CN112516304A

CN112516304A - Nano microcapsule wrapping intravenous immunoglobulin, preparation method and application thereof in preparing medicine for treating cerebral arterial thrombosis

Info

Publication number: CN112516304A
Application number: CN202011590701.7A
Authority: CN
Inventors: 康春生; 原续波; 金维丽
Original assignee: Tianjin Medical University General Hospital
Current assignee: Tianjin Medical University General Hospital
Priority date: 2020-12-29
Filing date: 2020-12-29
Publication date: 2021-03-19
Anticipated expiration: 2040-12-29
Also published as: CN112516304B

Abstract

The invention discloses a nano microcapsule wrapping intravenous immunoglobulin, a preparation method and application thereof in preparing a medicine for treating cerebral arterial thrombosis. The invention uses 2-methacryloyloxyethyl phosphorylcholine as a monomer and ethylene glycol dimethyl methacrylate as a cross-linking agent to encapsulate human immunoglobulin for intravenous injection by adopting a nano technology, and prepares the nano microcapsule by in-situ polymerization. The nano microcapsule can obviously target ischemic brain areas and has good biocompatibility. In the early stage of stroke, the nano microcapsule with lower dose is injected statically to relieve the further damage of stroke brain inflammation to the brain, reduce the damage of nerve function and improve the prognosis of patients, and simultaneously can relieve the side effect and treatment cost of high-dose use of the medicine of the patients, thereby having huge transformation prospect and transformation value.

Description

Nano microcapsule wrapping intravenous immunoglobulin, preparation method and application thereof in preparing medicine for treating cerebral arterial thrombosis

Technical Field

The invention relates to the technical field of biology, in particular to a nano microcapsule wrapping intravenous immunoglobulin, a preparation method and application thereof in preparing a medicine for treating cerebral arterial thrombosis.

Background

Stroke is the second most common cause of death worldwide and has become a major source of permanent disability. Ischemic stroke accounts for 80-90% of all strokes and can lead to fatal brain injury and severe neurological impairment. In ischemic stroke, the sudden cessation of cerebral blood supply in the vascular area creates an ischemic core surrounded by an area that is hypoperfused but may be salvaged, called the ischemic penumbra. Within minutes after ischemic injury, neuronal cell death triggers an inflammatory response characterized by activation of focal glial cells, infiltration of peripheral immune cells, and release of cytokines and chemokines, further damaging the brain parenchyma and vasculature. In ischemic stroke, complement component 3(C3) is critical for the complement cascade and immune recognition. At present, various clinical tests and experimental researches show that inflammation after cerebral ischemia plays a key role in the pathogenesis of stroke. In addition, modulating immunity attempts to maintain immune homeostasis and promote inflammation resolution, which can reduce neurological deficit and improve stroke prognosis. Although various immunomodulatory agents have been shown to have potent anti-inflammatory and immunomodulatory effects in an increasing number of diseases, challenges remain in fully exploiting their ability to treat ischemic stroke due to the inability to adequately cross the Blood Brain Barrier (BBB) and tissue off-target effects of drugs.

Advances in science continue to provide potential new classes of immunomodulators. For example, anti-cytokine therapies have been developed to neutralize inflammation-associated cytokines, such as tumor necrosis factor-alpha (TNF- α) and interleukin 1 β (IL-1 β), during the inflammatory response. However, off-target effects of these therapies may lead to adverse effects, including undesirable toxicity and susceptibility to infection with common complications following stroke. Immunoglobulins are glycoproteins produced by cells of the immune system and protect the body from pathogens by binding to or forming an encapsulation. Purified polyclonal immunoglobulin G from human plasma (> 98% human immunoglobulin G (igg)), so-called intravenous immunoglobulin or IVIg, has been approved by the U.S. food and drug administration for the treatment of various inflammatory and autoimmune diseases, such as kawasaki disease, immune thrombocytopenia, humoral immunodeficiency and bone marrow transplantation, for example, at high doses. Furthermore, IVIg shows great potential in the treatment of ischemic stroke at high doses by directly targeting the immune system as well as neuronal cells. However, patients in the high dose group are more prone to thromboembolic events and skin reactions, among other problems, than in the low dose group.

Disclosure of Invention

The invention aims to solve the problems and provides a nano microcapsule wrapping intravenous immunoglobulin, a preparation method and application thereof in preparing a medicine for treating cerebral arterial thrombosis.

The invention is implemented according to the following technical scheme.

An intravenous immunoglobulin-encapsulating nanocapsule, the nanocapsule comprising an intravenous immunoglobulin, N- (3-aminopropyl) methacrylamide, 2-methacryloyloxyethyl phosphorylcholine and a cross-linking agent, the intravenous immunoglobulin, N- (3-aminopropyl) methacrylamide, 2-methacryloyloxyethyl phosphorylcholine and cross-linking agent being present in a molar ratio of 1: (100-10000): (300-30000): (50-5000). Preferably, the intravenous immunoglobulin, N- (3-aminopropyl) methacrylamide, 2-methacryloyloxyethyl phosphorylcholine and the cross-linking agent are present in a molar ratio of 1: 1000: 3000: 500.

further, ethylene glycol dimethyl methacrylate is used as the crosslinking agent.

A preparation method of the nanocapsule for encapsulating intravenous immunoglobulin comprises the following steps:

a. weighing a specified amount of intravenous immunoglobulin, N- (3-aminopropyl) methacrylamide, 2-methacryloyloxyethyl phosphorylcholine, a cross-linking agent, ammonium persulfate and N, N, N ', N' -tetramethyl ethylenediamine, and reacting for 2 hours at 4 ℃;

b. dialyzing the solution obtained in the step a by using a PBS buffer solution to remove unreacted monomers and byproducts;

c. passing the solution obtained after dialysis in step B through a hydrophobic interaction column (Phenyl-Sepharose CL-4B) to remove any unencapsulated proteins and obtain nanocapsules, MPC-n (IVIg), encapsulating the intravenous immunoglobulin.

Further, in the step a, the molar ratio of ammonium persulfate to intravenous immunoglobulin is 500: 1.

further, in the step a, the mass ratio of the N, N, N ', N' -tetramethylethylenediamine to the ammonium persulfate is 2: 1.

further, in step b, the pH of the PBS buffer was 7.4.

An application of the nano microcapsule wrapping the intravenous immunoglobulin in preparing a medicine for treating cerebral arterial thrombosis.

The present invention achieves the following advantageous effects.

The nanocapsule wrapping the intravenous immunoglobulin can obviously target ischemic brain areas and has good biocompatibility. In the early stage of stroke, the further damage of brain caused by stroke brain inflammation can be relieved by intravenous injection of MPC-n (IVIg) with lower dose (one fifth of the lowest effective dose reported at present) so as to reduce the damage of nerve function and improve the prognosis of patients, and meanwhile, the side effect and treatment cost of high-dose use of medicines of the patients can be reduced, so that the method has great transformation prospect and transformation value.

Drawings

FIG. 1 is a graph showing the results of the test in example 1 of the present invention;

FIG. 2 is a graph showing the results of the test in example 2 of the present invention;

FIG. 3 is a graph showing the results of the test in example 3 of the present invention;

FIG. 4 is a graph showing the results of the test in example 4 of the present invention;

FIG. 5 is a graph showing the results of the test in example 5 of the present invention;

FIG. 6 is a graph showing the results of the test in example 6 of the present invention.

Detailed Description

The present invention will be further explained with reference to the drawings and examples.

Example 1 Synthesis and characterization of MPC-n (IVIg)

The invention uses N- (3-aminopropyl) methacrylamide (APM), 2-Methacryloyloxyethyl Phosphorylcholine (MPC) and degradable cross-linking agent (EGDMA) in acid environment to encapsulate IVIg in MPC nano microcapsule. The molar ratio of IVIg/APM/MPC/cross-linking agent is 1: 1000: 3000: 500. the free radical polymerization was initiated by adding together ammonium persulfate (APS/IVIg, 500: 1, N/N) and N, N, N ', N' -tetramethylethylenediamine (TEMED/APS, 2: 1, w/w) at 4 ℃ for 2 hours. Subsequently, the solution was dialyzed against PBS buffer (pH 7.4) to remove unreacted monomers and byproducts, and then any unencapsulated protein was removed by passing through a hydrophobic interaction column (Phenyl-Sepharose CL-4B). The column was prepared by transferring 5mL of phenyl-Sepharose CL-4B to a glass column, which was then pre-equilibrated with 2.5M sodium sulfate. The sample was first mixed with sodium sulfate to reach a final concentration of 2.5M sodium sulfate. The sample was then loaded onto a chromatography column and eluted with 2.5M sodium sulfate. The eluate with 2.5M sodium sulfate was collected and concentrated using centrifugal filtration. The samples were then dialyzed against PBS to remove sodium sulfate and MPC nanocapsules were stored at 4 ℃ until use.

To encapsulate IVIg in nanocapsules and to formulate an effective strategy to achieve its targeted ischemic penumbra, pH sensitive MPC nanocapsules were constructed by in situ polymerization using MPC monomers with EGDMA as degradable cross-linking agent (fig. 1 a-b). EGDMA is stable at neutral pH, but degradable in an acidic environment. Transmission Electron Microscopy (TEM) and Dynamic Light Scattering (DLS) showed MPC-n (IVIg) to be spherical and 28.61nm in average diameter, respectively (FIG. 1 c). During electrophoresis, MPC-n (IVIg) was retained on the upper layer of the separation gel (FIG. 1d), indicating that IVIg were successfully encapsulated in MPC nanocapsules. Next, it is discussed whether MPC-n (IVIg) reacts to an acidic environment to release IVIg in vitro. MPC-n (IVIg) was incubated in Phosphate Buffered Saline (PBS) at pH 7.4 or 6.5. The time course of IVIg release from MPC nanoparticles was quantified using an enzyme-linked immunosorbent assay (ELISA) at 37 ℃ under different pH conditions (7.4 and 6.5) (FIG. 1 e). The MPC-n (IVIg) solution remained stable at a pH of 7.4 for 48 hours. In contrast, MPC-n (IVIg) released IVIg rapidly (66.5% release at pH 6.5) within the first 24 hours. These findings indicate that encapsulation of IVIg enhances its stability and achieves controlled release.

FIG. 1, a) schematic representation of the synthesis of MPC-n (IVIg); b) schematic representation of MPC-n (IVIg) and a delivery process that enhances IVIg targeting to ischemic brain regions by intravenous injection of MPC-n (IVIg) upon ischemic stroke; c) representative transmission electron micrographs of MPC-n (IVIg) and hydrodynamic size distribution of free IVIg and MPC-n (IVIg) as measured by dynamic light scattering; d) gel electrophoresis of free IVIg and MPC-n (IVIg); e) IVIg was released from MPC-n (IVIg) in phosphate buffered saline at different pH's (6.5 and 7.4).

Example 2 Large accumulation of MPC-n (IVIg) in ischemic areas

Because of the stability and efficient release of MPC-n (IVIg), it was evaluated whether MPC nanocapsules could efficiently deliver IVIg to ischemic areas. Biodistribution studies were performed via the tail vein and the duration and ability of MPC-n (ivig) to target an ischemic hemisphere in mice was studied. 45 minutes after occlusion of the middle cerebral artery in the mouse, the occluded filaments were pulled out for reperfusion. Mice were injected intravenously with nanocapsules 2 hours after reperfusion. The accumulation and biodistribution of MPC-n (IVIg) (10mg/kg body weight) in the ischemic hemisphere of the MCAO model was evaluated using an In Vivo Imaging System (IVIS). MPC-n (IVIg) showed stronger fluorescence in the MCAO model 2h after injection compared to free IVIg and n (IVIg) (nanocapsules without MPC) and remained as long as 24h (fig. 2 a). The accumulation of MPC-n (IVIg) is promoted by ischemic stroke-induced damage. To further study accumulation in the ischemic hemisphere (damage from MCAO surgery), mice were heart perfused 2 and 24 hours after injection and their brains were harvested. After administration of MPC-n (ivig) in MCAO mice, ipsilateral hemispheres had higher Cy5 fluorescence intensity, indicating selective accumulation of MPC-n (ivig) in the ischemic hemispheres (fig. 2 b). In addition, the right (no damage caused by MCAO surgery) and left (no damage caused by MCAO surgery) brains were homogenized and the accumulation of IVIg was quantitatively measured using ELISA (fig. 2 c). Administration of MPC-n (IVIg) in the left hemisphere showed 3.22-fold and 5.17-fold higher. The accumulation in the MCAO model was higher than in the sham (sham) model at both 2 and 24h post-injection. In contrast, there was no significant difference in IVIg accumulation after MPC-n (IVIg) administration in the right hemisphere of the MCAO and Sham models. There was also no significant difference in IVIg accumulation between the left and right hemispheres in the sham model at 2h and 24h after MPC-n (IVIg) injection, respectively. In the MCAO model injected with MPC-n (IVIg), there was no reduction in IVIg accumulation in the left hemisphere after 24 hours. This indicates that MPC-n (IVIg) can last for several hours in ischemic brain tissue. These results indicate that MPC nanoparticles enhance the selective accumulation of IVIg in ischemic regions.

In FIG. 2, a) representative images of Cy5 fluorescence whole animal images in MCAO mice 2h after post-ischemic reperfusion after intravenous injection of free IVIg, n (IVIg) or MPC-n (IVIg) (10mg/kg)2, 4 and 24h in vivo. The histogram summarizes the relative fluorescence intensity of brain tissue; b) representative ex vivo Cy5 fluorescence images of isolated brain tissue from a) treated mice 2 hours and 24 hours after injection. The histogram summarizes the relative fluorescence intensity of ex vivo brain tissue; c) the concentration of IVIg in the brain hemispheres of treated mice was determined by enzyme-linked immunosorbent assay (ELISA) 2 hours and 24 hours after injection.

Example 3 MPC-n (IVIg) Selective targeting of overexpression of ChT1 in endothelial cells following ischemic area-dependent acute ischemic stroke

Although acute ischemic injury transiently disrupts the blood brain barrier, only a small amount of free IVIg or n (IVIg) accumulates in the ischemic hemisphere. However, the brain injury and surgical procedures that lead to the initial accumulation of free IVIg, n (IVIg) and MPC-n (IVIg) at the site of ischemia reperfusion are very limited. Thus, efficient BBB penetration is essential for effective drug entry into the ischemic hemisphere. The underlying mechanism of selective targeting of MPC-n (ivig) to ischemic areas remains to be studied, as MPC-n (ivig) does not enter ischemic areas directly through the BBB as the primary route to the brain. It has previously been shown that BBB permeability of MPC nanocapsules containing choline and acetylcholine analogues is due to ChT1 mediated endocytosis. mRNA levels of ChT1 in b end.3 cells after OGD treatment increased in vitro. However, as the reperfusion time increased, the expression of ChT1 decreased gradually in the bEND.3 cells (FIG. 3 a). Next, coronal sections from the MCAO model were used for immunofluorescence at different durations of ischemia reperfusion. ChT1 positive cells were elevated in the ischemic region of MCAO compared to sham group. Using the vascular endothelial cell marker (CD31), it was further demonstrated that ChT1 is highly expressed in endothelial cells following ischemic stroke. However, prolonged reperfusion gradually reduced the expression of ChT1 in endothelial cells, consistent with in vitro results (fig. 3 b). These data confirm overexpression of ChT1 in the endothelium during ischemia. Reperfusion decreases ChT1 expression.

MCAO model

0, 5 and 10. mu.g/kg of the ChT1 inhibitor choline-3 (hemicellium-3; HC-3) were injected intraperitoneally 20 minutes prior to the injection of Cy 5-labeled MPC-n (IVIg). 4 hours after MPC-n (IVIg) injection, in vivo and ex vivo fluorescence tests were performed on mouse brains (FIG. 3 c). An increase in HC-3 dose reduced the fluorescence intensity in the ischemic brain, confirming that ChT1 mediates transport of Cy 5-labeled MPC-n (IVIg) into the brain. In addition, ex vivo imaging of brains collected from mice 24h after injection, the fluorescence of mice 24h after MPC-n (IVIg) administration was enhanced in the MCAO model compared to the sham model and the MCAO model pre-injected with 10. mu.g/kg HC-intraperitoneally (FIG. 3 d). These findings confirm that overexpression of ChT1 in the ischemic hemisphere during early ischemia-reperfusion may provide an effective delivery strategy for the selective accumulation of MPC-n (IVIg) during ischemic stroke.

In FIG. 3, a) qRT-PCR analysis of ChT1 mRNA levels in bEND.3 cells treated with OGD for 12h and reinfused for 0, 4, 6, 12 and 24 h; b) immunofluorescence mapping ChT1 and CD31 on the surface of MCAO mice endothelial cells. The histogram summarizes the mean fluorescence intensity of ChT 1; c) cy 5-fluorescing in vivo C57 mice were imaged 4 hours after intravenous injection of MPC-n (IVIg) (10mg/kg) into MCAO mice previously injected with various doses of HC-3 intraperitoneally. Ex vivo Cy5 fluorescence images of ex vivo brain tissue from differently treated mice 4h after injection. The histogram summarizes the relative fluorescence intensity of ex vivo brain tissue; d) ex vivo Cy5 fluorescence images of ex vivo brain tissue 24 hours after injection from differently treated mice (sham surgery + MPC-n (IVIg)), MCAO + MPC-n (IVIg)) and MCAO + HC-3 (10. mu.kg) + MPC-n (IVIg)). The histograms summarize the relative fluorescence intensity of ex vivo brain tissue.

Example 4 early administration of MPC-n (IVIg) promotes IVIg accumulation in ischemic areas

Time-dependent cellular uptake of MPC-n (IVIg) during reperfusion was studied to assess the time of administration of MPC-n (IVIg) during ischemic stroke. Due to overexpression of ChT1 in the cell membranes during ischemia, bEND.3 cells incubated with FITC-labeled MPC-n (IVIg) for 4h showed strong FITC fluorescence in the OGD/R0 h group as confirmed by oxyboom 0(OGD/R0 h) or 12h (OGD/R12 h) after OGD12 h pretreatment and flow cytometry (FIG. 4 a). The flow cytometry data of bEND.3 cells incubated with Cy 5-labeled MPC-n (IVIg) for 4h was highly consistent with immunofluorescence (FIG. 4 b). bEND.3 cells in the OGD/R0 h group were incubated with MPC-n (IVIg) 2.22-4.04 times higher than the mean fluorescence intensity in the OGD/R12 h group. In vivo small animal imaging, MPC-n (IVIg) fluorescence intensity in ischemic brain was higher than 24h after 2h reperfusion. In vitro imaging and quantitative analysis of the brain showed a 2.45-fold increase in ischemic hemispheres 24 hours after administration 2 hours after reperfusion compared to administration 24 hours after reperfusion (fig. 4 c-d). Immunofluorescence of brain sections showed that CY5 fluorescence was stronger in brain parenchyma after 2h of reperfusion than after 24h (fig. 4 e). These data demonstrate that early intervention promotes cellular uptake of MPC-n (ivig), likely due to a decrease in ChT1 expression in endothelial cells during reperfusion, and indicate that early administration of MPC-n (ivig) promotes selective accumulation of MPC-n (ivig). IVIg selectively targets ischemic regions, thereby preventing the progression of stroke-induced damage.

In FIG. 4, a, b) immunofluorescence and flow cytometry showed that the OGD was reaerated for 12h and either 0 or 12h before incubation with MPC-n (IVIg) for 6h before bEND.3 cells took up MPC-n (IVIg) (0.5 mg/mL). Histograms represent the fluorescence intensity of MPC-n (ivig) in bned.3 cells using flow cytometry at a scale bar of 20 μm; c) in vivo C57 mice were imaged for

Cy5 fluorescence

2 or 24 hours after intravenous injection of MPC-n (IVIg) (10mg/kg)2, 4 or 24 hours after ischemia reperfusion; d) ex vivo Cy5 fluorescence images of ex vivo brain tissue from differently treated mice 24 hours after injection. The histograms summarize the relative fluorescence intensity of C57 mice and ex vivo brain tissue; e) immunofluorescence was used to co-localize Cy 5-labeled IVIg, CD31 and DAPI-stained nuclei in brain tissue of differently treated mice 24h after injection, with a scale bar of 20 μm.

Example 5 Low doses of MPC-n (IVIg) can enhance neuroprotective effects and reduce injury in acute ischemic stroke

High doses of IVIg (. gtoreq.500 mg/kg) effectively protect nerves by modulating a number of key biological processes during ischemic stroke. To determine whether early administration of low doses of MPC-n (ivig) could improve for recovery of function, a treatment window (2 hour post-reperfusion administration) was used and the dose of MPC-n (ivig) was reduced to 100mg/kg, and the neurological deficit score was recorded in mice. In addition, brain tissue was harvested 3 days after treatment with several formulations and sections were stained with 2% triphenyltetrazolium zinc chloride (TTC) to quantify infarcted brain tissue. Low doses of IVIg (100mg/kg) did not disrupt the evolution of acute infarct volume and had little to no reduction in neurological score before the third day. MPC-n (IVIg) (100mg/kg) treatment showed neurological deficit and reduction in infarct volume after 3 days (FIGS. 5 a-b). Treatment with MPC-n (IVIg) (100mg/kg) reduced TUNEL positive apoptotic cells in the ischemic penumbra (FIGS. 5c-d), indicating that early administration of low doses of MPC-n (IVIg) was effective in reducing apoptosis in the MCAO model. Next, it was demonstrated that MPC-n was administered 2 hours after reperfusion in MCAO model

(IVIg) (100mg/kg), treatment of stroke animals with recovery of motor function was verified using a corner test (FIG. 5 e). The above findings indicate that low doses of MPC-n (IVIg) are effective in enhancing neuroprotection and reducing acute injury during early ischemia-reperfusion.

In FIG. 5, a) neurological deficit score 24 hours or 72 hours after injection of solvent (vehicle) (same volume) in mice 2h after ischemia reperfusion, free IVIg (500 and 100mg/kg) or MPC-n (IVIg) (100 mg/kg); b) representative images of TTC staining of brain 3d after injection. Infarcted areas are indicated by white and black outlines. Infarct volume was measured over the hemisphere and corrected for contralateral structures. A significant reduction in infarct volume of the whole hemisphere of MPC-n (IVIg) (100mg/kg) compared to the solvent (vehicle) and free IVIg (500 and 100mg/kg) controls; c) the fluorescence of TUNEL in 3d ischemic penumbra after 2h of mice injection of solvent (vehicle) (same volume) after ischemia reperfusion, free IVIg (500 and 100mg/kg) or MPC-n (IVIg) (100mg/kg), scale bar 20 μm; d) histogram quantization TUNEL analysis (n ═ 3); e) the cornering test was analyzed using the laterality index (right turns-left turns)/10.

Example 6 early use of Low dose MPC-n (IVIg) inhibits complement C3 deposition, glial activation, and induces a protective phenotype in microglia

Following ischemic injury, neuronal cell death leads directly to inflammation, characterized by induction of the complement system, activation of focal glial cells and infiltration of peripheral immune cells, leading to damage to the brain parenchyma and vasculature. High dose IVIg treatment may inhibit stroke-induced elevation of C3 levels during ischemic stroke. To validate the therapeutic effect of low doses of MPC-n (IVIg) on complement inhibition, MCAO mice were injected with solvent (vehicle), free IVIg (500 and 100mg/kg) or MPC-n (IVIg) (100mg/kg) 2 hours after reperfusion, and 3 days later brain tissues were taken for immunofluorescent double-stained neurons (NeuN) and complement C3 (FIG. 6 a). The reduced levels of C3 in the MPC-n (IVIg) -treated group compared to the free IVIg-treated group indicates that administration of a low dose of MPC-n (IVIg) (100mg/kg) is effective in reducing stroke-the induction of C3 during early ischemia-reperfusion. In addition, the MCAO model was treated 2 hours after reperfusion. Brain tissue was obtained 3d after treatment to analyze inflammation. Immunofluorescence studies were performed to investigate the relative levels of GFAP, Iba-1 and CD206 (marker for M2) in the ischemic penumbra with different treatment methods. MPC-n (IVIg) treated mice showed lower GFAP + astrocyte and Iba1+ microglial densities (100 and 500mg/kg, FIGS. 6b-c) compared to free IVIg. MPC-n (IVIg) increased CD206 in ischemic penumbra compared to using free IVIg (100 and 500mg/kg, FIG. 6c)⁺The abundance of microglia. The above results indicate that early administration of low doses of MPC-n (ivig) enhances neuroprotection by inhibiting deposition of C3, inhibiting activation of glial cells (astrocytes and microglia), reducing inflammatory responses and inducing a protective phenotype in microglia.

In fig. 6, a) immunofluorescence revealed the expression of C3 in the ischemic penumbra. Staining with neuronal nuclear markers NeuN and C3 indicated co-localization. Scale bar 20 μm. The grey scale value is displayed in a line graph; b) fluorescence images of astrocytes in the ischemic penumbra 3d after injection; c) fluorescence images of microglia (Iba-1) and M2(CD206) in the ischemic penumbra 3d after injection. Scale bar 20 μm.

The applicant declares that the above description is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be understood by those skilled in the art that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are within the scope and disclosure of the present invention.

Claims

1. a nano-capsule for encapsulating intravenous immunoglobulin, characterized in that: the nano-capsule comprises intravenous immunoglobulin, N-(3-aminopropyl) methacrylamide, 2-methylpropene Acyloxyethylphosphorylcholine and cross-linking agent, the intravenous immunoglobulin, N-(3-aminopropyl)methacrylamide, 2-methacryloyloxyethylphosphorylcholine The molar ratio with the crosslinking agent is 1:(100-10000):(300-30000):(50-5000).

2 . The nanocapsule of claim 1 , wherein the cross-linking agent adopts ethylene glycol methyl dimethacrylate. 3 .

3. a kind of preparation method of the described nanometer capsule of claim 1 or 2, it is characterized in that: comprise the following steps:

a. Weigh the prescribed amount of intravenous immunoglobulin, N-(3-aminopropyl) methacrylamide, 2-methacryloyloxyethylphosphorylcholine and cross-linking agent and ammonium persulfate and N,N,N',N'-tetramethylethylenediamine, react at 4°C for 2 hours;

b. The solution obtained in step a is dialyzed with PBS buffer to remove unreacted monomers and by-products;

c. Pass the solution obtained after the dialysis in step b through a hydrophobic interaction column to remove any unencapsulated protein to obtain nanocapsules encapsulated with intravenous immunoglobulin.

4. The method for preparing a nanocapsule encapsulating intravenous immunoglobulin according to claim 3, wherein in step a, the molar ratio of ammonium persulfate and intravenous immunoglobulin is 500:1.

5. the preparation method of the nano-capsule of a kind of package intravenous immunoglobulin according to claim 3 or 4, is characterized in that: in step a, N,N,N',N'-tetramethyl ethylenedi The mass ratio of amine to ammonium persulfate was 2:1.

6 . The method for preparing a nanocapsule for intravenous injection of immunoglobulin according to claim 3 , wherein in step b, the pH of the PBS buffer is 7.4. 7 .

7. A use of the nanocapsules of claim 1 or 2 encapsulating intravenously injected immunoglobulins in the preparation of a medicine for treating ischemic stroke.