An orally administered brain-targeted nanoemulsion, its preparation method and application

Abstract

The present invention discloses an orally administered brain-targeted nanoemulsion, its preparation method and application, belonging to the field of biomedical preparations. In the present invention, drug molecules and stabilizers are dispersed in oil-phase molecules, and then surfactants and water are added to prepare the nanoemulsion. 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 realized, the drug loading capacity of the drug molecules can be effectively guaranteed, and it can cross the intestinal barrier and blood-brain barrier and be successfully delivered to the brain region to achieve multi-target therapy, thereby realizing the efficient treatment of multi-target brain diseases such as neurodegenerative diseases and central nervous system tumors.

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 increased sharply. However, 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 will 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 with mutual influence and promotion. In addition to the accumulation of toxic proteins, the onset of AD is also related to various factors such as dysfunction of glial cells in the brain, neuroinflammation, oxidative stress, and disruption of the blood-brain barrier (BBB). This suggests that 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 simultaneously, 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 actual 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 liposome-structured nanocarrier, the drug delivery technology of nanocarriers has experienced rapid development. Due to its stability, easy surface modification, and controllability, nanocarriers have played a huge role in improving the targeting, stability and therapeutic effect of drugs, and have shown great application potential in the fields of cancer treatment, gene therapy, and central nervous system disease treatment. However, in past studies, in order to avoid the reduction of drug delivery efficiency caused by multi-barrier crossing as much as possible, some nano-delivery systems for AD are mainly intravenous injection. However, in actual clinical applications, long-term intravenous injection not only causes inconvenience to 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 cost and challenges required for mass production are more severe. Its clinical transformation is more difficult than that of oral dosage forms. Therefore, the development of new oral AD drugs is urgent. However, since drugs face multiple challenges when they enter the brain orally, including five complex physiological barriers: biochemical barrier, mucus barrier, intestinal epithelial barrier (IEB), blood barrier, and blood-brain barrier, this process will greatly reduce the delivery efficiency of drugs. At present, some new nano-delivery systems with oral brain-targeting functions have improved the efficiency of oral drug delivery into the brain to a certain extent. However, since most of them use lipid nanoparticle surface modification technology or coated nanoparticles, their drug loading capacity is low, and due to the characteristics of the carrier molecules themselves, the range of drug molecules that can be encapsulated is relatively narrow, resulting in limited clinical applications.

[0005] In the prior art, Chinese patent CN117224503A reports a cinnamic acid black phosphorus nanocomposite targeting Aβ, which is composed of a drug-carrying body BP (black phosphorus nanosheet), a modifying material C18-PEG-NH2 (amino polyethylene 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 of Cur, solving the problem of low drug loading of existing nanoparticles, and using Tar as a group targeting Aβ to avoid diffuse distribution of drugs in the brain, thereby increasing the drug utilization rate of Cur and having a positive effect on the treatment of Alzheimer's disease. However, the drug-carrying body (black phosphorus nanosheet) used in this patent still has inherent limitations, which makes it difficult to further increase the maximum loading capacity of its 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 necessary to develop and study a new 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 achieved by 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.

[0009] The drug molecules are drugs for treating neurodegenerative diseases and / or central nervous system tumors.

[0010] The stabilizers include PLGA-PEG copolymers and mannose-modified PLGA-PEG copolymers.

[0011] 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.

[0012] 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.

[0013] The mass ratio of the drug molecules, PLGA-PEG copolymers, and mannose-modified PLGA-PEG copolymers is 1 - 30∶1 - 120∶1 - 120.

[0014] The mass ratio of the drug molecules to the oil-phase molecules is 1 - 20∶1 - 100.

[0015] The mass ratio of the oil-phase molecules to the surfactant is 1 - 10∶10 - 1.

[0016] 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.

[0017] Preferably, the drug molecule and the stabilizer are dissolved in an organic solvent to prepare a thin film, and then the thin 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.

[0018] An orally administered brain-targeted nanoemulsion is obtained by the above preparation method, and the obtained nanoemulsion satisfies the following:

[0019] Particle size: 70 - 150 nm;

[0020] PDI: < 0.3;

[0021] Stability: The change range of the particle size of the nanoemulsion within three days < 10%.

[0022] The application of the orally administered brain-targeted nanoemulsion provided by the present invention is to use the above-mentioned orally administered brain-targeted nanoemulsion to prepare an oral drug for the treatment and / or prevention of 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.

[0023] 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.

[0024] 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).

[0025] 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.

[0026] The pharmaceutical molecules for treating Huntington's disease include at least one of tetrabenazine, tritiated tetrabenazine, and nucleic acid drugs;

[0027] The pharmaceutical molecules for treating amyotrophic lateral sclerosis include at least one of celastrol, curcumin, ginsenoside, icariin, riluzole, edaravone, baclofen, diazepam, trihexyphenidyl, amitriptyline, sodium phenylbutyrate, and tauroursodeoxycholic acid;

[0028] The pharmaceutical molecules for treating Parkinson's disease include at least one of artane, amantadine, madopar, clozapine, quetiapine, ziprasidone, rivastigmine, donepezil, pimavanserin, aripiprazole, and piribedil;

[0029] The pharmaceutical molecules for treating multiple sclerosis include at least one of dexamethasone, methylprednisolone, cyclophosphamide, and prednisone;

[0030] The pharmaceutical drugs for treating depression include at least one of curcumin, ginsenoside, bupleurum saponin, spinosin, morinda officinalis oligosaccharide, polygala saponin, ginkgolide B, hesperidin, pachymic acid, cannabidiol, paroxetine, sertraline, fluoxetine, fluvoxamine, and ketamine;

[0031] The pharmaceutical molecules for treating neuroinflammation include at least one of mecobalamin, adenosylcobalamin, vitamin B12, and aspirin;

[0032] The pharmaceutical molecules for treating cerebrovascular diseases include at least one of astragaloside IV, hydroxysafflor yellow A, emodin, lovastatin, aspirin, ropizole, mannitol, and piracetam;

[0033] The pharmaceutical molecules for treating hydrocephalus include at least one of acetazolamide, mannitol, and glycerol fructose.

[0034] 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 brain metastases, oligodendroglioma, astrocytoma, and meningioma.

[0035] The pharmaceutical 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.

[0036] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0037] (1) Compared with the single-drug-loaded nano-delivery system, the nanoemulsion of the present invention can significantly improve the therapeutic effect of drugs through the combined action of two or more drug molecules, and has an efficient therapeutic effect in neurodegenerative diseases or central nervous system tumor diseases.

[0038] (2) The present invention designs for the first time a nanoemulsion with drug-carrier homology. In the treatment of AD, oil-phase molecules such as cinnamaldehyde, vanillin, vitamin E or fish oil can not only serve as the oil-phase components of the nanoemulsion, but also act as medicinal molecules for anti-AD, achieving a multi-functional effect.

[0039] (3) Compared with the existing oral or intravenous nano-delivery systems, the nanoemulsion of the present invention significantly increases the drug loading capacity, thereby reducing the dosing frequency and enhancing the compliance of patients with long-term diseases.

[0040] (4) By using the functional polymer fragment PLGA-PEG-Man (such as PLGA 5000 -PEG 3400 -Man), the nanoemulsion of the present invention not only improves the stability of the nanoemulsion, but also endows the nanoemulsion with the targeting ability to GLUT1 expressed on intestinal epithelial cells and brain endothelial cells, increasing the oral brain uptake efficiency of the nanoemulsion.

[0041] (5) Compared with the existing multi-barrier-crossing nano-targeted drug delivery systems, the nanoemulsion drug delivery system prepared by the present invention has simple and safe components, an easy synthesis method, and has good application prospects.

[0042] In summary, the present invention provides a stable (drug-carrier homologous) multi-drug system nanoemulsion co-delivery 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), realize the oral administration method, significantly improve the patient compliance, and can achieve a 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

[0043] Figure 1 It is a schematic diagram of the treatment principle of the nanoemulsion of the present invention.

[0044] Figure 2 It is the characterization of different nanoemulsions.

[0045] Figure 3 It is the particle size and polydispersity index PDI of different nanoemulsions (n = 3).

[0046] Figure 4 It is the zeta potential of different nanoemulsions (n = 3).

[0047] Figure 5 For the particle size stability of different nanoemulsions in PBS (n = 3).

[0048] Figure 6 For the stability of different nanoemulsions in simulated gastric fluid (SGF) (n = 3).

[0049] Figure 7 For the stability of different nanoemulsions in simulated intestinal fluid (SIF) (n = 3).

[0050] Figure 8 For the relative mucus adsorption ratio of different nanoemulsions (n = 3).

[0051] Figure 9 For the cumulative release amount of curcumin from nanoemulsions in artificial gastrointestinal fluid (n = 3).

[0052] Figure 10 For the cumulative release amount of cinnamaldehyde from nanoemulsions in artificial gastrointestinal fluid (n = 3).

[0053] Figure 11 For the fluorescence intensity after Caco-2 cells uptake different nanoemulsions (using the CC NE group as a control, n = 3).

[0054] Figure 12 For the fluorescence intensity after bEnd.3 cells uptake different nanoemulsions (using the CC NE group as a control, n = 3).

[0055] Figure 13 For the fluorescence intensity after BV-2 cells uptake different nanoemulsions (using the CC NE group as a control, n = 3).

[0056] Figure 14 For the statistical data of 12 H nesting behavior of APP / PS1 transgenic mice after different treatments (n = 12).

[0057] Figure 15 For the nesting diagrams of APP / PS1 transgenic mice 12 H after different treatments (n = 12).

[0058] Figure 16 For the effects of different preparations on the levels of IL-6 inflammatory factors in the brain tissues of APP / PS1 transgenic mice.

[0059] Figure 17 For the effects of different preparations on the levels of TNF-α inflammatory factors in the brain tissues of APP / PS1 transgenic mice. Detailed implementation manners

[0060] The object of the invention, the technical solution and the beneficial effects of the present invention will be further described in detail below.

[0061] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the claimed invention. Unless otherwise noted, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which the present invention belongs.

[0062] The present invention is 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 combined with the characteristics of in vivo physiological barriers. A nanoemulsion with an orally administered brain-targeting function and a multi-drug system is designed for the multi-modal treatment of AD. The nanoemulsion innovatively adopts a drug-loaded homologous mode and selects cinnamaldehyde molecules (such as vanillin, vitamin E or fish oil as alternatives), which can not only play roles such as inhibiting Aβ generation, antioxidation, and antibacterial as drug molecules, but also act as the oil phase of the nanoemulsion as a carrier component. To further increase the anti-AD efficacy of the nanoemulsion and make up for the deficiencies in the regulation of AD pathological characteristics by cinnamaldehyde, other drugs for the treatment of AD can also be selected, such as curcumin, baicalein, paeoniflorin, apigenin, puerarin, quercetin, etc.

[0063] 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 cinnamaldehyde and curcumin, which are both poor in stability and water solubility, into a nanoemulsion, 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 a stabilizer component, 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 a 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 the active targeting ability of the intestinal barrier and the blood-brain barrier, so that the two drugs can be successfully delivered to the brain region to play their role in the multi-modal treatment of AD. For details, see Figure 1 .

[0064] It should be noted that in the present invention, by changing the dosage form of the existing nanoparticles and using the cinnamaldehyde 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, in combination with other drugs for treating 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, enabling the dispersion of drug molecules in the nanoemulsion and being unrestricted by the nature of the carrier itself. Therefore, the drug loading capacity and the drug selection range of drug molecules can be significantly improved, facilitating the selection of a multi-modal fusion treatment method during AD treatment and enhancing the AD treatment effect. Further, through the selection of the nanoemulsion dosage form, by using surfactants (such as Tween 80) to provide their specific interfacial properties in the oil phase molecules, the solubility of the drug in the oil and water phases can be significantly enhanced by forming micelles or reducing the interfacial tension. And through interfacial adsorption, the surface of the nanoemulsion droplets is covered with a dense surfactant layer, and the drug molecules are fixed through physical adsorption or chemical action to prevent drug leakage and improve the loading efficiency.

[0065] The following is a further detailed overview of the technical solution of the present invention:

[0066] Disperse drug molecules, PLGA-PEG copolymer, and PLGA-PEG-Man copolymer in the oil phase molecules, and then add surfactants and water to prepare the nanoemulsion. During preparation, it is necessary to control the mass ratio of drug molecules, PLGA-PEG copolymer, and PLGA-PEG-Man copolymer to be 1-30:1-120:1-120.

[0067] The drug molecules are drugs 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 brain metastases, oligodendroglioma, astrocytoma, and meningioma. When selecting drug molecules, 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 the above has been detailedly described in the present invention, it will not be elaborated here.

[0068] Taking the drug for treating AD as an example, the drug molecules can be hydrophobic drugs such as curcumin, apigenin, puerarin, quercetin, etc., or hydrophilic drugs 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. The organic solvents can be 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 evaporate to dryness 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.

[0069] 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. The selectable oil phase molecules are such as 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 amount. Usually, the mass ratio of the surfactant to the oil phase molecule is controlled to be 1 - 10∶10 - 1. The selectable surfactants are such as 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.

[0070] 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.

[0071] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

[0072] Embodiment 1:

[0073] Accurately weigh 10 mg of curcumin, PLGA 5000 -PEG 2000 20mg, PLGA 5000 -PEG 3400 -Man 20 mg was dissolved in EA (ethyl acetate) and then dried into a thin film using a rotary evaporator. Subsequently, 100 mg of cinnamaldehyde and 100 μL of Tween80 were added and the film was dissolved by continuing to rotate in a water bath or using ultrasound. Then, 3.8 mL of UP water was added and vortexed and mixed. The mixture was then placed in a probe ultrasound machine and ultrasounded at a power of 100 W for 5 min, with ultrasound staying for 5 s to obtain CCPM NE.

[0074] Comparative Example 1:

[0075] Accurately weigh 10 mg of curcumin, dissolve it in EA (ethyl acetate), and then use a rotary evaporator to dry it into a thin film. Then add 100 mg of cinnamaldehyde and 100 μL of Tween 80 and continue to rotate the water bath or use ultrasound to dissolve the film. Then add 3.8 mL of UP water and vortex to mix. Then place it in a probe ultrasound and sonicate at a power of 100 w for 5 min, with ultrasound staying for 5 s to obtain CC NE.

[0076] Comparative Example 2:

[0077] Accurately weigh 10 mg of curcumin, PLGA 5000 -PEG 2000 40 mg of both were dissolved in EA (ethyl acetate) and then dried into a thin film using a rotary evaporator. Subsequently, 100 mg of cinnamaldehyde and 100 μL of Tween 80 were added and the film was dissolved in a rotating water bath or by ultrasound. 3.8 mL of UP water was added and vortexed to mix well. The mixture was then placed in a probe ultrasound machine and ultrasounded at a power of 100 W for 5 min, with ultrasound staying for 5 s. CCP NE was obtained.

[0078] The operating conditions of rotary evaporation, rotary water bath, vortex, ultrasound, etc. involved in the above-mentioned Example 1, Comparative Example 1 and Comparative Example 2 are the same, and refer to the above-mentioned detailed overview of the technical solution of the present invention.

[0079] Further, the nanoemulsions involved in the above-mentioned Example 1, Comparative Example 1 and Comparative Example 2 are compared as follows:

[0080] 1. Surface characteristics of nanoemulsion

[0081] Figure 2Respectively shown are the undiluted nanoemulsion (A) and the solution (B) diluted with UP water of Example 1 (CCPM NE), Comparative Example 1 (CC NE), and Comparative Example 2 (CCP NE). From Figure 2 It can be seen that nanoemulsions can be prepared in Example 1, Comparative Example 1, and Comparative Example 2, and their apparent characteristics show as bright yellow emulsions, with opalescence after dilution with UP water.

[0082] (II) Physicochemical characteristics of nanoemulsion (particle size)

[0083] 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 nanoemulsion respectively.

[0084] The measurement results are shown in Figures 3 to 5 Among them, 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.

[0085] From Figure 3 It can be seen that the particle sizes of the nanoemulsions in Example 1, Comparative Example 1, and Comparative Example 2 are around 100 nm, and the PDI is less than 0.3, indicating good homogeneity.

[0086] From Figure 4 It can be seen 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.

[0087] From Figure 5 It can be seen 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.

[0088] (III) Stability experiment

[0089] 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 nanoemulsion was dispersed in simulated gastric fluid SGF (preparation method is shown in the Chinese Pharmacopoeia), and the particle size of the nanoemulsion was measured at 0 h, 6 h, and 24 h; the nanoemulsion was dispersed in simulated intestinal fluid SIF (preparation method is shown in the Chinese Pharmacopoeia), and the particle size of the nanoemulsion was measured at 0 h, 6 h, and 24 h.

[0090] The measurement results are shown in Figure 6 and Figure 7, where Figure 6 is the stability of different nanoemulsions in simulated gastric fluid (SGF), Figure 7 is the stability of different nanoemulsions in simulated intestinal fluid (SIF).

[0091] From Figure 6 it can be seen that the stabilities of the nanoemulsions of Example 1, Comparative Example 1 and Comparative Example 2 in simulated gastric fluid are different. The nanoemulsion CCNE (Comparative Example 1) without polymer fragments has poor stability.

[0092] From Figure 7 it can be seen that when the nanoparticles of Example 1, Comparative Example 1 and Comparative Example 2 are dispersed in simulated intestinal fluid, their particle sizes all increase to a certain extent. Generally speaking, within 6 h, their particle sizes do not change significantly.

[0093] (IV) Mucus adsorption experiment

[0094] For Example 1 (CCPM NE), Comparative Example 1 (CC NE) and Comparative Example 2 (CCP NE), their adsorption in mucus solution was measured respectively.

[0095] Experimental method: Porcine gastric mucin was configured in a buffer solution with pH = 6.5 prepared by disodium hydrogen phosphate and sodium dihydrogen phosphate at a concentration of 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).

[0096] The test results are shown in Figure 8 , Figure 8 is the relative mucus adsorption ratio of different nanoemulsions.

[0097] From Figure 8 it can be seen that the nanoemulsions of Example 1, Comparative Example 1 and Comparative Example 2 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 more easily penetrate the mucus barrier.

[0098] (V) Drug release amount

[0099] 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.

[0100] Experimental method: Different nanoemulsions were successively placed in artificial gastric juice and artificial intestinal juice (the preparation method is 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 incubated with shaking 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 destroy 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.

[0101] 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 fluid, Figure 10 is the cumulative release amount of cinnamaldehyde from the nanoemulsion in artificial gastrointestinal fluid.

[0102] From Figure 9 and Figure 10 it can be seen 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, the release amounts of the two drugs in CC NE are significantly higher than those in CCPM NE at 4 h. 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.

[0103] 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 functionalized 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.

[0104] (VI) Cell fluorescence experiment

[0105] For Example 1 (CCPM NE), Comparative Example 1 (CC NE), and Comparative Example 2 (CCP NE), Caco-2 cells, bEnd-3 cells, and BV-2 cells were used to measure the fluorescence intensity after taking up different nanoemulsions, respectively.

[0106] Experimental method: Inoculate Caco-2 cells at 3×10 5 cells / well in a 6-well plate and wait for the cells to grow for 24 h. Remove the culture medium, add nanoemulsions with different components (Cur 2.5 μg / mL), and incubate for 3 h. Then remove the culture medium, wash the cells 3 times with PBS, digest the cells into single-cell suspensions with trypsin, then centrifuge at 3000 rpm and 4 °C for 3 min to collect the cells and resuspend them in PBS, and measure the fluorescence intensity in the cells of each sample group by flow cytometry.

[0107] Inoculate bEnd.3 cells at 3×10 5 cells / well in a 6-well plate and grow them in a cell incubator at 37 °C and 5% CO2 for 24 h. Then remove the culture medium, add different nanoemulsions (Cur 2.5 μg / mL), and incubate for 3 h. Remove the culture medium, wash the cells 3 times with PBS, digest the cells into single-cell suspensions with trypsin, then centrifuge at 3000 rpm and 4 °C for 3 min to collect the cells and resuspend them in PBS, and measure the fluorescence intensity in the cells of each sample group by flow cytometry.

[0108] Inoculate BV-2 cells at 2×10 5 cells / well in a 6-well plate and grow them in a cell incubator at 37 °C and 5% CO2 for 12 h. Then remove the culture medium, add a culture medium containing LPS (2 μg / mL) and culture for 24 h. Then remove the culture medium, add different nanoemulsions (Cur 2.5 μg / mL), and incubate for 3 h. Remove the culture medium, wash the cells 3 times with PBS, digest the cells into single-cell suspensions with trypsin, then centrifuge at 3000 rpm and 4 °C for 3 min to collect the cells and resuspend them in PBS, and measure the fluorescence intensity in the cells of each sample group by flow cytometry.

[0109] The experimental results are shown in Figures 11 to 13 , where Figure 11 is the fluorescence intensity of Caco-2 cells after taking up different nanoemulsions, Figure 12 is the fluorescence intensity of bEnd.3 cells after taking up different nanoemulsions, Figure 13 is the fluorescence intensity of BV-2 cells after taking up different nanoemulsions.

[0110] It can be seen from Figure 11 that the cell uptake behaviors of nanoemulsions with different components have obvious differences, among which the uptake behavior of CC NE is the lowest. Adding the polymer PLGA 5000-PEG 2000 There was a certain increase in uptake after CCP NE, and PLGA 5000 -PEG 2000 and PLGA 5000 -PEG 3400 -Man polymer showed a significant increase in its uptake behavior in CCPM NE, which was 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 of intestinal epithelial cells and improve the endocytosis efficiency of nanoemulsion.

[0111] As can be seen from Figure 12 it, among different nanoemulsions, CCPM NE showed the strongest cell endocytosis ability, which was 2.1 times higher than that of CC NE. It shows that the addition of functional polymer fragments can achieve the active targeting of nanoemulsion to brain endothelial cells, thereby achieving the purpose of increasing the amount of nanoemulsion entering the brain.

[0112] As can be seen from Figure 13 it, the difference in uptake multiples of different nanoemulsions on BV-2 cells was not obvious, but generally speaking, CCPMNE still had the best cell targeting ability, indicating that the addition of functional polymer fragments further improved the uptake ability of nanoemulsion by brain target cells.

[0113] (VII) Mouse treatment experiment

[0114] 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).

[0115] After the above grouped treatment, the mice were compared for the treatment effects of different experimental groups through behavioral cognitive tests and inflammatory factor detection (IL-6 / TNF-α).

[0116] (1) Behavioral cognitive test

[0117] Test method: The mice were divided into single cages (1 mouse per cage). After allowing them to adapt to the single-cage environment for 48 h, 20 odorless thin papers (10x10 cm 2 ) were placed in one 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.

[0118] The test results are shown in Figure 14 and Figure 15 , where Figure 14 are the statistical data of the 12-hour nesting behavior of APP / PS1 transgenic mice after different treatments, Figure 15 is the nesting diagram of APP / PS1 transgenic mice 12 hours after different treatments.

[0119] From Figure 14 and Figure 15 it can be seen that after different treatments, the nesting behavior of APP / PS1 transgenic mice showed obvious differences. Among them, compared with the AD group, the Free Cur group showed a slight improvement, but the nesting behavior of the CCPMNE group was significantly improved. 50% of the mice were able to completely tear the paper and build obvious nest sites, getting a full score for nesting, and the overall nesting 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 mice was significantly improved.

[0120] (2) Detection of inflammatory factors

[0121] 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 the inflammatory factors IL-6 and TNF-α in the brain tissue proteins were measured by an ELISA kit.

[0122] The test results are shown in Figure 16 and Figure 17 , where Figure 16 is the effect of different preparations on the level of IL-6 inflammatory factor in the brain tissues of APP / PS1 transgenic mice, Figure 17 is the effect of different preparations on the level of TNF-α inflammatory factor in the brain tissues of APP / PS1 transgenic mice.

[0123] From Figure 16 it can be seen that after treatment, the pro-inflammatory factor IL-6 index in the brain tissues of 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.

[0124] From Figure 17 it can be seen that after treatment, the pro-inflammatory factor TNF-α index in the brain tissues of 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.

[0125] The above results together indicate that nanoemulsion can exert a good neuroprotective effect and play an anti-AD therapeutic effect.

[0126] The above are only the preferred embodiments of the present invention, and do not impose any formal limitations on the present invention. Any simple modifications and equivalent changes made to the above embodiments based on the technical essence of the present invention all fall within the protection scope of the present invention.

Claims

1. A preparation method of an orally-administered brain-targeted nanoemulsion, characterized in that: Disperse the drug molecule and the stabilizer in the oil-phase molecule, then add the surfactant and water to prepare an oil-in-water nanoemulsion. The drug molecule is a drug for treating neurodegenerative diseases and / or central nervous system tumors. The stabilizer includes a PLGA-PEG copolymer and a PLGA-PEG-mannose copolymer. The oil-phase molecule is selected from at least one of cinnamaldehyde, castor oil, olive oil, vitamin E, soybean oil, peanut oil, fish oil, coconut oil, sesame oil, squalene, and medium-chain triglycerides.

2. The preparation method according to claim 1, characterized in that: The molecular weight of PLGA is 2,000 to 40,000, and the molecular weight of PEG is 1,000 to 10,000.

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

4. The preparation method according to claim 1, wherein: The mass ratio of the drug molecule to the oil-phase molecule is 1 to 20:1 to 100.

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

6. The preparation method according to claim 1, characterized in that: The oil-phase molecule is selected from at least one of cinnamaldehyde, 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, sorbitan oleate, sorbitan laurate, polyoxyethylene castor oil, poloxamer 188, glycyrrhizic acid, and fatty alcohol polyoxyethylene ether.

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

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

9. Application of an oral brain-targeting nanoemulsion, characterized in that: Use the oral brain-targeted nanoemulsion according to claim 8 to prepare an oral drug for treating and / or preventing neurodegenerative diseases, and the neurodegenerative diseases include Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, Parkinson's disease, multiple sclerosis, and spinocerebellar ataxia. 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, celastrol, rapamycin, salidroside, resveratrol, berberine, huperzine A, fingolimod, dl-3-n-butylphthalide, 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 Huntington's disease include at least one of tetrabenazine, deutetrabenazine, nucleic acid drugs. The drug molecules for treating amyotrophic lateral sclerosis include at least one of celastrol, curcumin, ginsenoside, icariin, riluzole, edaravone, baclofen, diazepam, trihexyphenidyl, amitriptyline, sodium phenylbutyrate, and 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.

10. Application of the orally administered brain-targeted nanoemulsion, characterized in that: The oral brain-targeted nanoemulsion described in claim 8 is used for preparing an oral drug for treating and / or preventing central nervous system tumors, and the central nervous system tumors include primary glioma, secondary tumor brain metastasis, oligodendroglioma, astrocytoma, and meningioma. The drug molecules for treating central nervous system tumors include at least one of artemisinin, resveratrol, oridonin, nobiletin, paclitaxel, doxorubicin, temozolomide, methotrexate, fluorouracil, cytarabine, ibrutinib, and hydroxychloroquine.

Images