NA1-albumin nanoparticles as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to NA1-albumin nanoparticles as well as a preparation method and application thereof. According to the preparation method disclosed by the invention, NA1 is encapsulated inside and on the surface of albumin nanoparticles through electrostatic interaction between Nerinetide (NA1) and the albumin nanoparticles, so that NA1-albumin nanoparticles are obtained. The encapsulation efficiency of the NA1-albumin nanoparticles to NA1 can reach 80-90%, and the NA1-albumin nanoparticles have the characteristics of natural degradation and pH responsive degradation. The albumin nanoparticles are swallowed by immune cells through complement conditioning, are delivered by using methods such as skull marrow or intracerebral injection, bypass a blood-brain barrier by using a brain marginal immune pathway, improve the delivery efficiency and targeting of NA1 in the brain, have significant effects on reducing the volume of a stroke ischemic region, the volume of an ischemic core region, encephaledema and encephalatrophy, and are suitable for clinical application. The pharmaceutical composition can be used for treating acute brain injury, especially acute ischemic cerebral apoplexy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to NA1-albumin nanoparticles, their preparation method, and applications. Background Technology

[0002] The blood-brain barrier refers to the barrier between blood plasma and brain cells formed by the walls of brain capillaries and glial cells, and the barrier between blood plasma and cerebrospinal fluid formed by the choroid plexus. These barriers can prevent viruses, bacteria and other toxins from entering brain tissue from the blood, but they also block the delivery of most drugs.

[0003] Nerinetide (NA1) is a neuroprotective agent that interferes with postsynaptic density protein 95 (PSD-95), which causes excitotoxic cell death during acute ischemia. NA1 inhibits signal transduction leading to neuroexcitotoxicity and has been shown to reduce stroke injury in preclinical ischemic stroke models (mice, rats, and primates), especially in cases of rapid reperfusion after ischemia. NA1 has demonstrated significant efficacy in a macaque middle cerebral artery ischemia-reperfusion model. However, due to the blood-brain barrier and the rapid metabolic clearance from peripheral blood, effective delivery of NA1 to the central nervous system remains a significant challenge. The difficulty in crossing the blood-brain barrier or resisting rapid metabolic clearance from peripheral blood hinders the in vivo and clinical application of NA1. Summary of the Invention

[0004] The purpose of this invention is to provide NA1-albumin nanoparticles, their preparation method, and applications, to achieve high-concentration Nerinetide delivery in the brain, thereby improving Nerinetide delivery efficiency and targeting.

[0005] The present invention provides NA1-albumin nanoparticles, comprising Nerinetide and albumin, wherein the Nerinetide is encapsulated within and on the surface of the albumin-formed nanoparticle structure.

[0006] Preferably, the albumin includes one or more of human serum albumin, recombinant human serum albumin, and bovine serum albumin.

[0007] This invention provides a method for preparing NA1-albumin nanoparticles as described above, comprising the following steps: (1) Under stirring conditions, albumin solution, organic solvent and cross-linking agent are mixed. After stirring, the organic solvent, unreacted albumin and cross-linking agent are removed to obtain albumin nanoparticles. (2) Under stirring conditions, the Nerinetide solution and the albumin nanoparticle solution are mixed. After stirring, the unencapsulated Nerinetide is removed to obtain NA1-albumin nanoparticles.

[0008] Preferably, the organic solvent includes a water-soluble organic solvent; the crosslinking agent includes one or more of glutaraldehyde, paraformaldehyde, and genipin.

[0009] Preferably, the volume ratio of the albumin solution to the organic solvent is 1:1 to 5; the solvent of the albumin solution includes water, physiological saline, or PBS buffer; and the concentration of albumin in the albumin solution is 5 to 30 mg / ml. The total volume ratio of the albumin solution and organic solvent to the crosslinking agent is 100:0.5~2.

[0010] Preferably, the solvent of the Nerinetide solution includes water, physiological saline, or PBS buffer; the concentration of Nerinetide in the Nerinetide solution is 0.1~10 mg / ml; The volume ratio of the Nerinetide solution to the albumin nanoparticle solution is 1:2~10; The solvent of the albumin nanoparticle solution includes water, physiological saline or PBS buffer; the concentration of albumin nanoparticles in the albumin nanoparticle solution is 2~20 mg / ml.

[0011] Preferably, in both (1) and (2), the stirring speed is 500~2000 rpm; In (1), under stirring conditions, albumin solution and organic solvent are mixed and stirred for 0.5-1 h, and then mixed with crosslinking agent and stirred for 2-10 h. In step (2), the stirring time is 1 to 4 hours.

[0012] This invention provides the application of NA1-albumin nanoparticles as described in the above technical solution or NA1-albumin nanoparticles obtained by the preparation method described in the above technical solution in the preparation of drugs for treating acute brain injury.

[0013] Preferably, the acute brain injury includes acute ischemic stroke.

[0014] Preferably, the drug includes one or more of the following: intracranial bone marrow injection drug, intracerebral injection drug, and intravenous injection drug.

[0015] Beneficial effects: This invention provides NA1-albumin nanoparticles, comprising nerinetide and albumin, wherein the nerinetide is encapsulated within and on the surface of the albumin-formed nanoparticle structure. The NA1-albumin nanoparticles of this invention encapsulate nerinetide within and on the surface of the albumin nanoparticles through electrostatic interaction between the nerinetide and the albumin nanoparticles, achieving an encapsulation rate of 80-90%, and exhibiting both natural degradation and pH-responsive degradation characteristics. The albumin nanoparticles are phagocytosed by immune cells via complement opsonization and delivered via methods such as cranial bone marrow injection or intracerebral injection. Utilizing the limbic immune pathway to bypass the blood-brain barrier, they improve the delivery efficiency and targeting of nerinetide in the brain, demonstrating significant effects in reducing the volume of the ischemic area, the ischemic core, cerebral edema, and cerebral atrophy in stroke patients. They can be used for the treatment of acute brain injury, especially acute ischemic stroke.

[0016] Furthermore, this invention adjusts the particle size and surface potential of albumin nanoparticles by regulating parameters such as albumin concentration, organic phase ratio, stirring speed, and stirring time; it adjusts the degradation time of albumin nanoparticles by adjusting the ratio of crosslinking agent; and it adjusts the drug loading and encapsulation efficiency of NA1-albumin nanoparticles by adjusting the concentration of the Nerinetide phase, the dissolving medium, the mixing ratio with albumin nanoparticles, and the stirring time. Results show that the particle size of the NA1-albumin nanoparticles obtained by this invention can be adjusted within the range of 100~1000 μm, the monodispersity coefficient is less than 0.2, the surface potential can be adjusted between -30~-10 mV, the particle size and surface potential stability reaches more than 14 days, the encapsulation efficiency can reach 80~90%, the drug loading can be adjusted between 0~30%, and it exhibits both natural degradation and pH-responsive degradation characteristics. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0018] Figure 1 The albumin nanoparticles were characterized by scanning electron microscopy (A) and dynamic light scattering (B). Figure 2 FITC-albumin nanoparticles and Gr-1 + Confocal images of cells; Figure 3 Flow cytometry results (A) and statistical results (B) show the in vivo targeting efficiency of albumin nanoparticles on bone marrow cells and subsets in untreated and LPS-treated mice; among them, express P <0.05, expressP <0.01; Figure 4 Representative TEM images of mouse skull bone marrow cells after ICO injection with saline and albumin nanoparticles; Figure 5 Representative scanning electron microscope images of NA1-albumin nanoparticles; Figure 6 The particle size and surface potential of albumin nanoparticles with different NA1 loadings; Figure 7 Encapsulation efficiency of albumin nanoparticles with different NA1 loadings; Figure 8 The therapeutic effects of NA1-albumin nanoparticles on acute ischemic stroke are shown below. A represents the timeline of the treatment evaluation study; B represents the results of TTC staining sections; C represents the statistical results of infarct volume 24 hours after stroke induction; D represents representative T2-weighted images from 3 to 28 days after stroke induction; E-G represent the statistical results of representative infarct volume, infarct core, and cerebral edema 3 days after stroke induction; H represents changes in brain volume after stroke induction; I represents representative images of the brains of mice treated differently 35 days after stroke induction; J represents Nissl staining sections of the brains of mice treated differently 35 days after stroke induction; K represents a representative DTI axial view of the FA image acquired 28 days after stroke induction, with yellow dots representing the corpus callosum; I represents the survival curves of MCAO mice under different treatments; M and N represent sensorimotor function assessed by neurological deficit assessment and the rotating rod test; O and P represent cognitive function assessed by the Morris water maze test and objective recognition test. express P <0.05, express P <0.01; express P <0.001; Figure 9 The dose-dependency, timing, and efficacy of ICO-NA1 NPs and IV-NA1 NPs are presented. A shows representative images of stained mouse brain sections, indicating the stroke-induced infarct area; B and C show the infarct area and neurological score 24 hours after stroke; D and E show the results of administration at 4.5 h and 6 h after stroke in MCAO mice; F shows the infarct area shown by TTC staining 72 h after stroke; G and H show the infarct volume and cerebral edema 72 h after stroke induction; I shows the brain volume changes after stroke induction; and J shows the survival curves of MCAO mice under different treatments. express P <0.05, expressP <0.01; express P <0.001, # indicates P <0.05; Figure 10 The drug weight ratio (A), brain-to-liver ratio (B), brain-to-spleen ratio (C), brain-to-lung ratio (D), and brain-to-kidney ratio (E) in the ipsilateral and contralateral cerebral hemispheres were measured 24 hours after stroke onset, depending on the route of administration. Detailed Implementation

[0019] The present invention provides an NA1-albumin nanoparticle comprising NA1 and albumin, wherein the NA1 is encapsulated within and on the surface of the albumin-formed nanoparticle structure.

[0020] In one embodiment, the albumin described in this invention includes one or more of human serum albumin, recombinant human serum albumin, and bovine serum albumin. This invention utilizes albumin nanoparticles because albumin can be phagocytosed by immune cells through complement opsonization, and immune cells can rapidly internalize albumin nanoparticles after injection into the skull and bone marrow. This invention encapsulates NA1 within and on the surface of the albumin nanoparticles through electrostatic interaction between NA1 and the nanoparticles.

[0021] This invention provides a method for preparing NA1-albumin nanoparticles as described above, comprising the following steps: (1) Under stirring conditions, albumin solution, organic solvent and cross-linking agent are mixed. After stirring, the organic solvent, unreacted albumin and cross-linking agent are removed to obtain albumin nanoparticles. (2) Under stirring conditions, the NA1 solution and the albumin nanoparticle solution are mixed. After stirring, the unencapsulated NA1 is removed to obtain NA1-albumin nanoparticles.

[0022] In this invention, albumin solution, organic solvent and cross-linking agent are mixed under stirring conditions. After stirring, the organic solvent, unreacted albumin and cross-linking agent are removed to obtain albumin nanoparticles.

[0023] In one embodiment, the stirring speed of the present invention is 500~2000 rpm; in another embodiment, the stirring speed of the present invention is 800~1500 rpm; in yet another embodiment, the stirring speed of the present invention is 1000~1200 rpm. The present invention's limitation of the stirring speed has the advantage of adjusting the morphology, particle size, and distribution width of nanoparticles. In one embodiment, the present invention, under stirring conditions, mixes albumin solution and organic solvent, stirs for 0.5~1 h, and then mixes with a crosslinking agent, stirring for 2~10 h; in another embodiment, the present invention, under stirring conditions, mixes albumin solution and organic solvent, stirs for 1 h, and then mixes with a crosslinking agent, stirring for 10 h. The present invention's method of mixing albumin solution and organic solvent for a certain period of time before mixing with a crosslinking agent has the advantage of stabilizing the morphology, particle size, and distribution width of nanoparticles.

[0024] In one embodiment, the solvent of the albumin solution of the present invention includes water, physiological saline, or PBS buffer. In one embodiment, the concentration of albumin in the albumin solution of the present invention is 5-30 mg / ml; in another embodiment, the concentration of albumin in the albumin solution of the present invention is 10-25 mg / ml; in yet another embodiment, the concentration of albumin in the albumin solution of the present invention is 15-20 mg / ml. In one embodiment, the volume ratio of the albumin solution to the organic solvent of the present invention is 1:1-5; in another embodiment, the volume ratio of the albumin solution to the organic solvent of the present invention is 1:2-4; in yet another embodiment, the volume ratio of the albumin solution to the organic solvent of the present invention is 1:3. In one embodiment, the organic solvent of the present invention includes a water-soluble organic solvent. In one embodiment, the water-soluble organic solvent of the present invention includes anhydrous ethanol and / or dimethyl sulfoxide. The present invention utilizes water-soluble organic solvents, which, compared with other organic solvents, have the advantages of good water solubility, good biocompatibility, and easy water washing.

[0025] As one embodiment, the crosslinking agent of this invention includes one or more of glutaraldehyde, paraformaldehyde, and genipin. Compared with other crosslinking agents, this invention uses glutaraldehyde, paraformaldehyde, and genipin as crosslinking agents, which has advantages such as a clear crosslinking principle, small molecular weight, high crosslinking efficiency, easy water washing and removal, and good biocompatibility.

[0026] In one embodiment, the volume ratio of the total volume of the albumin solution and organic solvent to the crosslinking agent is 100:0.5~2; in another embodiment, the volume ratio of the total volume of the albumin solution and organic solvent to the crosslinking agent is 100:0.6~1.8; in another embodiment, the volume ratio of the total volume of the albumin solution and organic solvent to the crosslinking agent is 100:0.7~1.5; in another embodiment, the volume ratio of the total volume of the albumin solution and organic solvent to the crosslinking agent is 100:0.8~1.2; in yet another embodiment, the volume ratio of the total volume of the albumin solution and organic solvent to the crosslinking agent is 100:1.0.

[0027] After obtaining the albumin nanoparticles, the present invention mixes the NA1 solution and the albumin nanoparticle solution under stirring conditions. After stirring, the unencapsulated NA1 is removed to obtain NA1-albumin nanoparticles.

[0028] In one embodiment, the stirring speed of the present invention is 500~2000 rpm; in another embodiment, the stirring speed of the present invention is 800~1500 rpm; in yet another embodiment, the stirring speed of the present invention is 1000~1200 rpm. In one embodiment, the stirring time of the present invention is 1~4 h; in yet another embodiment, the stirring time of the present invention is 2~3 h. The present invention's limitation of stirring time and speed has the advantage of regulating the drug loading process and stabilizing drug encapsulation efficiency.

[0029] In one embodiment, the solvent of the NA1 solution of the present invention includes water, physiological saline, or PBS buffer. In one embodiment, the concentration of NA1 in the NA1 solution of the present invention is 0.1~10 mg / ml; in another embodiment, the concentration of NA1 in the NA1 solution of the present invention is 0.2~8 mg / ml; in another embodiment, the concentration of NA1 in the NA1 solution of the present invention is 0.4~5 mg / ml; in another embodiment, the concentration of NA1 in the NA1 solution of the present invention is 0.5~3 mg / ml; in another embodiment, the concentration of NA1 in the NA1 solution of the present invention is 0.6~2 mg / ml; in another embodiment, the concentration of NA1 in the NA1 solution of the present invention is 0.8~1 mg / ml. In one embodiment, the volume ratio of the NA1 solution to the albumin nanoparticle solution of the present invention is 1:2 to 10; in another embodiment, the volume ratio is 1:3 to 8; in yet another embodiment, the volume ratio is 1:4 to 6; and in yet another embodiment, the volume ratio is 1:5. In one embodiment, the solvent for the albumin nanoparticle solution of the present invention includes water, physiological saline, or PBS buffer. In one embodiment, the concentration of albumin nanoparticles in the albumin nanoparticle solution of the present invention is 2 to 20 mg / ml; in yet another embodiment, the concentration is 3 to 18 mg / ml; in yet another embodiment, the concentration is 5 to 15 mg / ml; and in yet another embodiment, the concentration is 10 to 12 mg / ml.

[0030] This invention provides the application of NA1-albumin nanoparticles as described in the above technical solution or NA1-albumin nanoparticles obtained by the preparation method described in the above technical solution in the preparation of drugs for treating acute brain injury.

[0031] In one embodiment, the acute brain injury described in this invention includes acute ischemic stroke. In another embodiment, the drug described in this invention includes intracranial injection drugs and / or intracerebral injection drugs.

[0032] The NA1-albumin nanoparticles provided by this invention can be administered via intracranial or intracerebral injection, bypassing the blood-brain barrier through the limbic immune pathway to deliver NA1 to the brain, thereby improving the delivery efficiency and targeting of NA1 in the brain. They have significant effects in reducing the volume of the ischemic area, the volume of the ischemic core, cerebral edema, and brain atrophy in stroke, and can be used for the treatment of acute brain injury, especially acute ischemic stroke.

[0033] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes an NA1-albumin nanoparticle, its preparation method, and its application, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0034] Example 1 Preparation of albumin nanoparticles Under magnetic stirring (1000 rpm), 7.5 mL of anhydrous ethanol was continuously added to 3 mL of a 10 mg / mL bovine serum albumin solution. After stirring for 1 h, 80 μL of 2.5% glutaraldehyde was added for cross-linking fixation. After stirring for 10 h, anhydrous ethanol, unreacted bovine serum albumin, and glutaraldehyde were removed by centrifugation and washing with water. The solution was then reconstituted with physiological saline to a concentration of 5 mg / mL to obtain an albumin nanoparticle solution.

[0035] Example 2 Preparation of albumin nanoparticles Under magnetic stirring (8000 rpm), 15 mL of anhydrous ethanol was continuously added to 5 mL of a 10 mg / mL bovine serum albumin solution. After stirring for 2 h, 200 μL of 5% genipin solution was added for cross-linking fixation. After stirring for 12 h, the organic solvent, unreacted bovine serum albumin, and cross-linking agent were removed by centrifugation and washing with water. The solution was then reconstituted with physiological saline to 10 mg / mL to obtain an albumin nanoparticle solution.

[0036] Example 3 Preparation of albumin nanoparticles Under magnetic stirring (500 rpm), 15 mL of dimethyl sulfoxide was continuously added to 3 mL of a 20 mg / mL bovine serum albumin solution. After stirring for 3 h, 100 μL of 4% paraformaldehyde was added for cross-linking fixation. After stirring for 1 h, the organic solvent, unreacted bovine serum albumin, and cross-linking agent were removed by centrifugation and washing with water. The solution was then reconstituted with physiological saline to 10 mg / mL to obtain an albumin nanoparticle solution.

[0037] Test Example 1 1. The morphology of the albumin nanoparticles (NP) obtained in Example 1 was observed using a scanning electron microscope (Hitachi, SU8600), and the particle size distribution was determined using a dynamic light scattering instrument (Malvern). The results showed that the obtained albumin nanoparticles had a uniform diameter and good stability. Figure 1 ).

[0038] 2. To confirm the phagocytic activity of albumin nanoparticles by cranial tectal bone marrow cells, the albumin nanoparticles obtained in Example 1 were transplanted with fluorescein isothiocyanate (FITC), and characterized using confocal microscopy, flow cytometry, and transmission electron microscopy (TEM). Co-culture with cranial and tibial bone marrow cells in vitro showed an absorption efficiency of up to 90%. In vivo, 2 h after intracranial injection (ICO), confocal imaging revealed the interaction between FITC-NP and Gr-1. + Clear colocalization of cells ( Figure 2 This confirmed the phagocytic activity of calcaneal medullary cells. Flow cytometry further quantified the in vivo phagocytic activity of myeloid cell subsets, with FITC+ neutrophils and Ly6Chi monocytes accounting for 29.7% and 49.4%, respectively. Figure 3 In contrast, intravenous (IV) administration resulted in FITC labeling of 12.6% of peripheral neutrophils and 8.3% of peripheral Ly6Chi monocytes, further supporting the improved targeting efficiency of the ICO pathway.

[0039] 3. To investigate whether central nervous system perturbation enhances NP uptake, a lipopolysaccharide (LPS)-induced neuroinflammation model was used for verification. In this model, mice received intraperitoneal injections of LPS at a dose of 5 mg / kg body weight, prepared in a sterile, non-pyrogenic 0.9% saline solution. The injection volume was standardized to 10 μL per gram of body weight. For the control group, an equal volume of sterile saline solution was injected intraperitoneally. One hour after LPS or saline injection, fluorescently labeled FITC-NP was injected intracranially, and the calcaneal bone was collected 2 hours later. For brain tissue analysis, mice were sacrificed 24 hours after LPS or saline injection, and their brains without meninges were dissected, isolated, and stained for flow cytometry analysis. The results showed that, compared with the blank control group, flow cytometry analysis revealed a significant increase in the subset of calcaneal medullary cells co-stained with NP in the neuroinflammation group, and FITC-NP was also significantly increased. + Neutrophils and Ly6Chi monocytes increased to 70.1% and 76.1%, respectively. Figure 3 The proportion of FITC-positive neutrophils showed a more significant increase than that of Ly6Chi monocytes, which may be attributed to their late maturation phenotype and rapid immune response. Furthermore, TEM images clearly demonstrated the presence of a large number of NP-loaded cells in the bone marrow. Figure 4 No decline in the viability of bone marrow cells was observed after in vitro co-culture or in vivo injection of albumin nanoparticles, indicating that albumin nanoparticles have good biocompatibility.

[0040] Example 5 Preparation of NA1-albumin nanoparticles Under magnetic stirring (800 rpm), 1 mL of 2 mg / ml NA1 was continuously added to 10 mL of the albumin nanoparticle solution obtained in Example 1 to make the final concentration of NA1 7%. After stirring for 1 h, the unencapsulated NA1 was removed by centrifugation and washing with water. The solution was then reconstituted with physiological saline to 20 mg / ml to obtain the NA1-albumin nanoparticle solution for later use.

[0041] Example 6 Preparation of NA1-albumin nanoparticles Under magnetic stirring (800 rpm), 2 mL of 2 mg / ml NA1 was continuously added to 10 mL of the albumin nanoparticle solution obtained in Example 1 to make the final concentration of NA1 10.5%. After stirring for 1 h, the unencapsulated NA1 was removed by centrifugation and washing with water. The solution was then reconstituted with physiological saline to 20 mg / ml to obtain the NA1-albumin nanoparticle solution for later use.

[0042] Example 7 Preparation of NA1-albumin nanoparticles Under magnetic stirring (800 rpm), 3 mL of 2 mg / ml NA1 was continuously added to 10 mL of the albumin nanoparticle solution obtained in Example 1 to make the final concentration of NA1 14%. After stirring for 1 h, the unencapsulated NA1 was removed by centrifugation and washing with water. The solution was then reconstituted with physiological saline to 20 mg / ml to obtain the NA1-albumin nanoparticle solution for later use.

[0043] Example 8 Preparation of NA1-albumin nanoparticles Under magnetic stirring (800 rpm), 2 mL of 4 mg / ml NA1 was continuously added to 10 mL of the albumin nanoparticle solution obtained in Example 1 to make the final concentration of NA1 17.5%. After stirring for 1 h, the unencapsulated NA1 was removed by centrifugation and washing with water. The solution was then reconstituted with physiological saline to 20 mg / ml to obtain the NA1-albumin nanoparticle solution for later use.

[0044] Test Example 2 1. The morphology of the NA1-albumin nanoparticles obtained in Example 5 was observed using a scanning electron microscope (Hitachi, SU8600). The results showed that NA1 was surface-loaded onto the albumin nanoparticles through electrostatic interactions between its positive charge and the negative charge of the albumin nanoparticles. Figure 5 ).

[0045] 2. The size and surface zeta potential of the albumin nanoparticles obtained in Example 1 and Examples 5-8 were determined using a dynamic light scattering (Malvern) instrument. The results showed that the albumin nanoparticles loaded with different concentrations of NA1 exhibited minimal size change and stable surface potential (>-20 mV), indicating good colloidal stability of the obtained NA1-albumin nanoparticle solutions. Figure 6 ).

[0046] 3. The encapsulation efficiency of NA1 was determined by high-performance liquid chromatography (HPLC) (Agilent) to measure the concentration of free NA1 in the supernatant solutions of NA1-albumin nanoparticles obtained in Examples 5-8. The results showed that the encapsulation efficiency at each NA1 concentration exceeded 85%. Figure 7 ).

[0047] Application Example 1 according to Figure 8 The procedure shown in A evaluates the therapeutic effect and dose-response relationship of NA1-albumin nanoparticles administered via ICO injection. Specifically: 1. Ischemic Stroke Induction: Experimental stroke was induced using a filament-based middle cerebral artery occlusion (MCAO) model for transient and focal cerebral ischemia. Under anesthesia (isoflurane), a silicon-coated 6 / 0 filament was inserted into the external carotid artery (ECA) of mice until it occluded the MCA. The external carotid artery was then ligated with an occlusion suture 1 h prior to reperfusion. Sham-operated mice underwent the same procedure but without filament insertion. Administration was performed immediately after ischemia-reperfusion. As a positive control, NA1 was administered intravenously in mice at an effective dose of 10 nmol / g. Furthermore, to compare the cell-mediated delivery strategy with direct ICO injection, free NA1 was administered at an optimized dose equivalent to half the intravenous dose. For the NA1 NPs group, the NA1-albumin nanoparticles (NA1-NPs) obtained in Example 6 were administered at a dose adjusted to 1 / 30 to 1 / 3.75 of the intravenous dose.

[0048] 2. Twenty-four hours after MCAO or sham surgery onset, the brains of mice without meninges were dissected and stained with TTC. Results showed that, compared to placebo, mice treated with NA1 or NA1-NPs exhibited a significant reduction in infarct volume, at least 25.6%. All groups receiving NA1-NPs via ICO injection showed a greater reduction in infarct volume compared to intravenous NA1 (IV-NA1), with the highest efficacy observed in the group receiving NA1-NPs at 1 / 15th of the intravenous dose, showing an additional 26.7% reduction in infarct size and improved neurological function. Figure 8 B and C in the middle, Figure 9 (Among A, B, and C). Therefore, subsequent studies selected a 1 / 15 dose regimen.

[0049] 3. To assess the therapeutic time window, NA1-NPs were administered at delayed time points of 4.5 and 6 hours post-stroke. Results showed that while ICO delivery of NA1-NPs at 6 hours did not provide a therapeutic benefit, administration at 4.5 hours still produced a significant neuroprotective effect. In contrast, intravenous NA1 did not achieve significant efficacy at either delayed time point, likely due to limited drug crossing of the blood-brain barrier and insufficient accumulation at the ischemic site. These findings highlight the advantage of the ICO route in maintaining efficacy within the clinically relevant therapeutic window. Figure 9 (D and E in the middle).

[0050] 4. For long-term evaluation, continuous MRI analysis and behavioral observation were performed for up to 28 days. Results showed that 24-hour TTC staining revealed comparable infarct volumes between NA1-NPs administered via ICO injection and free NA1. However, by 72 hours, MRI and TTC staining showed a significantly smaller ischemic area in the ICO-NA1 NP group compared to all other treatments. The ICO group received only 1 / 15th the NA1 dose of the IV group. Despite the lower dose, animals in the ICO-NA1 NPs group exhibited significantly smaller infarct volumes compared to the IV-NA1 NPs group, as shown by TTC staining. Figure 9 (Middle F). MRI analysis confirmed these findings, showing a significant reduction in infarct volume and perilesional edema as early as day 3, confirming a more effective neuroprotective response. Figure 9 (G and H). These advantages lasted for up to 28 days, with the ICO group showing a sustained reduction in secondary tissue loss and improved survival compared to animals treated with intravenous injection. Figure 9 I and J). Apparent diffusion coefficient (ADC) imaging further demonstrated that, regardless of the route of administration, ICO-administered NA1 NPs were superior to free NA1 in relieving the infarct core. Figure 8(Middle F). Furthermore, compared to other groups, ICO-NA1 NP significantly reduced stroke-induced cerebral edema ( Figure 8 The D and G pathways highlighted their multifaceted neuroprotective capabilities in the acute phase. In contrast, while IV-NA1 and ICO-NA1 were effective within 24 hours, they failed to provide these protections within 72 hours, which may reflect the transient nature of free NA1 and the fact that NPs are not released continuously. To assess this, NA1 concentrations in the brain were quantified by liquid chromatography-tandem mass spectrometry (LC-MS / MS) 24 hours after stroke induction. The results showed that NA1-NPs delivered via the ICO pathway achieved the highest NA1 concentrations in the brain, and even at significantly lower doses, the accumulation in the ipsilateral hemisphere was significantly higher than that in the contralateral hemisphere. Figure 10 (A) This confirms the efficiency and targeting of NA1-NPs delivered by ICO. Furthermore, the brain-organ ratios, especially the brain-spleen, brain-lung, and brain-kidney ratios, were significantly higher in the ICO-NA1 NPs treatment group than in other treatment groups. Figure 10 (B~E). These beneficial effects persisted during a follow-up imaging observation period of up to 28 days, collectively highlighting their durable efficacy in improving ischemic stroke. Figure 8 (D).

[0051] 5. To assess the long-term neuroprotective effect against tissue damage, brain atrophy and white matter integrity were evaluated 28 days after stroke induction. Specifically, brain atrophy was quantified by measuring the reduction in tissue volume on T2-weighted images at 14, 21, and 28 days after stroke induction. Data showed that ICO administration of NA1-NPs was more effective in alleviating brain atrophy than intravenous or intracranial administration of free NA1, demonstrating its long-term histological neuroprotective effect. Figure 8 In mice injected intravenously or intracranially with NA1 35 days after stroke induction, the brains of mice showed severe brain atrophy on the affected side, similar to the placebo group. Delivery of NA1 NPs with ICO significantly reduced stroke-induced brain atrophy, and in some cases even demonstrated brain integrity indistinguishable from healthy controls. Figure 8 I and J). Diffusion tensor imaging (DTI) further revealed that, compared to the damaged structures seen in the saline or free NA1 groups, ICO-NA1 NPs preserved the integrity of the corpus callosum, maintaining continuous FA signals and fiber bundles ( Figure 8 35 days after stroke induction, MCAO mice treated with NA1 NPs showed improved survival compared to MCAO mice treated with intravenous or direct ICO injection of free NA1. Figure 8 (L).

[0052] 6. Neurological assessments were performed using the Neurological Severity Scale (NSS) and behavioral evaluations (including the rotating rod test, water maze test, and target recognition test) throughout the 28-day observation period. Animals treated with ICO-NA1 NPs showed improved NSS scores in the initial 24 hours after ischemic attack and throughout the entire 28-day period. Figure 8 (M). During the continuous observation period, this group also performed significantly better than other groups in the rotating rod task (M). Figure 8 NA1 NPs administered via the skull significantly improved the performance of mice in the water maze and object recognition tasks. Improvements in learning, memory, and object discrimination abilities indicated a significant and comprehensive improvement in cognitive abilities. Figure 8 (O and P).

[0053] As can be seen from the above, the NA1-albumin nanoparticles provided by this invention have significant effects on reducing the volume of the ischemic area, the volume of the ischemic core area, cerebral edema, and cerebral atrophy in stroke. They can be used for the treatment of acute ischemic stroke and acute brain injury. Compared with intravenous injection, ICO shows a sustained reduction in secondary tissue loss and an improved survival rate, resulting in better treatment effects.

[0054] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A type of NA1-albumin nanoparticle, characterized in that, It includes Nerinetide and albumin, wherein the Nerinetide is encapsulated inside and on the surface of the albumin-formed nanoparticle structure.

2. The NA1-albumin nanoparticles according to claim 1, characterized in that, The albumin includes one or more of human serum albumin, recombinant human serum albumin, and bovine serum albumin.

3. The method for preparing NA1-albumin nanoparticles according to claim 1 or 2, characterized in that, Includes the following steps: (1) Under stirring conditions, albumin solution, organic solvent and cross-linking agent are mixed. After stirring, the organic solvent, unreacted albumin and cross-linking agent are removed to obtain albumin nanoparticles. (2) Under stirring conditions, the Nerinetide solution and the albumin nanoparticle solution are mixed. After stirring, the unencapsulated Nerinetide is removed to obtain NA1-albumin nanoparticles.

4. The preparation method according to claim 3, characterized in that, The organic solvent includes water-soluble organic solvents; the crosslinking agent includes one or more of glutaraldehyde, paraformaldehyde, and genipin.

5. The preparation method according to claim 3 or 4, characterized in that, The volume ratio of the albumin solution to the organic solvent is 1:1 to 5; the solvent of the albumin solution includes water, physiological saline, or PBS buffer; the concentration of albumin in the albumin solution is 5 to 30 mg / ml. The total volume ratio of the albumin solution and organic solvent to the crosslinking agent is 100:0.5~2.

6. The preparation method according to claim 3, characterized in that, The solvent of the Nerinetide solution includes water, physiological saline, or PBS buffer; the concentration of Nerinetide in the Nerinetide solution is 0.1~10 mg / ml; The volume ratio of the Nerinetide solution to the albumin nanoparticle solution is 1:2~10; The solvent of the albumin nanoparticle solution includes water, physiological saline or PBS buffer; the concentration of albumin nanoparticles in the albumin nanoparticle solution is 2~20 mg / ml.

7. The preparation method according to claim 3, characterized in that, In both (1) and (2), the stirring speed is 500~2000 rpm; In (1), under stirring conditions, albumin solution and organic solvent are mixed and stirred for 0.5-1 h, and then mixed with crosslinking agent and stirred for 2-10 h. In step (2), the stirring time is 1 to 4 hours.

8. The use of the NA1-albumin nanoparticles according to claim 1 or 2 or the NA1-albumin nanoparticles obtained by the preparation method according to any one of claims 3 to 7 in the preparation of a drug for treating acute brain injury.

9. The application according to claim 8, characterized in that, The acute brain injury includes acute ischemic stroke.

10. The application according to claim 8 or 9, characterized in that, The drug includes one or more of the following: intracranial bone marrow injection drugs, intracerebral injection drugs, and intravenous injection drugs.

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