A brain-targeting nano-preparation loaded with ergosterol and a preparation method and application thereof

Abstract

The application provides a brain-targeting nano preparation loaded with ergosterol and a preparation method and application thereof, relates to the fields of nanometer materials and nanometer biological medicine, and the active ingredient ergosterol of the nano preparation is encapsulated in DSPE-PEG-pinacol boronate nanometer material, the nanometer material has ROS response characteristics, the nano preparation loaded with ergosterol is regular spherical, has uniform particle size, has good dispersibility and stability, has good ability of targeted treatment of ischemic stroke diseases, effectively solves the problems of poor targeting of ordinary drugs, low bioavailability and the like, enhances the treatment effect of ergosterol on ischemic stroke, can effectively improve brain damage caused by ischemic stroke, has good treatment effect, meanwhile, the active ingredient ergosterol plays a role in treating ischemic stroke by activating the PI3K / AKT / mTOR pathway, and provides a new direction for the treatment of ischemic stroke.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of nanomaterials and nanobiomedicine, and in particular to a brain-targeting nano-preparation loaded with ergosterol, and a preparation method and application thereof. BACKGROUND

[0002] Stroke, commonly known as "apoplexy", is an acute disease caused by blood vessel diseases that deliver blood to the brain, and has the characteristics of high incidence, high disability rate, high recurrence rate and high mortality rate, and is one of the most important fatal diseases in the world. Stroke is mainly divided into two categories: ischemic stroke (IS) and hemorrhagic stroke, among which ischemic stroke accounts for about 75% to 85% of the total number of stroke patients, and seriously affects the quality of life of patients.

[0003] The IS pathological state refers to the interruption of blood flow in a certain part of the brain, and the mitochondrial energy metabolism is converted to anaerobic glycolysis, the generation of ATP is reduced, the oxygen-sugar supply to the ischemic center is interrupted, causing a large amount of excitatory amino acid glutamate to be released in the central nervous system, which accumulates in the synaptic cleft, produces excitatory toxicity to the neuron cells, and triggers intracellular calcium overload, causing mitochondrial dysfunction and the generation of excessive free radicals such as ROS. Excessive ROS can damage intracellular protein molecules, causing autophagy and apoptosis of cells, and further damaging tissues.

[0004] IS treatment mainly includes surgical treatment and drug treatment, but faces the following problems: (1) Surgical treatment: through surgical methods to restore blood perfusion in the ischemic area of the brain, but the time window is strictly limited, and needs to be performed within a short time after the onset, otherwise the optimal treatment opportunity may be missed, resulting in poor surgical effect or even inability to perform surgery; high risk, the surgical process itself may cause additional damage to the brain tissue, increasing the patient's pain and the difficulty of postoperative recovery; uncertainty of prognosis, even if the surgery is successful, the prognosis of the patient is difficult to accurately predict, and different degrees of neurological dysfunction may still be left; (2) Drug treatment: ① Poor drug targeting: ordinary drugs are difficult to accurately reach the brain lesion site when treating ischemic stroke, resulting in limited treatment effect. ② Low bioavailability: during the absorption, distribution, metabolism and excretion of drugs in the body, the drugs cannot fully exert their efficacy, resulting in waste of drugs, and at the same time, the risk of adverse reactions may be increased by increasing the dose of drugs; (3) Lack of effective treatment drugs: the treatment drugs for ischemic stroke are limited in clinic, and the current focus of drug treatment is to quickly dissolve thrombus by giving thrombolytic drugs to restore blood perfusion in the ischemic area of the brain, but the only effective treatment drug t-PA (tissue plasminogen activator) in the acute phase certified by FDA has a short optimal treatment time window (≤4.5h), and is prone to complications such as reperfusion injury and cerebral hemorrhage, so new treatment drugs and methods are urgently needed. SUMMARY

[0005] In order to solve the above technical problems, the present application provides a brain-targeting nano-preparation loaded with ergosterol, a preparation method and application thereof, it is found through bioinformatics analysis and in-vivo and in-vitro verification experiments that ergosterol can prevent and treat IS by activating the PI3K / AKT / mTOR signal pathway, combined with the pathological state of IS, a ROS-responsive nanomaterial DSPE-PEG-fenretinide borate is synthesized, ergosterol is loaded to obtain an ergosterol nano-preparation, and targeted treatment of IS is achieved, the nano-preparation can effectively solve the problems of poor drug targeting, low bioavailability and the like, a new treatment method for ischemic stroke is developed, and the targeted treatment capability of the drug is enhanced, and it is found that ergosterol plays a role in treating ischemic stroke by activating the PI3K / AKT / mTOR pathway.

[0006] In order to achieve the above object, the present application firstly provides a brain-targeting nano-preparation loaded with ergosterol, the nano-preparation comprises ergosterol, fenretinide borate and DSPE-PEG, the fenretinide borate modifies the DSPE-PEG to form a DSPE-PEG-fenretinide borate nanomaterial loaded with ergosterol.

[0007] Preferably, the potential of the nano-preparation is -23.88±0.38, the particle size is 30 nm, the drug loading amount is 8.05% to 18.33%, and the encapsulation rate is 85.20% to 93.77%.

[0008] Based on a general inventive concept, the present application further provides a preparation method of the nano-preparation, comprising the following steps:

[0009] S1, activating fenretinide borate: 4-hydroxymethylphenylboronic acid fenretinide is mixed with N,N carbonyl diimidazole and added to anhydrous dichloromethane for stirring reaction, after the reaction is completed, anhydrous dichloromethane is added for extraction, deionized water is washed three times, the organic phase is further extracted with a saturated sodium chloride solution, and then anhydrous sodium sulfate is used for drying, thereby obtaining the activated fenretinide borate;

[0010] S2, preparing the nanomaterial DSPE-PEG-fenretinide borate: the activated fenretinide borate prepared in S1 is dissolved in dimethyl sulfoxide together with DSPE-PEG, beta-cyclodextrin, 4-dimethylaminopyridine and DSPE-PEG for reaction, the reaction solution is added with anhydrous ether for precipitation, anhydrous ether is used for washing, the precipitate is collected through centrifugation, and vacuum freeze-drying is performed under a vacuum environment, thereby obtaining the nanomaterial DSPE-PEG-fenretinide borate;

[0011] S3, preparation of nano-preparation: taking the ergosterol and the DSPE-PEG-phenylboronic acid ester prepared in S2, placing in a round-bottom flask, adding an organic solvent, stirring until completely dissolved, connecting to a rotary evaporator for slow rotary evaporation until the organic solvent is completely evaporated, forming a lipid film on the inner wall of the flask, adding PBS buffer, stirring in a 37℃ water bath to make the lipid film fully hydrated to form a suspension, after ultrasonic treatment and dialysis purification, freeze-drying to obtain the nano-preparation.

[0012] As a preferred, the mass ratio of the 4-hydroxymethyl phenylboronic acid phenol ester and N,N carbonyl diimidazole in the step S1 is 1-2:1-2.5, and the stirring time is 1h.

[0013] As a preferred, the mass ratio of the activated phenylboronic acid phenol ester, β-cyclodextrin, 4-dimethylamino pyridine in the step S2 is 1-2:0.1-1:0.5-1.

[0014] As a preferred, the mass ratio of the ergosterol and the DSPE-PEG-phenylboronic acid phenol ester in the step S3 is 1:4-12.

[0015] As a preferred, the organic solvent in the step S3 is selected from any one or several of methanol, dichloromethane, and trichloromethane.

[0016] As a preferred, the temperature of the slow rotary evaporation in the step S3 is room temperature to 45℃, and the pressure is 0.05-0.15MPa.

[0017] Based on one overall inventive concept, the scheme also provides an application of the ergosterol-loaded brain-targeting nano-preparation in targeted treatment of brain diseases.

[0018] As a preferred, the brain diseases include ischemic stroke.

[0019] The treatment mechanism of the nano-preparation of the scheme is as follows:

[0020] This study, for the first time, discovered that ergosterol can prevent and treat ischemic stroke by activating the PI3K / AKT / mTOR signaling pathway, and based on this discovery, a nanoformulation was constructed. During ischemic stroke, the ergosterol in the nanoformulation precisely targets this signaling pathway, thereby protecting neurons, reducing stroke-related neuronal cell death, maintaining a stable intracellular environment, and safeguarding normal neuronal function. It also regulates VEGF expression and signaling, promoting endothelial cell proliferation, migration, and lumen formation, thereby facilitating angiogenesis. Furthermore, it can provide a cell source for neuroregeneration, helping damaged neurons reconnect and restore neuronal function. Existing drugs or treatments that precisely regulate this pathway are lacking. This study, based on the novel discovery of ergosterol's mechanism of action and the innovative design of the nanoformulation, offers a novel mechanistic approach for the treatment of ischemic stroke. Unlike existing treatments that primarily rely on relatively single mechanisms, such as thrombolysis or surgery, this approach integrates multiple approaches, including cell signaling pathway regulation, neuroprotection, angiogenesis, and neuroregeneration, offering greater innovation and potential therapeutic value.

[0021] In this approach, the nanoformulation ergosterol-targeted micelles, made by encapsulating the active drug ergosterol with the nanomaterial DSPE-PEG-pinacol boronate, exhibit ROS-responsive properties. At the site of ischemic stroke, due to the presence of excessive ROS, the nanoformulation can rapidly respond and release ergosterol. The released ergosterol promptly activates the PI3K / AKT / mTOR pathway. This synergistic effect makes the treatment more precise and effective. Existing drugs or formulations often lack this responsive release mechanism adapted to the pathological environment, making it difficult to exert its effects at the appropriate time and location. Furthermore, the nanoformulation's particle size, potential, and other properties facilitate its interaction with cell membranes, promoting cellular uptake of ergosterol. With increased cellular uptake, ergosterol can more effectively enter cells and activate the PI3K / AKT / mTOR pathway. This provides more opportunities for ergosterol to exert its effects and activate the pathway within the cell, resulting in a higher cellular uptake efficiency than conventional drugs in the existing art.

[0022] Bioinformatics analysis found that the phosphate inositol 3-kinase (PI3K) / protein kinase B (Akt) / mTOR signaling pathway plays an important role in the process of IS. PI3K / AKT / mTOR signaling pathway plays an important role in central nervous system injury. When stimulated by external stimuli, PI3K is activated to produce PIP3, which recruits and activates AKT. AKT phosphorylates downstream proteins such as Bad protein and caspase-9, prevents cytochrome C release, and inhibits apoptosis caused by ischemia. This pathway can also regulate ion channels and transport proteins on the cell membrane, helping to maintain the stability of the intracellular environment, protect the normal function of nerve cells, and avoid intracellular calcium overload caused by ischemia. In addition, by activating eNOS to produce NO, it promotes endothelial cell migration and proliferation, and also regulates VEGF expression and signaling, promoting vascular endothelial cell proliferation, migration and lumen formation, which is beneficial to angiogenesis. The PI3K / AKT / mTOR pathway can stimulate the proliferation and differentiation of neural stem cells, guide the differentiation of neural stem cells to neurons, and provide a source of cells for nerve regeneration. Help damaged nerve cells re-establish connections and restore nerve function.

[0023] Compared with the prior art, the present application has the following beneficial effects:

[0024] 1. The nano material DSPE-PEG-fenretinide borate and the brain-targeting nano preparation loaded with ergosterol prepared by the scheme are regular spherical or spherical, have good dispersibility, uniform particle size distribution, are beneficial to circulation and distribution in the body, are more easily transported to the brain lesion site, and the brain-targeting nano preparation loaded with ergosterol has a higher absolute value of Zeta potential, effectively increasing the stability of the nano preparation, so that it can maintain structural integrity in storage and in vivo environment and is not easy to aggregate or decompose, thereby helping to improve the stability of the drug.

[0025] 2. The nano preparation has ROS response characteristics, and in the simulated ischemic stroke environment, the cumulative release rate of the nano preparation loaded with ergosterol is higher, and a high release level can be maintained, which shows that when ischemic stroke occurs, the ROS generated locally can trigger the rapid decomposition of the nano preparation and release the drug, realize the targeted release of the drug, increase the concentration of the drug at the lesion site, enhance the treatment effect, and at the same time reduce the toxic and side effects on normal tissues.

[0026] 3. The scheme first finds that ergosterol and its nano preparation can play a therapeutic role on ischemic stroke by activating the PI3K / AKT / mTOR pathway, can better regulate intracellular signal transduction, inhibit cell apoptosis, promote cell survival and functional recovery, and thus plays a therapeutic role on ischemic stroke.

[0027] 4、The scheme is expected to solve the problems of poor targeting, low bioavailability and large side effects of ordinary preparations when administered, enhance the therapeutic effect of ergosterol on ischemic stroke, and show better efficacy, safety and application prospect in the treatment of ischemic stroke, which is expected to improve the therapeutic effect and prognosis of patients, improve the quality of life of patients, provide a new idea and method for the treatment of ischemic stroke, and open up the treatment direction based on nano-preparation and specific signal pathway regulation, and provide an important reference for subsequent related research and drug development. BRIEF DESCRIPTION OF DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0029] Figure 1 The synthesis route of the nanomaterial DSPE-PEG-phenylboronic acid ester in Example 1;

[0030] Figure 2 The nuclear magnetic H spectrum of the nanomaterial DSPE-PEG-phenylboronic acid ester in Experimental Example 1, Figure 2 The nuclear magnetic C spectrum of DSPE-PEG-phenylboronic acid ester (A), the ultraviolet spectrum of DSPE-PEG-OH / phenylboronic acid ester (B), the infrared spectrum of DSPE-PEG-OH / phenylboronic acid ester (C), and the infrared spectrum of DSPE-PEG-OH / phenylboronic acid ester (D) in Experimental Example 1; Figure 2 Figure 2 Figure 2 The DLS particle size results of the nanomaterial DSPE-PEG-phenylboronic acid ester in Experimental Example 1;

[0031] Figure 3 The DLS particle size results of the nanomaterial DSPE-PEG-phenylboronic acid ester in Experimental Example 1;

[0032] Figure 4 The transmission electron micrographs of the blank nanometer preparation, the nanometer preparation loaded with the drug ergosterol, and the nanometer preparation loaded with ergosterol after H2O2 treatment in Experimental Example 2; A is the blank nanometer preparation, B is the nanometer preparation loaded with the drug ergosterol, and C is the nanometer preparation loaded with ergosterol after H2O2 treatment;

[0033] Figure 5 ​​Figure 2 shows the particle size test results of the blank nanoformulation formed by nanomaterials, the nanoformulation prepared by loading the drug ergosterol, and the nanoformulation loaded with ergosterol after H2O2 treatment in Experiment 2; A is the blank nanoformulation, B is the nanoformulation prepared by loading the drug ergosterol, and C is the nanoformulation loaded with ergosterol after H2O2 treatment;

[0034] Figure 6 Cellular uptake test results of ergosterol-loaded nanoformulation prepared in Experiment 2;

[0035] Figure 7 Figure 2 shows the particle size, potential detection and encapsulation efficiency of the nanoformulation in Experimental Example 2. Figure A shows the particle size detection results of the nanoformulation loaded with ergosterol at different time points; Figure B shows the potential detection results of the nanoformulation loaded with ergosterol at different time points; Figure C shows the encapsulation efficiency detection results of the nanoformulation loaded with ergosterol at different time points.

[0036] Figure 8 Figure 2 shows the drug release results of ergosterol drug and ergosterol-loaded nanoformulation in pH 7.4 and pH 7.4 + 100 μM H2O2 media, respectively; A is the drug release result in pH 7.4 medium, and B is the drug release result in pH 7.4 + 100 μM H2O2 medium;

[0037] Figure 9 This is a picture of the rat model of ischemic stroke established in Experimental Example 3 ( Figure 9 A), Effects of different treatments on Longa scores of rats ( Figure 9 B);

[0038] Figure 10 Figure 3: Brain tissue, cerebral infarction volume, and HE staining of rats in experimental example 3. A is the TTC staining result of rat brain tissue in different treatment groups, B is the cerebral infarction rate of rats in different treatment groups, and C is the HE staining result of brain tissue morphology of rats in different treatment groups.

[0039] Figure 11 Effects of ergosterol and its nanoformulation on the expression of related proteins in Experimental Example 3: A comparative study based on sham surgery and model groups; A is the expression level of related proteins detected by Western blot, B is the quantitative expression graph of p-mTOR / mTOR protein, C is the quantitative expression graph of p-AKT / AKT protein; D is the quantitative expression graph of p-PI3K / PI3K protein;

[0040] Figure 12 The ergosterol nanoparticles prepared in Comparative Example 1 ( Figure 12 A. Figure 12 B) and the ergosterol-loaded nanoformulation prepared in Example 2 ( Figure 12 C. Figure 12D) The results of in vivo imaging of rats at 1 hour and 4 hours after administration. DETAILED DESCRIPTION

[0041] In order to make the technical problems, technical solutions and advantages of the present application clearer, the following will be described in detail with reference to the drawings and specific embodiments.

[0042] The following examples are used to illustrate the present application, but are not used to limit the scope of the present application. Modifications or replacements of the methods, steps or conditions of the present application, without departing from the spirit and essence of the present application, all belong to the scope of the present application.

[0043] If not specifically indicated, the technical means used in the examples are conventional means known to those skilled in the art; if not specifically indicated, the reagents used in the examples are commercially available.

[0044] Example 1 Preparation of nanomaterial DSPE-PEG-fenretinide borate

[0045] S1, activation of fenretinide borate: 1.8g of 4-hydroxymethylphenyl borate fenretinide (PBAP) and 2.2g of N,N carbonyl diimidazole (CDI) were precisely weighed and placed in a 50mL double-necked flask, 20mL of anhydrous dichloromethane (DCM) was added, after it was dissolved, it was reacted on a constant temperature magnetic stirrer for one hour, then double DCM was added for extraction, deionized water was washed for 3 times, the organic phase was further extracted with saturated NaCl solution, and finally dried with an appropriate amount of anhydrous sodium sulfate, and the activated fenretinide borate CDI-PBAP was obtained;

[0046] S2, preparation of nanomaterial DSPE-PEG-fenretinide borate: 1.6g of CDI-PBAP, 0.35g of β-cyclodextrin (β-CD), 0.9g of 4-dimethylaminopyridine (DMAP), and 0.4g of DSPE-PEG prepared in step S1 were added to DMSO for reaction, the reaction solution was precipitated by adding anhydrous ether, washed with anhydrous ether for 3 times, centrifuged to collect the precipitate, and freeze-dried in a vacuum environment for about 24h, and the nanomaterial DSPE-PEG-fenretinide borate was obtained.

[0047] The synthesis route of the nanomaterial DSPE-PEG-fenretinide borate is shown in Figure 1 .

[0048] Example 2 Preparation of brain-targeting nanoformulation loaded with ergosterol

[0049] S1, 5mg of ergosterol and 40mg of DSPE-PEG-fenretinide borate prepared in Example 1 were weighed into a round-bottom flask, an appropriate amount of chloroform was added, and a magnetic stirrer was used to stir at room temperature until the material and drug were completely dissolved. The round-bottom flask was connected to a rotary evaporator, and slowly evaporated at 40°C and 0.1MPa until the organic solvent was completely evaporated, forming a uniform, thin and transparent lipid film on the inner wall of the flask;

[0050] S2, an appropriate amount of PBS buffer was slowly added to the flask with the lipid film, and the lipid film was fully hydrated under the condition of 37°C water bath and gentle stirring. After ultrasonic treatment and dialysis purification of the obtained suspension, freeze-drying was carried out to obtain the brain-targeting nano-preparation loaded with ergosterol.

[0051] Preparation of nano-preparation loaded with ergosterol without fenretinide borate

[0052] S1, the nanomaterial DSPE-PEG was prepared: 0.35g of β-cyclodextrin (β-CD), 0.9g of 4-dimethylaminopyridine (DMAP) and 0.4g of DSPE-PEG were added to DMSO for reaction, the reaction solution was precipitated with anhydrous ether, washed with anhydrous ether for 3 times, centrifuged to collect the precipitate, and freeze-dried in a vacuum environment for about 24h to obtain the nanomaterial DSPE-PEG;

[0053] S2, 5mg of ergosterol and 40mg of DSPE-PEG prepared in Example 1 were weighed into a round-bottom flask, an appropriate amount of chloroform was added, and a magnetic stirrer was used to stir at room temperature until the material and drug were completely dissolved. The round-bottom flask was connected to a rotary evaporator, and slowly evaporated at 40°C and 0.1MPa until the organic solvent was completely evaporated, forming a uniform, thin and transparent lipid film on the inner wall of the flask;

[0054] S2, an appropriate amount of PBS buffer was slowly added to the flask with the lipid film, and the lipid film was fully hydrated under the condition of 37°C water bath and gentle stirring. After ultrasonic treatment and dialysis purification of the obtained suspension, freeze-drying was carried out to obtain the brain-targeting nano-preparation loaded with ergosterol.

[0055] Experimental Example 1: Investigation of the characterization of nanomaterial DSPE-PEG-fenretinide borate

[0056] (1) Nuclear magnetic H spectrum, nuclear magnetic C spectrum and ultraviolet, infrared characterization

[0057] An appropriate amount of nanomaterial DSPE-PEG-fenretinide borate prepared in Example 1 was taken and dissolved in deuterated dimethyl sulfoxide to make the sample concentration reach 1-10mM, and then transferred to a nuclear magnetic tube. The nuclear magnetic tube was placed in a nuclear magnetic resonance spectrometer for nuclear magnetic H spectrum and C spectrum determination.

[0058] The nanomaterial DSPE-PEG-pinnacol borate is ground into fine powder, mixed with dry potassium bromide powder in proportion, and then placed in a tablet press mold. A tablet press is used to apply pressure for several minutes to make transparent thin slices, which can be used for infrared detection.

[0059] The results of nuclear magnetic detection are shown in Figure 2 A, Figure 2 B, the ultraviolet spectrum is shown in Figure 2 C, and the infrared detection results are shown in Figure 2 D. According to the above detection results, it can be determined that the nanomaterial DSPE-PEG-pinnacol borate is successfully prepared.

[0060] (2) Particle size

[0061] An appropriate amount of nanomaterial DSPE-PEG-pinnacol borate is diluted with ultrapure water to an appropriate concentration and then added to a sample cell to detect the particle size of the prepared nanomaterial DSPE-PEG-pinnacol borate. The detection results are shown in Figure 3 According to the detection results, the average particle size of the prepared nanomaterial is 26.92 nm, the PDI is 0.234, and the particle size distribution is uniform.

[0062] Experimental Example 2: Investigation of the basic characterization of brain-targeting nanomaterial loaded with ergosterol

[0063] (1) Morphological characterization

[0064] The copper mesh for transmission electron microscopy is hydrophilized, and then solutions of blank material DSPE-PEG-pinnacol borate, nanomaterial loaded with ergosterol prepared in Example 2, and H2O2-treated nanomaterial loaded with ergosterol with a concentration of about 250 μg / mL are added dropwise to the front of the copper mesh. After standing for 2 min, an appropriate amount of phosphotungstic acid dye is added, the liquid on the copper mesh is absorbed with filter paper, and the phosphotungstic acid dye is added again. After standing for 2 min, the liquid is absorbed with filter paper. After repeating the phosphotungstic acid staining once, the copper mesh is dried under an incandescent lamp. The copper mesh is placed in a transmission electron microscope (TEM) to investigate the morphology of the sample.

[0065] The detection results are shown in Figure 4 The nanomaterial DSPE-PEG-pinnacol borate and the nanomaterial loaded with ergosterol are regular spherical or spherical, and have good dispersibility. After loading ergosterol, the structure of the nanomaterial does not change significantly, indicating that the nanomaterial has good loading capacity for ergosterol. Compared with the above two, the H2O2-treated nanomaterial loaded with ergosterol shows obvious changes. The distribution of particles is no longer uniform, and the morphology of some particles has changed, showing signs of decomposition, providing direct evidence for studying the drug release mechanism and the stability of the nanomaterial.

[0066] (2) Particle size, potential and PDI determination

[0067] The particle size, potential and PDI of the blank material DSPE-PEG-phenanthroline borate, the ergosterol-loaded nanofomulation and the ergosterol-loaded nanofomulation treated by H2O2 were characterized by using a dynamic light scattering particle size analyzer. After the instrument was preheated for 30 min, the quartz cuvette was washed with purified water for 2-3 times, and the nanofomulation was diluted with purified water according to a certain proportion and then added into the cuvette. The measurement was set to be repeated for 3 times for each sample and the single cycle time was set, the test solvent was selected as "Water", and the measurement was started after the instrument was stabilized. The particle size test results are shown in Figure 5 The PDI result of the ergosterol-loaded nanofomulation was 0.196±0.002. According to the characterization results, the particle size of the nanomaterial DSPE-PEG-phenanthroline borate was uniformly distributed and concentrated at about 30 nm, and there was no obvious change before and after drug loading. However, the particle size of the ergosterol-loaded nanofomulation treated by H2O2 was significantly larger, indicating that H2O2 treatment can simulate the rapid decomposition of the targeted nanofomulation caused by ischemic stroke environment to release the drug, thereby possibly enhancing the treatment of ischemic stroke and reducing the toxic side effects on normal tissues.

[0068] Potential determination: After the instrument was preheated for 30 min, the quartz cuvette was washed with purified water for 2-3 times, the ergosterol nanofomulation was diluted with purified water according to a certain proportion and then added into the cuvette, the electrode sheet was inserted into the cuvette and connected to the instrument, the cycle time and cycle number were set, the test solvent was selected as "Water", and the measurement was started after the instrument was stabilized. The potential of the ergosterol-loaded nanofomulation was-23.88±0.38, and the absolute value of the Zeta potential was high, which effectively increased the stability of the ergosterol-loaded nanofomulation and improved the transmembrane activity of the drug, thereby promoting the improvement of the bioavailability of the drug.

[0069] (3) Cell uptake experiment

[0070] Cy2 (between 480 nm, emission wavelength at 508 nm) labeled ergosterol was prepared into Cy2-ergosterol-loaded nanofomulation according to the above method. The PC12 cells in the logarithmic growth phase were inoculated on a six-well plate (1×10 5 / mL, 2 mL), and the culture solution was discarded after the cells adhered. The two formulations with a labeling concentration of 20 nM were incubated with HCT116 cells in a confocal culture dish. The cells were cultured for 2, 4 and 12 h. The cells were washed with pre-cooled PBS for 3 times, and then incubated with DAPI (5 μg / mL) to facilitate the observation of the cells under a fluorescence microscope.

[0071] The detection results are shown in Figure 6As shown, a small amount of green fluorescence appeared around the nucleus after 1 h of incubation, and the Cy2-loaded ergosterol nanofomulation was absorbed by PC12 cells and the fluorescence intensity increased with the extension of incubation time after 2 h, 4 h and 6 h. This indicates that the Cy2-loaded ergosterol nanofomulation can be effectively taken up by cells to exert a therapeutic effect.

[0072] (4) Stability test of the ergosterol-loaded nanofomulation

[0073] The stability of the ergosterol-loaded nanofomulation at different storage times was investigated. The samples were stored in a sealed Schott bottle at 4°C for 30 days. The samples were taken at the corresponding time (1, 15 and 30 days) for determination of the particle size distribution, Zeta potential and encapsulation efficiency to evaluate the storage stability of the ergosterol-loaded nanofomulation.

[0074] The determination results are shown in Table 1. Figure 7 As shown in Table 1, after 30 days of preliminary stability test, the particle size distribution, encapsulation efficiency and Zeta potential of the ergosterol-loaded nanofomulation all increased in value within 30 days, with a drug loading of 8.05% to 18.33% and an encapsulation efficiency of 85.20% to 93.77%. However, there was no statistically significant change (P>0.05), indicating that the nanofomulation has good stability and can be stably stored in the above environment.

[0075] (5) In vitro release of the nanofomulation

[0076] The in vitro release of ergosterol and the ergosterol-loaded nanofomulation was determined by dialysis method. Ergosterol and the ergosterol-loaded nanofomulation were placed in a dialysis device with a molecular weight cut-off of 100 kDa. The buffer medium was pH 7.4 PBS and pH 7.4 PBS + 100 μM H2O2, respectively. At the time points of 15, 30, 45, 60, 90, 120, 180, 240, 360, 480, 600, 720, 1440, 2160 and 2880 min, 1 mL of the release solution was taken and the corresponding solution was added to make up. The content of the released ergosterol was determined by HPLC.

[0077] The results are shown in Table 2. Figure 8 As shown in Table 2, the cumulative release rate of the ergosterol-loaded nanofomulation was significantly higher than that of ergosterol in the medium with pH 7.4, and the cumulative release rate of the ergosterol-loaded nanofomulation was higher in the environment with pH 7.4 and containing 100 μM H2O2, and maintained a high release level throughout the process, while the cumulative release rate of ergosterol itself was relatively low. This indicates that the ergosterol-loaded nanofomulation can effectively improve the stability of the drug and reduce the influence of the external environment, and H2O2 treatment can simulate the rapid decomposition of the nanofomulation caused by ischemic stroke environment to release the drug.

[0078] Experimental Example 3 investigates the application of the ergosterol-loaded nano-preparation in the treatment of ischemic stroke disease

[0079] Construction of rat ischemic stroke model: SD rats weighing 300g±10g and 11-12 weeks old were used. After being anesthetized with chloral hydrate, the rats were fixed on a mouse plate in a supine position. The right common carotid artery (CCA), internal carotid artery (ICA) and external carotid artery (ECA) were bluntly separated with an ophthalmic forceps, and a thread was hung for standby. The ECA and the proximal end of the CCA were ligated, and the ICA was temporarily clamped with an artery clamp. The CCA was cut with an ophthalmic forceps to form a small opening, and a prepared thread was quickly inserted along the CCA to the ICA. When the tip of the thread felt resistance, the CCA standby thread was tightened to prevent the thread from moving out and bleeding. The standby thread was ligated and the blocking time was recorded. After 1h of ischemia, the thread was pulled out and perfusion was performed. The sham operation group had the same operation steps as above, but the depth of the inserted thread was only 15mm, and the middle cerebral artery blood flow was not blocked. When the rat's tail was lifted and suspended, the left forelimb was flexed and the activity showed a typical tail-chasing sign, indicating successful modeling.

[0080] 1. The effect of ergosterol and ergosterol-loaded nano-preparation on the neurological function and pathological state of rats

[0081] (1) The Longa scoring method was used to quantify the degree of neurological dysfunction of animals through a series of behavioral observations. The score ranged from 0 to 4, where 0 indicated no neurological defects, 1 indicated that the paralyzed forepaw could not fully extend, 2 indicated that the animal walked in a circle towards the paralyzed side, 3 indicated that the animal leaned towards the paralyzed side when walking, and 4 indicated that the animal could not walk automatically and showed signs of loss of consciousness. The higher the score, the worse the animal's neurological behavior and the more severe the neurological dysfunction. In the experiment, the Longa scoring method was used to preliminarily evaluate the success of model making. Animals with scores of 1-3 were included in the next experiment, while animals with scores of 4-5 were excluded because their condition was not suitable for participating in the experiment. The rats in each group were scored before treatment and on days 1, 3, 7, 14 and 28 after treatment.

[0082] The results of the determination are as follows Figure 9As shown, the Longa score of the model group was the highest, indicating that the neurological function of the model group was more severely damaged. Compared with the model group, the score of the ergosterol group was reduced, and the difference was significant (P<0.01), indicating that ergosterol could improve the neurological function damage caused by ischemic stroke. Compared with the ergosterol group, the score of the nanometer preparation loaded with ergosterol prepared by the present application was significantly reduced (P<0.01), indicating that the nanometer preparation loaded with ergosterol could significantly improve the neurological function damage caused by ischemic stroke, and was more effective than pure ergosterol.

[0083] (2) The appearance change of the rat brain is one of the important indicators for evaluating the severity of brain injury. At the end of the experiment, the rats were euthanized, the rat brain was separated, and the brain of each group of rats was dried on filter paper and photographed. The results show that Figure 10 A) The brain tissue color of the model group was uneven, and some white or light-colored areas appeared, which represented the cerebral infarction site and ischemic injury. The model group successfully induced brain injury, while ergosterol and nanometer preparation loaded with ergosterol both had different degrees of reduction in ischemic area, suggesting that there was an injury improvement effect, and the protection effect of the nanometer preparation loaded with ergosterol was more significant, closest to the sham operation group. According to the cerebral infarction volume determination results show Figure 10 B), the cerebral infarction volume of the model group was the largest, indicating that the brain injury of the model group was more severe. Compared with the model group, the cerebral infarction volume of the ergosterol group was reduced, and the difference was significant (P<0.01), indicating that ergosterol had a certain improvement effect on the cerebral infarction caused by ischemic stroke. Compared with the ergosterol group, the cerebral infarction volume of the nanometer preparation loaded with ergosterol prepared by the present application was significantly reduced (P<0.01), indicating that the nanometer preparation loaded with ergosterol could significantly improve the cerebral infarction caused by ischemic stroke.

[0084] (3) The HE staining results of the brain tissue directly reflect the brain injury, Figure 10 C can be seen, the tissue section of the sham operation group shows a relatively normal tissue structure, the cells are arranged in order, the intercellular space is uniform, and there is no obvious inflammation or damage; the model group shows obvious tissue damage. The cells are arranged in disorder, the intercellular space is increased, and a large number of inflammatory cells can be seen. Some cavities or necrotic areas appear in the tissue. The ergosterol group shows a certain degree of tissue repair. Compared with the model group, the cells are arranged in order, the inflammatory cell infiltration is reduced, and the necrotic area is reduced. The nanometer preparation loaded with ergosterol shows the best tissue repair effect: the tissue structure is normal, the cells are arranged in order, the inflammatory cell infiltration is significantly reduced, and the necrotic area is the smallest. This indicates that compared with ergosterol, the nanometer preparation loaded with ergosterol prepared by the present application has more significant effect in the treatment of ischemic stroke,

[0085] 2. Western Blot experiment for detecting:

[0086] The expression levels of p-PI3K, p-AKT, p-mTOR, mTOR, PI3K, AKT, etc. in the brain tissue of rats were detected by Western Blot experiment, so as to further verify that ergosterol and its preparation can treat ischemic stroke by activating the PI3K / AKT / mTOR pathway.

[0087] (1) Sample preparation: after the end of the animal experiment, the brain tissue samples of rats were collected, RIPA lysis buffer was added, and they were placed on ice for 50 min to fully lyse. The lysis buffer was transferred to a 1.5 mL centrifuge tube by blowing, and the supernatant was collected by centrifugation at 4°C and 12000 rpm for 20 min; the total protein concentration of each sample was determined by BCA method, and the RIPA lysis buffer was diluted to the same concentration; an appropriate amount of 5xLoading Buffer was added according to the volume of each sample, and it was mixed and denatured in boiling water for 15 min, then vortexed briefly and centrifuged, and it could be directly used for sample detection or stored at -20°C.

[0088] (2) Western Blot experiment was performed to detect the changes of p-PI3K, p-AKT, p-mTOR, mTOR, PI3K, AKT proteins in the brain tissue of rats.

[0089] The results are shown in Figure 11 Compared with the sham operation group, the expression levels of p-PI3K, p-AKT, p-mTOR, mTOR, PI3K, AKT proteins in the model group were significantly reduced (P<0.01), and after drug treatment, ergosterol and the nano-preparation loaded with ergosterol could significantly increase the expression levels of the above proteins, and compared with ergosterol, the nano-preparation loaded with ergosterol had a more obvious increase in the expression levels of the above proteins (P<0.01). The results of Western Blot experiment showed that ergosterol and its preparation could treat ischemic stroke by activating the PI3K / AKT / mTOR pathway, and the nano-preparation loaded with ergosterol had a better therapeutic effect.

[0090] 3. In vivo imaging experiment of rats

[0091] Through in vivo imaging experiment, the distribution of drug preparations in the body of rats was detected, and the brain targeting advantage of the nano-preparation loaded with ergosterol for treating ischemic stroke was intuitively displayed.

[0092] The results are shown in Figure 12As shown, the tail vein of 2 groups of rats was injected with the no pinacol borate nanometer reagent prepared by Comparative Example 1 and the ergosterol-loaded nanometer reagent prepared by Example 2, respectively, and it can be obviously seen that the nanometer reagent prepared by Comparative Example 1 mainly concentrates in the tail and lower abdomen of the rats after tail vein injection, and does not show the targeting property of moving to the brain whether for 1 hour or 4 hours Figure 12 A、 Figure 12 B), on the contrary, the ergosterol-loaded nanometer reagent prepared by the present scheme can be seen to mainly concentrate in the tail and lower abdomen of the rats within 1 hour after injection, indicating that the drug mainly concentrates in these parts within 1 hour after injection Figure 12 C). After 4 hours of tail vein injection of the ergosterol-loaded nanometer reagent, the red fluorescence obviously moves and enriches to the brain region of the rat Figure 12 D), this result directly indicates that the ergosterol-loaded nanometer reagent prepared by the present scheme has good brain targeting property, that is, it can effectively deliver the drug to the brain, and has unique advantages in the treatment of ischemic stroke.

[0093] In summary, the present scheme successfully constructs a new type of ergosterol-loaded nanometer reagent for targeted treatment of ischemic stroke, first successfully synthesizes a new type of nanometer material DSPE-PEG-pinacol borate, and provides the nanometer reagent, its preparation method and application, which can effectively solve the problems of poor drug targeting, low bioavailability and the like, opens up a new treatment method for ischemic stroke, enhances the targeted treatment ability of the drug, at the same time, it is found that ergosterol plays a role in treating ischemic stroke by activating the PI3K / AKT / mTOR pathway, and the above findings provide a new direction for the treatment of ischemic stroke.

[0094] The above only describes the preferred embodiments of the present application patent, and the protection scope of the present application patent is not limited to the above examples. For those skilled in the art, the improvements and changes obtained without departing from the technical concept of the present application patent should also be considered as the protection scope of the present application patent.

Claims

1. A brain-targeted nanoformulation loaded with ergosterol, characterized in that: The nanoformulation comprises ergosterol, pinacol borate and DSPE-PEG; the pinacol borate modifies DSPE-PEG to form a DSPE-PEG-pinacol borate nanomaterial encapsulating ergosterol; The preparation method for forming the DSPE-PEG-pinacol borate nanomaterial comprises the following steps: S1. Activated pinacol borate: 4-hydroxymethylphenylboronic acid pinacol ester and N,N'-carbonyldiimidazole were mixed and added to anhydrous dichloromethane for reaction, and anhydrous dichloromethane was added for extraction after the reaction was completed. The organic phase was further extracted with a saturated sodium chloride solution and dried over anhydrous sodium sulfate to obtain activated pinacol borate; the mass ratio of the 4-hydroxymethylphenylboronic acid pinacol ester to the N,N'-carbonyldiimidazole was (1-2): (1-2.5), and the stirring time was 1 hour; S2. Preparation of nanomaterial DSPE-PEG-pinacol borate: The activated pinacol borate prepared in S1 is dissolved in dimethyl sulfoxide with β-cyclodextrin, 4-dimethylaminopyridine, and DSPE-PEG for reaction. Anhydrous ether is added to the reaction solution for precipitation, and the mixture is washed with anhydrous ether. The precipitate is collected by centrifugation and freeze-dried under vacuum to obtain the nanomaterial DSPE-PEG-pinacol borate. The mass ratio of the activated pinacol borate, the β-cyclodextrin, and the 4-dimethylaminopyridine is (1-2): (0.1-1): (0.5-1).

2. The brain-targeted nanoformulation loaded with ergosterol according to claim 1, characterized in that The nanoformulation has a potential of -23.88±0.38 mV, a particle size of 30 nm, a drug loading of 8.05% to 18.33%, and an encapsulation efficiency of 85.20% to 93.77%.

3. A method for preparing the brain-targeted nanoformulation loaded with ergosterol according to any one of claims 1 to 2, characterized in that: The following steps are involved: S1. Activated pinacol borate: 4-hydroxymethylphenylboronic acid pinacol ester and N,N'-carbonyldiimidazole were mixed and added to anhydrous dichloromethane for reaction, and anhydrous dichloromethane was added for extraction after the reaction was completed. The organic phase was further extracted with a saturated sodium chloride solution and dried over anhydrous sodium sulfate to obtain activated pinacol borate; the mass ratio of the 4-hydroxymethylphenylboronic acid pinacol ester to the N,N'-carbonyldiimidazole was (1-2): (1-2.5), and the stirring time was 1 hour; S2. Preparation of nanomaterial DSPE-PEG-pinacol borate: The activated pinacol borate prepared in S1 is dissolved in dimethyl sulfoxide with β-cyclodextrin, 4-dimethylaminopyridine, and DSPE-PEG for reaction. Anhydrous ether is added to the reaction solution for precipitation, and the reaction solution is washed with anhydrous ether. The precipitate is collected by centrifugation and freeze-dried under vacuum to obtain the nanomaterial DSPE-PEG-pinacol borate. The mass ratio of the activated pinacol borate, the β-cyclodextrin, and the 4-dimethylaminopyridine is (1-2): (0.1-1): (0.5-1). S3. Preparation of nanoformulation: ergosterol and DSPE-PEG-pinacol borate prepared in S2 are placed in a round-bottom flask, wherein the mass ratio of ergosterol to DSPE-PEG-pinacol borate is 1:(4-12). An organic solvent is added and stirred until completely dissolved. The mixture is connected to a rotary evaporator and slowly rotary evaporated until the organic solvent is completely evaporated and a lipid film is formed on the inner wall of the flask. PBS buffer is added and stirred in a 37°C water bath to fully hydrate the lipid film to form a suspension. The suspension is purified by ultrasound and dialysis and then freeze-dried to obtain the nanoformulation.

4. The preparation method according to claim 3, characterized in that In step S3, the organic solvent is selected from any one or more of methanol, dichloromethane, and chloroform.

5. The preparation method according to claim 4, characterized in that The temperature of the slow rotary evaporation in step S3 is from room temperature to 45° C., and the pressure is 0.05-0.15 MPa.

6. Use of the ergosterol-loaded brain-targeted nanoformulation according to claim 1 or the ergosterol-loaded brain-targeted nanoformulation prepared by the preparation method according to any one of claims 3 to 5 in the preparation of a drug for the targeted treatment of brain diseases; the brain disease is ischemic stroke.

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