Application of microglial cell targeted IL-10 mRNA (messenger Ribonucleic Acid) nanoparticles in ischemic cerebral apoplexy immunotherapy

Abstract

The invention provides application of microglial cell targeted IL-10mRNA (messenger Ribonucleic Acid) nano particles in preparation of a preparation or a composition or a medicine box for immunotherapy of cerebral arterial thrombosis. Specifically, the microglial cell targeting IL-10mRNA nanoparticle can specifically target M2 type microglial cells, so that IL-10mRNA is selectively delivered to the microglial cells at the cerebral arterial thrombosis occurrence part, and the microglial cells are driven to be polarized to M2 phenotype, thereby treating cerebral arterial thrombosis with blood-brain barrier injury.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to the application of microglia-targeted IL10 mRNA nanoparticles in the immunotherapy of ischemic stroke. Background Art

[0002] The recent Global Burden of Disease study has shown that ischemic stroke has jumped to the top three of the global disease burden, and has become the leading cause of death and disability among adults in China. Currently, the most effective treatment for acute ischemic stroke approved by the FDA is recombinant human tissue plasminogen activator (rt-PA) thrombolysis and endovascular mechanical thrombectomy. However, rt-PA thrombolysis has a short treatment time window. In China, only about 20% of stroke patients receive thrombolysis within 3 hours, and serious complications may occur. Therefore, there is an urgent need to establish other potential therapies for stroke.

[0003] Since the inflammatory response is the main pathological cause of the development of stroke, immunotherapy after stroke has become the mainstream research direction in the field of stroke in recent years. Some studies have proven that targeting immune cells or inflammatory factors after stroke and adjusting the immune microenvironment in the brain after stroke play an important role in the prognosis of stroke. However, due to the limitations of targeting efficiency and pathways, the current intervention methods for immunotherapy after stroke have not been truly used in clinical practice.

[0004] Therefore, there is an urgent need in this field to develop effective drugs for immunotherapy after stroke to promote the research and development of new drugs in the field of stroke. Summary of the Invention

[0005] The purpose of the present invention is to provide the use of microglia-targeted IL-10 mRNA nanoparticles in the immunotherapy of ischemic stroke, as well as a method for treating ischemic stroke, especially ischemic stroke accompanied by blood-brain barrier damage and neuronal apoptosis, using the microglia-targeted IL-10 mRNA nanoparticles.

[0006] In the first aspect of the present invention, there is provided the use of microglia-targeted IL-10 mRNA nanoparticles in the preparation of a preparation or composition, or a kit for immunotherapy of ischemic stroke.

[0007] In another preferred embodiment, the microglia-targeted IL-10 mRNA nanoparticles specifically target M2 microglia, thereby selectively delivering IL-10 mRNA to M2 microglia at the site of ischemic stroke.

[0008] In another preferred example, the microglia-targeted IL-10 mRNA nanoparticles specifically target the CD206 molecule on the surface of M2 microglia.

[0009] In another preferred example, the microglia-targeted IL-10 mRNA nanoparticles can be endocytosed by microglia.

[0010] In another preferred example, the M2 microglia are CD206-positive and arginase 1 (Arg1)-positive microglia.

[0011] In another preferred example, the preparation or composition, or kit is administered to a subject with ischemic stroke, and the ischemic stroke has the following classification characteristics: accompanied by blood-brain barrier damage and / or neuronal apoptosis.

[0012] In another preferred example, the ischemic stroke also has one or more classification characteristics selected from the following group:

[0013] (1) The expression level of tight junction protein (zonula occlusionns-1, ZO-1) in the brain tissue is significantly reduced; wherein, the "significantly reduced" means that the ratio of the expression level F1 of ZO-1 in the brain tissue of the subject to the expression level F0 of ZO-1 in the normal brain tissue, F1 / F0 ≤ 1 / 3, preferably ≤ 1 / 2;

[0014] (2) The terminal deoxynucleotidyl transferase dUTP nick in brain tissue neurons is significantly increased; wherein, the "significantly increased" means that the ratio of the amount T1 of the terminal deoxynucleotidyl transferase dUTP nick in the brain tissue neurons of the subject to the amount T0 of the terminal deoxynucleotidyl transferase dUTP nick in the normal brain tissue neurons, T1 / T0 ≥ 1.2, preferably ≥ 1.5, more preferably ≥ 2.

[0015] In another preferred example, the preparation or composition, or kit is administered to a subject with ischemic stroke for:

[0016] (a) Altering the microglial polarity and driving the polarization of microglia towards the M2 phenotype;

[0017] (b) Remodeling the pathological microenvironment and reducing neuroinflammation after ischemic stroke;

[0018] (c) Improving the blood-brain barrier (BBB) damage after ischemic stroke;

[0019] (d) Inhibiting neuronal apoptosis after ischemic stroke; and / or

[0020] (e) Preventing long-term neurological dysfunctions caused by long-term cerebral ischemia (permanent cerebral ischemia), including axonal injury, demyelinating stroke, sensorimotor and cognitive neurological dysfunctions.

[0021] In another preferred example, the microglia-targeted IL-10 mRNA nanoparticles are lipid nanoparticles (LNP).

[0022] In another preferred example, the microglia-targeted IL-10 mRNA nanoparticles comprise the following components: IL-10 mRNA, ionizable lipid, and co-lipid.

[0023] In another preferred example, the co-lipid includes co-phospholipid, sterol, polymer-conjugated lipid, or a combination thereof.

[0024] In another preferred example, the co-lipid is a combination of co-phospholipid, sterol, and polymer-conjugated lipid.

[0025] In another preferred example, the formulation or composition comprises: the microglia-targeted IL-10 mRNA nanoparticles, and a pharmaceutically acceptable carrier.

[0026] In another preferred example, the dosage form of the formulation or composition includes a liquid formulation or a solid formulation, preferably a liquid formulation.

[0027] In another preferred example, the formulation or composition is an injection.

[0028] In another preferred example, the administration routes of the formulation or composition include: intravenous injection, intravenous infusion, intracranial injection.

[0029] In another preferred example, the kit further comprises: a detection reagent for detecting the typing characteristics of ischemic stroke.

[0030] Preferably, the detection reagent for detecting the typing characteristics is selected from the group consisting of: a reagent for detecting blood-brain barrier damage, a reagent for detecting neuronal apoptosis, a reagent for detecting the polarization of microglia to the M2 phenotype, or a combination thereof.

[0031] In another preferred example, the detection reagent for detecting the typing characteristics is selected from the group consisting of: a reagent for detecting the expression level of Z0-1 in brain tissue, a reagent for detecting the terminal deoxynucleotidyl transferase dUTP nick in brain tissue neurons, a reagent for detecting the expression of CD206, Arg1, and / or CD86 in microglia, or a combination thereof.

[0032] In a second aspect of the present invention, a method for in vitro inducing the polarization of microglia to the M2 phenotype is provided, the method comprising the step of: administering microglia-targeted IL-10 mRNA nanoparticles to microglia.

[0033] In another preferred example, the method is non-therapeutic and non-diagnostic.

[0034] In another preferred example, the microglia of the M2 phenotype are CD206-positive and arginase 1 (Arg1)-positive microglia.

[0035] In a third aspect of the present invention, there is provided a method for immunotherapy of ischemic stroke, the method comprising administering microglia-targeted IL-10 mRNA nanoparticles to a subject in need thereof.

[0036] In another preferred example, the subject in need thereof is a subject suffering from ischemic stroke, and the ischemic stroke has the following classification characteristics: accompanied by blood-brain barrier damage and / or neuronal apoptosis.

[0037] In another preferred example, the ischemic stroke further has one or more classification characteristics selected from the following group:

[0038] (1) The expression level of tight junction protein (zonula occlusionns-1, ZO-1) in the brain tissue is significantly reduced; wherein, the "significantly reduced" means that the ratio of the expression level F1 of ZO-1 in the brain tissue of the subject to the expression level F0 of ZO-1 in the normal brain tissue, F1 / F0 ≤ 1 / 3, preferably ≤ 1 / 2;

[0039] (2) The terminal deoxynucleotidyl transferase dUTP nick in the brain tissue neurons is significantly increased; wherein, the "significantly increased" means that the ratio of the amount T1 of the terminal deoxynucleotidyl transferase dUTP nick in the brain tissue neurons of the subject to the amount T0 of the terminal deoxynucleotidyl transferase dUTP nick in the normal brain tissue neurons, T1 / T0 ≥ 1.2, preferably ≥ 1.5, more preferably ≥ 2.

[0040] In another preferred example, the subject in need thereof includes humans or non-human mammals.

[0041] In another preferred example, the administration routes of the microglia-targeted IL-10 mRNA nanoparticles include intravenous injection, intravenous infusion, and intracranial injection.

[0042] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. Description of the Drawings

[0043] Figure 1.Therapeutic validation of.mIL-10@MLNP on BBB integrity and neuron survival. In tMCAO mice. (A) Schematic diagram of the treatment protocol for tMCAO mice. (B and C) Representative images (B) of endogenous IgG extravasation and quantification (C) of endogenous IgG extravasation in brain sections of tMCAO mice treated with PBS, mIL-10 alone, mNC@MLNP, MLNP, or mIL-10@MLNP at 3 days after treatment, (n = 4). (D and E) Representative images (D) of MAP2 immunostaining and quantification (E) of infarct volume in brain sections obtained from tMCAO mice. (F and G) Representative confocal images (F) of zonula occludens-1 (ZO-1: red) and endothelial cell marker CD31 (green) and MFI quantification (G) in the designated brain tissue sections. Scale bar = 30 μm. (H) TUNEL (red) and NeuN (green) images in brain sections of the designated groups. Scale bar = 30 μm. All data are shown as mean ± SD, and statistical significance was determined using one-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

[0044] Figure 2 .Effect of.mIL-10@MLNP treatment on microglial phenotypic transformation and neuroinflammation in tMCAO mice. (A) Flow cytometry plot of IL-10 content in microglia. (B) Expression of IL-10 in CD45+CD11b+ microglia isolated from ischemic brain tissue. (C-D) Flow cytometry plot (C) and quantification (D) of CD206+ microglia isolated from cerebral ischemia. (E, F) Representative flow cytometry plot (E) and quantification (F) of CD86+ microglia isolated from cerebral ischemia. (G) Immunostaining of microglia Iba-1 (red) and Arg-1 (green) in brain sections of the designated brain tissue. Scale bar = 20 μm. (H) Quantification of Arg-1+ microglia. (I and J) Immunostaining of TNF-α (green) and Iba-1 (red) in brain sections of the designated brain tissue. Scale bar = 20 μn. All data are shown as mean ± SD, and statistical significance was determined using: one-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

[0045] Figure 3. Therapeutic effect of.mIL-I0@MLNPs in a mouse model of ischemic stroke induced by distal middle cerebral artery occlusion (dMCAO). (A) Schematic diagram of the treatment protocol for ischemic stroke in dMCAO mice. (B) Representative immunofluorescence images of endogenous IgG extravasation (green) and MAP2 (red) in mice treated with PBS, MLNP, or mIL-10@MLNP 14 days after dMCAO, and quantitative determination of IgG leakage MFI and infarct volume. (C) Expression of SMI32 (red) and MBP (green) in the external capsule and striatum. Scale bar = 20 μm. (D) Quantitative analysis of the SMI32 / MBP MFI ratio in the external capsule (F) and striatum. (E-F) Overall neurological assessment of mice using the Garcia score (n = 8); assessment of sensorimotor function of mice treated with different protocols using the tape removal test (n = 8). (G) Representative images of the movement trajectories in the Barnes maze and cognitive function assessment, including spatial learning and memory ability of mice treated with different protocols. All data are shown as mean ± SD and were statistically analyzed. Significance was determined using one-way or two-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). Detailed implementation manner

[0046] Through extensive and in-depth research, the present inventors have innovatively applied mRNA technology to the neuroimmunotherapy of ischemic stroke for the first time. The present invention uses a nano-therapy method (referred to as microglia-targeted IL-10 mRNA nanoparticles) that can selectively deliver IL-10 mRNA to M2 microglia in the ischemic brain, enhance the action of IL-10 mRNA in the ischemic area, drive the polarization of microglia in the ischemic area to the M2 phenotype, inhibit neuroinflammation, repair blood-brain barrier damage, and inhibit neuronal apoptosis, thereby effectively treating ischemic stroke accompanied by blood-brain barrier damage. On this basis, the present invention has been completed.

[0047] Term

[0048] Microglia

[0049] As used herein, the term "microglia" refers to immune cells resident in the mammalian brain that are derived from the brain and can be activated after stroke and participate in the occurrence and development of neuroinflammation. Microglia can be identified and differentiated from other cells by Iba-1.

[0050] M2 microglia

[0051] Microglia can promote cerebral edema and clearance of cell debris, promote angiogenesis, and improve neurological function by alternatively activating the M2 phenotype. The expression of M2 characteristic anti-inflammatory cytokines is also seen after intracerebral hemorrhage. IL-10 is secreted by microglia and macrophages, and can induce the differentiation of microglia and macrophages cultured in vitro into the M2c subtype, enhancing the phagocytosis of monocytes. In addition, IL-10 can also induce the production of SOCS1 and SOCS3, among which SOCS3 inhibits the production of macrophage pro-inflammatory cytokines through STAT3. STAT3 is a member of the STAT family and is involved in various abnormal pro-inflammatory responses in the central nervous system. The STAT3 signaling pathway is involved in the pro-inflammatory response associated with the M1 phenotype, which may lead to early brain injury in subarachnoid hemorrhage (SAH). Specific deletion of STAT3 in microglia can up-regulate the expression of major anti-inflammatory factors, enhance anti-inflammatory function by triggering the polarization of microglia from M1 to M2, thereby reducing the neuroinflammatory response after SAH and improving neurological dysfunction and neuronal apoptosis. IL-13 and IL-4 are also anti-inflammatory cytokines that promote the polarization of M2 microglia. IL-13 is secreted by Th2 cells and shares the receptor subunit with IL-4 secreted by Treg cells and Th2 cells. Both are through the JAK-STAT6 pathway to up-regulate the expression of M2 microglia, including the expression of microglia markers such as Arg-1 and YM1. In addition, IL-4 is considered to drive the polarization of microglia to the M2 type. Studies have shown that intracerebral injection of IL-4 can inhibit the polarization of M1 microglia and promote M2 microglia, thereby improving the recovery of neurological dysfunction after ICH. IL-4 and TGF-β1 have anti-inflammatory effects and promote the response and functional recovery of M2 microglia after intracerebral hemorrhage. In addition to IL-4 and IL-10, LRP1 also enhances the M2 polarization of microglia after acute brain injury and improves nerve injury. In addition, simultaneous administration of cAMP and IL-4 can lead to up-regulation of Arg-1 expression, which is a phenotypic marker of M2 in TBI mice, and also reduces the production of reactive oxygen species (ROS) in mice.

[0052] Ischemic stroke

[0053] Ischemic stroke refers to a disease in which insufficient blood supply caused by stenosis or occlusion of the blood supply arteries of the brain leads to necrosis of the corresponding brain tissue. Brain tissue necrosis triggers an immune response in microglia and activates the peripheral immune system, chemotaxing peripheral immune cells such as neutrophils and monocytes to infiltrate into the ischemic brain tissue, triggering neuroinflammation. The severity of neuroinflammation can be reflected by the size of the cerebral infarction volume and the degree of blood-brain barrier disruption. All MCAO used in this article are models simulating ischemic stroke, that is, the middle cerebral artery occlusion model (MCAO), which is the most widely used cerebral ischemia model currently.

[0054] Lipid nanoparticles (LNPs)

[0055] As used herein, the terms “lipid nanoparticles”, “lipid nanoparticles” or “LNPs” refer to particles having a diameter of about 5 to 500 nm. In some embodiments, the lipid nanoparticles contain one or more active agents (bioactive substances). In some embodiments, the lipid nanoparticles include nucleic acids. In some embodiments, the nucleic acids are condensed inside the nanoparticles with cationic lipids, polymers or multivalent small molecules and an external lipid coating that interacts with the biological environment. Due to the repulsive forces between phosphate groups, nucleic acids are naturally rigid polymers and tend to adopt an elongated configuration. In cells, in response to volume constraints, DNA can package itself with the help of ions and other molecules under appropriate solution conditions. Generally, DNA condensation is defined as the collapse of extended DNA strands into compact, ordered particles containing only one or a few molecules. By binding to phosphate groups, cationic lipids can condense DNA by neutralizing the phosphate charge and causing it to pack tightly.

[0056] In some embodiments, the bioactive substance is encapsulated into the LNP. In some embodiments, the bioactive substance can be an anionic compound, including but not limited to DNA, RNA (messenger RNA, transfer RNA, ribosomal RNA, microRNA, etc.), natural and synthetic oligonucleotides (including antisense oligonucleotides, interfering RNA and small interfering RNA), nucleoproteins, peptides, nucleic acids, ribozymes, DNA-containing nucleoproteins, such as intact or partially deproteinized virus particles (virions), oligomeric and polymeric anionic compounds other than DNA (e.g., acidic polysaccharides and glycoproteins)). In some embodiments, the bioactive substance can be mixed with an adjuvant.

[0057] In LNP products, the bioactive substance is typically contained within the interior of the LNP. In some embodiments, the bioactive substance includes nucleic acids. Generally, water-soluble nucleic acids are condensed inside the particles with cationic lipids or polycationic polymers, and the particle surface is rich in helper phospholipids or PEG lipid derivatives. Additional ionizable cationic lipids may also be located on the surface and, when entering the cell lysosome, the ionizable cationic lipids are ionized to be positively charged through the acidic environment of the lysosome and interact with the lysosomal membrane to promote endosomal escape.

[0058] LNPs are generally prepared by the following method: (a) mixing ionizable lipids (or ionizable cationic lipids) and co-lipids with an organic solvent to obtain a lipid organic phase; (b) mixing a bioactive substance with an aqueous solvent to obtain an aqueous phase containing the bioactive substance; (c) mixing the lipid organic phase in step (a) with the aqueous phase in step (b) to obtain a lipid nanoparticle drug. Further, the method also includes step (d): purifying and concentrating the lipid nanoparticle drug obtained in step (c), filtering and sterilizing, and physicochemical quality control testing. The microglia-targeted IL-10 mRNA nanoparticles of the present invention can be prepared by the above method.

[0059] In some embodiments, the organic solvent includes (but is not limited to) ethanol, methanol, isopropanol, acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane, or tetrahydrofuran, or a combination thereof. In some embodiments, the lipid organic phase includes a small percentage of water or a pH buffer. The lipid organic phase may contain up to 60% by volume of water, such as up to about 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% by volume of water. In one embodiment, the lipid organic phase contains from about 0.05% to 60% by volume of water, for example, from about 0.05% to 50%, from about 0.05% to 40%, or from about 5% to 20% by volume of water.

[0060] In some embodiments, the lipid organic phase includes a single type of lipid, for example, an ionizable cationic lipid, a co-phospholipid, a sterol, or a polymer-conjugated lipid. In some embodiments, the lipid organic phase includes multiple lipids. In one embodiment of the present invention, the lipid organic phase contains an ionizable cationic lipid, a co-phospholipid (e.g., DSPC, DOPE, DOPC, or a combination thereof), a sterol (e.g., cholesterol or a cholesterol derivative, or a phytosterol such as β-sitosterol), and a polymer-conjugated lipid (e.g., DMG-PEG2000).

[0061] In some embodiments, the aqueous solvent is water. In some embodiments, the aqueous solvent is an aqueous buffer having a pH between 3 - 8 (e.g., a pH of about 3, about 4, about 5, or about 6, etc.). A bioactive substance such as a nucleic acid (e.g., mRNA) is dissolved in the aqueous solvent to obtain an aqueous phase containing the bioactive substance. The aqueous phase may contain a small percentage of a water-miscible organic solvent. The aqueous phase may contain up to 60% by volume of at least one water-miscible organic solvent, e.g., up to about 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or any volume % between the two of an organic solvent (e.g., a water-miscible organic solvent). In one embodiment, the aqueous phase contains an organic solvent in the range of about 0.05% to 60% by volume, e.g., an organic solution (e.g., a water-miscible organic solvent) in the range of about 0.05% and 50% by volume, about 0.05% to 40% by volume, or about 5% to 20% by volume. The aqueous phase buffer can be a citrate buffer, a Tris-Hcl buffer solution, a sodium acetate buffer solution, a PBS buffer solution, or a combination thereof, etc. In some embodiments, the aqueous buffer is a citrate buffer having a pH between 4 - 6 (e.g., a pH of about 4, about 5, or about 6). In one embodiment, the aqueous buffer solution is a citrate buffer having a pH of about 4.

[0062] In some embodiments, a solution comprising a mixture of a lipid organic phase and an aqueous phase containing a bioactive substance, which mixture comprises an LNP suspension, can be diluted. In some embodiments, the pH of a solution comprising a mixture of a lipid organic phase and an aqueous phase containing a bioactive substance, which mixture comprises an LNP suspension, can be adjusted. The pH of the LNP suspension can be diluted or adjusted by adding water, an acid, a base, or an aqueous buffer. In some embodiments, the pH of the LNP suspension is neither diluted nor adjusted. In some embodiments, the pH of the LNP suspension is both diluted and adjusted.

[0063] In some embodiments, excess reagents, solvents, unencapsulated nucleic acids can be removed from the LNP suspension by tangential flow filtration (TFF) (e.g., diafiltration). Organic solvents (e.g., ethanol) and buffers can also be removed from the LNP suspension using TFF. In some embodiments, the LNP suspension is dialyzed. In some embodiments, the LNP suspension is subjected to TFF. In some embodiments, the LNP suspension is both dialyzed and subjected to TFF.

[0064] Applications of the present invention

[0065] The present invention provides the use of microglia-targeted IL-10 mRNA nanoparticles in the preparation of a preparation or composition for immunotherapy of ischemic stroke. The microglia-targeted IL-10 mRNA nanoparticles of the present invention specifically target M2 microglia, thereby selectively delivering IL-10 mRNA to M2 microglia at the site of ischemic stroke, and are particularly suitable for ischemic stroke accompanied by blood-brain barrier damage and neuronal apoptosis. The microglia-targeted IL-10 mRNA nanoparticles can be administered to a subject in need by intravenous injection or infusion, pass through the damaged blood-brain barrier, and are selectively delivered to M2 microglia at the infarct site, thereby changing the microglial polarity, driving the polarization of microglia to the M2 phenotype, remodeling the pathological microenvironment, reducing neuroinflammation after ischemic stroke, improving blood-brain barrier damage after ischemic stroke, inhibiting neuronal apoptosis, and thus effectively treating this type of ischemic stroke. Further, it can also prevent long-term neurological dysfunctions caused by prolonged cerebral ischemia (permanent cerebral ischemia), including axonal injury, demyelinating stroke, sensorimotor and cognitive neurological dysfunctions, etc.

[0066] The main advantages of the present invention include:

[0067] (1) The present invention first applies mRNA delivery technology to the neuroimmunotherapy of ischemic stroke, which is beneficial to promoting the research and development of new drugs in the field of stroke;

[0068] (2) The microglia-targeted IL-10 mRNA nanoparticles of the present invention can prolong the half-life of IL-10 mRNA encapsulated in LNP, and selectively deliver IL-10 mRNA to M2 microglia through the damaged blood-brain barrier. Therefore, it can be administered intravenously and is particularly suitable for ischemic stroke accompanied by blood-brain barrier damage.

[0069] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually in accordance with conventional regulations, such as the conditions described in "Molecular Cloning: A Laboratory Manual" (Fourth Edition) (Science Press Co., Ltd., 2017) by J. Sambrook et al., or the conditions described in the product manuals provided by the product manufacturers. The materials, reagents, instruments, etc. used in the embodiments can be obtained from commercial channels without special instructions.

[0070] Experimental methods

[0071] (1) Animals and models

[0072] This program was approved by the Institutional Animal Ethics and Use Committee of Renji Hospital, Shanghai Jiao Tong University, and was conducted in accordance with the guidelines of the Institutional Animal Ethics and Use Committee. Male C57 / BL6 mice (8-10 weeks old) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. and housed under standard laboratory conditions (22 °C and 12-hour light-dark cycle), with unrestricted access to food and water. The mice were randomized and assigned by concealment. Briefly, the mice were marked using the animal facility labeling system and randomly assigned to the experimental groups. The surgeries and outcome evaluations were performed by the researchers in a "blinded" manner.

[0073] (2) Animal models of ischemic stroke and drug administration

[0074] Establishment of a mouse model of focal cerebral ischemia: Male C57 / BL6 mice (8-10 weeks old) received transient middle cerebral artery occlusion (tMCAO). Briefly, a monofilament was inserted into the common carotid artery of the mouse and advanced to the origin of the middle cerebral artery and left in place for 60 minutes until reperfusion. The mice were maintained at a temperature of 37 ± 0.5 °C using a heating pad during the surgery, and laser Doppler flowmetry (PERIMED-PeriCam PSI, Sweden) was used to confirm vessel occlusion and reperfusion. Control surgical animals received the same anesthesia and surgical incision but without middle cerebral artery occlusion. Two hours after tMCAO, the mice were injected with PBS, mIL-10 alone, mNC@MLNP, MLNP, or mIL-10@MLNP. The dose of mIL-10@MLNPs was 0.12 or 0.6 mg / kg, and the mice were sacrificed 3 days later to evaluate the therapeutic effect.

[0075] The mice were anesthetized with 2% isoflurane in 30% O2 / 68% N2O. A skin incision was made in the neck to expose and ligate the left common carotid artery. After suturing the neck incision, another skin incision was made between the left eye and the ear. The temporalis muscle was dissected and a burr hole was opened to expose the distal portion of the MCAO. Then, the dura mater was incised and the dMCAO (Shanghai Hutong Electronics Co., Ltd.) was coagulated with low-intensity bipolar electrocautery in the directly lateral part of the rhinal fissure. Sham-operated animals received anesthesia and surgical exposure of the artery but without artery occlusion. Three days and 7 days after dMCAO, the mice were injected with a certain dose of PBS, MLNP, and mIL-10@MLNP, 0.3 mg / kg mRNA per mouse, and sacrificed 7 days after the last injection to evaluate the therapeutic outcome. The mice were sacrificed with high-concentration carbon dioxide. According to the number of animals previously determined to have undergone surgery, the experiments in our laboratory were designed to minimize animal suffering and the use of animals.

[0076] (3) Immunofluorescence staining

[0077] Slices were blocked with 3% BSA for 30 minutes, incubated with the primary antibody overnight at 4°C, then incubated with the appropriate fluorescently labeled secondary antibody for 60 minutes at room temperature, and counterstained with DAPI for 5 minutes. Histological images were captured by microscope (Olympus) or CLSM (Olympus Fluoview FV3000, Olympus, Center Valley, PA) and analyzed using ImageJ software. Immunopositive cell counts were expressed as the mean percentage; three randomly selected regions in the cortex of three consecutive slices of each brain were analyzed by blinded researchers.

[0078] (3) TUNEL staining

[0079] Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was performed as previously described (4) to observe apoptosis. Sections were incubated for 1 hour in a humidified chamber at 37°C using a TUNEL kit (Roche). Double-label immunofluorescence for NeuN and TUNEL was performed by confocal imaging.

[0080] (4) Flow cytometry

[0081] The corresponding cells in the ischemic lesions or sham animals of the cerebral hemispheres of tMCAO mice were homogenized using a gentle MACS separator (Miltenyi Biotec). Microglial surface-specific antibody markers (anti-CD45-PE-Cy7, Invitrogen; anti-CD11b-FITC, BD), a marker of the M2 phenotype (anti-CD206-APC, Invitrogen), and a marker of the M1 phenotype (anti-CD86-BV510, BD), and IL10-bv421 (BD) were used for flow cytometry to characterize immune cells. Data were analyzed using FlowJo software (TreeStar).

[0082] (5) Neurological function assessment

[0083] The modified Garcia Score (5, 6) and the tape removal test (7). The modified Garcia Score is a well-established sensorimotor assessment system consisting of five individual tests, one measuring sensor function and four measuring motor function. Each test is scored from 0 to 3 points (maximum score = 15), including body proprioception, forelimb walking, limb symmetry, lateral turning, and climbing.

[0084] In the tape - stripping test, two strips of tape (0.3 x 0.4 cm) were applied to each paw. The order of tape placement (right or left) was randomized. Then the mouse was gently placed into a plexiglass box, and after a few seconds, each tape was removed and recorded. Mice were trained once a day for three consecutive days before the test, and the test was conducted regularly (3 trials per day, with a 15 - minute interval between each mouse).

[0085] (6) Barnes maze

[0086] The Barnes maze protocol was adopted from a previous study as a behavioral method to evaluate mouse neurological function (8). The maze consisted of a white PVC circular plate platform with a diameter of 36 inches and a thickness of 8 mm. There were 20 holes with a diameter of 3 inches on this platform, 1 inch away from the edge. One of the holes led to an escape box where the animal could find security, while the other holes led to dead - end holes without an escape box. The target location led to a small, dark recess under the platform, which was not visible from the platform. The task of the test animal was to find the hole that served as the target location, usually using cues both inside and outside the maze to help the test animal find the target hole. The platform was mounted on a rotating stool 30 inches above the ground. The outside of the cage wall was covered with black tape to prevent light from entering the escape cage. The maze was placed in a dedicated room in the center, and two 120W lights were placed at the edge facing the room to provide aversive stimuli for the mice. Eight colored paper shapes (squares, rectangles, and circles) were mounted on the room walls as visual cues. After each mouse was tested, the maze was cleaned with 70% ethanol and rotated clockwise for each mouse to avoid the smell or visual cues inside the maze. All experiments were recorded using SMART 3.0 video - tracking software.

[0087] Preparation of microglia - targeted IL - 10 mRNA nanoparticles in Preparation Example 1

[0088] IL - 10 mRNA was encapsulated into LNP to assemble microglia - targeted IL - 10 mRNA nanoparticles, hereinafter named mIL - 10@MLNP. After passing the physicochemical quality control, it was used for subsequent experiments.

[0089] Example 1 Treatment with mIL - 10@MLNP can improve blood - brain barrier damage and neuronal death after ischemic stroke

[0090] The therapeutic effect of MLNP targeted delivery was investigated, and mIL-10 was delivered to M2 microglia in the infarcted brain tissue using the tMCAO mouse model. Different treatment methods (PBS, pure IL-10 mRNA, non-targeting group without IL-10 mRNA specific target CD206 (mNC@MLNP), group with IL-10 mRNA specific target CD206 (MLNP), group with IL-10 mRNA specific target CD206 (mIL-10@MLNP)) were intravenously injected 2 hours after reperfusion in tMCAO mice. Brains were collected 3 days after injection to evaluate the treatment outcome ( Figure 1 in A).

[0091] The disruption of the BBB associated with ischemia-reperfusion injury after tMCAO was evaluated by staining brain sections for endogenous IgG extravasation into the brain parenchyma. Treatment with mIL-10@MLNP, especially at the dose of 0.6 mg / kg mRNA, resulted in a decrease in the green fluorescence signal corresponding to IgG ( Figure 1 in B and C), indicating a significant improvement in blood-brain barrier disruption. In addition, compared with the PBS-treated control group, both low-dose and high-dose mIL-10@MLNP treatments showed a decrease in microtubule organizing center (MAP-2), i.e., a significant reduction in the average infarct volume (from 44.92 ± 12.32 mm3 (PBS control) to 34.41 ± 8.26 mm 3 (at the low dose of 0.12 mg / kg mRNA (0.065 mg / infarct volume)) and 16.11 ± 0.61 mm 3 (at the high dose of 0.6 mg / kg mRNA (0.33 mg / infarct volume)) ( Figure 1 in B-E). The tight junction protein (zonula occlusionns-1, ZO-1) can be used as a reliable marker of endothelial cells, reflecting BBB integrity (25). ZO-1-positive immunofluorescence staining images showed that tight junction staining was disrupted in brain sections of tMCAO mice, revealing ischemia-reperfusion injury after stroke. Consistent with the IgG staining results, the application of intravenous mIL-10@MLNPs led to an increase in ZO-1 expression, indicating an improvement in BBB integrity ( Figure 1 in F and G). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was also performed to determine the apoptosis of NeuN+ neurons ( Figure 1In Figures H and I). The results showed that mIL-10@MLNP treatment significantly inhibited neuronal apoptosis after tMCAO in a dose-dependent manner. After treatment with mIL-10@MLNP, especially at the mRNA dose of 0.6 mg / kg, the survival of neurons in brain sections was restored to a level comparable to that of the control group, indicating that mIL-10@MLNP could improve neuronal apoptosis after tMCAO.

[0092] Example 2 mIL-10@MLNP treatment promotes microglial M2 polarization and resolves neuroinflammation after ischemic stroke

[0093] To gain an in-depth understanding of the mechanism of the therapeutic effect of mIL-10@MLNP on ischemic stroke, the expression level of IL-10 in the brains of tMCAO mice and the phenotypic changes of microglia were tested. Microglia were isolated from the ischemic-lesioned cerebral hemispheres three days after tMCAO. Compared with the groups treated with PBS or MLNP, the expression of microglial IL-10 in the ischemic brain tissue was significantly increased in the mIL-10@MLNPs group ( Figure 2 in Figures A and B). At the same time, compared with mice treated with PBS or blank MLNP, the number of CD206-positive microglia was significantly increased in the ischemic hemispheres of mice treated with mIL-10@MLNP ( Figure 2 in Figures C and D). Compared with the other two treatments, the number of CD86+ microglia in the brain was also significantly reduced in the mIL-10@MLNP treatment group ( Figure 2 in Figures E and F). These results indicate that mIL-10@MLNP targeting M2-phenotype microglia can effectively change the microglial polarity, reshape the pathological microenvironment into an anti-inflammatory environment, and promote the alleviation of neuroinflammation.

[0094] Next, the efficacy of mIL-10@MLNP in resolving neuroinflammation was investigated by the following method: quantifying the expression of key inflammation-related biomarkers in brain sections. Arginase 1 (Arg1) is a typical marker of M2-polarized microglia, which greatly promotes tissue repair and neuroprotection. In contrast, tumor necrosis factor α (TNF-α) plays a key role in the neuroinflammatory responses associated with various neurological diseases. As expected, compared with the PBS or MLNP treatment groups, mIL-10@MLNP treatment significantly increased the expression of microglial Arg1 in the infarct area ( Figure 2 in Figures G and H). In addition, compared with the PBS or MLNP treatment groups, mIL-10@MLNP treatment significantly reduced the expression of microglial TNF-α ( Figure 2 in Figures I and J), indicating that mIL-10@MLNP has a strong anti-inflammatory effect.

[0095] Example 3 mIL-10@MLNP treatment can prevent long-term neurological deficits in dMCAO mice

[0096] A dMCAO mouse model was established to evaluate the tissue damage and functional deficits caused by permanent cerebral ischemia by mIL-10@MLNP. The indicated drugs were intravenously injected twice (at 3 days and 7 days) after dMCAO ( Figure 3 in A). The brain tissues were used for histological analysis after 14 days. Compared with PBS or MLNP, mIL-10@MLNP treatment significantly reduced the volume of MAP2 and IgG extravasation after stroke, that is, mIL-10@MLNP treatment significantly improved the late infarct volume and blood-brain barrier disruption after dMCAO ( Figure 3 in B). Prolonged ischemia often occurs axonal injury and demyelinating stroke, leading to sensorimotor and cognitive neurological deficits. We first evaluated the expression of the axonal marker non-phosphorylated neurofilament H (SMI32) and the loss of the myelin integrity marker - myelin basic protein (MBP). Immunofluorescence staining results showed that the expression of SMI32 in the external capsule and striatum was significantly decreased in mice of the PBS or M-HNP group. The application of mIL-10@MLNP was beneficial to the repair of axons in the external capsule and striatum of dMCAO mice ( Figure 3 in C-D).

[0097] Then the following methods were used to evaluate the neurological deficits after permanent cerebral ischemia in mice: including Garcia Score, tape stripping test and Barnes maze. Consistent with the aforementioned immunofluorescence results, compared with the control group, the total neurological assessment score of mice treated with mIL-10@MLNP was significantly increased 14 days after stroke. In the tape stripping test, compared with the other two control groups, the mice in the mIL-10@MLNP treatment group showed higher proficiency in sensing and removing the adhesive, such as relatively faster response time ( Figure 3 in E-F). The Barnes maze was used to quantify the spatial cognitive ability of dMCAO mice receiving different treatments. Compared with PBS or blank MLNP treatment, the positioning time of the target hole leading to the escape cage in mice administered intravenous mIL-10@MLNP was reduced, indicating that mIL-10@MLNPs significantly enhanced the spatial learning ability of mice. In addition, the mice treated with mIL-10@MLNP spent more time training to find the hole in the target quadrant where they were before, indicating that their spatial memory ability was improved ( Figure 3 in G). These results together demonstrate that MLNP containing mIL-10 targeting M2 microglia exerts an effective neuroprotective effect by reducing infarcts and improving the infarct area, improving sensorimotor and cognitive functions after stroke.

[0098] All documents mentioned in this invention are cited herein by reference as if each individual document was cited by reference. In addition, it should be understood that after reading the above teachings of this invention, those skilled in the art can make various changes or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. Use of microglia-targeted IL-10 mRNA nanoparticles in the preparation of a preparation or composition, or a kit, for immunotherapy of ischemic stroke.

2. The use according to claim 1, characterized in that, The microglia-targeted IL-10 mRNA nanoparticles specifically target M2 microglia, thereby selectively delivering IL-10 mRNA to M2 microglia at the site of ischemic stroke.

3. The use according to claim 1, wherein The preparation or composition, or the kit, is administered to a subject suffering from ischemic stroke, and the ischemic stroke has the following classification characteristics: Accompanied by blood-brain barrier damage and / or neuronal apoptosis.

4. The use according to claim 3, characterized in that, The ischemic stroke also has one or more classification characteristics selected from the following group: (1) The expression level of tight junction protein (zonula occlusionns-1, ZO-1) in brain tissue is significantly reduced; wherein, the "significant reduction" means that the ratio of the expression level F1 of ZO-1 in the brain tissue of the subject to the expression level F0 of ZO-1 in normal brain tissue, F1 / F0 ≤ 1 / 3, preferably ≤ 1 / 2; (2) The terminal deoxynucleotidyl transferase dUTP nick in brain tissue neurons is significantly increased; wherein, the "significant increase" means that the amount T1 of the terminal deoxynucleotidyl transferase dUTP nick in brain tissue neurons of the subject is compared with the amount T0 of the terminal deoxynucleotidyl transferase dUTP nick in normal brain tissue neurons, and the ratio T1 / T0 ≥ 1.2, preferably ≥ 1.5, more preferably ≥ 2.

5. The use according to claim 1, characterized in that, The preparation or composition, or the kit, is administered to a subject suffering from ischemic stroke, thereby being used for: (a) Changing the polarity of microglia and driving microglia to polarize towards the M2 phenotype; (b) Remodeling the pathological microenvironment and reducing neuroinflammation after ischemic stroke; (c) Improving blood-brain barrier (BBB) damage after ischemic stroke; (d) Inhibiting neuronal apoptosis after ischemic stroke; and / or (e) Preventing long-term neurological dysfunctions caused by long-term cerebral ischemia (permanent cerebral ischemia), including axonal injury, demyelinating stroke, sensorimotor and cognitive neurological dysfunctions.

6. The use according to claim 1, characterized in that, The preparation or composition is an injection.

7. The use according to claim 1, wherein, The administration routes of the preparation or composition include: intravenous injection, intravenous infusion, intracranial injection.

8. The use according to claim 1, characterized in that, The kit further comprises: a detection reagent for detecting the classification characteristics of ischemic stroke; Preferably, the detection reagent for detecting the classification characteristics is selected from the following group: a reagent for detecting blood-brain barrier damage, a reagent for detecting neuronal apoptosis, a reagent for detecting the polarization of microglia towards the M2 phenotype, or a combination thereof.

9. The use according to claim 8, characterized in that, The detection reagent for detecting the classification characteristics is selected from the following group: a reagent for detecting the expression level of Z0-1 in brain tissue, a reagent for detecting the terminal deoxynucleotidyl transferase dUTP nick in brain tissue neurons, a reagent for detecting the expression of CD206, Arg1, and / or CD86 in microglia, or a combination thereof.

10. A method for in vitro inducing microglia to polarize into the M2 phenotype, characterized in that, The method comprises the step of: administering microglia-targeted IL-10 mRNA nanoparticles to microglia.

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