CN112516304B - Nanocapsule wrapping intravenous immunoglobulin, preparation method and application of nanocapsule in preparation of medicine for treating cerebral arterial thrombosis - Google Patents
Nanocapsule wrapping intravenous immunoglobulin, preparation method and application of nanocapsule in preparation of medicine for treating cerebral arterial thrombosis Download PDFInfo
- Publication number
- CN112516304B CN112516304B CN202011590701.7A CN202011590701A CN112516304B CN 112516304 B CN112516304 B CN 112516304B CN 202011590701 A CN202011590701 A CN 202011590701A CN 112516304 B CN112516304 B CN 112516304B
- Authority
- CN
- China
- Prior art keywords
- ivig
- mpc
- intravenous immunoglobulin
- nanocapsule
- preparation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/5005—Wall or coating material
- A61K9/5015—Organic compounds, e.g. fats, sugars
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Dermatology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Urology & Nephrology (AREA)
- Vascular Medicine (AREA)
- Cardiology (AREA)
- Heart & Thoracic Surgery (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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 dosage is injected for intravenous injection, so that the further damage of brain caused by stroke brain inflammation can be relieved, the damage of nerve function is reduced, the prognosis of a patient is improved, the side effect and the treatment cost of high-dosage use of a medicine of the patient can be reduced, and the nano microcapsule has huge conversion prospect and conversion value.
Description
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 damage 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 resolution of inflammation, 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 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 the usual complications that follow 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 (> 98% human immunoglobulin G (IgG)) from human plasma, so-called intravenous immunoglobulin or IVIg, has been approved by the U.S. food and drug administration for use in high doses for the treatment of various inflammatory and autoimmune diseases, such as kawasaki disease, immune thrombocytopenia, humoral immunodeficiency and bone marrow transplantation, for example. Furthermore, IVIg shows great potential in the treatment of ischemic stroke by directly targeting the immune system as well as neuronal cells at high doses. However, patients in the high dose group are more prone to problems such as thromboembolic events and skin reactions 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. and (c) passing the solution obtained after dialysis in the step B through a hydrophobic interaction column (Phenyl-Sepharose CL-4B) to remove any unencapsulated protein, so as to 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 nanocapsule 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 injection of MPC-n (IVIg) with lower dose (one fifth of the lowest effective dose reported at present) can relieve the further damage of stroke brain area inflammation to the brain, reduce the damage of nerve function and improve the prognosis of patients, and simultaneously can reduce the side effect and treatment cost of high-dose drug use of patients, thereby having huge transformation prospect and transformation value.
Drawings
FIG. 1 is a graph showing the test results of 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 test results of example 4 of the present invention;
FIG. 5 is a graph showing the test results of 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 acidic 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 proteins were 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 in the upper layer of the separation gel (FIG. 1 d), 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 release of IVIg 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% at pH 6.5) within the first 24 hours. These findings indicate that encapsulation of IVIg enhances its stability and achieves controlled release.
In FIG. 1, a) a schematic of the synthesis of MPC-n (IVIg); b) Schematic representation and delivery process of MPC-n (IVIg), which enhances IVIg targeting to ischemic brain areas 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 were released from MPC-n (IVIg) in phosphate buffered saline at different pH's (6.5 and 7.4).
Example 2 MPC-n (IVIg) accumulation in large amounts 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, investigating the duration and ability of MPC-n (IVIg) to target an ischemic hemisphere in mice. 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) (10 mg/kg body weight) in the ischemic hemisphere of the MCAO model was evaluated using an In Vivo Imaging System (IVIS). Compared to free IVIg and n (IVIg) (MPC-free nanocapsules), MPC-n (IVIg) showed stronger fluorescence in the MCAO model 2h after injection and remained as long as 24h (fig. 2 a). The accumulation of MPC-n (IVIg) is promoted by ischemic stroke-induced injury. 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 harvested. After administration of MPC-n (IVIg) in MCAO mice, the ipsilateral hemisphere had higher Cy5 fluorescence intensity, indicating selective accumulation of MPC-n (IVIg) in the ischemic hemisphere (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 measured quantitatively using ELISA (fig. 2 c). MPC-n (IVIg) dosing in the left hemisphere showed 3.22-fold and 5.17-fold higher. The accumulation in both 2h and 24h post injection, MCAO, models was higher than that in sham (sham) models. 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 after 2, 4 and 24h in vivo Cy5 fluorescence whole animal imaging of free IVIg, n (IVIg) or MPC-n (IVIg) (10 mg/kg) intravenously in MCAO mice 2h after post-ischemia reperfusion. The histogram summarizes the relative fluorescence intensity of brain tissue; b) Representative ex vivo Cy5 fluorescence images of isolated brain tissue from mice treated in a) at 2 and 24 hours post 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 Selective targeting of MPC-n (IVIg) to ChT1 overexpression in endothelial cells following ischemic regiodependent 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, 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 by which MPC-n (IVIg) is selectively targeted to ischemic regions remains to be studied, as direct access to ischemic regions by MPC-n (IVIg) through the BBB is not the primary pathway to the brain. It has previously been shown that BBB permeability of MPC nanocapsules containing choline and acetylcholine analogs is due to ChT 1-mediated endocytosis. ChT1 mRNA levels in bEND.3 cells after OGD treatment were increased in vitro. However, chT1 expression in bned.3 cells gradually decreased with increasing reperfusion time (fig. 3 a). Next, coronal sections from the MCAO model were used for immunofluorescence at different durations of ischemia reperfusion. ChT 1-positive cells were elevated in the ischemic region of the MCAO group compared to the sham group. Using the vascular endothelial cell marker (CD 31), it was further demonstrated that ChT1 is highly expressed in endothelial cells after ischemic stroke. However, prolonged reperfusion gradually reduced ChT1 expression 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 (hemicholenium-3. 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 the transport of Cy 5-labeled MPC-n (IVIg) into the brain. In addition, ex vivo imaging of brain collected from mice 24h after injection, fluorescence enhancement of mice 24h after MPC-n (IVIg) administration in MCAO model compared to sham model and MCAO model with pre-intraperitoneal injection of 10. Mu.g/kg HC- (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 to localize ChT1 and CD31 on the surface of the endothelial cells of MCAO mice. The histogram summarizes the mean fluorescence intensity of ChT 1; c) Cy 5-fluorescent in vivo C57 mice were imaged 4 hours after intravenous injection of MPC-n (IVIg) (10 mg/kg) in 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 isolated brain tissue from mice treated differently (sham + MPC-n (IVIg), MCAO + MPC-n (IVIg), and MCAO + HC-3 (10 μ/kg) + MPC-n (IVIg)) 24 hours after injection. The histograms summarize the relative fluorescence intensity of ex vivo brain tissue.
Example 4 early administration of MPC-n (IVIg) promotes the accumulation of IVIg 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 membrane 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 oxyful 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 were highly consistent with immunofluorescence (FIG. 4 b). Incubation of bEND.3 cells with MPC-n (IVIg) in the OGD/R0 h group was 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 reperfusion than after 24h (fig. 4 e). These data confirm that early intervention promotes cellular uptake of MPC-n (IVIg), which may be attributed 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 cells of bEND.3 took up MPC-n (IVIg) (0.5 mg/mL) after 12h reoxygenation of OGD 0 or after 12h incubation with MPC-n (IVIg). Histograms represent fluorescence intensity of MPC-n (IVIg) in bned.3 cells using flow cytometry, scale bar =20 μm; c) In vivo C57 mice were imaged for Cy5 fluorescence 2 or 24 hours after ischemia-reperfusion after 2 or 24 hours intravenous injection of MPC-n (IVIg) (10 mg/kg) 2, 4 or 24 hours; 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 post-injection, scale bar =20 μm.
Example 5 Low dose MPC-n (IVIg) enhances neuroprotective effects and reduces 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 the recovery of function, a therapeutic window (2 hour dosing after reperfusion) 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 (100 mg/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) (100 mg/kg) treatment showed neurological deficit and reduction in infarct volume after 3 days (FIGS. 5 a-b). Treatment with MPC-n (IVIg) (100 mg/kg) reduced TUNEL positive apoptotic cells in the ischemic penumbra (FIGS. 5 c-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) (100 mg/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 or 72 hours after injection of solvent (vehicle) (same volume) in mice 2h after ischemia reperfusion, free IVIg (500 and 100 mg/kg) or MPC-n (IVIg) (100 mg/kg); b) Representative images of brain TTC staining 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 the infarct volume of MPC-n (IVIg) (100 mg/kg) over the hemisphere compared to the solvent (vehicle) and free IVIg (500 and 100 mg/kg) controls; c) Fluorescence of TUNEL in 3d ischemic penumbra after 2h injection of solvent (vehicle) (same volume) after ischemia reperfusion, free IVIg (500 and 100 mg/kg) or MPC-n (IVIg) (100 mg/kg), scale bar =20 μm; d) Histogram quantification TUNEL analysis (n = 3); e) The turn test was analyzed using the laterality index (number of right turns-number of left turns)/10.
Example 6 early use of Low dose MPC-n (IVIg) inhibits complement C3 deposition, glial activation, and induces protective phenotype of 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, resulting in damage to the brain parenchyma and vasculature. High dose IVIg treatment can 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 either solvent (vehicle), free IVIg (500 and 100 mg/kg) or MPC-n (IVIg) (100 mg/kg) 2 hours after reperfusion and 3 days later brain tissue was taken for immunofluorescent double-stained neurons (NeuN) and complement C3 (FIG. 6 a). The C3 levels were reduced in the MPC-n (IVIg) treated group compared to the free IVIg treated group, indicating that administration of a low dose of MPC-n (IVIg) (100 mg/kg) was effective in reducing stroke-C3 induction during early ischemia reperfusion. In addition, the MCAO model was treated 2 hours after reperfusion. 3d after treatmentBrain tissue was obtained to analyze inflammation. Immunofluorescence studies were performed to investigate the relative levels of GFAP, iba-1 and CD206 (M2 marker) in ischemic penumbra with different treatment methods. MPC-n (IVIg) treated mice showed lower GFAP + astrocyte and Iba1+ microglia densities (100 and 500mg/kg, FIGS. 6 b-c) compared to free IVIg. MPC-n (IVIg) increased CD206 in ischemic penumbra compared to using free IVIg (100 and 500mg/kg, FIG. 6 c) + Microglial cell abundance. The above results indicate that early administration of low doses of MPC-n (IVIg) enhances neuroprotection by inhibiting C3 deposition, 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 gray 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 (CD 206) 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 (3)
1. An application of a nano microcapsule wrapping intravenous immunoglobulin in preparing a medicine for treating cerebral arterial thrombosis is characterized in that: the 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. b, dialyzing the solution obtained in the step a by using a PBS buffer solution, and removing unreacted monomers and byproducts;
c. b, passing the solution obtained after dialysis in the step b through a hydrophobic interaction column to remove any unencapsulated protein, thereby obtaining nanocapsules wrapping intravenous immunoglobulin;
the nanocapsule comprises intravenous immunoglobulin, N- (3-aminopropyl) methacrylamide, 2-methacryloyloxyethyl phosphorylcholine and a cross-linking agent, wherein the molar ratio of the intravenous immunoglobulin, the N- (3-aminopropyl) methacrylamide, the 2-methacryloyloxyethyl phosphorylcholine and the cross-linking agent is 1:1000:3000:500.
2. the use of the nanocapsule encapsulating intravenous immunoglobulin according to claim 1 for the preparation of a medicament for treating cerebral arterial thrombosis, wherein: in the step a, the molar ratio of ammonium persulfate to intravenous immunoglobulin is 500: the mass ratio of 1, N' -tetramethylethylenediamine to ammonium persulfate is 2:1.
3. the use of the nanocapsule encapsulating intravenous immunoglobulin according to claim 1 or 2 for the preparation of a medicament for treating cerebral arterial thrombosis, characterized in that: the cross-linking agent adopts ethylene glycol dimethyl methacrylate.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202011590701.7A CN112516304B (en) | 2020-12-29 | 2020-12-29 | Nanocapsule wrapping intravenous immunoglobulin, preparation method and application of nanocapsule in preparation of medicine for treating cerebral arterial thrombosis |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202011590701.7A CN112516304B (en) | 2020-12-29 | 2020-12-29 | Nanocapsule wrapping intravenous immunoglobulin, preparation method and application of nanocapsule in preparation of medicine for treating cerebral arterial thrombosis |
Publications (2)
Publication Number | Publication Date |
---|---|
CN112516304A CN112516304A (en) | 2021-03-19 |
CN112516304B true CN112516304B (en) | 2023-02-03 |
Family
ID=74976855
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202011590701.7A Active CN112516304B (en) | 2020-12-29 | 2020-12-29 | Nanocapsule wrapping intravenous immunoglobulin, preparation method and application of nanocapsule in preparation of medicine for treating cerebral arterial thrombosis |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN112516304B (en) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN116920116A (en) * | 2022-04-14 | 2023-10-24 | 上海巴久巴生物技术有限公司 | Catalase-encapsulated nanocapsule, preparation method and application thereof |
CN115554272B (en) * | 2022-09-28 | 2024-03-22 | 昆明理工大学 | Preparation method and application of nano particles |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10722475B2 (en) * | 2014-11-26 | 2020-07-28 | The Regents Of The University Of California | Stealth nanocapsules for protein delivery |
US10345302B2 (en) * | 2017-02-19 | 2019-07-09 | Sheng-He Huang | Circulating astrocytes and MFSD2A as biomarkers |
CN108159394B (en) * | 2018-02-07 | 2020-04-03 | 天津医科大学总医院 | Application of PR-957 in preparing medicine for treating cerebral ischemia reperfusion injury |
-
2020
- 2020-12-29 CN CN202011590701.7A patent/CN112516304B/en active Active
Also Published As
Publication number | Publication date |
---|---|
CN112516304A (en) | 2021-03-19 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Xiao et al. | Dihydrolipoic acid–gold nanoclusters regulate microglial polarization and have the potential to alter neurogenesis | |
Papa et al. | Selective nanovector mediated treatment of activated proinflammatory microglia/macrophages in spinal cord injury | |
Lu et al. | Targeted therapy of brain ischaemia using Fas ligand antibody conjugated PEG-lipid nanoparticles | |
Wang et al. | Combinational protective therapy for spinal cord injury medicated by sialic acid-driven and polyethylene glycol based micelles | |
CN112516304B (en) | Nanocapsule wrapping intravenous immunoglobulin, preparation method and application of nanocapsule in preparation of medicine for treating cerebral arterial thrombosis | |
Zhan et al. | The three-phase enriched environment paradigm promotes neurovascular restorative and prevents learning impairment after ischemic stroke in rats | |
Fu et al. | Value of functionalized superparamagnetic iron oxide nanoparticles in the diagnosis and treatment of acute temporal lobe epilepsy on MRI | |
WO2016025745A1 (en) | Dendrimer compositions and use in treatment of neurological and cns disorders | |
Huang et al. | The effect of lipid nanoparticle PEGylation on neuroinflammatory response in mouse brain | |
WO2012061695A1 (en) | Compositions and methods for the treatment of traumatic brain injury | |
Chen et al. | Surface-fill H2S-releasing silk fibroin hydrogel for brain repair through the repression of neuronal pyroptosis | |
Chen et al. | Etanercept, an inhibitor of TNF-a, prevents propofol-induced neurotoxicity in the developing brain | |
Hu et al. | Tetramethylpyrazine facilitates functional recovery after spinal cord injury by inhibiting MMP2, MMP9, and vascular endothelial cell apoptosis | |
Mu et al. | Ligustrazine nanoparticle hitchhiking on neutrophils for enhanced therapy of cerebral ischemia‐reperfusion injury | |
Fang et al. | Antineuroinflammatory and neurotrophic effects of CNTF and C16 peptide in an acute experimental autoimmune encephalomyelitis rat model | |
KR102015197B1 (en) | The use of dextran sulfate having an average molecular weight below 10000 da for inducing angiogenisis in a subject | |
Wang et al. | Nano-integrated cascade antioxidases opsonized by albumin bypass the blood–brain barrier for treatment of ischemia-reperfusion injury | |
Morales-Zavala et al. | In vivo micro computed tomography detection and decrease in amyloid load by using multifunctionalized gold nanorods: a neurotheranostic platform for Alzheimer's disease | |
US20120148488A1 (en) | Targeting Kidney Mesangium With Nanoparticles of Defined Diameter | |
Zhu et al. | C-176 loaded Ce DNase nanoparticles synergistically inhibit the cGAS-STING pathway for ischemic stroke treatment | |
Fleming et al. | Timing and duration of anti–α4β1 integrin treatment after spinal cord injury: effect on therapeutic efficacy | |
US20230233705A1 (en) | Gold nanoparticle-containing medicine | |
Mu et al. | Neutrophil Targeting Platform Reduces Neutrophil Extracellular Traps for Improved Traumatic Brain Injury and Stroke Theranostics | |
Zhang et al. | Functional nanoassemblies for the diagnosis and therapy of Alzheimer's diseases | |
EP3508214A1 (en) | Therapeutic agent for ischemic diseases |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |