Oral brain-targeted nanoemulsion as well as preparation method and application thereof

Abstract

The invention discloses oral brain-targeted nanoemulsion as well as a preparation method and application thereof, and belongs to the field of biological medicine preparations. Drug molecules and a stabilizer are dispersed in oil phase molecules, and then a surfactant and water are added to prepare the nano-emulsion. Drug molecules are dispersed in oil-phase molecules and then form an oil-in-water nanoemulsion with water, so that the problems of poor stability and poor water solubility of current drug molecules are solved, the oral bioavailability of the drug is improved, fusion of different drug molecules of multiple targets is realized, the drug loading capacity of the drug molecules can be effectively ensured, and the bioavailability of the drug is improved. In addition, the drug can pass through an intestinal barrier and a blood brain barrier to be successfully delivered to a brain region to realize multi-target treatment, so that efficient treatment of multi-target brain diseases such as neurodegenerative diseases and central nervous system tumors is realized.

Description

Technical Field

[0001] The present invention belongs to the field of biopharmaceutical preparations, and particularly relates to an orally administered brain-targeted nanoemulsion, a preparation method thereof, and an application thereof. Background Art

[0002] Alzheimer's disease (AD) is a degenerative disease of the central nervous system. With the increase in the average life expectancy of the population and the acceleration of the social aging process, the number of AD patients in China has risen sharply, but there is still no effective treatment method so far. Currently, the clinical treatment methods for AD are mostly cholinesterase inhibitors (such as donepezil, rivastigmine, galantamine, etc.). Since these drugs do not change the pathological characteristics of AD itself and focus on symptom management rather than changing the disease progression, and some drugs also bring many side effects such as nausea and vomiting. In recent years, hundreds of treatment methods targeting the most significant pathological mechanisms of AD, namely Aβ deposition and Tau protein, have been developed. Unfortunately, most of the drugs have failed in the middle and late stages of clinical trials. Only 3 Aβ monoclonal antibodies have been approved for use, but they still face problems such as poor efficacy and large side effects. The reason is that AD has a complex pathogenic mechanism that interacts with and promotes each other. In addition to the accumulation of toxic proteins, the onset of AD is also related to various factors such as dysfunction of brain glial cells, neuroinflammation, oxidative stress, and disruption of the blood-brain barrier (BBB). This suggests that a single inhibition of Aβ deposition or reduction of excessive phosphorylation of Tau protein may not be sufficient to reverse the progression of AD. Therefore, the development of multi-target AD treatment has potential research value.

[0003] With the further verification of the active ingredients and action mechanisms of traditional Chinese medicinal materials from natural sources in modern research, some drug molecules have received extensive attention due to their advantages such as multi-target action, mild mechanism, low toxicity and side effects, and suitability for long-term use. For example, cinnamaldehyde and curcumin molecules can act on multiple targets of AD at the same time, such as interfering with the generation and deposition of toxic proteins, inhibiting the NF-κB inflammatory pathway to relieve neuroinflammation, and reacting with reactive oxygen species to reduce oxidative stress, showing strong anti-AD potential. However, in practical applications, their efficacy is mostly limited by their own physicochemical properties, such as poor water solubility, low bioavailability, insufficient metabolic stability, obstacles of various barriers in the body, and toxic and side effects caused by lack of targeting.

[0004] Since the emergence of the first nanocarrier with a liposome structure, the drug delivery technology of nanocarriers has experienced rapid development. Due to its stability, easy surface modification, and controllability, nanocarriers have demonstrated great advantages in enhancing the targeting, stability, and therapeutic efficacy of drugs, and have shown extremely high application potential in the fields of cancer treatment, gene therapy, and central nervous system disease treatment. However, in past research, to minimize the reduction in drug delivery efficiency caused by crossing multiple barriers, some nanodelivery systems for AD mainly relied on intravenous injection. Nevertheless, in actual clinical applications, long-term intravenous injection not only inconveniences patients' lives but also increases the risk of infection. In addition, the safety of intravenous injection is worse than that of oral administration, and the costs and challenges required for mass production are more severe. Its clinical translation is more difficult than that of oral dosage forms. Therefore, it is urgent to develop new oral drugs for AD. However, since oral drug delivery to the brain faces multiple challenges, including five complex physiological barriers: biochemical barriers, mucus barriers, intestinal epithelial barriers (IEBs), blood barriers, and blood-brain barriers, this process significantly reduces the drug delivery efficiency. Currently, some novel nanodelivery systems with oral brain targeting functions have somewhat improved the oral drug delivery efficiency to the brain. However, due to mostly using lipid nanoparticle surface modification technology or coated nanoparticles, their drug loading capacity is low, and the range of drug molecules that can be encapsulated is narrow due to the characteristics of the carrier molecules themselves, resulting in limited clinical applications.

[0005] In the prior art, Chinese Patent CN117224503A reported a cinnamic acid black phosphorus nanocomposite targeting Aβ, which consists of a drug-loading host BP (black phosphorus nanosheets), a modifying material C18-PEG-NH2 (aminopolyethylene glycol stearic acid), a targeting material 4-(dimethylamino)cinnamic acid (Tar), and a therapeutic material Cur (curcumin). This patent uses black phosphorus nanosheets to load Cur to increase the drug loading capacity of Cur, solving the problem of low drug loading capacity of existing nanoparticles. Moreover, it uses Tar as a group targeting Aβ to avoid the diffuse distribution of drugs in the brain and improve the drug utilization rate of Cur, which has a positive effect on the treatment of Alzheimer's disease. However, the drug-loading host (black phosphorus nanosheets) used in this patent still has inherent limitations, resulting in difficulty in further increasing the maximum loading amount of therapeutic drugs. At the same time, there is only one therapeutic drug, curcumin, and its anti-AD effect still needs to be improved.

[0006] Therefore, it is very necessary to develop and study a new type of oral brain-targeted nanodelivery system that can load multi-target therapeutic materials and ensure its drug loading capacity. Summary of the Invention

[0007] The object of the present invention is to provide an orally administered brain-targeted nanoemulsion, its preparation method and application. By dispersing drug molecules in oil-phase molecules and then forming an oil-in-water nanoemulsion with water, the problems of poor stability and water solubility of current drug molecules are solved, the oral bioavailability of the drug is improved, the fusion of different drug molecules with multiple targets is achieved, the drug loading capacity of the drug molecules can be effectively guaranteed, and it can cross the intestinal barrier and blood-brain barrier to be successfully delivered to the brain region for multi-target treatment, thereby realizing the efficient treatment of multi-target brain diseases such as neurodegenerative diseases and central nervous system tumors.

[0008] The present invention is realized through the following technical solutions: drug molecules and stabilizers are dispersed in oil-phase molecules, and then a surfactant and water are added to prepare a nanoemulsion. The drug molecules are drugs for treating neurodegenerative diseases and / or central nervous system tumors. The stabilizers include PLGA-PEG copolymers and mannose-modified PLGA-PEG copolymers. The oil-phase molecules are selected from at least one of cinnamaldehyde, vanillin, castor oil, olive oil, vitamin E, soybean oil, peanut oil, fish oil, coconut oil, sesame oil, squalene, fatty acid triglyceride and medium-chain triglyceride.

[0009] The molecular weight of PLGA is 2000 - 40000, and the molecular weight of PEG is 1000 - 10000, such as PLGA 5000 -PEG 2000 and PLGA 5000 -PEG 3400 -Man.

[0010] The mass ratio of the drug molecules, PLGA-PEG copolymers and mannose-modified PLGA-PEG copolymers is 1 - 30∶1 - 120∶1 - 120.

[0011] The mass ratio of the drug molecules to the oil-phase molecules is 1 - 20∶1 - 100.

[0012] The mass ratio of the oil-phase molecules to the surfactant is 1 - 10∶10 - 1.

[0013] Furthermore, the oil-phase molecules are selected from at least one of cinnamaldehyde, vanillin, vitamin E and fish oil; the surfactant is selected from at least one of Tween 80, Tween 20, soybean lecithin, hydrogenated lecithin, cholesterol, sodium dodecyl sulfate, propylene glycol, ethanol, sorbitan oleate, sorbitan laurate, polyoxyethylene castor oil, poloxamer 188, glycyrrhizic acid and fatty alcohol polyoxyethylene ether.

[0014] Preferably, the drug molecule and the stabilizer are dissolved in an organic solvent to prepare a film, and then the film is dispersed with oil-phase molecules. The organic solvent is selected from at least one of ethyl acetate, ethyl lactate, dichloromethane, methanol, petroleum ether, acetone, tetrahydrofuran, and acetonitrile.

[0015] An oral brain-targeted nanoemulsion obtained by the above preparation method, and the prepared nanoemulsion satisfies: Particle size: 70 - 150 nm; PDI: < 0.3; Stability: The change range of the particle size of the nanoemulsion within three days < 10%.

[0016] The application of an oral brain-targeted nanoemulsion provided by the present invention is to use the above oral brain-targeted nanoemulsion to prepare an oral drug for treating and / or preventing neurodegenerative diseases, and the neurodegenerative diseases include Alzheimer's disease, traumatic brain injury, epilepsy, Huntington's disease, amyotrophic lateral sclerosis, Parkinson's disease, multiple sclerosis, depression, spinocerebellar ataxia, neuroinflammation, cerebrovascular diseases, hydrocephalus, and Dandy-Walker syndrome. Drug molecules for treating Alzheimer's disease include at least one of curcumin, baicalein, paeoniflorin, apigenin, puerarin, salvianolic acid B, ferulic acid, chlorogenic acid, ginsenoside, retinoic acid (ATRA), tea polyphenols, tanshinone, quercetin, celastrol, rapamycin, salidroside, resveratrol, berberine, huperzine A, fingolimod, butylphthalide (3-n-butylphthalide) and its derivatives, sildenafil, Exendin-4, Omega-3 polyunsaturated fatty acids, Malibatol A, donepezil, rivastigmine, galantamine, memantine hydrochloride, metformin, simvastatin, ibuprofen, dasatinib, mefloquine, masitinib. Drug molecules for treating traumatic brain injury include at least one of ligustrazine, notoginsenoside R1, breviscapine, aspirin, simvastatin, torvastatin, warfarin, mannitol, mecobalamin, trimetazidine (trimethoxybenzylamine), neurotrophic factors (such as BDNF or NGF). Drug molecules for treating epilepsy include at least one of gastrodin, rhynchophylline, baicalein, tanshinone, piperine, crocetin, phenytoin sodium, carbamazepine, phenobarbital, sodium valproate, ethosuximide, clonazepam, oxcarbazepine, lamotrigine, levetiracetam, topiramate, felbamate, gabapentin. Drug molecules for treating Huntington's disease include at least one of tetrabenazine, deutetrabenazine, nucleic acid drugs. Drug molecules for treating amyotrophic lateral sclerosis include at least one of celastrol, curcumin, ginsenoside, icariin, riluzole, edaravone, baclofen, diazepam, trihexyphenidyl, amitriptyline, sodium phenylbutyrate, tauroursodeoxycholic acid. The drug molecules for treating Parkinson's disease include at least one of artane, amantadine, madopar, clozapine, quetiapine, ziprasidone, rivastigmine, donepezil, pimavanserin, aripiprazole, and piribedil; The drug molecules for treating multiple sclerosis include at least one of dexamethasone, methylprednisolone, cyclophosphamide, and prednisone; The drugs for treating depression include at least one of curcumin, ginsenoside, saikosaponin, spinosin, morindaofficinalis oligosaccharide, polygala saponin, ginkgolide B, hesperidin, pachymic acid, cannabidiol, paroxetine, sertraline, fluoxetine, fluvoxamine, and ketamine; The drug molecules for treating neuroinflammation include at least one of mecobalamin, adenosylcobalamin, vitamin B12, and aspirin; The drug molecules for treating cerebrovascular diseases include at least one of astragaloside IV, hydroxysafflor yellow A, emodin, lovastatin, aspirin, ropizole, mannitol, and piracetam; The drug molecules for treating hydrocephalus include at least one of acetazolamide, mannitol, and glycerol fructose.

[0017] Another application of the oral brain-targeted nanoemulsion provided by the present invention is to use the above-mentioned oral brain-targeted nanoemulsion to prepare oral drugs for treating and / or preventing neurodegenerative diseases. The central nervous system tumors include primary glioblastoma, secondary tumor brain metastasis, oligodendroglioma, astrocytoma, and meningioma. The drug molecules for treating central nervous system tumors include at least one of artemisinin and its derivatives, resveratrol, oridonin, nobiletin, paclitaxel, doxorubicin, temozolomide, methotrexate, fluorouracil, cytarabine, ibrutinib, and hydroxychloroquine.

[0018] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) Compared with the nano-delivery system loaded with a single drug, the nanoemulsion of the present invention can significantly improve the therapeutic effect of the drug through the combined action of two or more drug molecules, and has a high therapeutic effect in neurodegenerative diseases or central nervous system tumor diseases.

[0019] (2) The present invention designs a nanoemulsion with drug-loaded homology for the first time. In the treatment of AD, the oil-phase molecules such as cinnamaldehyde, vanillin, vitamin E, or fish oil used can not only serve as the oil-phase components of the nanoemulsion but also as medicinal molecules for treating AD, realizing a multi-functional effect.

[0020] (3) Compared with the existing oral or intravenous nano-delivery system, the nanoemulsion of the present invention significantly improves the drug loading capacity, thereby reducing the dosing frequency and enhancing the compliance of patients with a long course of disease.

[0021] (4) The nanoemulsion of the present invention, by using the functional polymer fragment PLGA-PEG-Man (such as PLGA 5000 -PEG 3400 -Man), not only improves the stability of the nanoemulsion, but also endows the nanoemulsion with the targeting ability for GLUT1 expressed on intestinal epithelial cells and brain endothelial cells, increasing the oral brain uptake efficiency of the nanoemulsion.

[0022] (5) Compared with the existing multi-barrier crossing nano-targeted drug delivery system, the nanoemulsion drug delivery system prepared by the present invention has simple and safe components, an easy synthesis method, and has good application prospects.

[0023] In summary, the present invention provides a stable (drug-loaded homologous) nanoemulsion co-delivery system for a multi-drug system. This nanoemulsion co-delivery system can significantly increase the drug loading capacity, enhance the therapeutic effect, reduce the dosing frequency and the side effects of long-term drug administration; solve the problems of poor water solubility and low bioavailability of drugs (such as curcumin and cinnamaldehyde); overcome the obstacles of in vivo physiological barriers (intestinal barrier and blood-brain barrier), achieve the oral administration method, significantly improve the patient compliance, and can achieve the treatment mode for neurodegenerative diseases or central nervous system tumor diseases with multi-target fusion, and has more clinical transformation value. Brief Description of the Drawings

[0024] Figure 1 It is a schematic diagram of the treatment principle of the nanoemulsion of the present invention.

[0025] Figure 2 It is the characterization of different nanoemulsions.

[0026] Figure 3 It is the particle size and polydispersity index PDI of different nanoemulsions (n = 3).

[0027] Figure 4 It is the zeta potential of different nanoemulsions (n = 3).

[0028] Figure 5 It is the particle size stability of different nanoemulsions in PBS (n = 3).

[0029] Figure 6 It is the stability of different nanoemulsions in simulated gastric fluid SGF (n = 3).

[0030] Figure 7 It is the stability of different nanoemulsions in simulated intestinal fluid SIF (n = 3).

[0031] Figure 8 It is the relative mucus adsorption ratio of different nanoemulsions (n = 3).

[0032] Figure 9 is the cumulative release amount of curcumin from the nanoemulsion in artificial gastrointestinal fluid (n = 3).

[0033] Figure 10 is the cumulative release amount of cinnamaldehyde from the nanoemulsion in artificial gastrointestinal fluid (n = 3).

[0034] Figure 11 is the fluorescence intensity after different nanoemulsions were taken up by Caco-2 cells (using the CC NE group as the control, n = 3).

[0035] Figure 12 is the fluorescence intensity after different nanoemulsions were taken up by bEnd.3 cells (using the CC NE group as the control, n = 3).

[0036] Figure 13 is the fluorescence intensity after different nanoemulsions were taken up by BV-2 cells (using the CC NE group as the control, n = 3).

[0037] Figure 14 is the statistical data of the 12 H nesting behavior of APP / PS1 transgenic mice after different treatments (n = 12).

[0038] Figure 15 is the nesting diagram of APP / PS1 transgenic mice 12 H after different treatments (n = 12).

[0039] Figure 16 is the effect of different preparations on the level of IL-6 inflammatory factor in the brain tissue of APP / PS1 transgenic mice.

[0040] Figure 17 is the effect of different preparations on the level of TNF-α inflammatory factor in the brain tissue of APP / PS1 transgenic mice. Detailed implementation mode

[0041] The invention object, technical solution and beneficial effects of the present invention will be further described in detail below.

[0042] It should be noted that the following detailed description is exemplary and is intended to provide further illustration of the claimed invention. Unless otherwise noted, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0043] The present invention relates to an orally administered brain-targeted nanoemulsion. By selecting drug molecules, it can be used to treat neurodegenerative diseases and / or central nervous system tumor diseases. As a typical neurodegenerative disease, AD is based on multiple pathological characteristics of AD and combines the characteristics of in vivo physiological barriers. A nanoemulsion with an orally administered brain-targeted multi-drug system is designed for the multi-modal treatment of AD. This nanoemulsion innovatively adopts a drug-loaded homologous mode and selects cinnamaldehyde molecules (alternatively, vanillin, vitamin E, or fish oil). It can not only play roles such as inhibiting Aβ production, antioxidation, and antibacterial as drug molecules but also act as the oil phase of the nanoemulsion and serve as a carrier component. To further enhance the anti-AD efficacy of the nanoemulsion and complement the deficiencies in the regulation of AD pathological characteristics by cinnamaldehyde, other drugs for treating AD can be selected, such as curcumin, baicalein, paeoniflorin, apigenin, puerarin, quercetin, etc.

[0044] Taking curcumin as an example, although curcumin has extremely low oral bioavailability, it has multiple regulatory effects on AD pathological characteristics such as reducing Aβ aggregation and anti-inflammation. By preparing nanoemulsions from cinnamaldehyde and curcumin, both of which have poor stability and water solubility, the physicochemical properties of the two drugs can be changed simultaneously, greatly improving the oral bioavailability of the two drugs. At the same time, by adding stabilizer components, the present invention can significantly enhance the brain entry efficiency and bioavailability of curcumin and cinnamaldehyde molecules, while reducing the liver and gastrointestinal toxicity of the drugs. Among them, by adding the functional polymer fragment PLGA-PEG-Man (such as PLGA 5000 -PEG 3400 -Man), the oral brain-targeting ability of the nanoemulsion and the stability of the nanoemulsion are increased, and the nanoemulsion is also endowed with active targeting abilities for the intestinal barrier and blood-brain barrier, so that the two drugs can be successfully delivered to the brain region to play their roles in the multi-modal treatment of AD. For details, see Figure 1 .

[0045] It should be noted that by changing the dosage form of the existing nanoparticles and using the cinnamon aldehyde molecule with the same origin of medicine and carrier as the oil phase of the nanoemulsion, it can not only act as a carrier component but also as a drug molecule. At the same time, combined with other drugs for the treatment of AD, due to the nanoemulsion dosage form and the water-in-oil form of the nanoemulsion, the drug loading is not limited to hydrophobic or hydrophilic drugs. Hydrophobic drugs can be distributed in the oil phase, and hydrophilic drugs can be distributed in the water phase, both of which can achieve the dispersion of drug molecules in the nanoemulsion and are not restricted by the nature of the carrier itself. Therefore, it can significantly improve the drug loading capacity of drug molecules and the drug selection range, facilitating the selection of a multi-modal fusion treatment method during AD treatment and enhancing the curative effect of AD. Further, through the selection of the nanoemulsion dosage form, by using surfactants (such as Tween 80) to provide its specific interfacial properties in the oil phase molecules, the solubility of the drug in the oil and water phases can be significantly improved by forming micelles or reducing the interfacial tension, and the surface of the nanoemulsion droplets is covered with a dense surfactant layer through interfacial adsorption, fixing the drug molecules through physical adsorption or chemical action to prevent drug leakage and improve the loading efficiency.

[0046] The following is a further detailed overview of the technical solution of the present invention: Disperse the drug molecule, PLGA-PEG copolymer and PLGA-PEG-Man copolymer in the oil phase molecule, and then add a surfactant and water to prepare a nanoemulsion. During the preparation, it is necessary to control the mass ratio of the drug molecule, PLGA-PEG copolymer and PLGA-PEG-Man copolymer to be 1-30:1-120:1-120.

[0047] The drug molecule is a drug for treating neurodegenerative diseases and / or central nervous system tumors. It is generally recognized that neurodegenerative diseases include Alzheimer's disease, traumatic brain injury, epilepsy, Huntington's disease, amyotrophic lateral sclerosis, Parkinson's disease, multiple sclerosis, depression, spinocerebellar ataxia, neuroinflammation, cerebrovascular diseases, hydrocephalus and Dandy-Walker syndrome; central nervous system tumors include primary glioblastoma, secondary tumor brain metastasis, oligodendroglioma, astrocytoma and meningioma. When selecting the drug molecule, appropriate drug materials can be selected according to different diseases. These drug materials can be drugs approved by the FDA (US Food and Drug Administration), or drugs approved by other regulatory agencies, or drugs recorded in the Chinese Pharmacopoeia. Since it has been detailedly described above in the present invention, it will not be elaborated here.

[0048] Taking the drug for AD treatment as an example, the drug molecule can be a hydrophobic drug such as curcumin, apigenin, puerarin, quercetin, etc., or a hydrophilic drug such as salvianolic acid B, ferulic acid, etc. The PLGA-PEG copolymer can be PLGA 5000 -PEG 2000, the PLGA-PEG-Man copolymer can be PLGA 5000 -PEG 3400 -Man. Organic solvents can include ethyl acetate, ethyl lactate, dichloromethane, methanol, petroleum ether, acetone, tetrahydrofuran, acetonitrile, etc. If the drug molecule is a hydrophobic drug, the way to disperse it in the oil-phase molecules can be to dissolve the drug molecule, PLGA 5000 -PEG 2000 and PLGA 5000 -PEG 3400 -Man in an organic solvent, and then use a rotary evaporator (37 - 45 °C, 60 - 100 rpm / min) to dry it into a thin film, so as to have a better dispersion effect in the oil-phase molecules; if the drug molecule is hydrophilic, the drug molecule can be directly dissolved in water and directly form a nanoemulsion with the oil-phase molecules and the surfactant.

[0049] During its preparation, the mass ratio of the drug molecule to the oil-phase molecule can be selectively controlled to be 1 - 20∶1 - 100 according to the treatment mode. Optional oil-phase molecules include cinnamaldehyde, vanillin, castor oil, olive oil, vitamin E, soybean oil, peanut oil, fish oil, coconut oil, sesame oil, squalene, fatty acid triglyceride, and medium-chain triglyceride, etc. Taking AD treatment as an example, the drug molecule is selected as curcumin, the oil-phase molecule is selected as cinnamaldehyde, and the mass ratio of curcumin to cinnamaldehyde is controlled to be 1 - 10∶1 - 100, and further preferably 1∶10. The dosage of the surfactant is related to the particle size of the nanoemulsion and the drug loading. Usually, the mass ratio of the surfactant to the oil-phase molecule is controlled to be 1 - 10∶10 - 1. Optional surfactants include Tween 80, Tween 20, soybean lecithin, hydrogenated lecithin, cholesterol, sodium dodecyl sulfate, propylene glycol, ethanol, sorbitan oleate, sorbitan laurate, polyoxyethylene castor oil acid ester, poloxamer 188, glycyrrhizic acid (GA), fatty alcohol polyoxyethylene ether (AEO-9), etc. The dispersion method can adopt a rotary water bath (37 - 45 °C, 60 - 100 rpm / min) or use ultrasound (40 Hz), and dissolve the above-mentioned formed thin film in the oil-phase molecules and the surfactant.

[0050] Add ultrapure water (UP water) to the dispersed oil phase, vortex and mix well, and then after ultrasonic treatment, a nanoemulsion is prepared. Among them, the mass ratio of the oil phase to water is 1 - 40∶1 - 60, preferably 1∶20. The rotation speed can be controlled at 2000 rpm / min during vortex mixing. During ultrasonic treatment, the power can be controlled at 80 - 150 w, ultrasonic for 3 - 10 min, and stop for 0 - 10 s every 1 - 10 s of ultrasonic treatment. The purpose of vortex mixing and ultrasonic treatment is to make the drug molecules disperse evenly in the oil phase or water phase, so as to control the particle size distribution of the nanoemulsion to be uniform.

[0051] The present invention will be further described in detail below in conjunction with embodiments, but the embodiments of the present invention are not limited thereto.

[0052] Example 1: Accurately weigh 10 mg of curcumin, PLGA 5000 -PEG 2000 20 mg, PLGA 5000 -PEG 3400 -Man 20 mg. Dissolve them in EA (ethyl acetate), then dry them into a thin film with a rotary evaporator. Subsequently, add 100 mg of cinnamaldehyde and 100 μL of Tween 80, continue to rotate in a water bath or use ultrasound to dissolve the thin film. Then add 3.8 mL of UP water, vortex and mix well, and place it in a probe sonicator to sonicate at a power of 100 w for 5 min, with 5 s of sonication followed by 5 s of pause, to obtain CCPM NE.

[0053] Comparative Example 1: Accurately weigh 10 mg of curcumin, dissolve it in EA (ethyl acetate), and dry it into a thin film with a rotary evaporator. Subsequently, add 100 mg of cinnamaldehyde and 100 μL of Tween 80, continue to rotate in a water bath or use ultrasound to dissolve the thin film. Then add 3.8 mL of UP water, vortex and mix well, and place it in a probe sonicator to sonicate at a power of 100 w for 5 min, with 5 s of sonication followed by 5 s of pause, to obtain CC NE.

[0054] Comparative Example 2: Accurately weigh 10 mg of curcumin, PLGA 5000 -PEG 2000 40 mg. Dissolve the two in EA (ethyl acetate), and dry them into a thin film with a rotary evaporator. Subsequently, add 100 mg of cinnamaldehyde and 100 μL of Tween 80, continue to rotate in a water bath or use ultrasound to dissolve the thin film. Then add 3.8 mL of UP water, vortex and mix well, and place it in a probe sonicator to sonicate at a power of 100 w for 5 min, with 5 s of sonication followed by 5 s of pause, to obtain CCP NE.

[0055] The operating conditions such as rotary evaporation, rotary water bath, vortex, and sonication involved in Example 1, Comparative Example 1, and Comparative Example 2 are the same, and refer to the detailed overview of the technical solution of the present invention above.

[0056] Furthermore, the nanoemulsions involved in Example 1, Comparative Example 1, and Comparative Example 2 are compared as follows: (1) Apparent characteristics of nanoemulsions Figure 2 The nanoemulsion stock solutions (A) and solutions diluted with UP water (B) of Example 1 (CCPM NE), Comparative Example 1 (CC NE), and Comparative Example 2 (CCP NE) are respectively shown.Figure 2 It can be seen that nanoemulsions can be prepared in both Example 1, Comparative Example 1 and Comparative Example 2. Their apparent characteristics show as bright yellow emulsions, which have opalescence after dilution with UP water.

[0057] (II) Physicochemical characteristics of nanoemulsions (particle size) For Example 1 (CCPM NE), Comparative Example 1 (CC NE), and Comparative Example 2 (CCP NE), a ZEN3690 Malvern particle size analyzer was used to measure the particle size, polydispersity index PDI, zeta potential, and stability of the nanoemulsions respectively.

[0058] The measurement results are shown in Figures 3 to 5 , where Figure 3 are the particle sizes and polydispersity indices PDI of different nanoemulsions, Figure 4 are the zeta potentials of different nanoemulsions, Figure 5 are the particle size stabilities of different nanoemulsions.

[0059] It can be seen from Figure 3 that the particle sizes of the nanoemulsions in Example 1, Comparative Example 1 and Comparative Example 2 are about 100 nm, and the PDI is less than 0.3, indicating good homogeneity.

[0060] It can be seen from Figure 4 that the nanoemulsions in Example 1, Comparative Example 1 and Comparative Example 2 are all negatively charged and can repel mucin when crossing the oral barrier.

[0061] It can be seen from Figure 5 that compared with Comparative Example 1 and Comparative Example 2, the nanoemulsion in Example 1 has good stability within three days. The particle size change range of CCPM NE is 4.93%, and the particle size change ranges of CC NE and CCP NE are 17.54% and 11.94% respectively.

[0062] (III) Stability experiment For Example 1 (CCPM NE), Comparative Example 1 (CC NE), and Comparative Example 2 (CCP NE), their stabilities in simulated gastric fluid SGF and simulated intestinal fluid SIF were measured respectively. Specifically, the nanoemulsions were dispersed in simulated gastric fluid SGF (the preparation method is shown in the Chinese Pharmacopoeia), and the particle sizes of the nanoemulsions were measured at 0 h, 6 h, and 24 h; the nanoemulsions were dispersed in simulated intestinal fluid SIF (the preparation method is shown in the Chinese Pharmacopoeia), and the particle sizes of the nanoemulsions were measured at 0 h, 6 h, and 24 h.

[0063] The measurement results are shown in Figure 6 and Figure 7 , where Figure 6 are the stabilities of different nanoemulsions in simulated gastric fluid SGF, Figure 7 are the stabilities of different nanoemulsions in simulated intestinal fluid SIF.

[0064] As can be seen Figure 6 from Examples 1, Comparative Example 1 and Comparative Example 2, the stabilities of the nanoemulsions in artificial gastric juice are different, and the nanoemulsion CC NE (Comparative Example 1) without polymer fragments has poor stability.

[0065] As can be seen Figure 7 from Examples 1, Comparative Example 1 and Comparative Example 2, when the nanoparticles are dispersed in artificial intestinal fluid, their particle sizes all increase to a certain extent. Generally speaking, within 6 h, their particle sizes do not change significantly.

[0066] (IV) Mucus adsorption experiment For Example 1 (CCPM NE), Comparative Example 1 (CC NE), and Comparative Example 2 (CCP NE), their adsorption in mucus solution was measured respectively.

[0067] Experimental method: Porcine gastric mucin was configured in a buffer solution with pH = 6.5 prepared from disodium hydrogen phosphate and sodium dihydrogen phosphate at 4 mg / mL. Subsequently, the nanoemulsion was dispersed in the prepared mucus solution and incubated on a shaker at 37 °C and 60 rpm. After incubation for 1 h, 3 h, and 6 h, the mixture was centrifuged at 5000 rpm for 10 min, the adsorbed aggregates were discarded, the supernatant was collected, and acetonitrile was added to demulsify. The proportion of non-adsorbed nanoparticles was measured by an enzyme-labeled instrument (excitation wavelength: 495 nm, emission wavelength: 520 nm).

[0068] The test results are shown in Figure 8 , Figure 8 as the relative mucus adsorption ratios of different nanoemulsions.

[0069] As can be seen Figure 8 from Examples 1, Comparative Example 1 and Comparative Example 2, the nanoemulsions have little adsorption to mucin, and the nanoemulsions CCPM NE (Example 1) and CCP NE (Comparative Example 2) containing polymer fragments have even less adsorption, indicating that they can penetrate the mucus barrier more easily.

[0070] (V) Drug release amount For Example 1 (CCPM NE) and Comparative Example 1 (CC NE), the cumulative release amounts of curcumin or cinnamaldehyde from the nanoemulsion in artificial gastrointestinal fluid were measured respectively.

[0071] Experimental method: Different nanoemulsions were successively placed in artificial gastric juice and artificial intestinal juice (the preparation methods are shown in the Chinese Pharmacopoeia). 2 mL of each of the synthesized CC NE and CCPM NE nanoemulsions (Cur 2.5 mg / mL) was placed in a tubular dialyzer and placed in 50 mL of artificial gastric juice. It was shaken and incubated at 37 °C and 100 rpm to simulate the environment where nanoparticles are first distributed in the stomach after oral administration. After 2 h, the external medium was replaced with 50 mL of artificial intestinal juice and incubated at 37 °C for another 6 h to simulate the environment where nanoparticles enter the intestine after the gastric emptying cycle. At different time points (0.5 h, 1 h, 2 h, 2.5 h, 3 h, 5 h, 8 h), 50 μL of nanoemulsion was taken from the dialysis tube, 50 times the volume of acetonitrile was added to break the emulsion, and it was sonicated for 5 min to fully disrupt the nanoemulsion and completely release curcumin and cinnamaldehyde. Finally, the drug content was determined by high-performance liquid chromatography, and the release percentage was calculated.

[0072] The experimental results are shown in Figure 9 and Figure 10 , where Figure 9 is the cumulative release amount of curcumin from the nanoemulsion in artificial gastrointestinal juice, Figure 10 is the cumulative release amount of cinnamaldehyde from the nanoemulsion in artificial gastrointestinal juice.

[0073] It can be seen from Figure 9 and Figure 10 that within 2 h of artificial gastric juice, there is no significant difference in the drug release percentages of the two drugs in the two nanoemulsions. Among them, the release of curcumin is about 30%, and the release rate of cinnamaldehyde is about 40%. However, in intestinal juice, there are relatively obvious changes in the release percentages of the two nanoparticles. Among them, for the release rate of curcumin, at 4 h, the release amounts of the two drugs in CC NE are significantly higher than those in CCPM NE. Among them, the release amount of curcumin reaches about 70%, and the release amount of cinnamaldehyde reaches 80%. While the release rates of the two drugs in CCPM at 4 h are 35% and 45% respectively.

[0074] The above results indicate that nanoemulsions can endow drugs with a sustained-release effect, thereby increasing the retention time of drugs in the body. Moreover, the nanoemulsion of CCPM NE (Example 1) can further improve the gastrointestinal release behavior of the simple drug nanoemulsion due to the addition of functional polymer components, making its release rate lower, playing a better role in protecting the drug from degradation by the acidic environment and various enzymes in the gastrointestinal tract and maintaining the stability of the drug.

[0075] (VI) Cell fluorescence experiment For Example 1 (CCPM NE), Comparative Example 1 (CC NE), and Comparative Example 2 (CCP NE), the fluorescence intensities were measured after Caco-2 cells, bEnd-3 cells, and BV-2 cells were used to uptake different nanoemulsions respectively.

[0076] Experimental method: Caco-2 cells were seeded at 3×10 5 cells per well in a 6-well plate and allowed to grow for 24 h. The medium was removed, and nanoemulsions with different components (Cur 2.5 μg / mL) were added, followed by a 3-h co-incubation. Subsequently, the medium was removed, and the cells were washed three times with PBS. The cells were then digested into single-cell suspensions with trypsin, and the cells were collected by centrifugation at 3000 rpm for 3 min at 4 °C and resuspended in PBS. The fluorescence intensity of the cells in each sample group was measured by flow cytometry.

[0077] bEnd.3 cells were seeded at 3×10 5 cells per well in a 6-well plate and grown in a cell incubator at 37 °C and 5% CO 2 for 24 h. Subsequently, the medium was removed, and different nanoemulsions (Cur 2.5 μg / mL) were added, followed by a 3-h co-incubation. The medium was removed, the cells were washed three times with PBS, the cells were then digested into single-cell suspensions with trypsin, and the cells were collected by centrifugation at 3000 rpm for 3 min at 4 °C and resuspended in PBS. The fluorescence intensity of the cells in each sample group was measured by flow cytometry.

[0078] BV-2 cells were seeded at 2×10 5 cells per well in a 6-well plate and grown in a cell incubator at 37 °C and 5% CO 2 for 12 h. Subsequently, the medium was removed, and the cells were cultured with medium containing LPS (2 μg / mL) for 24 h. Then the medium was removed, and different nanoemulsions (Cur 2.5 μg / mL) were added, followed by a 3-h co-incubation. The medium was removed, the cells were washed three times with PBS, the cells were then digested into single-cell suspensions with trypsin, and the cells were collected by centrifugation at 3000 rpm for 3 min at 4 °C and resuspended in PBS. The fluorescence intensity of the cells in each sample group was measured by flow cytometry.

[0079] The experimental results are shown in Figures 11 to 13 , where Figure 11 is the fluorescence intensity of Caco-2 cells after uptake of different nanoemulsions, Figure 12 is the fluorescence intensity of bEnd.3 cells after uptake of different nanoemulsions, and Figure 13 is the fluorescence intensity of BV-2 cells after uptake of different nanoemulsions.

[0080] As can be seen from Figure 11 , there are obvious differences in the cellular uptake behaviors of nanoemulsions with different components. Among them, the uptake behavior of CC NE is the lowest. After adding the polymer PLGA 5000 -PEG 2000 to form CCP NE, there is a certain increase in uptake. After adding PLGA 5000 -PEG2000 and PLGA 5000 -PEG 3400 The uptake behavior of CCPM NE after the -Man polymer was significantly increased, 1.4 times higher than that of CC NE. It shows that the functional polymer PLGA 5000 -PEG 2000 and PLGA 5000 -PEG 3400 -Man can indeed increase the targeting ability to intestinal epithelial cells and improve the endocytosis efficiency of nanoemulsion.

[0081] From Figure 12 It can be seen that among different nanoemulsions, CCPM NE shows the strongest cell endocytosis ability, 2.1 times higher than that of CC NE, indicating that the addition of functional polymer fragments can achieve the active targeting of nanoemulsion to brain endothelial cells, thus achieving the purpose of increasing the amount of nanoemulsion entering the brain.

[0082] From Figure 13 It can be seen that the difference multiple of uptake of different nanoemulsions on BV-2 cells is not obvious, but generally speaking, CCPMNE still has the best cell targeting ability, indicating that the addition of functional polymer fragments further improves the uptake ability of nanoemulsion by brain target cells.

[0083] (VII) Mouse treatment experiment Grouped treatment was carried out on AD transgenic model mice (APP / PS1 transgenic mice). The experimental grouping was healthy control mice (WT), APP / PS1 transgenic mice without any treatment (AD), APP / PS1 transgenic mice treated with free curcumin (Free Cur), and APP / PS1 transgenic mice treated with CCPM NE nanoemulsion (CCPM NE).

[0084] The mice after the above grouped treatment were respectively compared for the treatment effects of different experimental groups through behavioral cognitive tests and inflammatory factor detection (IL-6 / TNF-α). (1) Behavioral cognitive test Test method: The mice were divided into single cages (1 mouse per cage). After they adapted to the single cage environment for 48 h, 20 odorless thin papers (10x10 cm 2 ) were placed on one side of the corner of the cage to provide conditions for the mice to build nests. The mice were evaluated by double-blind scoring according to the biting situation of the papers and the state of the nest position, and the final nest-building score of the mice was statistically analyzed.

[0085] The test results are shown in Figure 14 and Figure 15 , among which, Figure 14 are the statistical data of the 12 H nest-building behaviors of APP / PS1 transgenic mice after different treatments,Figure 15 Nest building diagram of APP / PS1 transgenic mice 12 h after different treatments.

[0086] From Figure 14 and Figure 15 It can be seen that after different treatments, the nest building behavior of APP / PS1 transgenic mice showed significant differences. Compared with the AD group, the Free Cur group showed a slight improvement, but the nest building behavior of the CCPMNE group was significantly improved. 50% of the mice were able to completely tear the paper and build obvious nest sites, obtaining a full score for nest building, and the overall nest building performance within the group was consistent with that of the healthy control mice in the WT group, indicating that after treatment with nanoemulsion, the cognition of the mice was significantly improved.

[0087] (2)Inflammatory factor detection After sacrificing the mice at the end of the treatment, the brain tissues were quickly removed, the brain tissue proteins were extracted by a total protein extraction kit, and the expression levels of inflammatory factors IL-6 and TNF-α in the brain tissue proteins were measured by an ELISA kit.

[0088] The test results are shown in Figure 16 and Figure 17 , among which, Figure 16 shows the effect of different preparations on the level of IL-6 inflammatory factor in the brain tissue of APP / PS1 transgenic mice, Figure 17 shows the effect of different preparations on the level of TNF-α inflammatory factor in the brain tissue of APP / PS1 transgenic mice.

[0089] From Figure 16 It can be seen that after treatment, the pro-inflammatory factor IL-6 index in the brain tissue of the mice was down-regulated to a certain extent, and the CCPM NE nanoemulsion had the lowest IL-6 level, indicating that the CCPM NE nanoemulsion exerted a good anti-inflammatory treatment effect.

[0090] From Figure 17 It can be seen that after treatment, the pro-inflammatory factor TNF-α index in the brain tissue of the mice was down-regulated to a certain extent, and the CCPM NE nanoemulsion had the lowest TNF-α level, indicating that the CCPM NE nanoemulsion exerted a good anti-inflammatory treatment effect.

[0091] The above results together indicate that nanoemulsion can exert a good neuroprotective effect and play an anti-AD therapeutic effect.

[0092] The above is only a preferred embodiment of the present invention, and does not impose any form of limitation on the present invention. Any simple modification or equivalent change made to the above embodiments based on the technical essence of the present invention falls within the protection scope of the present invention.

Claims

1. A method for preparing an oral brain-targeting nanoemulsion, characterized in that: The drug molecules and stabilizers are dispersed in the oil phase molecules, and then surfactants and water are added to prepare nanoemulsion. The drug molecule is a drug for treating neurodegenerative diseases and / or central nervous system tumors, The stabilizer includes PLGA-PEG copolymer and mannose-modified PLGA-PEG copolymer, The oil phase molecules are selected from at least one of cinnamaldehyde, vanillin, castor oil, olive oil, vitamin E, soybean oil, peanut oil, fish oil, coconut oil, sesame oil, squalene, fatty acid triglycerides and medium chain triglycerides.

2. The preparation method according to claim 1, characterized in that: The molecular weight of PLGA is 2000-40000, and the molecular weight of PEG is 1000-10000.

3. The preparation method according to claim 1, characterized in that: The mass ratio of the drug molecule, the PLGA-PEG copolymer and the mannose-modified PLGA-PEG copolymer is 1-30:1-120:1-120.

4. The preparation method according to claim 1, characterized in that: The mass ratio of the drug molecules to the oil phase molecules is 1-20:1-100.

5. The preparation method according to claim 1, characterized in that: The mass ratio of the oil phase molecules to the surfactant is 1-10:10-1.

6. The preparation method according to claim 1, characterized in that: The oil phase molecule is selected from at least one of cinnamaldehyde, vanillin, vitamin E and fish oil; the surfactant is selected from at least one of Tween 80, Tween 20, soybean lecithin, hydrogenated lecithin, cholesterol, sodium lauryl sulfate, propylene glycol, ethanol, sorbitan oleate, sorbitan laurate, polyethylene glycol ricinoleate, poloxamer 188, glycyrrhizic acid and fatty alcohol polyoxyethylene ether.

7. The preparation method according to claim 1, characterized in that: The drug molecules and stabilizer are dissolved in an organic solvent to obtain a film, and then the film is dispersed with oil phase molecules. The organic solvent is selected from at least one of ethyl acetate, ethyl lactate, dichloromethane, methanol, petroleum ether, acetone, tetrahydrofuran and acetonitrile.

8. An oral brain-targeted nanoemulsion, characterized in that: The nanoemulsion is obtained by the preparation method according to any one of claims 1 to 7, and the obtained nanoemulsion satisfies: Particle size: 70~150nm; PDI: <0.3; Stability: The particle size change of the nanoemulsion is less than 10% within three days.

9. Application of oral brain-targeted nanoemulsion, characterized in that: The oral brain-targeted nanoemulsion of claim 8 is used to prepare an oral drug for treating and / or preventing neurodegenerative diseases, wherein the neurodegenerative diseases include Alzheimer's disease, traumatic brain injury, epilepsy, Huntington's disease, amyotrophic lateral sclerosis, Parkinson's disease, multiple sclerosis, depression, spinocerebellar ataxia, neuroinflammation, cerebrovascular disease, hydrocephalus and Dandy-Walker syndrome, The drug molecules for treating Alzheimer's disease include at least one of curcumin, baicalein, paeoniflorin, apigenin, puerarin, salvianolic acid B, ferulic acid, chlorogenic acid, ginsenoside, retinoic acid, tea polyphenols, tanshinone, quercetin, triptolide, rapamycin, salidroside, resveratrol, berberine, huperzine A, fingolimod, butylphthalide and its derivatives, sildenafil, exendin-4, omega-3 polyunsaturated fatty acids, Malibatol A, donepezil, rivastigmine, galantamine, memantine hydrochloride, metformin, simvastatin, ibuprofen, dasatinib, mefloquine, and masitinib; The drug molecules for treating traumatic brain injury include at least one of chuanxiongqin, notoginseng saponin R1, breviscapine, aspirin, simvastatin, atorvastatin, warfarin, mannitol, methylcobalamin, trimetazidine, and neurotrophic factor; The drug molecules for treating epilepsy include at least one of gastrodin, rhynchophylline, baicalein, tanshinone, piperine, crocetin, phenytoin sodium, carbamazepine, phenobarbital, sodium valproate, ethosuximide, clonazepam, oxcarbazepine, lamotrigine, levetiracetam, topiramate, felbamate, and gabapentin; The drug molecule for treating Huntington's disease includes at least one of tetrabenazine, tritiated tetrabenazine, and nucleic acid drugs; The drug molecule for treating amyotrophic lateral sclerosis includes at least one of tripterygium wilfordii, curcumin, ginsenoside, icariin, riluzole, edaravone, baclofen, diazepam, trihexyphenidyl, amitriptyline, sodium phenylbutyrate, and taurine diol; The drug molecules for treating Parkinson's disease include at least one of antan, amantadine, madopar, clozapine, quetiapine, ziprasidone, rivastigmine, donepezil, pimaseline, aripiprazole, and piribedil; The drug molecule for treating multiple sclerosis includes at least one of dexamethasone, methylprednisolone, cyclophosphamide, and prednisone; Drugs for the treatment of depression: including at least one of curcumin, ginsenoside, saikosaponin, spinosum, Morinda officinalis oligosaccharides, polygala saponin, ginkgolide B, hesperidin, pachymic acid, cannabidiol, paroxetine, sertraline, fluoxetine, fluvoxamine, and ketamine; The drug molecules for treating neuroinflammation include at least one of methylcobalamin, adenosylcobalamin, vitamin B12, and aspirin; The drug molecules for treating cerebrovascular diseases include at least one of flavonoids, hydroxysafflor yellow A, rhubarb, lovastatin, aspirin, ropyrazole, mannitol, and piracetam; The drug molecules for treating hydrocephalus include at least one of acetazolamide, mannitol, and glycerol fructose.

10. Application of oral brain-targeted nanoemulsion, characterized in that: The oral brain-targeted nanoemulsion of claim 8 is used to prepare an oral drug for treating and / or preventing central nervous system tumors, wherein the central nervous system tumors include primary brain gliomas, secondary tumor brain metastases, oligodendrogliomas, astrocytomas and meningiomas, The drug molecules for treating central nervous system tumors include at least one of artemisinin and its derivatives, resveratrol, oridonin, nobiletin, paclitaxel, doxorubicin, temozolomide, methotrexate, fluorouracil, cytarabine, ibrutinib, and hydroxychloroquine.

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