (25, 50, 100 nM). Data are mean ± SEM, n = 5, **p < 0.01, ***p < 0.001.

[0052] Figure 3 D is a representative bright field photograph of mixed cultures of neurons and astrocytes treated with different concentrations of SNP-siBest1 (100, 200, 400 nM). Scale bar: 20 μm.

[0053] Figure 3 E is a statistical diagram of the activity of mixed cultured neural cells detected by CCK-8. Data are mean ± SEM, n = 5, *p < 0.05.

[0054] Figure 3 F is a representative image of immunofluorescence staining of neuronal marker β-111-Tubulin, astrocyte marker GFAP, nanoparticle marker Cy5 and nuclear Hoechst 33258 after nanoparticle treatment of co-cultured cells. Scale bar: 20 μm.

[0055] Figure 4 The biodistribution of SNP-siBest1 in vivo of Example 3 of the present application, wherein:

[0056] Figure 4 A shows the knockdown effect of SNP-siBest1 on BEST1 in healthy mouse brain. The doses of SNP-siBest1 administered via tail vein were 0.25, 0.5, and 1 mg / kg, respectively.

[0057] Figure 4 BE is the IVIS imaging result, which shows that SNP-siBest1 significantly prolonged the circulation and residence time of siBest1 in vivo. NS, SNP-siBest1-Cy5, or Naked siBest1-Cy5 (1 mg / kg) were injected into the tail vein on the 5th day after ischemia modeling. Figure 4 B: Representative images of brain fluorescence in mice 6 hours and 24 hours after injection of nanoparticles (left), siBest1-Cy5 (λex / λem=649 / 680nm); statistical graph of particle brain fluorescence (right). Data are mean ± SEM, n=5, *p<0.05, ***p<0.001. Figure 4C: Representative IVIS imaging of mouse brain tissue in vitro 24 hours after nanoparticle injection.

[0058] Figure 4 D: Representative images of fluorescence distribution of isolated mouse organs (heart, liver, spleen, lung, and kidney). Figure 4 E is the statistical graph of liver and kidney fluorescence intensity. Data are mean ± SEM, n = 5, **p < 0.01, ***p < 0.001.

[0059] Figure 4 F is the tissue distribution of SNP-rhodamine in the cortex around the infarct, immunofluorescence labeled with Rhodamine and Hoechst. Scale bar: upper, 50μm; lower, 20μm.

[0060] Figure 4 G is the distribution of SNP-rhodamine in the cortical cells around the infarct. Immunofluorescence staining, GFAP marks astrocytes, NeuN marks neurons, and Rhodamine shows SNP. Scale bar: 10 μm.

[0061] Figure 5 The SNP-siBest1 of Example 4 of the present application can improve the motor function defects of PT mice, wherein

[0062] Figure 5 A is a schematic diagram of the experimental process.

[0063] Figure 5 BC is a typical band showing the expression level of BEST1 in the peri-infarct tissue on the 11th and 39th days of modeling ( Figure 5 B) and statistics ( Figure 5 C), data are mean±SEM, n=5, ***p<0.001.

[0064] Figure 5 D is the forelimb asymmetry index of mice detected by cylinder test. Data are mean ± SEM, n = 15, sex ratio of PT group and sham group, ***p < 0.001, PT-SNP-siBest1 group compared with PT-SNP-siScrambled group, # p<0.05, ### p<0.001.

[0065] Figure 5 E and F are grid experiments to detect the stumble rate of the affected and healthy forelimbs of mice. Data are mean ± SEM, n = 15, sex ratio of PT group and sham group, ***p < 0.001, PT-SNP-siBest1 group compared with PT-SNP-siScrambled group, # p<0.05, ##p<0.01, ### p<0.001.

[0066] Figure 6 The SNP-siBest1 of Example 4 of the present application can improve the motor function defects of MCAO mice, wherein:

[0067] Figure 6 A is a schematic diagram of the experimental process.

[0068] Figure 6 B is the rotarod test to detect the latency of mice to fall. Data are mean ± SEM, n = 13-15, sex ratio of PT group and sham group, ***p < 0.001, PT-SNP-siBest1 group compared with PT-SNP-siScrambled group, # p<0.05, ## p<0.01, ### p<0.001.

[0069] Figure 6 C and D are grid experiments to detect the stumble rate of the affected and healthy forelimbs of mice. Data are mean ± SEM, n = 13-15, sex ratio of PT group and sham group, ***p < 0.001, PT-SNP-siBest1 group compared with PT-SNP-siScrambled group, ## p<0.01, ### p<0.001.

[0070] Figure 7 The SNP-siBest1 of Example 5 of the present application cannot exert therapeutic effects on mice with specific knockdown of astrocyte BEST1, wherein,

[0071] Figure 7 A is a schematic diagram of the experimental process.

[0072] Figure 7 B is the behavior cylinder test to detect the forelimb asymmetry index of mice. Data are mean ± SEM, n = 14.

[0073] Figure 7 C and D show the behavior grid test to detect the stumble rate of the left and right forelimbs of mice. Data are mean ± SEM, n = 14.

[0074] Figure 8 The knockdown and functional inhibition of SNP-siBest1 on HA1800 human astrocyte BEST1 in Example 6 of the present application, wherein:

[0075] Figure 8 A is a picture of HA1800 human astrocytes labeled with GFAP fluorescence, scale bar: 50 μm.

[0076] Figure 8 B is Western blot detection of BEST1 expression in HA1800 human astrocytes treated with SNP-siBest1 (100 nM). Data are mean ± SEM, n = 5, ns, no significant difference, ***p < 0.001.

[0077] Figure 8 C is the effect of SNP-siBest1 (100 nM) on GABA permeability in HA1800 human astrocyte ion channels. Left: IV curve; Right: current density at -80 mV. Data are mean ± SEM, n = 8. DETAILED DESCRIPTION

[0078] In order to better understand the technical solution of the present application, the above technical solution will be described in detail below in conjunction with the accompanying drawings and specific implementation methods. The reagents or instruments used without indicating the manufacturer are regarded as conventional products that can be purchased on the market.

[0079] Example 1: Preparation of Tween 80-coated composite nanoparticles SNP-siBest1 carrying siBest1. The specific scheme is as follows:

[0080] 1. Preparation and characterization of polyethyleneimine-epoxydodecane (PEI-C12)

[0081] PEI and O-C12 were stirred at a molar ratio of 1:7 at 90 °C under nitrogen protection for 48 h. Figure 1 A), separated and purified by silica column to obtain PEI-C12. PEI-C12 was characterized by Fourier transform infrared spectroscopy, proving the successful synthesis of PEI-C12 ( Figure 1 B). The ability of PEI-C12 to bind siRNA was investigated by agarose gel electrophoresis (1%, w / v). It was found that when the weight ratio of PEI-C12 to siBest1 reached 10:1, the migration of siBest1 was completely blocked ( Figure 1 C), indicating that PEI-C12 can effectively compress siBest1 to form a complex through electrostatic interaction;

[0082] The sequence of siRNA is as follows:

[0083] sense: 5'-UUGCCAACUUGUCAAUGAATT-3', as shown in SEQ ID No: 3;

[0084] antisense: 5′-UUCAUUGACAAGUUGGCAATT-3′, as shown in SEQ ID No: 4.

[0085] 2. Preparation and Characterization of SNP-siBest1

[0086] Add 5mg PLGA and 1.25mg PEI-C12 to DMF, stir and add 50μg siRNA dissolved in DEPC water. The final volume of the system is 1mL. Mix well to form siBest1 / PEI-C12 / PLGA complex, which is used as the organic phase. Dissolve 27.8μL of Tween 80 in 15mL of distilled water, and this phase is used as the aqueous phase. Under high-speed stirring, slowly drip the organic phase into the aqueous phase to allow the siBest1 / PEI-C12 / PLGA complex in the organic phase and the Tween 80 in the aqueous phase to self-assemble to form SNP-siBest1. Use an ultrafiltration centrifuge tube with a molecular weight cutoff of 100kDa, wash the nanoparticle solution three times with purified water at 4°C and 7000rpm by ultrafiltration centrifugation and concentrate, and store at 4°C for later use ( Figure 2 A). SNP-siBest1 was stained with 1% uranyl acetate and air-dried before observation by transmission electron microscopy. The results showed that SNP-siBest1 nanoparticles were spherical and evenly dispersed ( Figure 2 B). The particle size of SNP-siBest1 was 110 nm and the polydispersity index (PDI) was less than 0.2 as determined by dynamic light scattering. The encapsulation efficiency was 91.4% as determined by ultrafiltration centrifugation. The Zeta potential was -3.09 mV ( Figure 2 C).

[0087] 3. Serum stability study of siBest1 encapsulated by SNP-siBest1

[0088] Naked siBest1 and SNP-siBest1 loaded with an equal amount of siBest1 were incubated with 100% mouse serum (1:1 v / v) at 37°C for a certain period of time (0h, 12h and 24h). After incubation, the mixture of SNP-siBest1 loaded with siBest1 and serum was ultrafiltrated and concentrated, and the concentrated sample was dissolved in chloroform to release the entrapped siBest1, and siBest1 was extracted with 0.1% (w / v) sodium dodecyl sulfate in 0.5M sodium chloride solution. The mixture of naked siBest1 and serum was centrifuged and the supernatant was taken, chloroform was added to extract siBest1, and isopropanol was added, and the supernatant was discarded after centrifugation. The precipitate was washed with 75% ethanol, dried and dissolved in DEPC water to obtain siBest1 solution. The siBest1 extract or naked siBest1 solution was loaded on a 1% (w / v) agarose gel, and the voltage was 100mV for 30min for separation, and the results were observed using a gel imaging system. The results are as follows: After the nanoparticles carrying siBest1 were incubated with mouse serum for 12h or 24h at 37℃, siBest1 was not degraded, while naked siBest1 incubated with serum under the same conditions was degraded ( Figure 2 D). This indicates that SNP-siBest1 has a good protective effect on the encapsulated siBest1 and can reduce its degradation by nucleases in serum.

[0089] 4. Blood compatibility study of SNP-siBest1

[0090] Fresh whole blood of mice was collected in an anticoagulant tube, and red blood cell pellets were obtained by centrifugation. Physiological saline was added to prepare a 10% (v / v) red blood cell suspension. 200 μL of red blood cell suspension and 800 μL of a solution containing different concentrations of SNP-siBest1 were mixed and incubated at 37°C for 4 hours. The incubated mixture was centrifuged at 2000 rpm for 10 minutes, the supernatant was collected in a 96-well plate, and the absorbance was measured at 570 nm using an enzyme reader. The results showed that when the concentration of siBest1 carried was in the range of 50-800 nM, the hemolysis rate caused by SNP-siBest1 was less than 2% ( Figure 2 E), indicating that SNP-siBest1 has high biocompatibility as a delivery vector of siBest1.

[0091] Example 2: In vitro knockdown effect and toxicity of SNP-siBest1 on astrocyte BEST1. The specific scheme is as follows:

[0092] 1. Astrocyte Culture

[0093] B6 mice born 1 day old were disinfected with 2% benzalkonium bromide solution and 75% ethanol solution, the brain was exposed, the bilateral cortex was taken, the meninges were peeled off, the tissue was cut into pieces, and transferred to 0.125% trypsin digestion solution, and digested at 37°C for 15 minutes. DMEM / F12+10% FBS was added to terminate the digestion, and the liquid was gently blown with a gun to disperse the tissue evenly. After centrifugation at 2500rpm for 3 minutes, the culture medium (DMEM / F12+10% FBS) was added to resuspend the cells, and the single cell suspension was obtained by filtering through a 400-mesh sieve. 5×10 4 cells / cm 2 The cells were seeded at a density of 10 μg / mL on a culture plate coated with poly-ornithine. The culture medium was changed every 2-3 days, and the cells were passaged every 4-6 days when they reached confluence. The 1st to 5th generations of in vitro cultured astrocytes were used for experiments.

[0094] 2. Mixed Culture of Primary Neurons and Astrocytes

[0095] The primary culture method was the same as that of astrocytes. Neurobasal medium containing 2% B27 and DMEM / F12 medium containing 10% FBS were mixed in a ratio of 1:1 and cultured at a rate of 5×10 4 cells / cm 2 The cells were inoculated at a density of 100 μg / mL on culture plates coated with 10 μg / mL poly-ornithine, and the mixed culture medium was replaced every 3-4 days.

[0096] 3. Knockdown effect of SNP-siBest1 on astrocyte BEST1 in vitro

[0097] 3.1. Astrocytes were cultured until about 90% confluence, and the normal culture medium was discarded. Serum-free and dual antibody-free culture medium containing SNP-siBest1-Cy5 (100nM) and Naked siBest1-Cy5 (100nM) were given respectively. After 6 hours of culture, the drug-containing culture medium was discarded, and the cells were washed three times with PBS, fixed with 4% paraformaldehyde, and immunofluorescence labeling was performed. The results are as follows: The red fluorescence of SNP-siBest1-Cy5 surrounds GFAP + The red fluorescence was not observed in the astrocytes treated with Naked siBest1-Cy5 ( Figure 3 A). The cell membrane fluorescent dye Dio labeling again shows that SNP-siBest1-Cy5 is distributed around the nucleus in astrocytes; however, no red fluorescence was observed in astrocytes treated with Naked siBest1-Cy5 ( Figure 3 B) The above results all indicate that SNP-siBest1 can be taken up by astrocytes cultured in vitro, while Naked siBest1 cannot be taken up.

[0098] 3.2. Astrocytes were cultured until about 90% confluence, and the normal culture medium was discarded. Serum-free and dual-antibody-free culture medium containing SNP-siBest1 (25, 50, 100 nM) and Naked siBest1 (100 nM) were given respectively. The drug was withdrawn after 6 hours of culture, and the cells were washed twice with D-Hank's medium and replaced with normal astrocyte culture medium. After further culture for 48 hours, the protein was extracted, and the expression level of BEST1 was semi-quantitatively detected by Western blot. Western blot results showed that Naked siBest1 did not affect the expression of BEST1 in astrocytes cultured in vitro, and its BEST1 / GAPDH value was similar to that of the control group cells; however, the expression level of BEST1 in astrocytes given SNP-siBest1 was significantly reduced, and as the concentration of SNP-siBest1 increased, the knockdown effect became more obvious ( Figure 3 C).

[0099] 4. Toxicity of SNP-siBest1 to in vitro cultured neural cells

[0100] When the fusion degree of the mixed culture of primary neurons and astrocytes reached about 90%, a serum-free and dual-antibody-free mixed culture medium containing SNP-siBest1 (100nM, 200nM and 400nM) was given. The drug was withdrawn after 6 hours and replaced with a normal mixed culture medium for a further 48 hours. Then CCK-8 reagent was added for co-incubation for 2 hours, and the absorbance was measured at a wavelength of 450nm to detect the activity of the nerve cells. The results showed that when the concentration of SNP-siBest1 was 100nM and 200nM, the activity of the nerve cells was not affected. Only when the concentration was as high as 400nM, the activity of the nerve cells decreased slightly ( Figure 3 D, E). This indicates that SNP-siBest1 has no obvious toxicity at effective concentrations.

[0101] 5. Selectivity of SNP-siBest1 for astrocytes in cultured neural cells

[0102] When the confluence of the mixed culture of primary neurons and astrocytes reached about 90%, a serum-free and dual-antibody-free mixed culture medium containing SNP-siBest1-Cy5 (100 nM) was given. After 6 hours of culture, the drug-containing culture medium was discarded, and the cells were washed three times with PBS and fixed with 4% paraformaldehyde for immunofluorescence labeling. The results showed that the fluorescence of SNP-siBest1-Cy5 was mainly distributed in GFAP. + Astrocytes, and β-Ⅲ-tubulin + Cy5 fluorescence is rare in neurons ( Figure 3F). This indicates that SNP-siBest1-Cy5 has good selectivity for astrocytes.

[0103] In summary, SNP-siBest1 is easily taken up by astrocytes and can effectively and safely knock down BEST1 in astrocytes in vitro.

[0104] Example 3: Investigation of the in vivo biodistribution of SNP-siBest1. The specific scheme is as follows:

[0105] 1. Photothrombosis (PT) model

[0106] Mice were anesthetized with 2% isoflurane and fixed in a stereotaxic apparatus. Rose Bengal (100 mg / kg) was injected intraperitoneally, the scalp was cut along the midline sagittal axis, the skull was exposed, and the connective tissue was removed with an ear wash bulb, and the skull surface was kept dry. A cap ring was placed with the bregma as the center of the circle, and a cold light source was fixed on the stereotaxic apparatus to form an aperture with a diameter of 2 mm. The cap ring was overlapped with the center of the aperture and moved to the right 1.5 mm from the bregma. The cold light source was turned on 5 minutes after the injection of Rose Bengal (light intensity 10000 Lux × 12 min for virus infection experiments, 12000 Lux × 15 min for other experiments) to irradiate the skull. Before illumination, cotton balls were dipped in physiological saline to completely wet it and then applied to the mouse eyes to avoid damage to the eyes caused by excessive light. Rose Bengal produces singlet oxygen after light excitation, which damages and occludes the vascular endothelium, leading to focal cortical ischemia. The mice in the sham operation group were intraperitoneally injected with the same dose of Rose Bengal but were not exposed to light.

[0107] 2. In vivo biodistribution experiment of SNP-siBest1

[0108] The mice were PT modeled as described above, and 1 mg / kg of SNP-siBest1-Cy5 (a dose that can effectively knock down BEST1) was injected into the tail vein on the 5th day after ischemia. Figure 4 A), and imaging was performed using the small animal in vivo imaging system (IVIS) at 6 hours and 24 hours after administration. The in vivo imaging results showed that the brains of mice in the Naked siBest1-Cy5 group showed weak fluorescence signals at 6 hours and 24 hours, while the fluorescence signals in the brains of mice in the SNP-siBest1-Cy5 group were significantly higher than those in the Naked siBest1-Cy5 group at both time points. Moreover, 24 hours after injection, the fluorescence signal of Naked siBest1-Cy5 in the mouse brain basically disappeared, while the same dose of SNP-siBest1-Cy5 still showed a strong fluorescence signal in the mouse brain ( Figure 4B). The above results suggest that nanoparticle encapsulation can reduce the clearance of siBest1 and prolong the circulation and brain residence time of siBest1. The mice were then euthanized, and IVIS imaging of the isolated brain tissue and organs was performed. The results showed that the brain tissue of mice injected with SNP-siBest1-Cy5 showed strong fluorescence, and the nanoparticles were enriched in the peri-infarction area. At the same time, their liver and kidneys also showed strong fluorescence; however, the mice injected with Naked siBest1 had almost no fluorescence in the brain infarction area, and the fluorescence in the liver and kidneys was also weak ( Figure 4 CE). The above results indicate that naked siBest1 is rapidly metabolized and cleared from the circulation and is difficult to reach the brain, while nanocarrier encapsulation can prolong the circulation of siBest1, increase its stability and biomembrane transport, and thus increase the accumulation of siBest1 in the brain, especially in the peri-infarct area.

[0109] Due to the limited amount of Cy5 labeled on siBest1, the in vivo fluorescence of SNP-siBest1-Cy5 is weak, making it difficult to perform microscopic observations on brain slices. We further used the fluorescent dye Rhodamine B with stronger luminescence properties to encapsulate it in Tween-modified nanoparticles to prepare SNP-rhodamine to observe the in vivo cellular distribution of nanoparticles. On the 5th day after PT ischemia in mice, SNP-rhodamine was injected into the tail vein, and 6 hours later, the brain tissue was fixed by transcardial perfusion of PBS and 4% PFA. The results of immunofluorescence staining of brain slices showed that a large number of nanoparticles accumulated in the cortex around the infarct, and the closer to the core area of ​​the infarct, the more particles there were ( Figure 4 F). We also found that the nanoparticles have better selectivity for activated astrocytes than neurons ( Figure 4 G), which is consistent with the phenomenon observed in cells co-cultured in vitro.

[0110] In summary, after cerebral ischemia in mice, the composite nanoparticles can selectively enter the astrocytes in the cortex around the infarction and knock down BEST1.

[0111] Example 4: In vivo knockdown effect of SNP-siBest1 on astrocyte BEST1 and its improvement on motor function defects in mice with cerebral ischemia. The specific scheme is as follows:

[0112] 1. SNP-siBest1 improves motor function in PT model mice

[0113] The photochemical ischemia model of mice was established according to the method in Example 3. SNP-siBest1 (1 mg / kg) and its control agent SNP-siScrambled were injected into the tail vein on days 5, 7, 9 and 11 after modeling, and then the motor function of mice was detected by cylinder and grid behavioral experiments on days 11, 18, 25 and 39 after modeling. Brain tissues were collected on days 11 and 39, and the cortex was separated. The expression of BEST1 was analyzed by Western blot ( Figure 5 A). The results showed that 11 days after PT, SNP-siBest1 significantly reduced the expression of BEST1 induced by ischemic injury, while SNP-siScrambled had no significant effect on the expression of BEST1 induced by ischemic injury; 39 days after PT, the knockdown effect of SNP-siBest1 on BEST1 was weakened ( Figure 5 B, C). Behavioral tests showed that from 11 days to 39 days after ischemia, the stumble rate and asymmetry index of the affected limbs of mice in the PT model group were significantly higher than those in the sham-operated group; compared with the SNP-siScrambled group, the stumble rate and asymmetry index of the affected limbs of mice treated with SNP-siBest1 were significantly decreased; there was no significant difference in the motor function of the healthy limbs of mice in each group ( Figure 5 DF). In summary, SNP-siBest1 can improve motor deficits in mice with focal cortical ischemia, and the effect can last for at least one month.

[0114] 2. SNP-siBest1 improves motor function in MCAO model mice

[0115] 2.1. Middle cerebral artery occlusion (MCAO) model

[0116] Adult male B6 mice were anesthetized with 2% isoflurane, the neck was cut to expose the internal carotid artery, and an 8 / 0 (mouse) surgical nylon monofilament was introduced into the left internal carotid artery through the stump of the external carotid artery, 10-11 mm forward through the carotid bifurcation until slight resistance was felt. The fiber was left in place for 75 minutes, then removed and reperfused. The local cerebral blood flow was monitored using a laser Doppler blood flow monitor to ensure that the local cerebral blood flow was reduced by 85% to 95%. The suture was disinfected with iodine, placed on an electric blanket and returned to the cage after awakening. Sham-operated mice underwent the same procedure as MCAO rats, except that the nylon monofilament was only pushed 7 mm above the carotid bifurcation.

[0117] 2.2. Behavioral test results

[0118] SNP-siBest1 (1 mg / kg) was injected into the tail vein on the 5th, 7th, 9th and 11th days of modeling, and the rotarod and grid tests were performed on the 11th, 18th, 25th and 39th days to detect the neuromotor function of the mice ( Figure 6 A). The experimental results were similar to those observed in the PT model. Compared with SNP-siScrambled treatment, SNP-siBest1 treatment significantly prolonged the time that stroke mice stayed on the rotarod and reduced the rate of slips on the affected side of the mice, but had no effect on the motor function of the ipsilateral limb of the infarction ( Figure 6 BD). This indicates that SNP-siBest1 has a therapeutic effect on the MCAO model.

[0119] In summary, regardless of the PT model or the MCAO model, SNP-siBest1 treatment of cerebral ischemia mice in the subacute phase can effectively improve motor function impairment and has long-term effects.

[0120] Example 5: Dependence of the therapeutic effect of SNP-siBest1 on BEST1. The specific scheme is as follows:

[0121] To further verify that astrocyte BEST1 is the target of SNP-siBest1, we injected AAV-Flex-shBest1-GFP virus into the motor cortex of GFAP-Cre mice 14 days before ischemia modeling to specifically knock down BEST1 in astrocytes. SNP-siBest1 (1 mg / kg) was injected into the tail vein on days 5, 7, 9, and 11 after modeling, and behavioral tests were performed on days 11, 18, 25, and 39. Figure 7 A). The grid and cylinder experiment showed that when BEST1 was specifically knocked down in astrocytes of mice, treatment with SNP-siBest1 did not further improve motor function impairment ( Figure 7 BD), suggesting that the effect of SNP-siBest1 in improving motor function is dependent on astrocyte BEST1.

[0122] Example 6: Knockdown and functional inhibition of BEST1 in HA1800 human astrocytes by SNP-siBest1. The specific scheme is as follows:

[0123] To investigate the feasibility of SNP-siBest1 in clinical use, we observed whether it could effectively knock down BEST1 in HA1800 human astrocytes and inhibit channel function.

[0124] 1. HA1800 human astrocyte culture

[0125] The revived cells were plated at 5×10 4 cells / cm2 The cells were inoculated in a culture plate at a density of 100 μg / cm2 and the culture medium was DMEM + 10% FBS mixed evenly. The culture medium was changed every 2-3 days. When the cells grew to 80%-90% confluence, they were digested and passaged with 0.25% trypsin. The passaged cells were diluted at a ratio of 1:3 and re-inoculated into a 12-well culture plate or a 1cm×1cm glass slide according to the needs of subsequent experiments. When they were cultured to 90% confluence, they were used for biochemical detection and electrophysiological experiments respectively. The identification results of HA1800 human astrocytes are shown in the figure. Figure 8 As shown in A.

[0126] 2. IV Curve Recording of Cell Permeability to GABA

[0127] Electrode external solution (mM): 150NaCl, 10HEPES, 3KCl, 2CaCl2, 2MgCl2, 5.5Glucose, recording glass electrode (4-6MΩ) internal solution (mM): 140GABA, 5CaCl2, 5EGTA-NMDG, 2MgCl2, 10HEPES, 10Sucrose, 4Mg-ATP and 0.3Na2-GTP (pH adjusted to 7.3 with CsOH, osmotic pressure adjusted to 275-285mOsmol with sucrose). The clamping potential was maintained at 0mV, and the voltage was increased stepwise from -100 to +100mV in 20mV increments to establish the IV curve. All recordings were made using patch clamp electrodes, low-pass filtered at 2kHz, and sampled at 10kHz. At any time during the recording, cells whose resistance (R) or capacitance deviated from the initial value by 20% or R>20MΩ were excluded from the analysis. Data were collected using pClamp 10.3 software and analyzed using Clamfit 10.3.

[0128] 3. Inhibitory effect of SNP-siBest1 on BEST1 function in HA1800 human astrocytes

[0129] HA1800 human astrocytes were cultured until about 90% confluence, and the normal culture medium was discarded. Serum-free and dual-antibody-free culture medium containing SNP-siBest1 (100nM) and Naked siBest1 (100nM) were given respectively. The drug was withdrawn after 6 hours of culture, and the cells were washed twice with D-Hank's medium and replaced with normal culture medium. The culture was continued for 48 hours, and the protein was extracted to semi-quantitatively detect the expression level of BEST1 by Western blot or to record the IV curve of ion channel permeability to GABA by electrophysiological recording. The results showed that Naked siBest1 did not affect the expression of BEST1 in HA1800 cells; however, SNP-siBest1 could significantly reduce the expression level of BEST1 in HA1800 cells ( Figure 8B). Consistent with the decrease in protein expression, electrophysiological recordings showed that the permeability of ion channels to GABA in HA1800 cells treated with SNP-siBest1 was significantly reduced ( Figure 8 C). This preliminarily proves that SNP-siBest1 can effectively knock down BEST1 in human astrocytes and inhibit their permeability to GABA, which has the possibility of clinical use.

[0130] Although some preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0131] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of this application and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Application of Bestrophin 1 as a therapeutic target for subacute ischemic stroke in the preparation of drugs for the treatment of subacute ischemic stroke.

2. The use according to claim 1, characterized in that: The drug is used to improve motor function.

3. Use of a drug for downregulating or knocking out Bestrophin 1 in the preparation of a drug for treating subacute ischemic stroke, wherein the active ingredient in the drug for downregulating or knocking out Bestrophin 1 is siRNA of Bestrophin 1 or a small molecule compound that can specifically inhibit Bestrophin 1.

4. The use according to claim 3, characterized in that: The nucleic acid sequence of the Bestrophin 1 siRNA is: UUGCCAACUUGUCAAUGAATT.

5. The use according to claim 4, characterized in that: The drug is a composite nano preparation, which takes polylactic acid hydroxyglycolic acid copolymer as the main core, encapsulates polyethyleneimine-epoxydodecane and Bestrophin1 siRNA, and Tween 80 is distributed on the surface of the core.

6. The use according to claim 5, characterized in that: The mass ratio of the polylactic acid hydroxyglycolic acid copolymer to the polyethyleneimine-epoxy dodecane is 3-5:1, preferably 4:1, the mass ratio of the polyethyleneimine-epoxy dodecane to the Bestrophin 1 siRNA is 20-30:1, preferably 25:1, and the concentration of Tween 80 in the aqueous phase is 1-5 mg / mL, preferably 2 mg / mL.

7. The use according to claim 6, characterized in that: The preparation method of the composite nano preparation is specifically as follows: (1) adding poly(lactic acid-hydroxyglycolic acid) copolymer and polyethyleneimine-dodecane oxide to N,N-dimethylformamide, stirring evenly, adding Bestrophin 1 siRNA dissolved in DEPC water, stirring to obtain an organic phase; (2) adding Tween 80 to water and stirring to obtain an aqueous phase; (3) The organic phase was added dropwise into the aqueous phase and stirred vigorously to obtain nanoparticles.

8. A drug, characterized in that It comprises a pharmaceutically acceptable carrier and an effective amount of an active ingredient, wherein the active ingredient is siRNA of Bestrophin 1; Preferably, the nucleic acid sequence of the Bestrophin 1 siRNA is: UUGCCAACUUGUCAAUGAATT.

9. The drug according to claim 8, characterized in that The drug is a composite nanoparticle preparation, which has a polylactic acid-hydroxyglycolic acid copolymer as the main core, encapsulating polyethyleneimine-epoxydodecane and Bestrophin1 siRNA, and Tween 80 is distributed on the surface of the core; Preferably, the mass ratio of the polylactic acid-hydroxyglycolic acid copolymer to the polyethyleneimine-epoxydodecane is 3-5:1, preferably 4:1, the mass ratio of the polyethyleneimine-epoxydodecane to the Bestrophin 1 siRNA is 20-30:1, preferably 25:1, and the concentration of Tween 80 in the aqueous phase is 1-5 mg / mL, preferably 2 mg / mL.

10. The drug according to claim 9, characterized in that The preparation steps of the drug are as follows: (1) adding poly(lactic acid-hydroxyglycolic acid) copolymer and polyethyleneimine-dodecane oxide to N,N-dimethylformamide, stirring evenly, adding Bestrophin 1 siRNA dissolved in DEPC water, stirring to obtain an organic phase; (2) adding Tween 80 to water and stirring to obtain an aqueous phase; (3) The organic phase was added dropwise into the aqueous phase and stirred vigorously to obtain nanoparticles.

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