WO2020024490A1 - 一种抗阿尔兹海默症的天然纳米粒-药物组合物及其制备方法和应用 - Google Patents

一种抗阿尔兹海默症的天然纳米粒-药物组合物及其制备方法和应用 Download PDF

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WO2020024490A1
WO2020024490A1 PCT/CN2018/118109 CN2018118109W WO2020024490A1 WO 2020024490 A1 WO2020024490 A1 WO 2020024490A1 CN 2018118109 W CN2018118109 W CN 2018118109W WO 2020024490 A1 WO2020024490 A1 WO 2020024490A1
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natural
drug
nanoparticle
pharmaceutical composition
natural nanoparticle
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周建平
丁杨
张华清
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中国药科大学
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
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    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
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    • A61K9/5107—Excipients; Inactive ingredients
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
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    • B82—NANOTECHNOLOGY
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    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • the invention belongs to the field of pharmaceutical preparations, in particular to the preparation and application of a natural nanoparticle-pharmaceutical composition against Alzheimer's disease.
  • AD Alzheimer's disease
  • senile dementia is a degenerative disease of the central nervous system characterized by progressive cognitive impairment and memory loss.
  • the World Alzheimer's Report states that the number of patients with dementia is expected to nearly double every 20 years. The number of patients will soon exceed 100 million by 2050. Therefore, Alzheimer's disease has become a major public health issue and an increasingly prominent social issue of common concern to countries around the world.
  • the bottleneck of treatment is mainly due to the lack of effective treatment methods designed for the disease process and the cause of the disease, and the difficulty of brain drug delivery caused by the presence of the blood-brain barrier (BBB).
  • BBB blood-brain barrier
  • the drugs for clinical AD treatment are only to relieve the symptoms of the disease.
  • Five drugs are widely used in clinical practice, of which four are cholinesterase inhibitors (donepezil, tacrine, galantamine, and lismin) And a glutamate NMDA receptor antagonist (memantine).
  • cholinesterase inhibitors cause the latter to temporarily lose activity by binding to the target enzyme to form a covalent complex, delaying the degradation of acetylcholine released by cholinergic neurons, and selectively enhancing the effects of acetylcholine in the cerebral cortex and hippocampus Promote the activity of cholinergic nerves, improve the decline of learning and memory ability of cholinergic deficiency;
  • NMDA receptor antagonists act on the glutamate neurotransmitter system in the brain to replace magnesium ions to occupy NMDA access and inhibit calcium influx, Improve cognition.
  • the above five drugs can only alleviate the symptoms to a certain extent and cannot effectively suppress the disease process.
  • AD Alzheimer's disease
  • the blood-brain barrier is a double barrier formed by brain capillary endothelial cells, glial cells, and choroid plexus, which can prevent 98% of drugs from penetrating into brain tissue.
  • NFT Neurofibrillary tangle formed by senile plaque (SP) formed by the deposition of ⁇ amyloid protein (A ⁇ ) in neurons and highly phosphorylated Tau protein in neurons
  • SP senile plaque
  • a ⁇ amyloid protein
  • a ⁇ has become the most important disease biomarker and therapeutic target for AD, and how to reduce the level of A ⁇ 1-42 in the brain has also become an important strategy for AD treatment.
  • Immunotherapy to clear brain A ⁇ deposits and improve AD pathology is by far the most effective treatment for AD drugs in preclinical studies.
  • due to poor brain targeting immunotherapy incorrectly recognizes APP fragments of neuronal cells and immune side effects.
  • Ended in failure in preclinical studies. Therefore, it is urgent to develop a new treatment strategy that can overcome the BBB and specifically accumulate in the brain lesions, and then recognize the neurotoxic A ⁇ accumulated in the extracellular space of the neurons, effectively eliminating A ⁇ without significant side effects.
  • Lipoprotein is an endogenous microparticle present in the plasma and responsible for important lipid metabolism. It is mainly composed of phospholipids, cholesterol, apolipoprotein, and non-polar lipids. Recent studies have shown that the level of lipoprotein expression in the brain plays an important role in A ⁇ clearance. Injecting apolipoprotein through the tail vein can clear A ⁇ deposition in the brain and improve the learning and memory ability of AD model mice. However, natural lipoproteins are scarce in source, cumbersome to prepare, and poor in quality control. Therefore, in vitro recombinant forms of endogenous lipoproteins-recombinant lipoproteins-have attracted much attention due to their unique "bionic" properties.
  • Recombinant lipoproteins have the unique amphiphilic (external hydrophilic-internal hydrophobic) structure of natural lipoproteins, and have achieved a variety of load forms (surface loading, covalent modification, core embedding, etc.), which is conducive to drug loading and targeted modification; At the same time, recombinant lipoproteins can be mediated by scavenger receptors (SR-BI receptors) and low-density lipoprotein-related receptors (LDLR-1) overexpressed by BBB endothelial cells to cross the BBB barrier and achieve efficient brain targeting Drug delivery is a good brain-targeted delivery vehicle.
  • SR-BI receptors scavenger receptors
  • LDLR-1 low-density lipoprotein-related receptors
  • CN 104324007A discloses a preparation technology and application of natural lipid nanoparticles, specifically extracting apolipoproteins and phospholipids from plasma component IV in vitro and recombining with antitumor drugs to prepare highly effective tumor-targeting nanoparticles, and the method is suitable for large Large-scale production, but the method has relatively limited raw material sources, and the purity of the extracted lipoproteins is not high. At the same time, it is used for the delivery of antitumor drugs. How to extract high-purity lipoproteins, expand the source of raw materials, and achieve anti-Alzheimer's disease needs to be improved.
  • CN104138595A discloses the preparation of a bionic recombinant high-density lipoprotein and the application of prevention and treatment of Alzheimer's disease.
  • the recombinant high-density lipoprotein is constructed using in vitro lipid and apolipoprotein recombination to achieve targeted elimination of A ⁇ for the treatment of AD
  • the lipoprotein composition involved in this method is only high-density lipoprotein and the composition is not exactly the same as natural lipoprotein, which cannot reproduce the physiological function of natural lipoprotein well.
  • the recombinant high-density lipoprotein is not used as a brain Targeted drug carriers address the problem of targeted drug delivery in the AD brain. Therefore, it has not been reported how to prepare recombinant lipoproteins that highly reduce the physiological functions of endogenous lipoproteins and efficiently carry drugs to achieve targeted brain delivery.
  • the technical problem to be solved by the present invention is to provide a process for preparing a natural nanoparticle-pharmaceutical composition against Alzheimer's disease and its application against Alzheimer's disease.
  • the present invention provides a natural nanoparticle-pharmaceutical composition for anti-Alzheimer's disease, a preparation process, and an application for anti-Alzheimer's disease.
  • the natural nanoparticle-pharmaceutical composition has brain-targeting and amyloid-targeting properties. Based on the total mass of the prescription, the natural nanoparticle content accounts for 50-99% of the total prescription mass, and the drug content accounts for the total prescription. 1 to 50% of the mass.
  • the natural nanoparticle is a natural nanoparticle with anti-Alzheimer's effect extracted from a plasma component or expressed by genetic engineering, and has a particle size range of 20-200 nm. .
  • the natural nanoparticle-pharmaceutical composition is characterized in that the natural nanoparticle is a natural lipoprotein nanoparticle, the lipoprotein is mainly composed of apolipoprotein and lipid, and is selected from high density lipoprotein , One or more of low density lipoprotein and very low density lipoprotein.
  • the apolipoprotein source is one or more selected from apolipoprotein apoA-I, apoA-II, apoE, apoC, apoB, and the apolipoprotein mimetic peptide. Species.
  • the lipid is selected from one or more of cholesterol, cholesterol ester, phosphatidylcholine, triglyceride, ceramide, ganglioside, and cerebral phospholipid.
  • the drug for treating Alzheimer's disease is selected from the group consisting of cholinesterase inhibitors, aspartate receptor antagonists, antioxidants, anti-inflammatory drugs, protein drugs, peptide drugs and gene drugs. Or a derivative selected from one or more of donepezil, huperzine A, memantine, curcumin, methylene blue, a NAP polypeptide, and siRNA.
  • the natural nanoparticle-pharmaceutical composition is prepared by an ultrasonic drug loading method (see Example 1 for details), and the specific steps are as follows:
  • the drug solvent is ultrapure water; when the drug is a fat-soluble drug, the drug solvent is ethanol.
  • the natural nanoparticle-pharmaceutical composition is prepared by a recombinant ultrasound method (see Example 1 for details), and the specific steps are as follows:
  • step (4) adding ultrapure water to the lipid film in step (4) and hydrating at 20 to 40 ° C for 5 to 20 minutes;
  • the probe is dispersed ultrasonically in an ice bath for 5-20 minutes to obtain drug-loaded nanoparticles
  • step (6) Add the apolipoprotein solution to the drug-loaded nanoparticles in step (6), and incubate with magnetic stirring at room temperature for 24-48 hours; after incubation, pass the 0.22 ⁇ m filter membrane and freeze-dry to obtain the natural nanoparticle-drug combination Thing.
  • the natural nanoparticle-pharmaceutical composition may be administered after being diluted with a buffer solution, the buffer solution including physiological saline, phosphate buffer solution, 5% glucose solution, HEPES buffer solution, and Tris buffer solution.
  • the method of administration is intravenous, or oral, or nasal, and is used for the treatment of Alzheimer's disease.
  • a natural nanoparticle-pharmaceutical composition was constructed by extracting natural lipoprotein nanoparticles and recombining anti-AD drugs in vitro.
  • the composition has high biomimeticity and biological safety, and can reduce endogenous lipoprotein high blood-brain barrier permeability and complete AD.
  • the therapeutic drug is delivered to the brain, the targeted clearance of A ⁇ at the lesion site is achieved, and the effect of multimodal AD treatment can be achieved. It can solve the problems of current AD treatment drug delivery difficulty and single treatment mode, and has a wide application prospect.
  • natural lipoproteins and anti-Alzheimer's chemicals are reconstituted in vitro to construct a natural nanoparticle-medicine composition against Alzheimer's disease, which can achieve targeted delivery of AD treatment drugs in the brain, and at the same time mimic natural fats in the body
  • the protein's A ⁇ clearance mechanism and the realization of multimodal combined therapy have important regulatory effects on AD disease process.
  • Recombinant natural lipoprotein and anti-Alzheimer's drug in vitro to construct a natural nanoparticle-pharmaceutical composition against Alzheimer's disease can achieve targeted delivery of AD treatment drugs in the brain, solve drug delivery difficulties in the brain, and combine lipid Targeted clearance of protein A ⁇ in the brain, to achieve multimodal AD treatment.
  • the invention has the following advantages:
  • the lipoprotein composition used is exactly the same as the natural lipoprotein, which guarantees its biological safety and biological function integrity, especially the endogenous lipoprotein brain targeting and Targeted A ⁇ clearance in the brain.
  • Multimodal combination therapy Aiming at the mechanism of AD formation and development, the use of anti-AD drugs to relieve symptoms combined with the A ⁇ targeted clearance function of lipoproteins was selected to achieve efficient combined treatment of AD disease nervous system.
  • natural lipoproteins and anti-AD drugs can be reconstituted in vitro by ultrasonic drug loading method or recombinant ultrasonic method to conveniently prepare natural nanoparticle-pharmaceutical compositions with high drug content and simple preparation
  • the invention provides a preparation process and application of a natural nanoparticle-pharmaceutical composition against Alzheimer's disease, which highly reduces the physiological functions of natural lipoproteins, achieves highly efficient brain-targeted drug delivery and targeted clearance of lesion A ⁇ , and realizes multiple The purpose of the model combined treatment of AD.
  • this technology has the following advantages:
  • the natural nanoparticles used in the present invention are natural lipoprotein nanoparticles, which highly reduce the physiological functions of endogenous lipoproteins and guarantee its high-efficiency brain targeting, high A ⁇ affinity and targeted clearance ability;
  • the present invention realizes highly efficient drug encapsulation on the basis of ensuring the physiological activity of natural nanoparticles, constructs a natural nanoparticle-drug composition, and solves the problem of difficult brain delivery of AD treatment drugs;
  • the natural nanoparticle source according to the present invention is extracted from plasma or expressed through genetic engineering, and has a wide range of sources, high safety, easy mass production, and great potential for clinical application;
  • Figure 1 Schematic illustration of preparation and structure of natural nanoparticle-pharmaceutical composition against Alzheimer's disease
  • Figure 2 Projection electron micrograph of natural nanoparticle-pharmaceutical composition against Alzheimer's disease
  • Figure 3 In vitro release profile of natural nanoparticle-pharmaceutical composition against Alzheimer's disease
  • Figure 4 Investigation of cellular uptake of natural nanoparticles-pharmaceutical composition against Alzheimer's disease
  • Fig. 5 Investigation chart of natural nanoparticle-pharmaceutical composition against Alzheimer's disease to promote A? Uptake by microglia.
  • Example 1 Preparation process of natural nanoparticle-pharmaceutical composition against Alzheimer's disease:
  • the donepezil-loaded natural nanoparticle-pharmaceutical composition is prepared according to the ultrasonic drug loading method. Weigh out a prescribed amount of natural nanoparticle-HDL 10mg dispersed in ultrapure water to form a natural HDL nanoparticle suspension;
  • the drug solvent ethanol was removed by rotary evaporation under reduced pressure at 37 ° C. for 20 min, and then passed through a 0.22 ⁇ m filter membrane and freeze-dried to obtain donepezil-containing natural nanoparticles-pharmaceutical composition.
  • a nanocurcumin-loaded natural nanoparticle-pharmaceutical composition is prepared according to an ultrasonic drug loading method. Weigh out the prescribed amount of natural nanoparticles-natural high-density lipoprotein, low-density lipoprotein, and very low-density lipoprotein dispersed in ultrapure water to form natural high-density lipoprotein, low-density lipoprotein, and ultra-low-density lipoprotein nano Granular suspension
  • the drug solvent ethanol was removed by rotary evaporation under reduced pressure at 37 ° C. for 20 min, and then passed through a 0.22 ⁇ m filter membrane and freeze-dried to obtain a curcumin-loaded natural nanoparticle-pharmaceutical composition.
  • a natural nanoparticle-pharmaceutical composition containing Huperzine A is prepared according to the ultrasonic drug loading method. Weigh out the prescribed amount of natural nano-particles-low-density lipoprotein in ultrapure water to form a natural low-density lipoprotein nanoparticle suspension;
  • the drug solvent ethanol was removed by rotary evaporation under reduced pressure at 37 ° C. for 20 min, and then passed through a 0.22 ⁇ m filter membrane and freeze-dried to obtain a natural nanoparticle-pharmaceutical composition containing Huperzine A.
  • a natural nanoparticle-pharmaceutical composition carrying siRNA was prepared by an ultrasonic drug loading method. Weigh out a prescribed amount of natural nanoparticle-HDL 10mg dispersed in ultrapure water to form a natural HDL nanoparticle suspension;
  • a natural nanoparticle-pharmaceutical composition carrying a NAP polypeptide is prepared by an ultrasonic drug loading method. Weigh out a prescribed amount of natural nanoparticles-high density lipoprotein 8mg and low density lipoprotein 2mg and disperse them in ultrapure water to form a suspension of natural high density lipoprotein and low density lipoprotein nanoparticles;
  • a memantine-loaded natural nanoparticle-pharmaceutical composition is prepared by an ultrasonic drug loading method. Weigh out a prescribed amount of natural nanoparticles-low density lipoprotein 5mg and very low density lipoprotein 10mg dispersed in ultrapure water to form a suspension of natural low density lipoprotein and very low density lipoprotein nanoparticles;
  • the drug solvent ethanol was removed by rotary evaporation under reduced pressure at 37 ° C for 20 min, and then passed through a 0.22 ⁇ m filter membrane and freeze-dried to obtain the memantine-containing natural nanoparticle-pharmaceutical composition.
  • a methylene blue-containing natural nanoparticle-pharmaceutical composition is prepared by an ultrasonic drug loading method. Weigh out a prescribed amount of natural nanoparticle-HDL 10mg dispersed in ultrapure water to form a natural HDL nanoparticle suspension;
  • the drug solvent ethanol was removed by rotary evaporation under reduced pressure at 37 ° C. for 20 min, and then passed through a 0.22 ⁇ m filter membrane and freeze-dried to obtain a natural nanoparticle-medicine composition containing methylene blue.
  • a prepared nanoparticle-pharmaceutical composition containing donepezil was prepared by a recombinant ultrasonic method. Weigh a prescribed amount of lipid phosphatidylcholine 5mg and cholesterol 1mg, add a film-forming solvent (chloroform: methanol, 1: 1) to form a lipid solution;
  • step (4) adding ultrapure water to the lipid film in step (4) and hydrating at 37 ° C. for 5 min;
  • the probe is dispersed ultrasonically for 5 min in an ice bath to obtain drug-loaded nanoparticles
  • step (7) Add the apolipoprotein solution to the drug-loaded nanoparticles in step (6), and magnetically incubate at room temperature for 24 hours; after the end of the incubation, pass through a 0.22 ⁇ m filter membrane and freeze-dry to obtain the donepezil-containing natural nanoparticles-drug combination.
  • a natural nano-curcumin-pharmaceutical composition containing curcumin was prepared by a recombinant ultrasonic method. Weigh a prescribed amount of lipid phosphatidylcholine 2mg, cholesterol 5mg, and cholesterol ester 1mg into a film-forming solvent (chloroform: methanol, 1: 1) to form a lipid solution;
  • step (4) adding ultrapure water to the lipid film in step (4) and hydrolyzing at 37 ° C for 10 min for hydration;
  • the probe is dispersed ultrasonically in an ice bath for 15 minutes to obtain drug-loaded nanoparticles
  • step (7) Add the apolipoprotein solution to the drug-loaded nanoparticles in step (6), and magnetically incubate at room temperature for 24 hours; after the end of the incubation, pass through a 0.22 ⁇ m filter membrane and freeze-dry to obtain natural curcumin-containing nanoparticles- Pharmaceutical composition.
  • a natural nanoparticle-pharmaceutical composition containing Huperzine A is prepared by a recombinant ultrasonic method. Weigh the prescribed amount of lipid triglyceride 0.5mg, ceramide 1mg, cerebrolipid 1mg, and ganglioside 1mg into the film-forming solvent (chloroform: methanol, 1: 1) to form a lipid solution;
  • step (4) adding ultrapure water to the lipid film in step (4) and hydrolyzing at 37 ° C for 15 min for hydration;
  • the probe is dispersed ultrasonically in an ice bath for 20 minutes to obtain drug-loaded nanoparticles
  • step (7) Add the apolipoprotein solution to the drug-loaded nanoparticles in step (6), and magnetically incubate at room temperature for 24 hours; after incubation, pass through a 0.22 ⁇ m filter membrane and freeze-dry to obtain the natural nanoparticle containing Huperzine A. Granules-pharmaceutical composition.
  • a natural nanoparticle-pharmaceutical composition carrying siRNA was prepared by a recombinant ultrasonic method. Weigh 1 mg of the prescribed amount of cholesterol and 4 mg of phosphatidylcholine into a film-forming solvent (chloroform: methanol, 1: 1) to form a lipid solution;
  • step (4) adding ultrapure water to the lipid film in step (4) and hydrolyzing at 37 ° C for 25 minutes for hydration;
  • the probe is dispersed ultrasonically for 10 min in an ice bath to obtain nanoparticles
  • step (7) Add the apolipoprotein solution to the nanoparticles in step (6), and magnetically incubate at room temperature for 24 hours; after incubation, pass a 0.22 ⁇ m filter membrane and freeze dry to obtain a natural nanoparticle-pharmaceutical composition containing siRNA .
  • a natural nanoparticle-pharmaceutical composition carrying a NAP polypeptide is prepared by a recombinant ultrasonic method. Weigh 5mg of the prescribed amount of cholesterol and 4mg of phosphatidylcholine into a film-forming solvent (chloroform: methanol, 1: 1) to form a lipid solution;
  • step (4) adding ultrapure water to the lipid film in step (4) and hydrolyzing at 37 ° C for 25 minutes for hydration;
  • the probe is dispersed ultrasonically for 10 min in an ice bath to obtain nanoparticles
  • step (7) Add the apolipoprotein solution to the nanoparticles in step (6), and magnetically incubate at room temperature for 24 hours; after incubation, pass a 0.22 ⁇ m filter membrane and freeze dry to obtain a natural nanoparticle-pharmaceutical combination containing NAP polypeptide Thing.
  • a memantine-containing natural nanoparticle-pharmaceutical composition is prepared by a recombinant ultrasonic method. Weigh a prescribed amount of 3 mg of phosphatidylcholine into a film-forming solvent (chloroform: methanol, 1: 1) to form a lipid solution;
  • step (4) adding ultrapure water to the lipid film in step (4) and hydrolyzing at 37 ° C for 15 min for hydration;
  • the probe is dispersed ultrasonically in an ice bath for 15 minutes to obtain drug-loaded nanoparticles
  • step (7) Add the apolipoprotein solution to the nanoparticles in step (6), and magnetically incubate at room temperature for 24 hours; after incubation, pass a 0.22 ⁇ m filter membrane and freeze dry to obtain the memantine-containing natural nanoparticles-drug combination Thing.
  • a methylene blue-containing natural nanoparticle-pharmaceutical composition scale is prepared by a recombinant ultrasonic method. Take a prescribed amount of 3mg of phosphatidylcholine and 2mg of cholesterol into a film-forming solvent (chloroform: methanol, 1: 1) to form a lipid solution;
  • step (4) adding ultrapure water to the lipid film in step (4) and hydrolyzing at 37 ° C for 25 minutes for hydration;
  • the probe is ultrasonically dispersed in an ice bath for 25 minutes to obtain drug-loaded nanoparticles
  • step (7) Add the apolipoprotein solution to the nanoparticles in step (6), and magnetically incubate at room temperature for 24 hours; after the incubation is completed, pass through a 0.22 ⁇ m filter membrane and freeze-dry to obtain the natural nanoparticle-medicine-drug-containing drug combination.
  • Example 2 Investigation of properties of natural nanoparticles-pharmaceutical composition against Alzheimer's disease:
  • the particle size of the natural nanoparticle-pharmaceutical composition measured by dynamic light scattering method was (65.8 ⁇ 4.45) nm, showing a spherical appearance under the projection electron microscope ( Figure 2), and it is consistent with the particle size measured by the laser particle size analyzer.
  • encapsulation rate (%) (Mass of donepezil in the composition / total mass of donepezil input) * 100%
  • drug loading (%) (mass of donepezil in the composition / total mass of the composition) * 100%.
  • the encapsulation rate of the donepezil-containing natural nanoparticle-pharmaceutical composition was measured to be 90%, and the drug loading amount was 5%.
  • Example 3 Preparation of donepezil-containing natural nanoparticles-pharmaceutical composition prepared according to Example 1.1: In vitro BBB model was constructed by Transwell co-culture, and human brain microvascular endothelial cells (hCMEC / D3) were seeded in the upper chamber of Transwell (6 ⁇ 10 5 cells / cm 2 ), mouse-derived microglia (BV-2) were seeded in the lower chamber of Transwell (2 ⁇ 10 5 cells / cm 2 ), and the transmembrane resistance of the cells was measured by a transmembrane resistance meter. When the seventh day of co-cultivation, the transmembrane resistance value was as high as 200 ⁇ / cm 2 , which proved that the Transwell BBB model had been successfully constructed.
  • hCMEC / D3 human brain microvascular endothelial cells
  • BV-2 mouse-derived microglia
  • the fluorescent dye coumarin 6 (C6) was used as a substitute for the drug, and C6 labeled natural nanoparticles-pharmaceutical compositions and liposomes were constructed respectively.
  • C6 labeled natural nanoparticles-pharmaceutical compositions and liposomes were constructed respectively.
  • 200 ⁇ L of C6 labeled natural nanoparticles-pharmaceutical composition and liposome were added respectively.
  • the pre-cooled PBS was added to the lower chamber and washed 3 times. The cells were digested with trypsin and collected. Quantitative statistics of fluorescence uptake of BV-2 cells by mini-flow cytometry.
  • the fluorescence intensity of cells co-incubated with C6 labeled natural nanoparticles-pharmaceutical composition is nearly twice that of C6 labeled liposome group, which proves that natural nanoparticle-pharmaceutical composition can cross BBB endothelial cells and be further Targeted uptake of lower glial cells has good brain targeting capabilities.
  • Example 4 Investigation on the targeted clearance ability of donepezil-containing natural nanoparticles-pharmaceutical composition A ⁇ prepared according to Example 1.1: Microglial cells BV-2 were seeded in a 24-well cell culture plate (2 ⁇ 10 5 cells / well ), Add fluorescently labeled A ⁇ (FAM-A ⁇ ), and respectively add natural nanoparticles-pharmaceutical composition or liposome and incubate for 4h. After incubation, the medium was aspirated, and the cells were washed with pre-chilled PBS and washed three times. After the cells were digested with trypsin, the cells were collected and the fluorescence of the BV-2 cells was quantified using a small flow cytometer.
  • FAM-A ⁇ fluorescently labeled A ⁇

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Abstract

一种抗阿尔兹海默症(AD)的天然纳米粒-药物组合物，及其制备方法和应用。该制备方法为通过提取天然脂蛋白纳米粒与AD治疗药物重组构建天然纳米粒-药物组合物。该天然纳米粒-药物组合物以静脉注射、口服、鼻腔给药等方式给药。

Description

一种抗阿尔兹海默症的天然纳米粒-药物组合物及其制备方法和应用 技术领域
本发明属药物制剂领域，尤其涉及一种抗阿尔兹海默症的天然纳米粒-药物组合物的制备及应用。
背景技术
阿尔茨海默病(Alzheimer's disease，AD)又称老年性痴呆症，是一种以进行性认知障碍和记忆能力减退为主要特征的中枢神经系统退行性疾病。随着全球老龄化进程的发展，AD的发病率持续增高且严重影响人类的健康和生活质量，《世界阿尔茨海默病报告》指出痴呆患者人数预计每20年增长近一倍，并且，到2050年患者人数即将突破1亿，因此，老年痴呆症已成为世界各国共同关注的重大公共卫生课题和越来越突出的社会问题。
然而，目前依旧缺乏有效的AD治疗手段，治疗瓶颈主要在于缺乏有效针对疾病进程、病变原因设计的治疗手段和血脑屏障(blood brain barrier，BBB)存在导致的脑部药物递送困难。目前，临床AD治疗的药物仅在于缓解疾病症状，在临床应用较为广泛的5种药物，其中四种是胆碱酯酶抑制剂(多奈哌齐，他克林，加兰他敏，利斯的明)和一种谷氨酸NMDA受体拮抗剂(美金刚)。其中，胆碱酯酶抑制剂通过与靶酶结合成共价复合物而使后者暂时丧失活性，延缓胆碱能神经元对释放的乙酰胆碱降解，选择性增强脑皮质和海马等部位乙酰胆碱的效应，促进胆碱能神经的活动，改善胆碱能缺失的学习记忆能力下降；NMDA受体拮抗剂是作用于脑内谷氨酸神经递质系统取代镁离子占据NMDA通达，抑制钙离子内流，改善认知。以上5种药物均只能在一定程度上缓解症状，无法有效抑制疾病进程，因此，针对AD病理特征的疾病治疗手段的设计和开发是非常有必要的。AD治疗的另一主要瓶颈，血脑屏障是由大脑毛细血管内皮细胞与神经胶质细胞、脉络丛形成的双重屏障，可阻止98％的药物渗透入脑组织，这也是目前临床AD治疗药物非特异性分布导致的胃肠道不良反应的问题所在。因此，从AD病变病理机制出发设计逆转疾病的治疗手段联合开发BBB高渗透性药物载体对AD治疗至关重要。
神经元细胞外β淀粉样蛋白(βamyloid protein，Aβ)沉积形成的老年斑(senile plaque，SP)和神经元细胞内高度磷酸化的Tau蛋白聚集形成的神经元纤维缠结(neurofibrillary tangle，NFT)是主要的AD的病理学特征。正常情况下，脑内Aβ的产生和清除(胞外降解，小胶质细胞介导的吞噬，转运清除)处于动态平衡状态，而当此平衡遭到破坏，Aβ在脑内过度产生和异常沉积，引起其周边神经元突触功能障碍、Tau蛋白过度磷酸化、氧化应激和继发炎性反应，导致神经元变性死亡，最终产生痴呆。因此，Aβ成为AD最重要的疾病生物标记物和治疗靶点，而如何降低脑内Aβ 1-42水平也成为AD治疗的重要策略。免疫疗法清除脑部Aβ 沉积，改善AD病理进程是迄今为止临床前研究AD药物中最有效的治疗手段，然而，免疫疗法因脑靶向性差，错误识别神经元细胞APP片段及免疫副反应等问题，在临床前研究中以失败告终。因此，迫切需要开发新型治疗策略，可以克服BBB，特异性蓄积于脑部病灶区，进而识别神经元胞外蓄积的神经毒性Aβ，有效清除Aβ并无明显副反应。Song等人利用体外磷脂与载脂蛋白E3重组构建重组高密度脂蛋白apoE3-rHDL纳米粒，该纳米粒经静脉注射后可以跨越BBB实现脑部Aβ清除，治疗AD。Yao等人设计了一种包载α倒捻子素的PEG-PLA纳米粒，通过上调低密度脂蛋白受体表达实现促进脑部Aβ清除，起到AD治疗效果。虽然针对Aβ清除策略设计的纳米粒已有文献报道，但是如何设计并制备兼具高效脑靶向性，高Aβ亲和力和靶向清除能力的纳米药物，实现药物-载体为一体，提高多模式治疗AD效果的技术却有待开发。
脂蛋白(lipoprotein)是一种存在于血浆中的承担重要脂质代谢功能的内源性微粒，主要由磷脂、胆固醇、载脂蛋白和非极性脂质等成分组成。近年研究表明，脑内脂蛋白表达水平在Aβ清除过程中扮演重要角色，通过尾静脉注射载脂蛋白可以清除脑内Aβ沉积，改善AD模型小鼠学习记忆能力。然而，天然脂蛋白来源稀缺、制备繁琐、质量可控性不强，因此，内源脂蛋白体外重组形式-重组脂蛋白因其独特的“仿生”特性备受关注。重组脂蛋白拥有天然脂蛋白的独特两亲性(外亲水-内疏水)结构，成就了多样的荷载形式(表面负载、共价修饰、内核包埋等)，利于药物荷载及靶向修饰；同时，重组脂蛋白可以通过BBB内皮细胞高表达的清道夫受体(SR-BI受体)和低密度脂蛋白相关受体(LDLR-1)介导，跨越BBB屏障，实现高效脑部靶向递送药物，是很好的脑靶向递送载体。基于重组脂蛋白的高效Aβ清除能力及脑靶向递送潜能，通过合理设计联合治疗模式，可以有效改善AD症状，抑制疾病进程，甚至逆转AD。重组脂蛋白制备方法已有文献和专利报道，但存在原料来源有限，制备过程复杂，不能很好模拟内源性脂蛋白等问题。CN 104324007A公开一种天然脂质纳米粒的制备技术及应用，具体为从血浆组分IV中提取载脂蛋白和磷脂在体外与抗肿瘤药物重组制备高效肿瘤靶向性纳米粒，该方法适合大规模生产，但是该方法原料来源较为局限，提取的脂蛋白纯度不高，同时应用为抗肿瘤药物递送，如何提取高纯度脂蛋白，扩大原料来源及实现抗阿尔兹海默病的应用有待改进。CN 104138595 A公开了一种仿生重组高密度脂蛋白的制备及预防和治疗阿尔兹海默病的应用，利用体外脂质和载脂蛋白重组构建重组高密度脂蛋白，实现靶向清除Aβ治疗AD，但该方法涉及的脂蛋白组成仅为高密度脂蛋白且组成成分与天然脂蛋白并不完全相同，不能很好再现天然脂蛋白生理功能，同时该重组高密度脂蛋白并未作为一种脑靶向药物载体解决AD脑部靶向递送药物问题。因此，如何制备高度还原内源脂蛋白生理功能的重组脂蛋白，并高效运载药物实现脑部靶向递送等尚未有报道。
发明内容
本发明所要解决的技术问题是提供一种抗阿尔兹海默症的天然纳米粒-药物组合物的制备工艺及抗阿尔兹海默症的应用。
为了解决上述问题，本发明提供了抗阿尔兹海默症的天然纳米粒-药物组合物的处方组成、制备工艺及提供了其在抗阿尔兹海默症的应用。
作为优选，所述天然纳米粒-药物组合物具有脑靶向性和淀粉样蛋白靶向性，以处方总质量计，天然纳米粒含量占处方总质量的50～99％，药物含量占处方总质量的1～50％。
作为优选，所述天然纳米粒是从血浆组分中提取或经基因工程表达的具有抗阿尔兹海默症作用的天然纳米颗粒，其粒径范围为20-200nm。。
作为优选，所述的天然纳米粒-药物组合物，其特征在于所述的天然纳米粒是天然脂蛋白纳米粒，所述脂蛋白主要由载脂蛋白和脂质组成，选自高密度脂蛋白、低密度脂蛋白和极低密度脂蛋白中的一种或多种。
作为优选，所述载脂蛋白来源为血浆中提取或基因工程获得，选自载脂蛋白apoA-I，apoA-II，apoE，apoC，apoB及所述载脂蛋白模拟肽中的一种或多种。
作为优选，所述脂质选自胆固醇，胆固醇酯，磷脂酰胆碱，甘油三酯，神经酰胺，神经节苷脂，脑磷脂中的一种或多种。
作为优选，所述阿尔兹海默症治疗药物选自胆碱酯酶抑制剂，天门冬氨酸受体拮抗剂，抗氧化剂，抗炎药物，蛋白质药物，多肽药物和基因药物中的任一物质或衍生物，选自多奈哌齐，石杉碱甲，美金刚，姜黄素，亚甲蓝，NAP多肽和siRNA中的一种或多种。
作为优选，所述天然纳米粒-药物组合物的制备工艺为超声载药法(详见实施例1)，具体步骤如下：
(1)将血浆中提取或经基因工程提取天然脂蛋白纳米粒分散于超纯水中，组成天然纳米粒混悬液；
(2)将处方量药物溶解于药物溶剂并逐滴加入(1)中所述天然纳米粒混悬液，置于冰浴下探头超声分散15～60min；
(3)超声结束后，当药物溶剂为有机溶剂时，于20～40℃减压旋蒸5～20min去除药物溶剂；当药物溶剂为水时，不旋蒸去除溶剂；随后过0.22μm滤膜，冷冻干燥即得。
作为优选，所述药物溶剂当所述药物为水溶性药物时，药物溶剂选择超纯水；当药物为脂溶性药物时，药物溶剂选择乙醇。
作为优选，所述天然纳米粒-药物组合物的制备工艺为重组超声法(详见实施例1)，具体 步骤如下：
(1)称取处方量的脂质，加入成膜溶剂，组成脂质溶液；
(2)称取处方量的载脂蛋白溶解于超纯水，组成载脂蛋白溶液；
(3)称取处方量药物，按“相似相溶”原则溶于上述对于组分：脂溶性药物溶解于脂质溶液，水溶性药物溶解于蛋白溶液；
(4)将上述脂质溶液于20～40℃旋蒸0.5～2h成膜，真空干燥过夜；
(5)在步骤(4)所述脂质膜中加入超纯水于20～40℃旋蒸5～20min水合；
(6)水合结束后，于冰浴下探头超声分散5～20min，得载药纳米粒；
(7)将所述载脂蛋白溶液加入步骤(6)所述载药纳米粒中，室温磁力搅拌孵育24～48h；孵育结束后过0.22μm滤膜，冷冻干燥即得天然纳米粒-药物组合物。
作为优选，所述天然纳米粒-药物组合物，可以经缓冲液稀释后给药，所述缓冲液包括生理盐水、磷酸盐缓冲液、5％葡萄糖溶液、HEPES缓冲液和Tris缓冲液，所述给药方式为静脉注射、或口服给药、或鼻腔给药方式，用于阿尔兹海默病的治疗。
本发明的特点在于：
通过提取天然脂蛋白纳米粒体外重组包载抗AD药物构建天然纳米粒-药物组合物，该组合物具有高度仿生性和生物安全性，可以还原内源脂蛋白高血脑屏障渗透性，完成AD治疗药物脑部递送的同时实现病灶部位Aβ的靶向清除，实现多模式AD治疗效果，可以解决目前AD治疗药物递送困难，治疗模式单一等问题，应用前景广泛。
本发明通过将天然脂蛋白与抗阿尔兹海默症化药体外重组构建抗阿尔兹海默症的天然纳米粒-药物组合物，可以实现脑部靶向递送AD治疗药物，同时模拟机体天然脂蛋白的Aβ清除机制，实现多模式联合治疗，对AD疾病进程具有重要的调节作用。
将天然脂蛋白与抗阿尔兹海默症药物体外重组构建抗阿尔兹海默症的天然纳米粒-药物组合物，可以实现脑部靶向递送AD治疗药物，解决药物脑部递送困难，联合脂蛋白脑内Aβ靶向清除，实现多模式AD治疗。本发明具有以下优势：
(1)载体的高度同源性和生物相容性：采用的脂蛋白组成与天然脂蛋白完全相同，保证保障其生物安全性和生物功能完整性，尤其是内源脂蛋白脑靶向性和脑内靶向清除Aβ能力。
(2)BBB高渗透性和AD主动靶向性：利用脂蛋白的BBB高渗透性和载脂蛋白的Aβ淀粉样斑块靶向性，可实现包载药物在AD病变区域的有效定位和释放，解决脑内药物的靶向递送难题。
(3)多模式联合治疗：针对AD形成与发展机制，选择利用抗AD药物缓解症状联合脂蛋白的Aβ靶向清除功能相结合，实现AD病变神经系统的高效联合治疗。
(4)工艺简单，载药量高：将天然脂蛋白与抗AD药物经超声载药法或重组超声法体外重组可方便地制备药物含量高的天然纳米粒-药物组合物，并且简单的制备工艺为后期工艺放大奠定了基础。
本发明提供了一种抗阿尔兹海默症的天然纳米粒-药物组合物的制备工艺及应用，高度还原天然脂蛋白生理功能，实现高效脑靶向递送药物并靶向清除病灶Aβ，实现多模式联合治疗AD的目的。
与现有技术相比，本技术具有以下优势：
(1)本发明采用的天然纳米粒为天然脂蛋白纳米粒，高度还原内源脂蛋白生理功能，保障其高效脑靶向性，高度Aβ亲和力及靶向清除能力；
(2)本发明在保障天然纳米粒的生理活性基础上实现高效包载药物，构建天然纳米粒-药物组合物，解决AD治疗药物脑部递送困难的问题；
(3)本发明所述天然纳米粒来源为血浆中提取或经基因工程表达，来源广泛，安全性高，易于大规模生产，具有极大的临床应用潜力；
(4)本发明提供的抗阿尔兹海默症的天然纳米粒-药物组合物的制备及抗AD应用，针对AD病变高效脑部递送抗AD药物，并实现病灶Aβ高效靶向清除的多模式联合治疗的目的，为AD治疗提供了新思路和新技术。
附图说明
图1：抗阿尔兹海默症的天然纳米粒-药物组合物的制备及结构示意图；
图2：抗阿尔兹海默症的天然纳米粒-药物组合物的投射电镜图；
图3：抗阿尔兹海默症的天然纳米粒-药物组合物的体外释放图；
图4：抗阿尔兹海默症的天然纳米粒-药物组合物的细胞摄取考察图；
图5：抗阿尔兹海默症的天然纳米粒-药物组合物促进小胶质细胞摄取Aβ考察图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合实施例，对本发明进一步详细的说明。应当理解，此处所述的具体实施例用以说明本发明，并不用于限定本发明的范围。
实施例1：抗阿尔兹海默症的天然纳米粒-药物组合物的制备工艺：
1.1超声载药法制备载多奈哌齐的天然纳米粒-药物组合物
Figure PCTCN2018118109-appb-000001
Figure PCTCN2018118109-appb-000002
(1)如图1所示，按超声载药法制备载多奈哌齐的天然纳米粒-药物组合物。称取处方量的天然纳米粒-高密度脂蛋白10mg分散于超纯水中，组成天然高密度脂蛋白纳米粒混悬液；
(2)称取处方量多奈哌齐5mg溶解于乙醇并逐滴加入(1)中所述天然纳米粒混悬液，置于冰浴下探头超声分散60min；
(3)超声结束后，于37℃减压旋蒸20min去除药物溶剂乙醇，随后过0.22μm滤膜，冷冻干燥即得载多奈哌齐的天然纳米粒-药物组合物。
1.2超声载药法制备载姜黄素的天然纳米粒-药物组合物
Figure PCTCN2018118109-appb-000003
(1)如图1所示，按超声载药法制备载姜黄素的天然纳米粒-药物组合物。称取处方量的天然纳米粒-天然高密度脂蛋白、低密度脂蛋白和极低密度脂蛋白分散于超纯水中，组成天然高密度脂蛋白、低密度脂蛋白和极低密度脂蛋白纳米粒混悬液；
(2)称取处方量姜黄素10mg溶解于乙醇并逐滴加入(1)中所述天然纳米粒混悬液，置于冰浴下探头超声分散15min；
(3)超声结束后，于37℃减压旋蒸20min去除药物溶剂乙醇，随后过0.22μm滤膜，冷冻干燥即得载姜黄素的天然纳米粒-药物组合物。
1.3超声载药法制备载石杉碱甲的天然纳米粒-药物组合物
Figure PCTCN2018118109-appb-000004
(1)如图1所示，按超声载药法制备载石杉碱甲的天然纳米粒-药物组合物。称取处方量的天然纳米粒-低密度脂蛋白分散于超纯水中，组成天然低密度脂蛋白纳米粒混悬液；
(2)称取处方量石杉碱甲3mg溶解于乙醇并逐滴加入(1)中所述天然纳米粒混悬液，置于冰浴下探头超声分散25min；
(3)超声结束后，于37℃减压旋蒸20min去除药物溶剂乙醇，随后过0.22μm滤膜，冷冻干燥即得载石杉碱甲的天然纳米粒-药物组合物。
1.4超声载药法制备载siRNA的天然纳米粒-药物组合物
Figure PCTCN2018118109-appb-000005
(1)如图1所示，按超声载药法制备载siRNA的天然纳米粒-药物组合物。称取处方量的天然纳米粒-高密度脂蛋白10mg分散于超纯水中，组成天然高密度脂蛋白纳米粒混悬液；
(2)称取胆固醇修饰的Chol-siRNA 3mg溶解于PBS缓冲液并逐滴加入(1)中所述天然纳米粒混悬液，置于冰浴下探头超声分散15min；
(3)超声结束后过0.22μm滤膜，冷冻干燥即得载siRNA的天然纳米粒-药物组合物。
1.5超声载药法制备载NAP多肽的天然纳米粒-药物组合物
Figure PCTCN2018118109-appb-000006
(1)如图1所示，按超声载药法制备载NAP多肽的天然纳米粒-药物组合物。称取处方量的天然纳米粒-高密度脂蛋白8mg和低密度脂蛋白2mg分散于超纯水中，组成天然高密度脂蛋白和低密度脂蛋白纳米粒混悬液；
(2)称取处方量多肽NAP 80μg溶解于Tris缓冲液并逐滴加入(1)中所述天然纳米粒混悬液，置于冰浴下探头超声分散15min；
(3)超声结束后过0.22μm滤膜，冷冻干燥即得载NAP的天然纳米粒-药物组合物。
1.6超声载药法制备载美金刚的天然纳米粒-药物组合物
Figure PCTCN2018118109-appb-000007
(1)如图1所示，按超声载药法制备载美金刚的天然纳米粒-药物组合物。称取处方量的 天然纳米粒-低密度脂蛋白5mg和极低密度脂蛋白10mg分散于超纯水中，组成天然低密度脂蛋白和极低密度脂蛋白纳米粒混悬液；
(2)称取处方量美金刚8mg溶解于乙醇并逐滴加入(1)中所述天然纳米粒混悬液，置于冰浴下探头超声分散10min；
(3)超声结束后，于37℃减压旋蒸20min去除药物溶剂乙醇，随后过0.22μm滤膜，冷冻干燥即得载美金刚的天然纳米粒-药物组合物。
1.7超声载药法制备载亚甲蓝的天然纳米粒-药物组合物
Figure PCTCN2018118109-appb-000008
(1)如图1所示，按超声载药法制备载亚甲蓝的天然纳米粒-药物组合物。称取处方量的天然纳米粒-高密度脂蛋白10mg分散于超纯水中，组成天然高密度脂蛋白纳米粒混悬液；
(2)称取处方量亚甲蓝6mg溶解于乙醇并逐滴加入(1)中所述天然纳米粒混悬液，置于冰浴下探头超声分散25min；
(3)超声结束后，于37℃减压旋蒸20min去除药物溶剂乙醇，随后过0.22μm滤膜，冷冻干燥即得载亚甲蓝的天然纳米粒-药物组合物。
1.8重组超声法制备载多奈哌齐的天然纳米粒-药物组合物
Figure PCTCN2018118109-appb-000009
(1)如图1所示，按重组超声法制备载多奈哌齐的天然纳米粒-药物组合物。称取处方量的脂质磷脂酰胆碱5mg，胆固醇1mg，加入成膜溶剂(氯仿:甲醇，1:1)，组成脂质溶液；
(2)称取处方量的载脂蛋白apoA-I 8mg溶解于超纯水，组成载脂蛋白溶液；
(3)称取处方量药物多奈哌齐，按“相似相溶”原则溶于上述脂质溶液；
(4)将上述脂质溶液于37℃旋蒸0.5h成膜，真空干燥过夜；
(5)在步骤(4)所述脂质膜中加入超纯水于37℃旋蒸5min水合；
(6)水合结束后，于冰浴下探头超声分散5min，得载药纳米粒；
(7)将所述载脂蛋白溶液加入步骤(6)所述载药纳米粒中，室温磁力搅拌孵育24h；孵育结束后过0.22μm滤膜，冷冻干燥即得载多奈哌齐的天然纳米粒-药物组合物。
1.9重组超声法制备载姜黄素的天然纳米粒-药物组合物
Figure PCTCN2018118109-appb-000010
(1)如图1所示，按重组超声法制备载姜黄素的天然纳米粒-药物组合物。称取处方量的脂质磷脂酰胆碱2mg，胆固醇5mg，胆固醇酯1mg加入成膜溶剂(氯仿:甲醇，1:1)，组成脂质溶液；
(2)称取处方量的载脂蛋白apoA-I 6mg，apoC 2mg，apoB100 2mg溶解于超纯水，组成载脂蛋白溶液；
(3)称取处方量药物姜黄素10mg，按“相似相溶”原则溶于上述脂质溶液；
(4)将上述脂质溶液于37℃旋蒸0.6h成膜，真空干燥过夜；
(5)在步骤(4)所述脂质膜中加入超纯水于37℃旋蒸10min水合；
(6)水合结束后，于冰浴下探头超声分散15min，得载药纳米粒；
(7)将所述载脂蛋白溶液加入步骤(6)所述载药纳米粒中，室温磁力搅拌孵育24h；孵育结束后过0.22μm滤膜，冷冻干燥即得载姜黄素的天然纳米粒-药物组合物。
1.10重组超声法制备载石杉碱甲的天然纳米粒-药物组合物
Figure PCTCN2018118109-appb-000011
Figure PCTCN2018118109-appb-000012
(1)如图1所示，按重组超声法制备载石杉碱甲的天然纳米粒-药物组合物。称取处方量的脂质甘油三酯0.5mg，神经酰胺1mg，脑磷脂1mg，神经节苷酯1mg加入成膜溶剂(氯仿:甲醇，1:1)，组成脂质溶液；
(2)称取处方量的载脂蛋白apoA-I 7mg，apoA-II 2mg，apoE 1mg溶解于超纯水，组成载脂蛋白溶液；
(3)称取处方量药物石杉碱甲3mg，按“相似相溶”原则溶于上述脂质溶液；
(4)将上述脂质溶液于37℃旋蒸0.5h成膜，真空干燥过夜；
(5)在步骤(4)所述脂质膜中加入超纯水于37℃旋蒸15min水合；
(6)水合结束后，于冰浴下探头超声分散20min，得载药纳米粒；
(7)将所述载脂蛋白溶液加入步骤(6)所述载药纳米粒中，室温磁力搅拌孵育24h；孵育结束后过0.22μm滤膜，冷冻干燥即得载石杉碱甲的天然纳米粒-药物组合物。
1.11重组超声法制备载siRNA的天然纳米粒-药物组合物
Figure PCTCN2018118109-appb-000013
(1)如图1所示，按重组超声法制备载siRNA的天然纳米粒-药物组合物。称取处方量的胆固醇1mg，磷脂酰胆碱4mg加入成膜溶剂(氯仿:甲醇，1:1)，组成脂质溶液；
(2)称取处方量的载脂蛋白apoA-I 5mg，apoB 3mg，apoE 2mg溶解于超纯水，组成载脂蛋白溶液；
(3)称取处方量药物Chol-siRNA 50μg，按“相似相溶”原则溶于上述载脂蛋白溶液；
(4)将上述脂质溶液于37℃旋蒸1h成膜，真空干燥过夜；
(5)在步骤(4)所述脂质膜中加入超纯水于37℃旋蒸25min水合；
(6)水合结束后，于冰浴下探头超声分散10min，得纳米粒；
(7)将所述载脂蛋白溶液加入步骤(6)所述纳米粒中，室温磁力搅拌孵育24h；孵育结束后过0.22μm滤膜，冷冻干燥即得载siRNA的天然纳米粒-药物组合物。
1.12重组超声法制备载NAP多肽的天然纳米粒-药物组合物
Figure PCTCN2018118109-appb-000014
(1)如图1所示，按重组超声法制备载NAP多肽的天然纳米粒-药物组合物。称取处方量的胆固醇5mg，磷脂酰胆碱4mg加入成膜溶剂(氯仿:甲醇，1:1)，组成脂质溶液；
(2)称取处方量的载脂蛋白apoA-I 10mg溶解于超纯水，组成载脂蛋白溶液；
(3)称取处方量药物NAP多肽100μg，按“相似相溶”原则溶于上述载脂蛋白溶液；
(4)将上述脂质溶液于37℃旋蒸1h成膜，真空干燥过夜；
(5)在步骤(4)所述脂质膜中加入超纯水于37℃旋蒸25min水合；
(6)水合结束后，于冰浴下探头超声分散10min，得纳米粒；
(7)将所述载脂蛋白溶液加入步骤(6)所述纳米粒中，室温磁力搅拌孵育24h；孵育结束后过0.22μm滤膜，冷冻干燥即得载NAP多肽的天然纳米粒-药物组合物。
1.13重组超声法制备载美金刚的天然纳米粒-药物组合物
Figure PCTCN2018118109-appb-000015
(1)如图1所示，按重组超声法制备载美金刚的天然纳米粒-药物组合物。称取处方量的磷脂酰胆碱3mg加入成膜溶剂(氯仿:甲醇，1:1)，组成脂质溶液；
(2)称取处方量的载脂蛋白apoE 10mg溶解于超纯水，组成载脂蛋白溶液；
(3)称取处方量药物美金刚8mg，按“相似相溶”原则溶于上述脂质溶液；
(4)将上述脂质溶液于37℃旋蒸0.5h成膜，真空干燥过夜；
(5)在步骤(4)所述脂质膜中加入超纯水于37℃旋蒸15min水合；
(6)水合结束后，于冰浴下探头超声分散15min，得载药纳米粒；
(7)将所述载脂蛋白溶液加入步骤(6)所述纳米粒中，室温磁力搅拌孵育24h；孵育结束后过0.22μm滤膜，冷冻干燥即得载美金刚的天然纳米粒-药物组合物。
1.14重组超声法制备载亚甲蓝的天然纳米粒-药物组合物
Figure PCTCN2018118109-appb-000016
Figure PCTCN2018118109-appb-000017
(1)如图1所示，按重组超声法制备载亚甲蓝的天然纳米粒-药物组合物称。取处方量的磷脂酰胆碱3mg，胆固醇2mg加入成膜溶剂(氯仿:甲醇，1:1)，组成脂质溶液；
(2)称取处方量的载脂蛋白apoB 10mg溶解于超纯水，组成载脂蛋白溶液；
(3)称取处方量药物亚甲蓝14mg，按“相似相溶”原则溶于上述脂质溶液；
(4)将上述脂质溶液于37℃旋蒸1h成膜，真空干燥过夜；
(5)在步骤(4)所述脂质膜中加入超纯水于37℃旋蒸25min水合；
(6)水合结束后，于冰浴下探头超声分散25min，得载药纳米粒；
(7)将所述载脂蛋白溶液加入步骤(6)所述纳米粒中，室温磁力搅拌孵育24h；孵育结束后过0.22μm滤膜，冷冻干燥即得载亚甲蓝的天然纳米粒-药物组合物。
实施例2：抗阿尔兹海默症的天然纳米粒-药物组合物的性质考察：
2.1按实施例1.1制备的载多奈哌齐的天然纳米粒-药物组合物，并表征其粒径和进行形态学考察：采用动态光散射法测得天然纳米粒-药物组合物的粒径为(65.8±4.45)nm，投射电镜下呈现出类球状外观(图2)，且与激光粒度仪测得的粒径一致。
2.2按实施例1.1制备的载多奈哌齐的天然纳米粒-药物组合物的包封率和载药量考察：量取200μL载多奈哌齐的天然纳米粒-药物组合物于10mL容量瓶中，加入甲醇定容至刻度，水浴超声30min，破乳后离心(10000rpm，20min)，取上清，上清液中多奈哌齐浓度经高效液相色谱测定，并按以下公式计算包封率和载药量：包封率(％)＝(组合物中多奈哌齐质量/投入的多奈哌齐总质量)*100％，载药量(％)＝(组合物中多奈哌齐质量/组合物总质量)*100％。测得载多奈哌齐的天然纳米粒-药物组合物的包封率为90％，载药量为5％。
2.3按实施例1.1制备的载多奈哌齐的天然纳米粒-药物组合物的体外释放的考察：分别取2mL载多奈哌齐的天然纳米粒-药物组合物置于透析袋(截留分子量3500)中，置于50mL释放介质(0.5％Tween-80,pH 7.4)，置于37℃恒温摇床，定点取出1mL释放介质并补充空白释放介质，释放药物。如图3所示，载多奈哌齐的天然纳米粒-药物组合物在1h内仅有0.5％的多奈哌齐释放，没有明显的药物突释，在48h的多奈哌齐累计释放为51％，显著低于脂质体组，可见，载多奈哌齐天然纳米粒-药物组合物具有很好的药物缓释效果。
实施例3：按实施例1.1制备的载多奈哌齐的天然纳米粒-药物组合物的：通过Transwell共培养构建体外BBB模型，人源脑微血管内皮细胞(hCMEC/D3)接种于Transwell上室(6×10 5cells/cm 2)，鼠源小胶质细胞(BV-2)接种于Transwell下室(2×10 5cells/cm 2)，通过跨膜电 阻仪测定细胞跨膜电阻。当共培养第七天时，跨膜电阻值高达200Ω/cm 2，证明Transwell BBB模型已经构建成功。用荧光染料香豆素6(C6)替代药物，分别构建C6标记的天然纳米粒-药物组合物和脂质体。在构建成功BBB模型的Transwell上室分别加入200μL C6标记的天然纳米粒-药物组合物和脂质体，孵育4h后，在下室加入预冷PBS洗3遍，用胰酶消化细胞后收集细胞用小型流式细胞仪对BV-2细胞摄取荧光定量统计。如图4所示，C6标记的天然纳米粒-药物组合物共孵育的细胞荧光强度是C6标记的脂质体组的近2倍，证明天然纳米粒-药物组合物可以跨越BBB内皮细胞进而被下层神经胶质细胞靶向摄取，具有很好的脑靶向能力。
实施例4：按实施例1.1制备的载多奈哌齐的天然纳米粒-药物组合物Aβ靶向清除能力考察：将小胶质细胞BV-2接种于24孔细胞培养板(2×10 5cells/孔)，加入荧光标记的Aβ(FAM-Aβ)，并分别加入天然纳米粒-药物组合物或脂质体孵育4h。孵育结束后，吸去培养基，预冷PBS水洗三遍后，用胰酶消化细胞后收集细胞用小型流式细胞仪对BV-2细胞摄取荧光定量统计。如图5所示，天然纳米粒-药物组合物共孵育的BV-2细胞靶向摄取Aβ比脂质体组有了很大提高，证明天然纳米粒-药物组合物具有很好的靶向清除Aβ能力，具有AD治疗潜能。

Claims (10)

  1. 一种抗阿尔兹海默症的天然纳米粒-药物组合物，其特征在于具有脑靶向性和淀粉样蛋白靶向性，以处方总质量计，天然纳米粒含量占处方总质量的50～99％，药物含量占处方总质量的1～50％。
  2. 根据权利要求1中所述的天然纳米粒-药物组合物，其特征在于所述天然纳米粒是从血浆组分中提取或经基因工程表达的具有抗阿尔兹海默症作用的天然纳米颗粒，其粒径范围为20-200nm。
  3. 根据权利要求2所述的天然纳米粒-药物组合物，其特征在于所述的天然纳米粒是天然脂蛋白纳米粒，所述脂蛋白主要由载脂蛋白和脂质组成，选自高密度脂蛋白、低密度脂蛋白和极低密度脂蛋白中的一种或多种。
  4. 根据权利要求3中所述的天然纳米粒-药物组合物，其特征在于所述载脂蛋白来源为血浆中提取或基因工程获得，选自载脂蛋白apoA-I，apoA-II，apoE，apoC，apoB及载脂蛋白模拟肽中的一种或多种。
  5. 根据权利要求3中所述的天然纳米粒-药物组合物，其特征在于所述脂质选自胆固醇，胆固醇酯，磷脂酰胆碱，甘油三酯，神经酰胺，神经节苷脂，脑磷脂中的一种或多种。
  6. 根据权利要求1中所述的天然纳米粒-药物组合物，其特征在于所述阿尔兹海默症治疗药物选自胆碱酯酶抑制剂，天门冬氨酸受体拮抗剂，抗氧化剂，抗炎药物，蛋白质药物，多肽药物和基因药物中的任一物质或衍生物，选自多奈哌齐，石杉碱甲，美金刚，姜黄素，亚甲蓝，NAP多肽或siRNA中的一种或多种。
  7. 根据权利要求1所述的天然纳米粒-药物组合物的制备方法，其特征在于所述工艺为超声载药法，具体包括以下步骤：
    (1)将血浆中提取或经基因工程提取天然脂蛋白纳米粒分散于超纯水中，组成天然纳米粒混悬液；
    (2)将处方量药物溶解于药物溶剂并逐滴加入(1)中所述天然纳米粒混悬液，置于冰浴下探头超声分散15～60min；
    (3)超声结束后，当药物溶剂为有机溶剂时，于20～40℃减压旋蒸5～20min去除药物溶剂；当药物溶剂为水时，不旋蒸去除溶剂；随后过0.22μm滤膜，冷冻干燥即得。
  8. 根据权利要求7所述的天然纳米粒-药物组合物的制备方法，其特征在于所述药物溶剂为：水溶性药物的溶剂选择超纯水，生理盐水、磷酸盐缓冲液和Tris缓冲液中的任一种；脂溶性药物的溶剂选择乙醇、甲醇和丙酮中的任一种。
  9. 根据权利要求1中所述的天然纳米粒-药物组合物的制备方法，其特征在于所述工艺为重组超声法：
    (1)称取处方量的脂质，加入成膜溶剂，组成脂质溶液；
    (2)称取处方量的载脂蛋白溶解于超纯水，组成载脂蛋白溶液；
    (3)称取处方量药物，按“相似相溶”原则溶于上述对应组分：脂溶性药物溶解于脂质溶液，水溶性药物溶解于载脂蛋白溶液；
    (4)将上述脂质溶液于20～40℃旋蒸0.5～2h成膜，真空干燥过夜；
    (5)在步骤(4)所述脂质膜中加入超纯水于20～40℃旋蒸5～20min水合；
    (6)水合结束后，于冰浴下探头超声分散5～20min，得载药纳米粒；
    (7)将所述载脂蛋白溶液加入步骤(6)所述载药纳米粒中，室温磁力搅拌孵育24～48h；孵育结束后过0.22μm滤膜，冷冻干燥即得天然纳米粒-药物组合物。
  10. 根据权利要求1所述抗阿尔兹海默症的天然纳米粒-药物组合物的使用方法，其特征在于可以经缓冲液稀释后给药，所述缓冲液包括生理盐水、磷酸盐缓冲液、5％葡萄糖溶液、HEPES缓冲液和Tris缓冲液，所述给药方式为静脉注射、或口服给药、或鼻腔给药方式，用于阿尔兹海默病的治疗。
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