Brain-targeted nano preparation loaded with ergosterol as well as preparation method and application of brain-targeted nano preparation

Abstract

The invention provides an ergosterol-loaded brain-targeted nano preparation and a preparation method and application thereof, and relates to the field of nano materials and nano biological medicine.The active ingredient ergosterol of the nano preparation is entrapped in a DSPE-PEG-pinacol borate nano material, and the nano material has the ROS response characteristic and can be used for preparing a brain-targeted nano preparation. The ergosterol-loaded nano preparation is in the shape of a regular sphere, is uniform in particle size, has good dispersibility and stability, has good ability of targeted therapy of cerebral arterial thrombosis diseases, effectively solves the problems of poor targeting property, low bioavailability and the like of common drugs, enhances the therapeutic effect of ergosterol on cerebral arterial thrombosis, and has good application prospects. The ergosterol can effectively improve brain injury caused by the cerebral arterial thrombosis and has a good treatment effect, meanwhile, the ergosterol serving as the active ingredient plays a role in treating the cerebral arterial thrombosis by activating a PI3K / AKT / mTOR pathway, and a new direction is provided for treatment of the cerebral arterial thrombosis.

Description

Technical Field

[0001] The present invention relates to the fields of nanomaterials and nanobiomedicine, and in particular to a brain-targeted nano preparation loaded with ergosterol, and a preparation method and application thereof. Background Art

[0002] Cerebral stroke, commonly known as "stroke", is an acute disease caused by a disease of the blood vessels that transport blood to the brain. It is characterized by high morbidity, high disability, high recurrence and high mortality, 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 them, ischemic stroke accounts for about 75% to 85% of the total number of stroke patients, seriously affecting the quality of life of patients.

[0003] The pathological state of IS refers to the interruption of blood flow in a part of the brain, which causes the mitochondrial energy metabolism to switch to anaerobic glycolysis, resulting in a decrease in the production of ATP, and an interruption of the oxygen and sugar supply to the cells in the ischemic center, causing a large release of the excitatory amino acid glutamate in the central nervous system, which accumulates in the synaptic cleft, produces excitotoxicity to neuronal cells, triggers intracellular calcium overload, causes mitochondrial dysfunction, and causes the production of excessive free radicals such as ROS. Excessive ROS can damage protein molecules in cells, causing autophagy and apoptosis of cells, further damaging tissues.

[0004] IS treatment mainly includes surgical treatment and drug treatment, but it faces the following problems: (1) Surgical treatment: The blood perfusion of the ischemic area of ​​the brain is restored by surgery, but the time window is strictly limited. The surgery needs to be performed within a short time after the onset of the disease, otherwise the best treatment time may be missed, resulting in poor surgical results or even inability to perform the surgery; the risk is high, and the surgical process itself may cause additional damage to the brain tissue, increasing the patient's pain and the difficulty of postoperative recovery. The prognosis is uncertain. Even if the operation is successful, the patient's prognosis is difficult to accurately predict, and different degrees of neurological dysfunction may still remain; (2) Drug treatment: ① Poor drug targeting: When ordinary drugs are used to treat ischemic stroke, it is difficult to accurately reach the lesion site of the brain, resulting in limited treatment effects. ② Low bioavailability: The drug cannot fully exert its efficacy during absorption, distribution, metabolism and excretion in the body, resulting in drug waste. It may also be necessary to increase the drug dosage, thereby increasing the risk of adverse reactions. (3) Lack of effective therapeutic drugs: There are limited clinical treatments for ischemic stroke. The current focus of drug treatment is to restore blood perfusion to the ischemic area of ​​the brain by giving thrombolytic drugs to quickly dissolve the blood. However, the only effective acute treatment drug certified by the FDA, tissue plasminogen activator t-PA, has a short optimal treatment time window (≤4.5h) and is prone to complications such as reperfusion injury and cerebral hemorrhage. New therapeutic drugs and methods are urgently needed. Summary of the invention

[0005] In order to solve the above technical problems, this scheme provides a brain-targeted nanoformulation loaded with ergosterol, and its preparation method and application. Through bioinformatics analysis and in vitro and in vivo verification experiments, it was found that ergosterol can prevent and treat IS by activating the PI3K / AKT / mTOR signaling pathway. Combined with the pathological state of IS, a ROS-responsive nanomaterial DSPE-PEG-pinacol borate was synthesized, and ergosterol was encapsulated to obtain an ergosterol nanoformulation to achieve targeted treatment of IS. The nanoformulation can effectively solve the problems of poor drug targeting and low bioavailability, open up a new treatment method for ischemic stroke, and enhance the targeted treatment ability of drugs. At the same time, it was found that ergosterol plays a role in treating ischemic stroke by activating the PI3K / AKT / mTOR pathway.

[0006] To achieve the above object, the present invention first provides a brain-targeted nanoformulation loaded with ergosterol, the nanoformulation comprising ergosterol, pinacol borate and DSPE-PEG, wherein the pinacol borate modifies DSPE-PEG to form a DSPE-PEG-pinacol borate nanomaterial encapsulating ergosterol.

[0007] Preferably, the nano-preparation has a potential of -23.88±0.38, 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%.

[0008] Based on a general inventive concept, the present invention also provides a method for preparing a nanoformulation, comprising the following steps:

[0009] S1. Activated pinacol borate: 4-hydroxymethylphenylboronic acid pinacol ester and N,N-carbonyldiimidazole were mixed and added into anhydrous dichloromethane for reaction. After the reaction, anhydrous dichloromethane was added for extraction. The mixture was washed with deionized water for three times. The organic phase was further extracted with a saturated sodium chloride solution and then dried with anhydrous sodium sulfate to obtain activated pinacol borate.

[0010] S2, preparation of nanomaterial DSPE-PEG-pinacol borate: the activated pinacol borate prepared in S1 is dissolved in dimethyl sulfoxide with DSPE-PEG, β-cyclodextrin, 4-dimethylaminopyridine and DSPE-PEG for reaction, anhydrous ether is added to the reaction solution for precipitation, anhydrous ether is washed, the precipitate is collected by centrifugation, and freeze-dried under vacuum to obtain the nanomaterial DSPE-PEG-pinacol borate;

[0011] S3. Preparation of nanoformulations: Take ergosterol and DSPE-PEG-pinacol borate prepared in S2, place them in a round-bottom flask, add an organic solvent, stir until completely dissolved, connect to a rotary evaporator and slowly rotate and evaporate until the organic solvent is completely evaporated and a lipid film is formed on the inner wall of the flask, add PBS buffer, stir in a 37°C water bath to fully hydrate the lipid film to form a suspension, purify the suspension by ultrasound and dialysis, and then freeze-dry to obtain the nanoformulation.

[0012] Preferably, in step S1, the mass ratio of 4-hydroxymethylphenylboronic acid pinacol ester to N,N-carbonyldiimidazole is 1-2:1-2.5, and the stirring time is 1 hour.

[0013] Preferably, in step S2, the mass ratio of activated pinacol borate, β-cyclodextrin and 4-dimethylaminopyridine is 1-2:0.1-1:0.5-1.

[0014] Preferably, in step S3, the mass ratio of ergosterol to DSPE-PEG-pinacol borate is 1:4-12.

[0015] Preferably, the organic solvent in step S3 is selected from any one or more of methanol, dichloromethane and chloroform.

[0016] Preferably, 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.

[0017] Based on a general inventive concept, the present invention also provides an application of a brain-targeted nanoformulation loaded with ergosterol in the targeted treatment of brain diseases.

[0018] Preferably, the brain disease includes ischemic stroke.

[0019] The therapeutic mechanism of the nanoformulation in this scheme is as follows:

[0020] This scheme discovered for the first time that ergosterol can prevent and treat ischemic stroke by activating the PI3K / AKT / mTOR signaling pathway, and based on this, a nano-preparation was constructed. When ischemic stroke occurs, ergosterol in the nano-preparation can accurately act on the signaling pathway, thereby protecting nerve cells, reducing nerve cell death caused by ischemic stroke, maintaining the stability of the intracellular environment, protecting the normal function of nerve cells, and regulating the expression and signal transduction of VEGF, promoting endothelial cell proliferation, migration and lumen formation, and facilitating angiogenesis. In addition, it can provide a cell source for nerve regeneration, help damaged nerve cells re-establish connections, and restore nerve function. However, there is a lack of drugs or treatment methods that can precisely regulate this pathway in the prior art. This scheme is based on the new discovery of the mechanism of action of ergosterol and the innovative design of nano-preparations, providing a new mechanism for the treatment of ischemic stroke. Unlike the relatively single treatment mechanisms mainly based on thrombolysis or surgery in the prior art, this scheme plays a comprehensive role in cell signaling pathway regulation, neuroprotection, angiogenesis and nerve regeneration, and has higher innovation and potential therapeutic value.

[0021] The nano-preparation ergosterol targeted micelles prepared by encapsulating the active drug ergosterol with the nanomaterial DSPE-PEG-pinacol borate in this scheme have ROS response characteristics. In the ischemic stroke site, due to the presence of excessive ROS, the nano-preparation can quickly respond and release ergosterol. The released ergosterol activates the PI3K / AKT / mTOR pathway in time, and this synergistic effect makes the treatment more accurate and effective. However, the drugs or preparations in the prior art often lack this responsive release mechanism adapted to the pathological environment, and it is difficult to play a role at the right time and location. In addition, the particle size, potential and other characteristics of the nano-preparation are conducive to its interaction with the cell membrane, promoting the cell's uptake of ergosterol. After the cell uptake increases, ergosterol can more effectively enter the cell to activate the PI3K / AKT / mTOR pathway, which provides more opportunities for ergosterol to play a role in the cell and activate the pathway, and the cell uptake efficiency of ordinary drugs in the prior art is higher.

[0022] Through bioinformatics analysis, it was found that the phosphatidylinositol 3-kinase (PI3K) / serine-threonine protein kinase (Akt) / -mTOR signaling pathway plays an important role in the process of IS. The 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 inhibiting Bad protein and caspase-9, preventing the release of cytochrome C, and inhibiting cell apoptosis caused by ischemia. This pathway can also regulate ion channels and transporters on the cell membrane, help 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, etc. It can also regulate the expression and signal transduction of VEGF, promote vascular endothelial cell proliferation, migration and lumen formation, and facilitate angiogenesis. The PI3K / AKT / mTOR pathway can stimulate the proliferation and differentiation of neural stem cells, guide neural stem cells to differentiate into neurons, provide a cell source for nerve regeneration, help damaged nerve cells reconnect, and restore nerve function.

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

[0024] 1. The nanomaterial DSPE-PEG-pinacol borate and the brain-targeted nanoformulation loaded with ergosterol prepared in this scheme are regular spherical or quasi-spherical, have good dispersibility, and uniform particle size distribution, which is conducive to circulation and distribution in the body and is easier to reach the lesion site of the brain. The absolute value of the Zeta potential value of the brain-targeted nanoformulation loaded with ergosterol is relatively high, which effectively increases the stability of the nanoformulation, enables it to maintain structural integrity during 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 nanoformulation has ROS response characteristics. In the simulated ischemic stroke environment, the cumulative release rate of the nanoformulation loaded with ergosterol is higher and can maintain a high release level. This indicates that when ischemic stroke occurs, the locally generated ROS can trigger the nanoformulation to quickly decompose and release the drug, achieve targeted release of the drug, increase the concentration of the drug in the lesion site, enhance the therapeutic effect, and reduce the toxic side effects on normal tissues.

[0026] 3. This study discovered for the first time that ergosterol and its nanoformulations can play a therapeutic role in ischemic stroke by activating the PI3K / AKT / mTOR pathway, which can better regulate intracellular signal transduction, inhibit cell apoptosis, promote cell survival and functional recovery, and thus play a therapeutic role in ischemic stroke.

[0027] 4. This solution is expected to effectively solve the problems of poor targeting, low bioavailability and large side effects when ordinary preparations are administered, enhance the therapeutic effect of ergosterol on ischemic stroke, and show better efficacy, safety and application prospects in the treatment of ischemic stroke. It is expected to improve the treatment effect and prognosis of patients and improve their quality of life; it provides new ideas and methods for the treatment of ischemic stroke, opens up a treatment direction based on nanopreparations and regulation of specific signal pathways, and provides important reference and reference for subsequent related research and drug development. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 The synthetic route of the nanomaterial DSPE-PEG-pinacol borate in Example 1;

[0030] Figure 2 is the H NMR spectrum of the nanomaterial DSPE-PEG-pinacol borate in Experimental Example 1 ( Figure 2 A) NMR C spectrum of DSPE-PEG-pinacol borate ( Figure 2 B) DSPE-PEG-OH / pinacol borate UV spectrum ( Figure 2 C) DSPE-PEG-OH / Pinacol borate infrared spectrum ( Figure 2 D)

[0031] Figure 3 The DLS particle size result of the nanomaterial DSPE-PEG-pinacol borate in Experimental Example 1;

[0032] Figure 4 The blank nanoformulation of the nanomaterial in Experimental Example 2, the nanoformulation prepared by loading the drug ergosterol, and H 2 O 2 Transmission electron microscopy images of the treated ergosterol-loaded nanoformulations; A is a blank nanoformulation, B is a nanoformulation loaded with ergosterol, and C is H 2 O 2 The treated ergosterol-loaded nanoformulations;

[0033] Figure 5 The blank nanoformulation formed by the nanomaterials in Experiment 2, the nanoformulation prepared by loading the drug ergosterol, and H2 O 2 The particle size test results of the treated ergosterol-loaded nanoformulations; A is the blank nanoformulation, B is the nanoformulation loaded with ergosterol, and C is the H 2 O 2 The treated ergosterol-loaded nanoformulations;

[0034] Figure 6 The results of cellular uptake assay of ergosterol-loaded nanoformulation prepared in Experiment 2;

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

[0036] Figure 8 The ergosterol drug and the ergosterol-loaded nanoformulation in Experimental Example 2 were respectively prepared at pH 7.4 and pH 7.4 + 100 μM H 2 O 2 Drug release results in the medium; A is the drug release result in pH 7.4 medium, B is the drug release result in pH 7.4 + 100 μM H 2 O 2 Drug release results in the medium;

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

[0038] Fig.10 Figure 3 shows the brain tissue, cerebral infarction volume and HE staining of rats in Experimental Example 3. A is the result of TTC staining 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 the brain tissue morphology of rats in different treatment groups;

[0039] Fig.11 The effect 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] Fig.12 The ergosterol nanoparticle preparation prepared in Comparative Example 1 ( Fig.12 A. Fig.12B) and the ergosterol-loaded nanoformulation prepared in Example 2 ( Fig.12 C. Fig.12 D) In ​​vivo imaging results of rats after 1 hour and 4 hours of drug administration. DETAILED DESCRIPTION

[0041] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0042] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention are within the scope of the present invention.

[0043] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art; unless otherwise specified, the reagents used in the examples are commercially available.

[0044] Example 1 Preparation of Nanomaterial DSPE-PEG-Pinacol Borate

[0045] S1. Activated pinacol borate: accurately weigh 1.8 g of 4-hydroxymethylphenylboronic acid pinacol ester (PBAP) and 2.2 g of N,N-carbonyldiimidazole (CDI), place in a 50 mL double-necked bottle, add 20 mL of anhydrous dichloromethane (DCM), wait for them to dissolve, react on a constant temperature magnetic stirrer for one hour, then add double DCM for extraction, wash with deionized water for 3 times, further extract the organic phase with a saturated NaCl solution, and finally dry with an appropriate amount of anhydrous sodium sulfate to obtain activated pinacol borate CDI-PBAP;

[0046] S2. Preparation of nanomaterial DSPE-PEG-pinacol borate: 1.6 g of CDI-PBAP, 0.35 g of β-cyclodextrin (β-CD), 0.9 g of 4-dimethylaminopyridine (DMAP), and 0.4 g of DSPE-PEG prepared in step S1 were added to DMSO for reaction. Anhydrous ether was added to the reaction solution for precipitation. The mixture was washed with anhydrous ether for 3 times. The precipitate was collected by centrifugation and freeze-dried under vacuum for about 24 h to obtain the nanomaterial DSPE-PEG-pinacol borate.

[0047] The synthetic route of the nanomaterial DSPE-PEG-pinacol borate is as follows: Figure 1 shown.

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

[0049] S1. Weigh 5 mg of ergosterol and 40 mg of DSPE-PEG-pinacol borate prepared in Example 1, place in a round-bottom flask, add an appropriate amount of chloroform, use a magnetic stirrer to stir at room temperature until the material and the drug are completely dissolved, connect the round-bottom flask to a rotary evaporator, and slowly perform rotary evaporation at 40° C. and 0.1 MPa until the organic solvent is completely evaporated, forming a uniform, thin and transparent lipid film on the inner wall of the flask;

[0050] S2. Slowly add an appropriate amount of PBS buffer to the flask where the lipid film is formed, gently stir in a 37°C water bath to fully hydrate the lipid film, and then subject the resulting suspension to ultrasonic treatment and dialysis purification, and then freeze-drying to obtain the brain-targeted nanoformulation loaded with ergosterol.

[0051] Comparative Example 1 Preparation of ergosterol-loaded nanoformulation without pinacol borate

[0052] S1. Preparation of nanomaterial DSPE-PEG: 0.35 g of β-cyclodextrin (β-CD), 0.9 g of 4-dimethylaminopyridine (DMAP) and 0.4 g of DSPE-PEG were added to DMSO for reaction, anhydrous ether was added to the reaction solution for precipitation, and the anhydrous ether was washed three times, and the precipitate was collected by centrifugation, and freeze-dried under vacuum for about 24 h to obtain the nanomaterial DSPE-PEG;

[0053] S2. Weigh 5 mg of ergosterol and 40 mg of DSPE-PEG prepared in Example 1, place in a round-bottom flask, add an appropriate amount of chloroform, stir at room temperature with a magnetic stirrer until the material and the drug are completely dissolved, connect the round-bottom flask to a rotary evaporator, and slowly rotary evaporate until the organic solvent is completely evaporated to form a uniform, thin and transparent lipid film on the inner wall of the flask;

[0054] S2. Slowly add an appropriate amount of PBS buffer to the flask where the lipid film is formed, gently stir in a 37°C water bath to fully hydrate the lipid film, and then subject the resulting suspension to ultrasonic treatment and dialysis purification, followed by freeze-drying to obtain the ergosterol-loaded nanoformulation without pinacol borate.

[0055] Experimental Example 1 Characterization of Nanomaterial DSPE-PEG-Pinacol Borate

[0056] (1) H NMR spectrum, C NMR spectrum, UV and IR characterization

[0057] Take an appropriate amount of the nanomaterial DSPE-PEG-pinacol borate prepared in Example 1, dissolve it in deuterated dimethyl sulfoxide to make the sample concentration reach 1-10 mM, transfer it to a nuclear magnetic resonance tube, put the nuclear magnetic resonance tube into a nuclear magnetic resonance spectrometer, and perform nuclear magnetic H spectrum and C spectrum determination;

[0058] The nanomaterial DSPE-PEG-pinacol borate is ground into fine powder, and then mixed evenly with dry potassium bromide powder in proportion. The mixture is then placed in a tablet pressing mold, and a certain pressure is applied using a tablet press. The pressure is maintained for several minutes to form a transparent sheet that can be used for infrared detection.

[0059] The results of nuclear magnetic resonance testing are as follows Figure 2 A. Figure 2 B shows the UV spectrum. Figure 2 C shows the infrared detection results. Figure 2 As shown in D, based on the above test results, it can be determined that the nanomaterial DSPE-PEG-pinacol borate was successfully prepared.

[0060] (2) Particle size

[0061] Take an appropriate amount of nanomaterial DSPE-PEG-pinacol borate, dilute it to a suitable concentration with ultrapure water and add it to the sample pool to detect the particle size of the prepared nanomaterial DSPE-PEG-pinacol borate. The test results are as follows: Figure 3 As shown, according to the test results, the average particle size of the prepared nanomaterial is 26.92nm, the PDI is 0.234, and the particle size distribution is uniform.

[0062] Experimental Example 2 Basic Characterization of Brain-Targeted Nanoformulations Loaded with Ergosterol

[0063] (1) Morphological characterization

[0064] After the copper mesh for transmission electron microscopy was hydrophilized, blank material DSPE-PEG-pinacol borate with a concentration of about 250 μg / mL, the ergosterol-loaded nanoformulation prepared in Example 2, and H 2 O 2 The treated ergosterol-loaded nanoformulation solution was dripped onto the front of the copper mesh; after standing for 2 minutes, an appropriate amount of phosphotungstic acid dye was dripped, and the liquid on the copper mesh was dried with filter paper, and the phosphotungstic acid dye was dripped again, and after standing for 2 minutes, the liquid was dried with filter paper; after repeating the above phosphotungstic acid staining once, it was placed under an incandescent lamp to dry; the copper mesh was placed in a transmission electron microscope (TEM) to examine the sample morphology.

[0065] Test results such as Figure 4 As shown in the figure, the nanomaterial DSPE-PEG-pinacol borate and the nanoformulation loaded with ergosterol are regular spherical or quasi-spherical, with good dispersibility. After loading ergosterol, the structure of the nanomaterial does not change significantly, indicating that the nanomaterial has a good encapsulation capacity for ergosterol. 2 O 2The treated ergosterol-loaded nanoformulations showed obvious changes. The distribution of particles was no longer uniform, and the morphology of some particles was observed to have changed, showing signs of decomposition, which provided intuitive evidence for studying the drug release mechanism and stability of the nanoformulations.

[0066] (2) Determination of particle size, potential and PDI

[0067] The blank material DSPE-PEG-pinacol borate, the nanoformulation loaded with ergosterol, H 2 O 2 The particle size, potential and PDI of the treated ergosterol-loaded nanoformulation were characterized. Determination of particle size and PDI: After the instrument is preheated for 30 minutes, wash the quartz cuvette 2-3 times with purified water, dilute the nanoformulation in a certain proportion with purified water and add it to the cuvette. Set each sample to cycle 3 times and set the single cycle time. Select "Water" as the test solvent. Start the measurement after the instrument is stable. The particle size test results are as follows: Figure 5 The PDI result of the nanoformulation loaded with ergosterol was 0.196±0.002. According to the characterization results, the particle size of the nanomaterial DSPE-PEG-pinacol borate was uniformly distributed around 30 nm, and there was no obvious change before and after drug loading. However, after H 2 O 2 The particle size of the treated ergosterol-loaded nanoformulations was significantly increased, indicating that H 2 O 2 The treatment can simulate the rapid decomposition of targeted nanoformulations caused by the ischemic stroke environment to release drugs, which may enhance the therapeutic effect on ischemic stroke and reduce the toxic side effects on normal tissues.

[0068] Determination of potential: After the instrument is preheated for 30 minutes, wash the quartz cuvette with purified water for 2-3 times, dilute the ergosterol nanoformulation in a certain proportion with purified water and add it to the cuvette, insert the electrode into the cuvette and connect it to the instrument, set the cycle time and number of cycles, select "Water" as the test solvent, and start measuring after the instrument stabilizes. The potential of the nanoformulation loaded with ergosterol is measured to be -23.88±0.38, and the absolute value of the Zeta potential is relatively high, which effectively increases the stability of the nanoformulation loaded with ergosterol and improves the transmembrane activity of the drug, thereby promoting the improvement of the bioavailability of the drug.

[0069] (3) Cell uptake experiment

[0070] Ergosterol labeled with Cy2 (480 nm, emission wavelength at 508 nm) was prepared according to the above method to prepare Cy2-loaded ergosterol nanoformulation. PC12 cells in the logarithmic growth phase were seeded on a six-well plate (1×105 / mL, 2mL), and discard the culture medium after the cells adhere to the wall. The two preparations with a labeled concentration of 20nM were incubated with HCT116 cells in a confocal culture dish. The cells were cultured for 2, 4, and 12 hours. The cells were washed three times with pre-cooled PBS and then incubated with DAPI (5μg / mL) to facilitate observation of the cells under a fluorescence microscope.

[0071] Test results such as Figure 6 As shown in the figure, after 1 hour of incubation, a small amount of green fluorescence can be detected around the cell nucleus. As the incubation time increases, the amount of Cy2-loaded ergosterol nanoformulation absorbed by PC12 cells increases after 2 hours, 4 hours, and 6 hours, and the fluorescence intensity increases. This indicates that the Cy2-loaded ergosterol nanoformulation can be effectively taken up by cells, thereby exerting a therapeutic effect.

[0072] (4) Stability testing of ergosterol-loaded nanoformulations

[0073] The stability of the nanoformulation loaded with ergosterol at different storage times was investigated. The samples were stored in sealed vials at 4°C for 30 days. Samples were taken at the corresponding time (1, 15, and 30 days) to measure their particle size distribution, Zeta potential, and encapsulation efficiency to evaluate the storage stability of the nanoformulation loaded with ergosterol.

[0074] The results of the test are as follows Figure 7 As shown in the results, after 30 days of preliminary stability test, the particle size distribution, encapsulation efficiency and Zeta potential of the ergosterol-loaded nanoformulation increased numerically within 30 days, with the drug loading being 8.05% to 18.33% and the encapsulation efficiency being 85.20% to 93.77%. However, there was no statistically significant change (P>0.05), indicating that the nanoformulation had good stability and could be stably stored in the above environment.

[0075] (5) In vitro release of nanoformulations

[0076] The in vitro release of ergosterol and ergosterol-loaded nanoformulations was determined by dialysis. Ergosterol and ergosterol-loaded nanoformulations were placed in a dialysis device with a molecular weight cutoff of 100 kDa. The buffer medium was pH 7.4 PBS and pH 7.4 PBS + 100 μM H 2 O 2 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 supplemented with the corresponding solution. The content of the released ergosterol was detected by HPLC.

[0077] The results are as follows Figure 8As shown in Figure 2, the cumulative release rate of the nanoformulation loaded with ergosterol in the medium with pH 7.4 was significantly higher than that of ergosterol. 2 O 2 Under the environment, the cumulative release rate of the ergosterol-loaded nanoparticles was higher and maintained at a high release level throughout the process, while the cumulative release rate of ergosterol itself was relatively low. This shows that the preparation of ergosterol-loaded nanoparticles can effectively improve drug stability and reduce the impact of the external environment. 2 O 2 The treatment can simulate the ischemic stroke environment, causing the nanoformulation to rapidly decompose and release the drug.

[0078] Experimental Example 3: Application of ergosterol-loaded nanoformulations in the treatment of ischemic stroke

[0079] Construction of rat ischemic stroke model: SD rats were selected, weighing 300 g ± 10 g, aged 11-12 weeks, anesthetized with chloral hydrate, and fixed on a mouse board in supine position. The right common carotid artery (CCA), internal carotid artery (ICA) and external carotid artery (ECA) were bluntly separated with ophthalmic forceps, and the hanging line was prepared. The proximal ends of ECA and CCA were ligated respectively, and the ICA was temporarily clamped with an artery clamp. The CCA was picked up with ophthalmic forceps to cut a small hole, and the prepared ligature was quickly inserted from CCA to ICA. When the top of the ligature felt a little resistance, it was stopped. At this time, the prepared line on CCA was tightened to prevent the ligature from moving out and bleeding. The spare hanging line was ligated and the occlusion time was recorded. After 1 hour of ischemia, the line plug was pulled out and reperfused. The surgical steps of the sham operation group were the same as above, but the insertion depth of the ligature was only 15 mm, and the blood flow of the middle cerebral artery was not blocked. When the rat's tail was lifted and suspended in the air, the left forelimb flexed and adducted, and a typical tail-chasing sign was present during movement, indicating that the model was successfully established.

[0080] 1. Effects of ergosterol and ergosterol-loaded nanoformulations on neurological function and pathological states in rats

[0081] (1) The Longa scoring method is used to quantify the degree of neurological deficit of animals through a series of behavioral observations. The scoring range is from 0 to 4 points, where 0 points indicates no neurological deficit, 1 point indicates that the paralyzed front paw cannot be fully extended, 2 points indicates turning in circles toward the paralyzed side when walking, 3 points indicates leaning toward the paralyzed side when walking, and 4 points indicates that the animal cannot walk automatically and has loss of consciousness. The higher the score, the worse the animal's neurobehavioral condition and the more severe the neurological deficit. In the experiment, the Longa scoring method can be used to preliminarily assess whether the model is successful. Usually, animals with scores of 1-3 are included in the next experiment, while animals with scores of 4-5 are excluded because this may indicate that the animal's condition is not suitable for participating in the experiment. Rats in each group were scored before treatment and on days 1, 3, 7, 14, and 28 after treatment.

[0082] The results of the test are as follows Fig. 9 As shown in the figure, the Longa score of the model group was the highest, indicating that the neurological damage of the rats in the model group was more serious. Compared with the model group, the score of the ergosterol group was lower, and the difference was significant (P < 0.01), indicating that ergosterol can improve the neurological damage caused by ischemic stroke to a certain extent. Compared with the ergosterol group, the score of the ergosterol-loaded nanoformulation group prepared in this scheme was significantly reduced (P < 0.01), indicating that the ergosterol-loaded nanoformulation can significantly and effectively improve the neurological damage caused by ischemic stroke, which is more effective than simple ergosterol.

[0083] (2) The changes in the appearance of the rat brain are one of the important indicators for assessing the severity of brain damage. At the end of the experiment, the rats were euthanized, the rat brains were separated, filtered and dried, and the brains of the rats in each group were photographed. The results showed that ( Fig.10 A) The color of brain tissue in the model group was uneven, with some white or light-colored areas, which represented the site of cerebral infarction and ischemic damage. The model group successfully induced brain damage, while ergosterol and ergosterol-loaded nanoformulations had different degrees of reduction in ischemic areas, indicating an improvement in damage. Among them, the protective effect of ergosterol-loaded nanoformulations was more significant and closest to the sham operation group. According to the results of cerebral infarction volume measurement, ( Fig.10 B), the cerebral infarction volume of the model group was the largest, indicating that the brain damage of the rats in the model group was more serious. 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 cerebral infarction caused by ischemic stroke. Compared with the ergosterol group, the cerebral infarction volume of the nanoformulation group loaded with ergosterol prepared by the present invention was significantly reduced (P < 0.01), indicating that the nanoformulation loaded with ergosterol can significantly improve cerebral infarction caused by ischemic stroke.

[0084] (3) HE staining results of brain tissue directly reflect the brain damage. Fig.10 C As can be seen, the tissue sections of the sham operation group showed relatively normal tissue structure, the cells were arranged more neatly, the gaps between cells were uniform, and there were no obvious signs of inflammation or damage; the model group showed obvious tissue damage. The cells were arranged in disorder, the cell gaps were enlarged, and more inflammatory cells infiltrated. Some cavities or necrotic areas appeared in the tissue. The ergosterol group showed a certain degree of tissue repair. Compared with the model group, the cells were arranged more neatly, the infiltration of inflammatory cells was reduced, and the necrotic area was reduced. The ergosterol-loaded nanoformulation group showed the best tissue repair effect: normal tissue structure, neat cell arrangement, significantly reduced inflammatory cell infiltration, and the smallest necrotic area. This shows that compared with ergosterol, the ergosterol-loaded nanoformulation prepared in this scheme has a more significant effect in the treatment of ischemic stroke.

[0085] 2. Western Blot assay:

[0086] Through Western Blot experiments, the expression levels of p-PI3K, p-AKT, p-mTOR, mTOR, PI3K, AKT and other proteins in rat brain tissue were measured to further verify that ergosterol and its preparations can treat ischemic stroke by activating the PI3K / AKT / mTOR pathway.

[0087] (1) Sample preparation: After the animal experiment, collect rat brain tissue samples, add RIPA lysis buffer, and place on ice for 50 minutes to fully lyse. Transfer the lysis buffer to a 1.5mL centrifuge tube by pipetting, centrifuge at 4℃, 12000rpm for 20 minutes, and collect the supernatant by centrifugation; use the BCA method to determine the total protein concentration of each sample, and dilute it to the same concentration with RIPA lysis buffer; add an appropriate amount of 5× Loading Buffer according to the volume of each sample, mix well, and continue to denature in boiling water for 15 minutes, vortex briefly, and centrifuge. It can be used directly for sample detection or stored at -20℃.

[0088] (2) Western Blot experiments were performed to determine the changes in p-PI3K, p-AKT, p-mTOR, mTOR, PI3K, and AKT proteins in rat brain tissue.

[0089] The results are as follows Fig.11As shown in the figure, compared with the sham operation group, the expression levels of p-PI3K, p-AKT, p-mTOR, mTOR, PI3K, and AKT proteins in the model group were significantly reduced (P < 0.01). After drug treatment, ergosterol and ergosterol-loaded nanoformulations can significantly increase the expression levels of the above proteins, and compared with ergosterol, the expression levels of the above proteins in the ergosterol-loaded nanoformulations increased more significantly after treatment (P < 0.01). The results of Western Blot experiments showed that ergosterol and its preparations can treat ischemic stroke by activating the PI3K / AKT / mTOR pathway, and the ergosterol-loaded nanoformulations have better therapeutic effects.

[0090] 3. Rat in vivo imaging test

[0091] Through in vivo imaging experiments, the distribution of drug preparations in rat body parts was detected, intuitively demonstrating the brain-targeted advantages of the ergosterol-loaded nanoformulations prepared in this scheme for the treatment of ischemic stroke.

[0092] The results are as follows Fig.12 As shown, two groups of rats were injected with the pinacol borate-free nanoagent prepared in Comparative Example 1 and the ergosterol-loaded nanoagent prepared in Example 2 through the tail vein, respectively. It can be clearly seen that the nanoagent prepared in Comparative Example 1 was mainly concentrated in the tail and lower abdomen of the rats, whether it was 1 hour or 4 hours after the tail vein injection, and did not show targeting to the brain ( Fig.12 A. Fig.12 B), on the contrary, the ergosterol-loaded nanoreagent prepared in this scheme showed red fluorescence mainly concentrated in the tail and lower abdomen of the rat within 1 hour after injection, indicating that the drug was mainly concentrated in these areas within 1 hour after injection ( Fig.12 C). 4 hours after the tail vein injection of ergosterol-loaded nanoformulation, the red fluorescence was significantly shifted and enriched in the brain area of ​​the rat ( Fig.12 D), this result intuitively shows that the ergosterol-loaded nanoreagent prepared by this scheme has good brain targeting performance, that is, it can effectively deliver drugs to the brain and has unique advantages in the treatment of ischemic stroke.

[0093] In summary, this scheme successfully constructed a new type of ergosterol-loaded nanoformulation for the targeted treatment of ischemic stroke. First, a new nanomaterial DSPE-PEG-pinacol borate was successfully synthesized, and this nanoformulation and its preparation method and application were provided. The nanoformulation can effectively solve the problems of poor drug targeting and low bioavailability, open up new treatment methods for ischemic stroke, and enhance the targeted treatment ability of drugs. At the same time, it was found that ergosterol plays a role in treating ischemic stroke by activating the PI3K / AKT / mTOR pathway. The above findings provide a new direction for the treatment of ischemic stroke.

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

Claims

1. A brain-targeted nanoformulation loaded with ergosterol, characterized in that: The nanoformulation comprises ergosterol, pinacol borate and DSPE-PEG, wherein the pinacol borate modifies DSPE-PEG to form a DSPE-PEG-pinacol borate nanomaterial encapsulating ergosterol.

2. The nanoformulation according to claim 1, characterized in that The nano preparation has a potential of -23.88±0.38, a particle size of 30 nm, a drug loading of 8.05% to 18.33%, and an encapsulation rate of 85.20% to 93.77%.

3. A method for preparing a brain-targeted nanoformulation loaded with ergosterol as claimed in 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 into anhydrous dichloromethane for reaction. After the reaction, anhydrous dichloromethane was added for extraction. The mixture was washed with deionized water for three times. The organic phase was further extracted with a saturated sodium chloride solution and then dried with anhydrous sodium sulfate to obtain activated pinacol borate. 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, anhydrous ether is washed, the precipitate is collected by centrifugation, and freeze-dried under vacuum to obtain the nanomaterial DSPE-PEG-pinacol borate; S3. Preparation of nanoformulations: Take ergosterol and DSPE-PEG-pinacol borate prepared in S2, place them in a round-bottom flask, add an organic solvent, stir until completely dissolved, connect to a rotary evaporator and slowly rotate and evaporate until the organic solvent is completely evaporated and a lipid film is formed on the inner wall of the flask, add PBS buffer, stir in a 37°C water bath to fully hydrate the lipid film to form a suspension, purify the suspension by ultrasound and dialysis, and then freeze-dry to obtain the nanoformulation.

4. The preparation method according to claim 3, characterized in that: In the step S1, the mass ratio of 4-hydroxymethylphenylboronic acid pinacol ester to N,N-carbonyldiimidazole is 1-2:1-2.5, and the stirring time is 1 hour.

5. The preparation method according to claim 3, characterized in that: The mass ratio of activated pinacol borate, β-cyclodextrin and 4-dimethylaminopyridine in step S2 is 1-2:0.1-1:0.5-1.

6. The preparation method according to claim 3, characterized in that: The mass ratio of ergosterol to DSPE-PEG-pinacol borate in step S3 is 1:4-12.

7. 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.

8. The preparation method according to claim 3, 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.

9. Use of the brain-targeted nanoformulation loaded with ergosterol as described in claims 1-2 or the brain-targeted nanoformulation loaded with ergosterol prepared by the preparation method according to any one of claims 3-8 in targeted treatment of brain diseases.

10. The use according to claim 9, characterized in that: The brain disease includes ischemic stroke.

Images