Fingolimod-loaded nanoparticles and application thereof in treatment of cerebral hemorrhage

Abstract

The invention relates to the technical field of biological medicine, and discloses fingolimod-loaded nanoparticles and a preparation method thereof, and the preparation method comprises the following steps: synthesizing CeO2 nanoparticles through a high-temperature organic solution phase method; the preparation method comprises the following steps: adding CeO2 nanoparticles into polyethylene glycol to obtain PEG-CeO2 nanoparticles, and modifying the surfaces of the CeO2 nanoparticles by the polyethylene glycol to form a coating layer; the fingolimod and the PEG-CeO2 nano particles are mixed, and the fingolimod is attached to the surface of the coating layer, so that CeO2-FTY 720 nano particles are prepared; the invention also discloses application of the medicine in treating cerebral hemorrhage. According to the present invention, the dual characteristics of CeO2 nanoparticles and fingolimod are inherited, the use of the single component is reduced, the characteristics such as antioxidant activity and the like are superior to the characteristics of the single component and the simple superposition of the components, and the functional synergistic interaction is achieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to fingolimod-loaded nanoparticles and applications thereof in treating cerebral hemorrhage. Background Art

[0002] Intracerebral hemorrhage (ICH), a subtype of hemorrhagic stroke, is triggered by intraparenchymal vascular rupture. ICH-induced white matter injury (WMI) manifests as oligodendrocyte damage and demyelination, leading to long-term neurological dysfunction. Following ICH surgery, residual small hematomas that cannot be removed and perihematomal edema (PHE) can lead to secondary WMI. Neuroinflammation plays a key role in secondary WMI, and these pathophysiological processes, such as neuroinflammation, cannot be blocked surgically. Therefore, the development of novel drugs to promote myelination and alleviate neuroinflammation is particularly important.

[0003] Fingolimod (FTY720), a potent sphingosine-1-phosphate (S1P) receptor agonist, can alleviate WMI in ischemic stroke models by regulating the transition of microglia from the M1 to the M2 phenotype. Small clinical trials have shown that oral fingolimod has a certain therapeutic effect in patients with acute stroke, including ICH and ischemic stroke.

[0004] However, the drug has the following defects: 1. The drug's delivery is poor, and high doses are required to improve its efficacy. High doses lead to adverse reactions, such as cardiovascular problems and increased risk of infection; 2. The drug's therapeutic effect is poor, and patients recover slowly.

[0005] Therefore, how to improve the therapeutic effect of FTY720 is an urgent problem to be solved in its clinical application in the treatment of ICH. Summary of the Invention

[0006] The purpose of the present invention is to provide a nanoparticle loaded with fingolimod and its application in treating cerebral hemorrhage, so as to solve the technical problem of how to better apply the drug fingolimod in the prior art.

[0007] In order to solve the above technical problems, the present invention specifically provides the following technical solutions:

[0008] The present invention provides a method for preparing fingolimod-loaded nanoparticles, comprising the following steps:

[0009] CeO2 nanoparticles were synthesized by a high-temperature organic solution phase method;

[0010] adding the CeO2 nanoparticles to polyethylene glycol to obtain PEG-CeO2 nanoparticles, wherein the polyethylene glycol modifies the surface of the CeO2 nanoparticles to form a coating layer;

[0011] Fingolimod is mixed with the PEG-CeO2 nanoparticles, and fingolimod is attached to the surface of the coating layer to prepare CeO2-FTY 720 nanoparticles.

[0012] As a preferred embodiment of the present invention, the high temperature organic solution phase method comprises the following steps:

[0013] In a nitrogen environment, oleylamine is added to octadecene to obtain a mixed solvent;

[0014] Cerium acetate hydrate is added to the mixed solvent to obtain cerium acetylacetonate hydrate, stirred evenly, heated at 280° C. for one hour, and filtered to obtain the CeO 2 nanoparticles.

[0015] The present invention provides fingolimod-loaded nanoparticles, comprising fingolimod and PEG-CeO2 nanoparticles; the concentration ratio of the fingolimod to the PEG-CeO2 nanoparticles is 1:2.91.

[0016] As a preferred embodiment of the present invention, in the CeO2-FTY 720 nanoparticles, Ce 3+ With Ce 4+ The ratio is 1:1.

[0017] The present invention provides use of fingolimod-loaded nanoparticles in treating cerebral hemorrhage.

[0018] As a preferred embodiment of the present invention, the CeO2-FTY720 nanoparticles are used in the preparation of a drug for treating secondary white matter damage caused by cerebral hemorrhage.

[0019] As a preferred embodiment of the present invention, the drug further comprises a pharmaceutically acceptable carrier.

[0020] As a preferred embodiment of the present invention, the dosage form of the drug is an injection.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) The CeO2-FTY720 nanoparticles prepared by the present invention inherit the dual properties of CeO2 nanoparticles and fingolimod, reducing the use of a single component, thereby reducing the side effects caused by the high-dose use of a single drug;

[0023] (2) The CeO2-FTY 720 nanoparticles disclosed in the present invention have achieved functional synergy based on their dual characteristics. First, CeO2 nanoparticles can deliver FTY 720 in a targeted manner, achieving controlled release of FTY 720 with minimal side effects, allowing for sustained treatment. Second, the loading of FTY 720 improves the CeO2 nanoparticles. 3+ and Ce 4+ The ratio of 20mg / ml to 10mg / ml makes its antioxidant activity better than that of a single ingredient, thus enhancing its strong antioxidant capacity;

[0024] (3) The present invention discloses the mechanism of CeO2-FTY720 nanoparticles in improving long-term neurological damage after ICH, which can be widely applied to the relief and treatment of diseases related to oligodendrocyte death, demyelination, neuroinflammation, and ROS;

[0025] (4) In the preparation method disclosed in the present invention, the surface of CeO2 is modified by polyethylene glycol, so that it can be combined with fingolimod-type drugs to achieve efficient drug loading; at the same time, the biocompatibility of the nano-loaded particles is enhanced, the water suspension stability of the nanoparticles is improved, the solubility is good, and it is not easy to aggregate. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the embodiments or the description of the prior art. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other implementation drawings based on the provided drawings without inventive effort.

[0027] Figure 1 The present invention provides a schematic flow chart of a method for preparing fingolimod-loaded nanoparticles;

[0028] Figure 2 Transmission electron microscopy (TEM) images of CeO2-FTY 720 nanoparticles are provided for the present invention;

[0029] Figure 3 Provided for the present invention are X-ray diffraction (XRD) patterns of CeO2 and CeO2-FTY 720;

[0030] Figure 4 The present invention provides a kinetic statistical graph of the release of FTY 720 from CeO2-FTY 720 in PBS within 48 hours;

[0031] Figure 5 The present invention provides an X-ray photoelectron spectrum of CeO2-FTY 720;

[0032] Figure 6 Provided is a graph showing the superoxide dismutase (SOD) activity assay of CeO2-FTY 720;

[0033] Figure 7 Provided are statistical graphs of the clearance of ABTS+· by ascorbic acid (AA), FTY 720, CeO2, and CeO2-FTY 720 at different concentrations;

[0034] Figure 8 Provided for the present invention are statistical graphs showing the clearance of TMB+OH· by ascorbic acid (AA), FTY 720, CeO2, and CeO2-FTY 720 at different concentrations;

[0035] Figure 9 Provided are statistical graphs of the time-dependent analysis of the ABTS+ scavenging ability of ascorbic acid (AA), FTY 720, CeO2, and CeO2-FTY 720;

[0036] Figure 10 The present invention provides a statistical graph of the time-dependent analysis of the TMB+OH scavenging ability of ascorbic acid (AA), FTY 720, CeO2 and CeO2-FTY 720;

[0037] Figure 11 The present invention provides a comparison chart of hemolysis of CeO2-FTY 720 in water and at different concentrations;

[0038] Figure 12 Provides a graphical representation of the solubility of CeO2-FTY 720 in different aqueous environments;

[0039] Figure 13 Provided for the present invention are internalization and transport markers of Cy7-labeled CeO2-FTY 720 in BV2 cells;

[0040] Figure 14 Provides statistical graphs of DCF levels in cells of different groups for the present invention;

[0041] Figure 15 Provides statistical graphs of the M2 / M1 ratios in different groups for the present invention;

[0042] Figure 16 Provided is a schematic diagram of CeO2-FTY 720 regulating microglial polarization in BV2 cells;

[0043] Figure 17 The present invention provides representative images of hematoma in a cerebral hemorrhage model after treatment with different concentrations of CeO2-FTY 720;

[0044] Figure 18 The present invention provides a statistical analysis of the hematoma volume of a cerebral hemorrhage model after treatment with different concentrations of CeO2-FTY 720;

[0045] Figure 19 The present invention provides an electron microscopic image of CeO2-FTY 720 endocytosis in mouse brain tissue 24 hours after cerebral hemorrhage;

[0046] Figure 20 The present invention provides fluorescence distribution diagrams of normal and cerebral hemorrhage mice after intravenous injection of Cy7-labeled CeO2-FTY 720 at 0, 6, 12 and 24 hours;

[0047] Figure 21 Provided for the present invention are statistical graphs of relative fluorescence intensity of normal and cerebral hemorrhage brain tissues at 6, 12 and 24 hours;

[0048] Figure 22 Provides statistical graphs of water content in each group of brain tissues for dry / wet weight ratio assessment in the present invention;

[0049] Figure 23 Provide representative immunofluorescence images of colocalization of Iba1 and CD16 / 32 for the present invention;

[0050] Figure 24 Provide representative immunofluorescence images of colocalization of Iba1 and CD206 for the present invention;

[0051] Figure 25 The present invention provides transmission electron microscopy images of mitochondrial ultrastructure in the brain tissue of each group of mice;

[0052] Figure 26 Provided are representative images of FITC-dextran (40 kDa) leakage in perihematomal vessels of mice with intracerebral hemorrhage;

[0053] Figure 27 The present invention provides a diagram of the internalization and transport of Cy7-labeled CeO2-FTY 720 in OLN-93 cells;

[0054] Figure 28 Provided herein are representative immunofluorescence images of the colocalization of Ki-67, NG2, and Dapi in OLN-93 cells;

[0055] Figure 29 The present invention provides a statistical analysis graph of Ki67+ / NG2+;

[0056] Figure 30 The present invention provides a 24-hour analysis statistical graph of CNPase in OLN-93 after each group of experiments;

[0057] Figure 31The present invention provides a 24-hour analysis statistical graph of MBP in OLN-93 cells after each group of experiments;

[0058] Figure 32 The present invention provides a comparison of the LFB staining results of the perihematoma area in each group 7 days after cerebral hemorrhage;

[0059] Figure 33 Provides a statistical analysis result graph of the G-ratio in each experimental group of the present invention;

[0060] Figure 34 Provides a statistical analysis result graph of damaged axons in each experimental group for the present invention;

[0061] Figure 35 The present invention provides Western blot analysis and quantitative evaluation statistical graphs of SMI 32 in the perihematoma area in each experimental group;

[0062] Figure 36 The present invention provides Western blot analysis and quantitative evaluation statistical graphs of NF 200 in the perihematoma area in each experimental group;

[0063] Figure 37 The present invention provides Western blot analysis and quantitative evaluation statistical graphs of MBP in the perihematoma area in each experimental group;

[0064] Figure 38 Provided are statistical graphs of the statistical analysis results of the rotarod test (blue for the sham-operated group, red for the blank-treated group, green for the FTY 720-treated group, purple for the CeO2 nanoparticle-treated group, orange for CeO2+FTY 720, and black for the CeO2-FTY 720 nanoparticle-treated group);

[0065] Figure 39 Provided are statistical graphs of the statistical analysis results of the staggered step test (blue for the sham operation group, red for the blank treatment group, green for the FTY 720 treatment group, purple for the CeO2 nanoparticle treatment group, orange for CeO2+FTY 720, and black for the CeO2-FTY 720 nanoparticle treatment group);

[0066] Figure 40 Provided are statistical analysis results of the sticker removal experiment of the present invention (blue for the sham operation group, red for the blank treatment group, green for the FTY 720 treatment group, purple for the CeO2 nanoparticle treatment group, orange for CeO2+FTY 720, and black for the CeO2-FTY 720 nanoparticle treatment group);

[0067] Figure 41The present invention provides a statistical analysis of the escape latency of each group on days 21-25 after cerebral hemorrhage (blue is the sham operation group, red is the blank treatment group, green is the FTY 720 treatment group, purple is the CeO2 nanoparticle treatment group, orange is CeO2+FTY 720, and black is the CeO2-FTY 720 nanoparticle treatment group). DETAILED DESCRIPTION

[0068] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0069] like Figure 1 As shown, the present invention provides a method for preparing fingolimod-loaded nanoparticles, comprising the following steps:

[0070] (1) CeO2 nanoparticles were synthesized by a high-temperature organic solution phase method using cerium acetate hydrate as a precursor and a mixed solvent system consisting of oleylamine (OM) and octadecene (ODE);

[0071] (2) mixing CeO2 nanoparticles with polyethylene glycol, wherein the polyethylene glycol modifies the surface of the CeO2 nanoparticles to form a coating layer to prepare PEG-CeO2 nanoparticles;

[0072] (3) Fingolimod is mixed with PEG-CeO2 nanoparticles, and fingolimod is attached to the surface of the coating layer to prepare CeO2-FTY 720 nanoparticles.

[0073] The high temperature organic solution phase method includes the following steps:

[0074] In a nitrogen environment, oleylamine is added to octadecene to obtain a mixed solvent;

[0075] Cerium acetate hydrate was added to the mixed solvent to obtain cerium acetylacetonate hydrate, which was stirred evenly, heated at 280° C. for one hour, and filtered to obtain CeO 2 nanoparticles.

[0076] Finally, FTY 720 was successfully loaded onto PEG-modified CeO2 nanoparticles to construct CeO2-FTY 720 nanoparticles.

[0077] CeO2 nanoparticles have excellent strong antioxidant capacity and can scavenge reactive oxygen species (ROS). They can effectively reduce neuroinflammation in the ICH mouse model. CeO2 nanoparticles can also alleviate WMI caused by ICH by promoting the maturation of oligodendrocyte precursor cells (OPCs) and myelin regeneration. Combining CeO2 nanoparticles with FTY720 can reduce the dosage of FTY720, reduce the side effects of FTY720, and improve the therapeutic effect on secondary white matter damage caused by cerebral hemorrhage.

[0078] This invention discloses a method for combining CeO2 nanoparticles with FTY720. Furthermore, the invention utilizes a high-temperature synthesis method to prepare the particles, followed by polyethylene glycol (PEG) modification, to achieve efficient loading of FTY720, resulting in CeO2-FTY720 nanoparticles. These CeO2-FTY720 nanoparticles possess the functionality of CeO2 nanoparticles and are capable of sustained release of FTY720 in solution.

[0079] CeO2 nanoparticles are a highly promising nanocarrier. As a polymeric nanoparticle, CeO2 possesses drug delivery capabilities after ICH. Under the action of CeO2 nanoparticles, FTY720 is delivered to the blood-brain barrier (BBB) in a targeted manner. The nanoparticles successfully cross the BBB and achieve controlled release of FTY720 in the brain region surrounding the hematoma. This allows for targeted delivery of FTY720, enhancing its therapeutic effects.

[0080] The antioxidant capacity of CeO2 nanoparticles comes from Ce 3+ and Ce 4+ The mutual conversion of electrons between CeO2-FTY 720 and CeO2-FTY 720 3+ With Ce 4+ The ratio of CeO2-FTY 720 will also change. 3+ With Ce 4+ The ratio of CeO2-FTY 720 nanoparticles is more uniform, and the antioxidant capacity of CeO2-FTY 720 nanoparticles is further enhanced. The antioxidant capacity is related to the removal of ROS. CeO2-FTY 720 nanoparticles can better promote the proliferation and differentiation of oligodendrocyte precursor cells (OPCs) and myelin regeneration, effectively reducing neuroinflammation.

[0081] It can be seen that the CeO2-FTY 720 nanoparticles prepared by the present invention inherit the dual characteristics of CeO2 nanoparticles and fingolimod. CeO2 nanoparticles and fingolimod can act on the secondary white matter damage of cerebral hemorrhage respectively, thereby improving the efficacy of the drug and reducing the use of a single component, thereby reducing the side effects caused by the large-dose use of a single drug; secondly, CeO2 nanoparticles achieve targeted delivery of FTY 720 and complete the controlled release of FTY 720, with little side effects and continuous treatment; further, the loading of FTY 720 improves the Ce content in CeO2 nanoparticles. 3+ and Ce 4+ The ratio of 20mg to 40mg is 20mg / 100mg, and its antioxidant activity is significantly better than that of a single ingredient, which improves the strong antioxidant capacity and achieves functional synergistic enhancement.

[0082] In the preparation method provided by the present invention, the surface of CeO2 is modified with polyethylene glycol (PEG), enabling it to be combined with drugs such as fingolimod to form integrated drug particles. This can also further enhance the biocompatibility of the nanoparticles, improve the water suspension stability of the nanoparticles, improve solubility, prevent aggregation, and enable uniform dispersion in solution. It can also be quickly dispersed into other drug carriers and is easily adaptable to various drug administration methods. At the same time, when the drug is released into the body environment, it is also easy to disperse quickly, allowing the active ingredients in the drug to be quickly dispersed into the body.

[0083] The characteristics of CeO2-FTY 720 nanoparticles are described below through examples and comparative examples:

[0084] Example 1: CeO2-FTY 720 group

[0085] In a nitrogen environment, oleylamine is added to octadecene to obtain a mixed solvent;

[0086] Adding cerium acetate hydrate to a mixed solvent to obtain cerium acetylacetonate hydrate, stirring evenly, heating at 280°C for one hour, and filtering to obtain CeO2 nanoparticles;

[0087] mixing CeO2 nanoparticles with polyethylene glycol, wherein the polyethylene glycol modifies the surface of the CeO2 nanoparticles to prepare PEG-CeO2 nanoparticles;

[0088] Fingolimod was mixed with PEG-CeO2 nanoparticles to prepare CeO2-FTY 720 nanoparticles.

[0089] (1) Physical representation

[0090] Figure 2Transmission electron microscopy (TEM) images of CeO2-FTY 720 nanoparticles. Figure 2 It can be seen that the CeO2-FTY720 nanoparticles synthesized in Example 1 present a uniform ultra-small nano-dot structure with an average diameter of about 5 nm.

[0091] (2) Structure

[0092] Figure 3 is the X-ray diffraction (XRD) pattern of CeO2 and CeO2-FTY 720 (Example 1), from Figure 3 As can be seen from the figure, all diffraction peaks of the nanoparticles correspond to the cubic crystal structure of CeO2. After loading with FTY 720, these peaks remain unchanged, indicating that CeO2-FTY720 has excellent stability.

[0093] (3) Composition

[0094] CeO2-FTY 720 was tested, and by analyzing the ultraviolet spectra of FTY 720 and CeO2 at different concentrations, the concentration ratio of FTY 720 to CeO2 was found to be 1:2.91.

[0095] (4) Sustained release performance

[0096] See also Figure 4 , Figure 4 Figure 3 is a kinetic statistical diagram of the release of FTY 720 from CeO2-FTY 720 in PBS within 48 hours. The figure shows that in the PBS environment, FTY 720 is rapidly released within the first 1.5 hours and then slowly released within the next 2 days.

[0097] It can be seen that compared with the direct use of FTY 720, the CeO2-modified nanoparticles can play the role of sustained drug release, allowing it to be released to the damaged area in a targeted and continuous manner.

[0098] (5) Antioxidant capacity

[0099] Figure 5 The X-ray photoelectron spectrum of CeO2-FTY 720 shows that Ce in CeO2-FTY 720 3+ With Ce 4+ The ratio of CeO2 is about 1:1, while the ratio of CeO2 is 1:1.26. 3+ and Ce 4+ The electrons between CeO2 and CeO2 can be converted into each other, so CeO2 nanoparticles have strong antioxidant capacity and can remove reactive oxygen species (ROS). 3+ With Ce 4+The ratio is more uniform, and in theory it has better antioxidant properties.

[0100] Figure 6 The results of superoxide dismutase (SOD) activity determination of CeO2-FTY 720 show that the SOD activity increased significantly with the increase of CeO2-FTY 720 concentration from 0.5 μg / mL to 4.0 μg / mL.

[0101] In order to more comprehensively illustrate the antioxidant properties of CeO2-FTY 720, the free radical scavenging ability of CeO2-FTY 720 at different concentrations and reaction times was characterized. Figures 7 to 10 As shown, Figures 7 to 10 This is the test of the free radical scavenging ability of CeO2, FTY 720 and CeO2-FTY720 at different concentrations and reaction times, and ascorbic acid (AA) was used as a positive control.

[0102] Figure 7 and Figure 8 The statistical graphs of ABTS+· and TMB+OH· scavenging by ascorbic acid (AA), FTY 720, CeO2 and CeO2-FTY 720 at different concentrations are shown; Figure 9 and Figure 10 Statistical graphs of the time-dependent analysis of the scavenging ability of ascorbic acid (AA), FTY 720, CeO2 and CeO2-FTY 720 on ABTS+· and TMB+OH·.

[0103] From the results, we can see that the free radical scavenging ability of FTY 720 itself is weak, and the free radical scavenging effect of CeO2-FTY 720 is slightly higher than that of CeO2, which is consistent with the above-mentioned CeO2-FTY 720. 3+ With Ce 4+ The proportional relationships can reflect each other.

[0104] These results indicate that CeO2-FTY 720 has excellent free radical scavenging ability and can be effectively used in the treatment of ROS-related diseases.

[0105] (4) Solubility

[0106] Figure 11 The figure shows the hemolysis comparison of CeO2-FTY 720 in water and at different concentrations. In the hemolysis experiment, the hemolysis rate of the H2O group was significantly higher than that of all CeO2-FTY 720 concentration groups, while the hemolysis rates of the latter were lower at concentrations of 50, 100 and 200 μg / mL, and there was no significant difference between the groups, indicating that CeO2-FTY 720 has excellent biocompatibility.

[0107] Figure 12The figure shows the solubility of CeO2-FTY 720 in different water environments (specifically H2O, phosphate buffered saline (PBS), 0.9% sodium chloride (NaCl) and Dulbecco's modified Eagle's medium). It can be seen from the figure that CeO2-FTY 720 exhibits excellent stability and does not aggregate for a long time.

[0108] The present invention further provides verification examples to verify the mechanism of action of CeO2-FTY 720 as a drug.

[0109] Verification Example 1: Alleviating neuroinflammation

[0110] To verify that Example 1 can alleviate neuroinflammation, the present invention illustrates that CeO2-FTY 720 alleviates neuroinflammation by regulating microglial polarization in two models: a BV2 microglial cell model (in vitro model) and an ICH (intracranial hemorrhage) model of C57BL / 6 mice.

[0111] (1) BV2 microglial cell model

[0112] ① Establish multiple experimental groups: experimental group 1 was the blank test group; experimental group 2 was the cerebral hemorrhage group (OxyHb stimulation, no subsequent intervention); experimental group 3 was the FTY 720 group; experimental group 4 was the CeO2 group; experimental group 5 was the CeO2+FTY 720 group; and experimental group 6 was the CeO2-FTY 720 group.

[0113] The concentrations of FTY 720, CeO2, and CeO2-FTY 720 were set at 4 μg / mL. The CeO2+FTY 720 group acted as an unbound drug, added immediately after the application of CeO2 (i.e., a simple combination). The concentrations of FTY 720 and CeO2 were influenced by the CeO2-FTY 720 adsorption ratio (1:1.26).

[0114] ② BV2 cells were stimulated with OxyHb. After OxyHb stimulation, the activity of BV2 cells was significantly reduced and ROS increased.

[0115] ③ The above experimental groups were combined with stimulated BV2 cells. LysoTracker, a green fluorescent probe that specifically labels lysosomes in BV2 cells, was used to verify the endocytosis of CeO2-FTY 720 nanoparticles through the endolysosomal system. Cy7 dye was attached to CeO2-FTY 720 for tracking (red), while Hoechst was used to label the cell nucleus (blue). The results are shown in Figure 3. Figure 13 , Figure 13 Figure 2 shows the internalization and trafficking of Cy7-labeled CeO2-FTY 720 in BV2 cells.

[0116] As shown in the figure, at 15 minutes, Cy7-labeled CeO2-FTY 720 nanoparticles (red) began to aggregate on the cell membrane and partially penetrated into lysosomes. After 30 minutes, the red fluorescence intensity gradually increased, indicating that sufficient CeO2 nanoparticles had entered the lysosomes. Subsequently, the red fluorescence strongly overlapped with the green fluorescence, indicating that BV2 cells had successfully taken up CeO2-FTY 720 through lysosome-mediated endocytosis.

[0117] ④ROS scavenging ability

[0118] After OxyHb stimulation, 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) probe was used to detect the ROS levels in cells of each group. The results are shown in Figure 14 , Figure 14 The following is a statistical graph showing DCF levels in cells from different groups. As can be seen, FTY 720 partially reduced ROS levels in BV2 cells. CeO2 and CeO2 + FTY 720 demonstrated superior ROS scavenging capabilities, effectively eliminating the majority of ROS.

[0119] However, compared with the CeO2+FTY 720 group, the CeO2-FTY 720 group exhibited stronger ROS scavenging ability. It can be seen that the CeO2-FTY 720 nanoparticles provided by the present invention have a strong advantage in antioxidant properties not only through combined use, but also through the combination, which improves the ion ratio distribution within the particles, thereby further improving the antioxidant capacity of the entire nanoparticles and achieving synergistic enhancement.

[0120] ⑤ Changes in cell polarity

[0121] After OxyHb stimulation, the classic M1 phenotype of BV2 cells was characterized by upregulation of CD16 / 32, while the alternative M2 phenotype was characterized by upregulation of CD206 (mannose receptor). Double labeling of Iba-1 with CD16 / 32 (M1 marker) and CD206 (M2 marker) was used to study the polarization of BV2 cells under OxyHb stimulation.

[0122] After 24 hours of OxyHb stimulation, the results were Figure 15 , Figure 15 The following is a statistical graph of the M2 / M1 ratio in different groups. As can be seen from the figure, FTY 720 can convert microglia from the M1 phenotype to the M2 phenotype. Furthermore, CeO2 treatment alone or CeO2 + FTY 720 significantly promoted M2 polarization. Among the CeO2 + FTY 720 and CeO2 - FTY 720 groups, CeO2 - FTY 720 had a more pronounced effect on promoting M2 polarization.

[0123] Figure 16 Schematic diagram of CeO2-FTY 720 regulating microglial polarization in BV2 cells.

[0124] Neuroinflammation occurs immediately after the formation of a hematoma in a patient's intracerebral hemorrhage, and is a key innate immune response to secondary brain injury after ICH. Microglia are resident macrophages in the central nervous system and act as key inflammatory cells after ICH. Normally, these microglia exist in a resting state, but they are activated after ICH. Activated microglia can be divided into two main phenotypes: the classic M1 phenotype and the alternative M2 phenotype. The M1 phenotype releases proinflammatory cytokines (TNF-α, IL-1β), chemokines, and ROS, significantly aggravating brain edema and blood-brain barrier (BBB) damage. The M2 phenotype exhibits anti-inflammatory properties by releasing anti-inflammatory factors (such as IL-4 and IL-10).

[0125] It can be seen that since CeO2-FTY 720 polarizes microglia to the alternative M2 phenotype and has a more significant effect on promoting M2 polarization compared with simple combination, guiding microglia polarization to an anti-inflammatory state has great potential in alleviating brain damage after ICH. After being used as a drug, it has the potential to significantly alleviate neuroinflammation.

[0126] (2) ICH (intracranial hemorrhage) model in C57BL / 6 mice

[0127] ① Group establishment: To further verify the anti-inflammatory properties of CeO2-FTY 720 in vivo, the ICH mouse model was established by stereotactic injection of 30 μL of autologous whole blood into the right basal ganglia, referring to the group establishment of the above model.

[0128] ②Administration method: To better simulate the clinical scenario of ICH patients, who are usually admitted to the hospital several hours after symptom onset, this validation case administered FTY 720, CeO2, CeO2+FTY 720, or CeO2-FTY720 by intravenous injection 6 hours after ICH surgery and administered once daily to ICH mice for 3 or 7 days.

[0129] ③Delivery mechanism of CeO2-FTY 720

[0130] Using the above-mentioned recent drug dose, the biodistribution of Cy7-labeled CeO2-FTY 720 in vivo was visualized using an imaging system. The results are shown in Figure 19 As shown, Figure 19 The electron micrograph of CeO2-FTY720 endocytosis in mouse brain tissue 24 hours after intracerebral hemorrhage is shown. Scale bar = 500 nm.

[0131] As can be seen from the figure, after ICH occurs, the blood-brain barrier (BBB) is destroyed, providing a therapeutic window for drug delivery. The nanoparticles successfully cross the BBB, and CeO2-FTY 720 is successfully delivered to the brain hematoma within 24 hours, and the controlled release of FTY 720 is achieved in the brain area around the hematoma.

[0132] ④Relationship between dosage and hematoma volume

[0133] Brain sections from different mouse groups were analyzed 3 days after ICH to evaluate the dose-dependent effect of CeO2-FTY 720 on hematoma volume. Figure 17 As shown in the statistical chart, see Figure 18 As shown, Figure 17 Representative images of hematoma in the cerebral hemorrhage model after treatment with different concentrations of CeO2-FTY 720. Figure 18 Statistical analysis of hematoma volume in cerebral hemorrhage model after treatment with different concentrations of CeO2-FTY 720.

[0134] The results showed that the concentration of CeO2-FTY 720 was significantly correlated with the reduction of hematoma volume, and that there was an optimal dosage, which in this validation case was 0.4 mg / kg. It is important to note that the CeO2-FTY 720 nanoparticles described above have only been used in mouse experiments and have not yet been validated in humans.

[0135] ⑤Relationship between drug administration time and hematoma volume

[0136] The biodistribution of Cy7-labeled CeO2-FTY 720 in vivo was visualized and analyzed using an imaging system. After intravenous tail injection, CeO2-FTY 720 entered the brain within 6 hours and preferentially accumulated in the tissue around the hematoma, where it could be retained for up to 24 hours.

[0137] Figure 20 Fluorescence distribution of normal and ICH mice at 0, 6, 12, and 24 hours after intravenous injection of Cy7-labeled CeO2-FTY 720. Figure 21 The relative fluorescence intensity of normal and ICH brain tissue at 6, 12, and 24 hours post-injection (n=3) is shown. As can be seen from the figure, compared with normal mice, the CeO2-FTY 720-treated group showed significant differences in brain tissue distribution at 6, 12, and 24 hours post-injection. CeO2-FTY 720 successfully delivered the most drug to the area surrounding the hematoma within 12 hours, demonstrating the best delivery effect.

[0138] ⑥CeO2-FTY 720 and cerebral edema

[0139] Figure 22The water content of brain tissue in each group was evaluated by dry / wet weight ratio. It can be seen from the figure that brain edema after ICH surgery will aggravate the injury. Compared with CeO2+FTY 720, intravenous injection of CeO2-FTY 720 can better reduce brain edema 3 days after ICH.

[0140] ⑦CeO2-FTY 720 and cell polarization

[0141] Immunofluorescence staining was used to assess the polarization status of microglia, where CD16 / 32 and CD206 represent the M1 and M2 phenotypes, respectively. Figure 23 Representative immunofluorescence images of Iba1 co-localization with CD16 / 32. Figure 24 This is a representative immunofluorescence image of co-localization of Iba1 and CD206. It can be seen from the figure that intravenous injection of CeO2-FTY 720 can significantly reduce M1 microglia and increase M2 microglia. CeO2-FTY 720 also has a higher level of anti-inflammatory factors.

[0142] ⑧CeO2-FTY 720 and mitochondrial damage

[0143] Normal neuronal mitochondria have regular and dense cristae structures, but ICH-induced perihematoma injury can lead to destruction of mitochondrial cristae structure and death. Figure 25 Transmission electron micrographs of mitochondrial ultrastructure in brain tissue from mice in each group show mitochondrial swelling, membrane damage, and reduced cristae 24 hours after ICH. Treatment with CeO₂+FTY 720 partially improved mitochondrial structure, while treatment with CeO₂-FTY 720 significantly protected mitochondrial integrity and function.

[0144] ⑨CeO2-FTY 720 and BBB damage

[0145] By co-staining with 40kDa FITC-dextran and vascular marker CD31, Figure 26 As shown, Figure 26 Representative images of FITC-dextran (40 kDa) leakage in perihematomal vessels in mice with intracerebral hemorrhage. Leakage of the fluorescent dye increased after ICH surgery, and FTY 720 or CeO2 treatment slightly improved it. The CeO2+FTY 720 group showed moderate improvement, while the CeO2-FTY 720 group significantly improved BBB disruption and repaired BBB damage.

[0146] Verification Example 1 demonstrates that CeO2-FTY 720 nanoparticles possess a stronger ability to scavenge ROS than CeO2, making them suitable for ROS-related diseases. Furthermore, compared to FTY 720, CeO2-FTY 720 nanoparticles are more effective in promoting the transition from the M1 to the M2 phenotype, alleviating neuroinflammation. Furthermore, after intravenous injection, compared to simple combination therapy, CeO2-FTY 720 nanoparticles can reach the site of brain injury within 24 hours, eliminating secondary hematoma and edema after intracerebral hemorrhage surgery, increasing M2 microglia, and boosting anti-inflammatory cytokine levels. Furthermore, they can better repair mitochondrial and BBB damage.

[0147] The following provides an in vitro model of oligodendrocyte precursor cells (OPCs) and an in vivo model of cerebral hemorrhage in C57BL / 6 mice to further verify that CeO2-FTY 720 nanoparticles can promote the proliferation and differentiation of OPCs and alleviate white matter damage.

[0148] Verification Example 2

[0149] (1) Oligodendrocyte precursor cell (OPCs) in vitro model

[0150] ①The experimental group was the same as the above experimental group, and both used the experimental group in the BV2 microglial cell model.

[0151] ② Compatibility test of CeO2-FTY 720 nanoparticles and OPCs

[0152] Oligodendrocyte cell line OLN-93 was selected, lysosomes were labeled with lysosome tracers, and Cy7-labeled CeO2-FTY 720 was added to the cell culture medium. The results are shown in the figure. Figure 27 Figure 2 shows the internalization and trafficking of Cy7-labeled CeO2-FTY 720 in OLN-93 cells; CeO2-FTY 720 (red), lysosomes (green), and nuclei (blue). After 15 minutes of incubation, a certain degree of colocalization between CeO2-FTY 720 (red) and lysosomes (green) was observed, indicating that CeO2-FTY 720 can be internalized by lysosomes. After 30 minutes of incubation, lysosomal uptake of CeO2-FTY 720 was significantly enhanced. After 1 hour, CeO2-FTY 720 accumulated significantly within the cells, demonstrating the good biocompatibility of CeO2-FTY 720.

[0153] ③CeO2-FTY 720 promotes the proliferation of OLN-93 cells

[0154] The proliferation of OLN-93 cells stimulated by OxyHb was investigated by double labeling of Ki67 and NG2. The immunofluorescence staining results are shown in Figure 28 , Figure 28 Representative immunofluorescence images of colocalization of Ki-67, NG2, and Dapi in OLN-93 cells. Figure 29 Statistical analysis chart of Ki67+ / NG2+.

[0155] FTY 720 promoted the proliferation of OLN-93 cells after 24 hours of OxyHb stimulation. The effects of CeO2 and CeO2+FTY 720 treatments were stronger. However, compared with other groups, CeO2-FTY 720 had the most significant promoting effect on the proliferation of OLN-93 cells.

[0156] ④CeO2-FTY 720 promotes OLN-93 cell differentiation

[0157] The differentiation of OLN-93 cells was assessed by measuring the levels of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) and myelin basic protein (MBP), which are markers of mature OLs.

[0158] Figure 30 This is a statistical chart of CNPase activity in mice for 24 hours after each group of experiments. Figure 31 The graph shows the statistical analysis of MBP in mice 24 hours after the experiment for each group.

[0159] As shown in the figure, FTY 720 alleviated these protein changes to some extent, while CeO2 or CeO2 + FTY 720 had a more pronounced effect, with the CeO2-FTY 720 group showing the best results. Notably, among all experimental groups, CeO2-FTY 720 had the most significant effect in promoting OLN-93 cell differentiation. This suggests that CeO2-FTY 720 can alleviate WMI by promoting the proliferation and differentiation of OPCs.

[0160] (2) In vivo model - intracerebral hemorrhage model in C57BL / 6 mice

[0161] Verify the relationship between CeO2-FTY 720 and repair of demyelination and axonal damage, CeO2-FTY 720 repairs demyelination and axonal damage

[0162] Figure 32 The figure shows a comparison of the LFB staining results of the perihematoma area in each group 7 days after cerebral hemorrhage. As can be seen from the figure, compared with the sham operation group, the myelin coverage was significantly reduced 7 days after ICH. FTY 720 treatment can moderately restore myelin coverage. The groups treated with CeO2 alone or CeO2+FTY 720 significantly restored myelin coverage after ICH. In addition, the myelin coverage in the CeO2-FTY 720 treatment group was more obvious than that in the CeO2+FTY 720 treatment group.

[0163] Figure 33 Figure 2 is the statistical analysis result of G-ratio in each experimental group; Figure 34 The figure shows the statistical analysis results of damaged axons in each experimental group. It can be seen from the figure that compared with the sham operation group, the G ratio increased significantly on the 7th day after ICH, indicating that obvious demyelination occurred in the area around the hematoma and the number of damaged axons also increased significantly. However, mice treated with FTY 720 showed a slight decrease in the G ratio and the number of damaged axons. Compared with CeO2 and CeO2+FTY 720, the changes in the CeO2-FTY 720 group were more obvious, indicating that the CeO2-FTY 720 group had a more prominent effect in alleviating demyelination and axonal damage.

[0164] Western blot (WB) analysis was used to evaluate the expression levels of major myelin and axon-related proteins (MBP, SMI 32, NF 200). Figure 35 The WB analysis and quantitative evaluation statistical graph of SMI 32 in the perihematoma area in each experimental group; Figure 36 The WB analysis and quantitative evaluation statistical graph of NF 200 in the perihematoma area in each experimental group; Figure 37 The WB analysis and quantitative evaluation statistical graph of MBP in the perihematoma area in each experimental group.

[0165] As can be seen from the figure, CeO2-FTY 720 is superior to other treatment groups in promoting myelin and axon recovery after ICH.

[0166] Existing studies have shown that intracerebral hemorrhage can lead to the death of oligodendrocytes (OLs) and demyelination, which in turn causes white matter injury (WMI).

[0167] Verification Example 2 shows that compared with CeO2 and CeO2+FTY 720, CeO2-FTY 720 nanoparticles can better promote the proliferation of OLN-93 cells and promote the differentiation of OLN-93 cells. OPCs promote remyelination through proliferation and differentiation, thereby reducing white matter damage.

[0168] It can be seen that CeO2-FTY 720 nanoparticles can be used as drugs to participate in the treatment of OPCs and myelin-related diseases, and have a wide range of applications.

[0169] Verification Example 3: Cytotoxicity Verification

[0170] (1) In the BV2 microglial cell model, the cytotoxicity of CeO2-FTY 720 in BV2 cells was evaluated using the methylthiazolium (MTT) assay. After 24 hours of treatment, the treatment had no significant effect on the cells themselves, demonstrating the excellent compatibility and low cytotoxicity of the synthesized CeO2-FTY 720. At the same time, in the OPCs in vitro model, CeO2-FTY 720 also had no significant effect on OLN-93 cells.

[0171] (2) In the ICH (intracerebrovascular hemorrhage) model of C57BL / 6 mice, hematoxylin-eosin (H&E) staining was performed on several important organs 14 days after tail vein injection. No significant changes occurred in the function and morphology of the spleen, heart, lungs, liver, and kidneys, indicating that the biotoxicity of CeO2-FTY 720 was low. At the same time, blood samples were collected before injection of CeO2-FTY 720 and on the 1st and 14th days after ICH, and biochemical indicators (CR, CKMB, ALT, AST, BUN, and LDH-1) were analyzed. The results showed that compared with the level before CeO2-FTY720 injection, these biochemical indicators did not change significantly on the 1st and 14th days after injection.

[0172] The present invention further provides Verification Example 4, which demonstrates that CeO2-FTY 720 can significantly alleviate long-term neurological damage after ICH in mice.

[0173] Verification Example 4:

[0174] Behavioral assessments were performed for 4 weeks after sham surgery or ICH modeling. The experiments included the rotarod test, the stagger test, and the sticker removal test. Mice received intravenous drug injections at a dose of 0.4 mg / kg 3 days after ICH for 7 days. In addition, water maze testing was performed from day 21 to day 25 after ICH.

[0175] The rotarod test was used to assess motor coordination and balance ability. The experimental results are shown in Figure 38 As shown, Figure 38 The statistical analysis results of the rod test are shown in the figure. As shown in the figure, the latency to fall of mice in the ICH group was significantly shorter than that in the sham-operated group. The CeO2+FTY 720 group showed a moderate improvement in latency, while the intravenous CeO2-FTY 720 group showed the most significant increase in latency.

[0176] The stagger test was used to assess sensory and motor function. The results are shown in Figure 39 As shown, Figure 39This is a statistical analysis result of the gait error test. It can be seen from the figure that mice 3 days after ICH had a higher foot fault rate than the sham operation group, indicating that their sensory and motor functions were impaired. The foot fault rate in the CeO2+FTY 720 group was reduced, and the CeO2-FTY 720 group showed the most significant improvement.

[0177] The sticker removal test measured fine motor skills by requiring mice to use their teeth and right forepaw to remove an adhered sticker from their left forepaw. Figure 40 As shown, Figure 40 This is a statistical analysis of the sticker removal test. It can be seen from the figure that the time taken by mice after ICH to remove the stickers was significantly longer than that of the sham operation group. The removal time of the CeO2+FTY 720 group was shortened, and the CeO2-FTY 720 group showed the most significant improvement, indicating that their fine motor skills recovered better.

[0178] The water maze test, used to assess spatial learning and memory, involves training mice to locate a hidden platform and then testing their memory by removing the platform and measuring the time the mice remain in the target quadrant. Figure 41 As shown, Figure 41 This figure shows a statistical analysis of the escape latency of each group on days 21-25 after ICH. Compared with the sham-operated group, the escape latency of mice in the ICH group was significantly prolonged. The CeO2+FTY 720 group showed some improvement in escape latency, while the CeO2-FTY 720 group showed the most significant reduction in escape latency, indicating enhanced memory retention.

[0179] Verification Example 4 evaluated the long-term effects of different drug treatments on the neurobehavioral and cognitive functions of mice 3 days after ICH. In the rotarod test, stagger test, sticker removal test, and water maze test, the mice in the CeO2-FTY 720 group had a better recovery rate.

[0180] It can be seen that CeO2-FTY 720 achieves synergistic enhancement of substances and has low toxicity. It can be used as a drug to alleviate the sequelae of cerebral hemorrhage and relieve long-term neurological damage.

[0181] The above embodiments are merely exemplary embodiments of the present application and are not intended to limit the scope of the present application. The scope of protection of the present application is defined by the claims. Those skilled in the art may make various modifications or equivalent substitutions to the present application within the essence and scope of protection of the present application, and such modifications or equivalent substitutions shall also be deemed to fall within the scope of protection of the present application.

Claims

1. A method for preparing fingolimod-loaded nanoparticles, characterized in that: The steps include: CeO2 nanoparticles were synthesized by a high-temperature organic solution phase method; adding the CeO2 nanoparticles to polyethylene glycol to obtain PEG-CeO2 nanoparticles, wherein the polyethylene glycol modifies the surface of the CeO2 nanoparticles to form a coating layer; Fingolimod is mixed with the PEG-CeO2 nanoparticles, and fingolimod is attached to the surface of the coating layer to prepare CeO2-FTY 720 nanoparticles.

2. The method for preparing fingolimod-loaded nanoparticles according to claim 1, wherein: The high temperature organic solution phase method comprises the following steps: In a nitrogen environment, oleylamine is added to octadecene to obtain a mixed solvent; Cerium acetate hydrate is added to the mixed solvent to obtain cerium acetylacetonate hydrate, stirred evenly, heated at 280° C. for one hour, and filtered to obtain the CeO 2 nanoparticles.

3. Fingolimod-loaded nanoparticles prepared by the preparation method according to any one of claims 1 to 2.

4. The fingolimod-loaded nanoparticles according to claim 3, characterized in that: In the CeO2-FTY720 nanoparticles, Ce 3+ With Ce 4+ The ratio is 1:

1.

5. Use of the fingolimod-loaded nanoparticles according to any one of claim 4 in treating cerebral hemorrhage, characterized in that: The invention also includes application of the CeO2-FTY 720 nanoparticles in preparing drugs for treating secondary white matter damage caused by cerebral hemorrhage.

6. The use according to claim 5, characterized in that The drug further includes a pharmaceutically acceptable carrier.

7. The use according to claim 5, characterized in that The dosage form of the medicine is injection.

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