DE212018000131U1 - Nano-drugs with delayed release against a neurodegenerative disease - Google Patents

Abstract

Nanoparticles comprising a carrier material, an active agent and a targeting molecule, wherein the carrier material is human serum albumin (HSA) and the active ingredient can prevent and / or treat a neurodegenerative disease;
preferably, wherein the drug is loaded into the carrier material to form a drug-loaded HSA nanoparticle;
in which the drug-loaded HSA nanoparticle is formed by a process comprising the following steps:
Step (1-1): dissolving or dispersing the human serum albumin and the drug in a good solvent to form a solution (1);
Step (1-2): adding dropwise the solution (1) to a poor solvent and adding a crosslinking agent to conduct a reaction to obtain a drug-loaded HSA nanoparticle;
preferably, wherein the nanoparticle has a drug loading rate of 15% to 25%.

Description

Field of the invention

The present invention relates to the technical field of medicine, in particular to a nanoparticle comprising a carrier material, an active substance and a targeting molecule. The carrier material is human serum albumin and the active ingredient can prevent and / or treat a neurodegenerative disease. The invention also relates to a process for the preparation of the nanoparticle, a pharmaceutical composition comprising the nanoparticle and a use of the nanoparticle or the pharmaceutical composition in the manufacture of a medicament for the prevention and / or treatment of a neurodegenerative disease in an individual.

Background of the invention

Cholinergic hypothesis of Alzheimer's disease

Alzheimer's disease (AD) is a neurodegenerative disease characterized by a progressive decline in memory and cognitive ability. At present no definitive conclusion has been drawn on the etiology of AD, and there are mainly two theories: the cholinergic theory and the β-amyloid protein (Aβ) theory.

The central cholinergic system is closely linked to learning and memory. Acetylcholine is one of the important neurotransmitters in the central cholinergic system and its main function is to maintain awareness and it plays an important role in learning and memory. Although the aetiology of AD is still unknown, the massive loss of neurons in certain brain regions is a direct cause of the progressive decline in cognitive and memory capabilities, particularly in the loss of cholinergic neurons in the hippocampus. Currently, the US Food and Drug Administration (FDA) has approved five drugs for the treatment of AD, tacrine, neostigmine, galantamine, donepezil, and memantine. The drugs other than memantine are reversible acetylcholinesterase inhibitors. The acetylcholinesterase inhibitor can tightly interact with the acetylcholinesterase in the body to occupy the enzyme site and allow the enzyme to lose the function of acetylcholine degradation, thereby indirectly increasing the concentration of acetylcholine in the synaptic cleft and increasing the effect of the acetylcholinesterase Relief of AD is achieved.

Restrictions on existing medicines

At present, the drugs used to treat AD are still limited, not only because the mechanism is unclear, but also because the brain's complex defense system prevents the drug from penetrating the blood-brain barrier (BBB) to aim at the center. Even if some of the drugs cross the BBB, they will also be pumped out under the action of an efflux transporter (eg, p-glycoprotein). Although these limitations can be overcome by chemical modification for common drugs, reduced efficiency may occur after modification, introduction of toxic byproducts during the modification, and complicated modification processes and other problems. Proteins and peptide drugs are rapidly degraded and inactivated by the action of peripheral proteases, which limit the use of a large number of effector molecules.

The neurodegenerative disease represented by AD is a type of chronic disease. After diagnosis, patients should take long-term uninterrupted medicines or even take medicines for life. Existing drugs for the treatment of AD have the problems of short plasma half-life, poor blood-brain barrier penetration and fast metabolic rate, etc., so that patients need to take medicines at a reasonable dose every day in a timely manner, however Patient is very difficult, whose memory decreases. Therefore, it is urgently necessary to prepare a medicine for the treatment of AD with high efficiency, sustained release and high objective.

Progress in the research of nano-pharmaceuticals

Most existing nano-drugs targeting the central nervous system use synthetic polymeric materials as the carrier, that is, a carrier material is incorporated into the outer layer of the target. Wrapped in medicine. When the carrier material is degraded at the target site, drug is released in the nucleus. This type of nano-pharmaceuticals is easy to produce, to some extent non-toxic and readily degradable with high stability.

However, the neurodegenerative disease represented by AD is a type of chronic disease of the elderly. Patients with this disease have a decreased brain metabolism. These exogenous synthetic polymer materials and their degradation products will undoubtedly increase the burden on the brain. Therefore, in the manufacture of nano-drugs for AD, emphasis should be placed on the biocompatibility of materials and the toxicity of material metabolites.

For the above reasons, the research group of Shrinidh A. Joshi of India used a biocompatible polylactic acid-glycolic acid copolymer (PLGA) as a carrier to load rivastigmine for the treatment of AD and prepared encapsulated drugs by sedimentation method. In terms of material safety, PLGA, while improving biocompatibility to some extent, has poor centralized targeting ability and rapid drug release, and can not meet the therapeutic needs of chronic disease (Shrinidh A. Joshi, Sandip S. Chavhan et al., Rivastigmine. loaded PLGA and PBCA nanoparticles: Preparation, optimization, characterization, in vitro and pharmacodynamic studies, European Journal of Pharmaceutics and Biopharmaceutics 76 (2010) 189-199).

The research group of Helen F. Stanyon in the UK found that human serum albumin (HSA) can not only bind to 95% Aβ in peripheral plasma, but also binds to almost half of the Aβ monomers in the cerebrospinal fluid at physiological level effectively inhibits the formation of β-amyloid fibers and significantly reduces the total amount of fibrils; In addition, HSA can also remove Cu (II) in the Aβ-Cu (II) complex in order to reduce the catalytic activity of metal ions, thereby inhibiting the formation of fibrils by a double mechanism ( Helen F. Stanyon and John H. Viles, Human Serum Albumin Can Regulate Amyloid-β Peptides Fiber Growth in the Brain Interstitium, J. Biol. Chem. 2012, 287: 28163-28168 ).

In summary, neither molecular drugs nor nano-pharmaceuticals currently meet the requirements for efficient, sustained release and targeted treatment of neurodegenerative diseases. Human serum albumin as a natural protein has good material properties and Aß targeting properties. Therefore, there is an urgent need to develop nanoparticles using HSA nanoparticles as a carrier material to meet the requirements for efficient sustained release and targeted treatment.

Summary of the invention

In the present invention, the scientific and technical terms used herein have the meanings that are well known to those skilled in the art, unless otherwise specified. Moreover, laboratory procedures involved herein are routine steps that are widely used in the art. For a better understanding of the present invention, the definitions and explanations of related terms are provided below.

As used herein, the term "nanoparticle" (NP) refers to nanoscale sized particles, such as particles having a size of not more than 1000 nm, for example, having a size of 10 nm to 100 nm, 100 nm to 200 nm, 200 nm to 300 nm, 300 nm to 400 nm, 400 nm to 500 nm, 500 nm to 600 nm, 600 nm to 700 nm, 700 nm to 800 nm, 800 nm to 900 nm or 900 nm to 1000 nm.

The term "particle size" or "equivalent particle size" as used herein means that when a physical property or behavior of a particle to be measured comes closest to a homogeneous sphere (or combination) of a given diameter, the diameter of the sphere (or combination) is taken as the equivalent particle size (or particle size distribution) of the particle to be measured.

The term "average particle size" as used herein means that when the total length of the particle size of a group of actual particles consisting of particles of different sizes and shapes is the same as that of a group of hypothetical particles consisting of uniformly spherical particles , the diameter of the spherical particle is the average particle size of the actual particle group. The methods for measuring the average particle size are the Skilled in the art, such as light scattering methods; and the instruments for measuring the average particle size include, but are not limited to, light scattering particle size analyzers.

The term "cholinergic transmitter supplement" as used herein refers to an agent (eg, a drug) that exogenously supplements a cholinergic neurotransmitter (cholinergic transmitter).

The term "prodrug" as used herein refers to a compound that is inactive or less active in vitro and exerts a pharmaceutical activity by releasing an active drug after enzymatic or non-enzymatic conversion in vivo, and is obtained by modifying an active compound , The design principles and methods of preparation of prodrugs are known to those skilled in the art.

The term "pharmaceutically acceptable salt" as used herein refers to (1) a salt represented by an acidic functional group (e.g., -COOH, -OH, -SO ₃ H, etc.) contained in a compound is formed, and a suitable inorganic or organic cation (base) is formed, for example, a salt which is formed by a compound with an alkali metal or alkaline earth metal, an ammonium salt of a compound, and a salt which is formed by a compound having a nitrogen-containing organic Base is formed; and (2) a salt formed by a basic functional group (e.g., -NH _2, etc.) present in the compound and a suitable inorganic or organic anion (acid), for example, a salt, which is represented by a compound is formed with an inorganic acid or an organic carboxylic acid.

Thus, in the present application, the "pharmaceutically acceptable salts" include, but are not limited to, alkali metal salts such as sodium salt, potassium salt, lithium salt, etc .; Alkaline earth metal salts such as calcium salt, magnesium salt, etc .; other metal salts such as aluminum salt, iron salt, zinc salt, copper salt, nickel salt, cobalt salt, etc .; inorganic alkali salt, such as ammonium salt; organic alkali salt such as t-octylamine salt, dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycinalkyl ester salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N, N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine Salt, N-benzylphenethylamine salt, piperazine salt, tetramethylamine salt, tris (hydroxymethyl) aminomethane salt; Hydrohalide salt such as hydrofluoride salt, hydrochloride salt, hydrobromide salt, hydroiodide salt, etc .; inorganic acid salt such as nitrate, perchlorate, sulfate, phosphate, etc .; Lower alkanesulfonates such as methanesulfonate, triflate, ethanesulfonate, etc .; Arylsulfonates such as besylate, p-benzenesulfonate, etc .; organic acid salts such as acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, maleate, etc .; Amino acid salts such as glycinate, salt of trimethylglycine, salt of arginine, salt of ornithine, glutamate, aspartate, etc.

The term "neurodegenerative disease" as used herein refers to a disease caused by a progressive disease of the nervous system, including, but not limited to, Alzheimer's Disease (AD), Parkinson's Disease, Huntington's Disease and Amyotrophic Lateral Sclerosis and cerebrospinal multiple sclerosis.

As used herein, the term "good solvent" refers to a solvent capable of dissolving human serum albumin, including, but not limited to, water and sodium chloride solutions.

The term "poor solvent" as used herein refers to a solvent that does not dissolve human serum albumin, including, but not limited to, ethanol.

The term "room temperature" as used herein refers to 25 ± 5 ° C.

As used herein, the term "about" should be understood by those skilled in the art and will vary to some extent depending on the context in which it is used. If the term is not clear to a person skilled in the art, it does not mean more than ± 10% of a certain value or range.

In order to meet the therapeutic needs of neurodegenerative diseases such as Alzheimer's disease, the inventors have obtained the nanoparticles of the present application through intensive research and creative work that can be used to prepare the drugs having poor permeability to the blood brain. Barrier and / or short plasma half-life to deliver effectively the to achieve key objectives and delayed release of drugs. Thus, the following invention is provided.

In one aspect, the present invention provides a nanoparticle comprising a carrier material, an active agent and a targeting molecule, wherein the carrier material is human serum albumin (HSA) and the active ingredient can prevent and / or treat a neurodegenerative disease.

The inventors have found that the natural protein drug carrier human serum albumin nanoparticle can effectively meet the requirements of central targeted drug delivery. First, the human serum albumin has a high affinity for Aβ monomer and has a targeted property against Aβ; it can quickly target the lesion site while ensuring the encapsulation of a large amount of drug molecules to achieve excellent targeting performance of the material itself.

In addition, natural proteinaceous materials have high biosecurity to meet the requirements of the pharmaceutical field, and they are not immunogenic and their decomposition products are amino acids that can be reused as endogenous nutrients or converted to other nitrogenous substances in order to control the injury and strain of exogenous organic or inorganic polymer materials to minimize the central nervous system.

In some embodiments, the drug is loaded into a carrier material to form a drug-loaded HSA nanoparticle.

• Schritt (1-1): Lösen oder Dispergieren von humanem Serumalbumin und Wirkstoff in einem guten Lösungsmittel, um eine Lösung (1) zu bilden;
• Schritt (1-2): tropfenweise Zugabe der Lösung (1) zu einem schlechten Lösungsmittel und Zugabe eines Vernetzungsmittels, um eine Reaktion durchzuführen, um ein Arzneimittel-beladenes HSA-Nanopartikel zu erhalten.

In some embodiments, the drug-loaded HSA nanoparticle is formed by a process comprising the steps of:

• Step ( 1-1 ): Dissolving or dispersing human serum albumin and active ingredient in a good solvent to form a solution ( 1 ) to build;
• Step ( 1-2 ): dropwise addition of the solution ( 1 ) to a poor solvent and adding a crosslinking agent to perform a reaction to obtain a drug-loaded HSA nanoparticle.

In some embodiments, the nanoparticle has a drug loading rate of 15% to 25%, for example 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%. or 25%.

In some embodiments, the active agent is selected from a group consisting of a cholinergic transmitter supplement and a cholinesterase inhibitor.

In some embodiments, the cholinergic transmitter supplement is selected from a group consisting of acetylcholine, a prodrug of acetylcholine, or a pharmaceutically acceptable salt of acetylcholine (for example, hydrochloride).

In some embodiments, the cholinergic transmitter supplement is acetylcholine or acetylcholine chloride.

In some embodiments, the cholinesterase inhibitor is selected from a group consisting of rivastigmine, galantamine, or donepezil, or a pharmaceutically acceptable salt thereof (for example, tartrate).

In some embodiments, the cholinesterase inhibitor is rivastigmine hydrogen tartrate.

In order to ensure a sufficient central input dose and to improve the central targeting of the nanoparticle, the surface of the nanoparticle is modified by targeting molecules in order to improve the permeability of the nanoparticle to the blood-brain barrier and to achieve efficient central targeted delivery.

In some embodiments, the targeting molecule is selected from a group consisting of a surfactant, a polyethylene glycol polymer, and a blood brain barrier specific antibody.

In some embodiments, the targeting molecule is a surfactant.

In some embodiments, the targeting molecule is Tween-80.

In some embodiments, the targeting molecule is modified on the surface of the nanoparticle.

In some embodiments, the targeting molecule is modified on the surface of the drug-loaded HSA nanoparticle.

In some embodiments, the targeting molecule on the surface of the nanoparticle is modified by adsorption.

In some embodiments, the nanoparticle has a diameter of 150 nm to 250 nm; such as 150 nm to 180 nm, 180 nm to 200 nm, 200 nm to 220 nm or 220 nm to 250 nm; for example 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm, 210 nm, 220 nm, 230 nm, 240 nm or 250 nm.

In some embodiments, the nanoparticle has a zeta potential of from -30 mV to +20 mV, such as -30 mV to -20 mV, -20 mV to -10 mV, -10 mV to 0, 0 to +10 mV or + 10 mV to +20 mV; for example -30mV, -25mV, -20mV, -15mV, -10mV, -5mV, 0, + 5mV, + 10mV, + 15mV or + 20mV.

In some embodiments, the nanoparticle has an entrapment efficiency of 40% to 50%, for example 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49% or 50%. ,

1 exemplifies a structure of the nanoparticles of the present application. In the nanoparticle shown in the figure, HSA having Aβ targeting property is used as a nanocarrier for encapsulating and loading a drug capable of treating AD, moreover, a target molecule on the surface of the HSA nanoparticle is modified to to improve the permeability of the blood-brain barrier (BBB) of the HSA nanoparticle and to prolong the circulatory time in vivo.

• Schritt (1): Herstellen eines Arzneimittel-beladenen HSA-Nanopartikels;
• Schritt (2): Modifizieren eines Targeting-Moleküls auf der Oberfläche des Arzneimittel-beladenen HSA-Nanopartikels.

In one aspect, the present application provides a method of making the above nanoparticles, comprising the following steps:

• Step (1): preparing a drug-loaded HSA nanoparticle;
• Step (2): Modifying a targeting molecule on the surface of the drug-loaded HSA nanoparticle.

In some embodiments, the method of making the drug-loaded HSA nanoparticle is an anti-solvent method in step (1).

• Schritt (1-1): Lösen oder Dispergieren des humanen Serumalbumins und Wirkstoffs in einem guten Lösungsmittel, um eine Lösung (1) zu bilden;
• Schritt (1-2): tropfenweise Zugabe der Lösung (1) in ein schlechtes Lösungsmittel und Zugabe eines Vernetzungsmittels, um eine Reaktion durchzuführen, um ein Arzneimittel-beladenes HSA-Nanopartikel zu erhalten.

In some embodiments, step (1) includes the following steps:

• Step ( 1-1 ): Dissolving or dispersing the human serum albumin and drug in a good solvent to give a solution ( 1 ) to build;
• Step ( 1-2 ): dropwise addition of the solution ( 1 ) in a poor solvent and adding a crosslinking agent to perform a reaction to obtain a drug-loaded HSA nanoparticle.

In some embodiments, the human serum albumin and the active agent in step ( 1-1 ) has a mass ratio of 1: 1 to 10: 1, for example 1: 1, 2: 1, 3: 1.4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1 or 10: 1.

In some embodiments, the good solvent is a sodium chloride solution in step ( 1-1 ).

In some embodiments, the sodium chloride solution in step ( 1-1 ) has a concentration of 1 mmol / l to 20 mmol / l, for example from 1 mmol / l to 5 mmol / l, from 5 mmol / l to 10 mmol / l and from 10 mmol / l to 15 mmol / l or from 15 mmol / L to 20 mmol / L.

In some embodiments, the step comprises 1-1 ) further: adjusting the pH of the solution ( 1 ).

In some embodiments, the poor solvent is ethanol.

In some embodiments, the step ( 1-2 ) with stirring.

In some embodiments, the crosslinking agent in step ( 1-2 ) Glutaraldehyde.

In the present invention, the addition of the crosslinking agent may cause the human serum albumin particles to crosslink and solidify so that they do not immediately swell and disperse in the aqueous solution, thereby avoiding rapid release of the drug encapsulated in the particles.

In some embodiments, the step comprises 1 ) continue the step ( 1 -3): Isolate the drug-loaded HSA nanoparticle.

In some embodiments, the drug-loaded HSA nanoparticle is centrifuged in step ( 1-3 ) isolated.

• Schritt (2-1): Dispergieren des Arzneimittel-beladenen HSA-Nanopartikels in physiologischer Kochsalzlösung, um eine Lösung (2) zu erhalten;
• Schritt (2-2): Mischen der Lösung (2) mit einem Targeting-Molekül oder einer Lösung davon.

In some embodiments, the step comprises 2 ) the following steps:

• Step ( 2-1 ): Dispersing the drug-loaded HSA nanoparticle in physiological saline to form a solution ( 2 ) to obtain;
• Step ( 2-2 ): Mixing the solution ( 2 ) with a targeting molecule or a solution thereof.

In some embodiments, the solution of the targeting molecule is a physiological saline solution of the targeting molecule.

In some embodiments, the solution of the targeting molecule has a mass percent concentration of the targeting molecule of 0.1% to 10%, for example 0.1% to 0.5%, 0.5% to 1%, 1% to 5 % or 5%. until 10%.

In some embodiments, mixing is accomplished by stirring and / or ultrasound in step ( 2-2 ) carried out.

In some embodiments, the drug loading rate and entrapment efficiency of the drug-loaded HSA nanoparticle after step (FIG. 1 ) tested.

2 shows by way of example a method for producing the nanoparticles of the present application. The method described in the figure comprises: mixing an active agent (for example acetylcholine or rivastigmine) with HSA and forming a drug-loaded HSA nanoparticle by HSA crosslinking under the action of glutaraldehyde; and modifying the targeting molecule (eg, Tween-80) on the surface of HSA-loaded drug to obtain the modified drug-loaded nanoparticles.

In one aspect, the present application provides a pharmaceutical composition comprising the above nanoparticle.

Optionally, in the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable adjuvant (eg, a carrier and / or adjuvant). In some embodiments, the carrier and / or excipient is selected from the group consisting of ion exchanger, alumina, aluminum stearate, lecithin, serum protein (e.g., human serum albumin), glycerol, sorbic acid, potassium sorbate, partial glyceride mixture of saturated vegetable fatty acids, water, protamine sulfate, Disodium hydrogenphosphate, potassium hydrogenphosphate, sodium chloride, zinc salt, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic material, polyethylene glycol, sodium carboxymethylcellulose, polyacrylate, beeswax, polyethylene-polyoxypropylene block polymer and lanolin.

In the present invention, the pharmaceutical composition can be formulated into any pharmaceutically acceptable dosage form. For example, the pharmaceutical composition of the present invention may be formulated into tablets, capsules, pills, granules, solutions, suspensions, syrups, injections (including liquid injections, powders for injection or tablets for injection), suppositories, inhalants or sprays.

In addition, the pharmaceutical composition of the present invention may also be administered to an individual by any suitable route of administration, such as oral, parenteral, rectal, pulmonary or topical, etc. In some preferred embodiments, the pharmaceutical composition is oral, parenteral (intravenous, intramuscular or subcutaneous), transdermal, translingual or respiratory administration.

When used for oral administration, the pharmaceutical composition may be formulated into an oral preparation, such as an oral solid preparation, for example a tablet, capsule, pill, granules, etc .; or an oral liquid preparation, for example, an oral solution or an oral suspension, syrup, etc. When formulated into an oral formulation, the pharmaceutical composition may also contain suitable fillers, binders, disintegrants, lubricants, etc. When used for parenteral administration, the pharmaceutical composition may be formulated into an injection, including a fluid injection, a powder for injection or a tablet for injection. When formulated as an injection, the pharmaceutical composition can be prepared by a conventional method in the existing pharmaceutical field. In the preparation of the injection, an additive may be added or not added depending on the type of the drug. When used for rectal administration, the pharmaceutical composition may be formulated into a suppository, etc. When used for transpulmonary administration, the pharmaceutical composition may be formulated as an inhalant or spray, etc.

In some preferred embodiments, the nanoparticles of the present invention are in a unit dosage form pharmaceutical composition.

In some preferred embodiments, individuals are administered an effective amount of the pharmaceutical composition. The term "effective amount" as used herein refers to an amount sufficient to achieve or at least partially achieve a desired effect. For example, a prophylactically effective amount refers to an amount sufficient to prevent, arrest, or delay the onset of a disease; a therapeutically effective amount refers to an amount sufficient to cure or at least partially arrest a disease and its complications in a patient suffering from the disease. The determination of such an effective amount is within the skill of those in the art. For example, the amount effective for therapeutic use will depend on the severity of the condition being treated, the overall condition of the patient's own immune system, the general condition of the patient such as age, body weight and sex, the route of drug administration, and concurrent Depend on treatments etc.

In one aspect, the application provides a use of the nanoparticle or pharmaceutical composition as described above in the manufacture of a medicament for the prevention and / or treatment of a neurodegenerative disease in a subject.

In another aspect, the application provides a method of preventing and / or treating a neurodegenerative disease in an individual, comprising administering the nanoparticle to an individual as described above.

In yet another aspect, the application provides the nanoparticle, as described above, for the prevention and / or treatment of a neurodegenerative disease in a subject.

In some embodiments, the neurodegenerative disease is selected from a group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis and cerebrospinal multiple sclerosis.

In some embodiments, the subject is a mammal, such as a cow, horse, sheep, pig, dog, cat, rodent, primate; For example, the individual is a human.

The nanoparticles of the present application are particularly useful for delivery of drugs that are difficult to penetrate the blood-brain barrier due to their high water solubility, or drugs that have a short plasma half-life and can not meet the long-term sustained release requirement. Thus, in another aspect, the application provides the use of the nanoparticles, as described above, to enhance the ability of an agent to pass through the blood-brain barrier of an individual, and / or the in vivo release time of an agent to extend an individual; wherein the active ingredient is capable of preventing and / or treating a neurodegenerative disease in a subject.

In some embodiments, the active agent is selected from a group consisting of a cholinergic transmitter supplement and a cholinesterase inhibitor.

In some embodiments, the cholinergic transmitter supplement is selected from a group consisting of acetylcholine, a prodrug of acetylcholine, or a pharmaceutically acceptable salt of acetylcholine (for example, hydrochloride).

In some embodiments, the cholinergic transmitter supplement is acetylcholine or acetylcholine chloride.

In some embodiments, the cholinesterase inhibitor is selected from a group consisting of rivastigmine, galantamine, or donepezil, or a pharmaceutically acceptable salt thereof (for example, tartrate).

In some embodiments, the cholinesterase inhibitor is rivastigmine hydrogen tartrate.

In some embodiments, the neurodegenerative disease is selected from a group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis and cerebrospinal multiple sclerosis.

In some embodiments, the subject is a mammal, such as a cow, horse, sheep, pig, dog, cat, rodent, primate; For example, the individual is a human.

Advantageous effects

1. 1. Die Nanopartikel der vorliegenden Anmeldung lösen die Probleme, dass ein Arzneimittel mit hoher Wasserlöslichkeit die Blut-Hirn-Schranke nur schwer passieren kann und/oder ein Arzneimittel mit einer kurzen Plasma-Halbwertszeit keine langfristige und langsame Freisetzung erzielen kann.
2. 2. Die Nanopartikel dieser Anwendung sind sehr sicher. Der in den Nanopartikeln der vorliegenden Anmeldung verwendete Nanoträger ist ein natürliches Proteinmaterial, weist keine Immunogenität auf und weist eine hohe Biokompatibilität auf. Seine Abbauprodukte sind eine Vielzahl von essentiellen Aminosäuren und weisen keine toxischen Effekte und Nebenwirkungen auf.
3. 3. Der Arzneimittel-Beladungsmodus von Nanopartikeln der vorliegenden Anmeldung ist modifiziert. Der in der Literatur berichtete Arzneimittel-Beladungsmodus konzentriert sich hauptsächlich auf die Adsorption von Arzneimittelmolekülen an der äußeren Schicht des Nanopartikels, und die Arzneimittelbeladungsrate ist niedrig und Nanopartikel sind leicht zu desorbieren. Die vorliegende Erfindung verwendet jedoch Nanopartikel, um Arzneimittelmoleküle in dem Nanoträger einzukapseln, und mit der allmählichen Auflösung werden die Arzneimittelmoleküle, die in dem Nanoträger eingeschlossen sind, langsam freigesetzt.
4. 4. Die Nanopartikel der vorliegenden Anmeldung können das räumliche Lernen und die Gedächtnisfähigkeit von Mäusen wirksam verbessern, die Aktivität der Enzyme, die mit dem cholinergen System in Verbindung stehen, und das dynamische Gleichgewicht des Acetylcholingehalts aufrechterhalten und das Level an oxidativem Stress verringern, insbesondere mit einer offensichtlichen Schutzwirkung auf den frontotemporalen Kortex.

The nanoparticles of the present application have one or more of the following advantageous effects.

1. 1. The nanoparticles of the present application solve the problems that a drug with high water solubility is difficult to cross the blood-brain barrier and / or a drug with a short plasma half-life can not achieve long-term and slow release.
2. 2. The nanoparticles of this application are very safe. The nanocarrier used in the nanoparticles of the present application is a natural protein material, has no immunogenicity, and has high biocompatibility. Its breakdown products are a variety of essential amino acids and have no toxic effects and side effects.
3. 3. The drug loading mode of nanoparticles of the present application is modified. The drug loading mode reported in the literature focuses primarily on the adsorption of drug molecules to the outer layer of the nanoparticle, and the drug loading rate is low and nanoparticles are easy to desorb. However, the present invention uses nanoparticles to encapsulate drug molecules in the nanocarrier, and with the gradual dissolution, the drug molecules entrapped in the nanocarrier are slowly released.
4. 4. The nanoparticles of the present application can effectively enhance the spatial learning and memory ability of mice, maintain the activity of the enzymes associated with the cholinergic system, and maintain the dynamic balance of acetylcholine content and reduce the level of oxidative stress, in particular an obvious protective effect on the frontotemporal cortex.

The embodiments of the present invention will be described below in detail with reference to the accompanying drawings and examples. However, it will be understood by those skilled in the art that the following drawings and examples are merely illustrative of the invention and are not intended to limit the scope of the invention. The various objects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description of the drawings and preferred embodiments.

list of figures

• 1 exemplifies a structure of the nanoparticles of the present application. In the nanoparticle shown in the figure, HSA having Aβ targeting property is used as a carrier material for encapsulating and loading the drug for the treatment of AD; moreover, the surface of the HSA nanoparticle is modified by a target molecule to enhance the BBB permeability of the HSA nanoparticle and to lengthen the circulation time in vivo of the HSA nanoparticle.
• 2 shows by way of example a method for producing the nanoparticles of the present application. The method described in the figure comprises: mixing an active agent (for example acetylcholine or rivastigmine) with HSA and forming a drug-loaded HSA nanoparticle by HSA crosslinking under the action of glutaraldehyde; and modifying the targeting molecule (eg, Tween-80) on the surface of drug-loaded HSA nanoparticles to obtain the modified drug-loaded nanoparticles.
• 3 is an SEM image of an ACh-loaded HSA nanoparticle that was not modified with Tween-80 in Example 1. As shown in the figure, the unmodified drug-loaded HSA nanoparticles are spherical and have a diameter of about 200 nm.
• 4 Figure 13 is an SEM photograph of an ACh-loaded HSA nanoparticle modified in Example 1 with Tween-80. As shown in the figure, the Tween-80 modified drug-loaded HSA nanoparticles are cubes, with a side length of about 200 nm.
• 5 Figure 13 is an SEM photograph of an ACh-loaded HSA nanoparticle modified in Example 1 with Tween-80. As shown in the figure, the Tween-80 modified drug-loaded HSA nanoparticles are cube with a side length of about 200 nm.
• 6 Figure 12 shows the UV absorption curves of the aqueous ACh solution, the aqueous HSA solution, the empty HSA nanoparticle without drug loading, and without modification with Tween-80 and the HSA-ACh nanoparticle without modification with Tween-80 in Example 3. In the Figure, the HSA solution has a distinct characteristic absorption peak at 280 nm (curve 1 ), while the ACh solution has no absorption peak at 280 nm (curve 2 ). After the formation of nanoparticles by HSA, the absorption peak at 280 nm is still clearly visible (curve 3 ); while for the Ach-loaded HSA nanoparticle the absorption peak at 280 nm is significantly reduced (curve 4 ). The results show that the drug molecules are successfully falsified and encapsulated in the nanocarrier.
• 7 Figure 12 shows the XRD patterns of the empty HSA nanoparticles without drug loading and without modification with Tween-80, the HSA Ach nanoparticles without modification with Tween-80, the Tween-80-modified unloaded HSA nanoparticles, and the Tween-80 modified HSA-ACh nanoparticles in Example 3. In the figure, the Tween-80 modified HSA nanoparticles show an apparent sharp peak at 20 = 31.6 ° (curve 3 and curve 4 ), while the unmodified nanoparticles show no peak (curve 1 and curve 2 ). The results show that the targeting molecule is tweened 80 was successfully modified on the surface of the nanoparticles.
• 8th is a standard curve of doxorubicin as determined by UV spectrophotometry in Example 4.
• 9 is an in vitro cumulative release rate curve for doxorubicin samples in Example 4. As shown in the figure, the doxorubicin solution group reaches the maximum cumulative release rate after about 10 hours and the recovery rate is over 90%, proving that doxorubicin was completely released; while the cumulative release rate in the doxorubicin-loaded nanoparticle group steadily increases within 80 h and the cumulative release rate in neutral saline solution is only 30% of the drug loading rate. It is concluded that the drug-loaded nanoparticles can maintain structural stability during transport of body fluid, thereby ensuring that nanoparticles entering the center can still maintain a high drug loading rate.
• 10 FIG. 12 is a trend graph showing the time for mice in each group to find the platform during the 4 days of the water labyrinth performance evaluation period in Example 5. FIG. During the training period, there is no significant difference in the time to find the platform for mice on the first day in each group; while on the third and fourth days, the time to find the platform in the blank control group is 20 s, and the time to find the platform in the model group, ACh solution group and RT solution group is significantly longer than that of the blank control group; the time for finding the platform in the HSA-ACh nanoparticle group and HSA-RT nanoparticle group does not differ significantly from that of the blank control group, but is significantly lower than that the model group. Experiments have shown that the short-term spatial learning ability of mice is improved after HSA-ACh nanoparticles or HSA-RT nanoparticles are administered.
• 11 Figure 4 shows the incubation time for mice to find the platform for the first time during the assessment of water labyrinth behavior in mice in each group. As shown in the figure, the mice in the blank control group have the shortest incubation time. Analysis of variance shows that the incubation time of the model group, ACh solution group and RT solution group is significantly longer than that of the blank control group (P <0.05), whereas no statistically significant difference between the HSA-ACh nanoparticle group, the HSA- RT nanoparticle group and the blank control group.
• 12 Figure 4 shows the frequency at which mice in each group cross the platform within 60 sec during the test period of the water labyrinth performance evaluation in Example 5. As shown in the figure, the frequency with which the platform was crossed for mice in the blank control group is significantly higher than that in the model group, ACh solution group and RT solution group (P <0.01), but does not differ significantly from in the HSA-ACh nanoparticle group and the HSA-RT nanoparticle group.
• 13 Figure 11 shows the time in which the mice of each group in the target quadrant swim during the test period of the water labyrinth behavior evaluation in Example 5. As shown in the figure, the swim time of the model group and ACh solution group shows a statistically significant difference compared to the blind control group (P <0.05), while there is no statistically significant difference between other groups and the blind control group.
• 14 Figure 6 shows images of HE staining of left brain left hemisphere pathologies in each group in Example 6. Figures A-L correspond to the hippocampus (FIG. A ) and cortex ( B ) of the blind control group, the hippocampus ( C ) and cortex ( D ) of the model group, the hippocampus ( e ) and cortex ( F ) of the ACh dissolution group, the hippocampus ( G ) and cortex ( H ) of the HSA-ACh nanoparticle group, the hippocampus ( I ) and cortex ( J ) of the RT solution group or the hippocampus ( K ) and cortex ( L ) of the HSA-RT nanoparticle group.
• 15 shows confocal laser microscope images in Example 7. Figures A to C correspond to the heart-injected saline group (control group), the acetylcholine nanoparticle group and the rivastigmine nanoparticle group, respectively. As can be seen from the figure, there is no fluorescence distribution in normal zebrafish; in the acetylcholine nanoparticle group and the rivastigmine nanoparticle group, in the brain of the zebrafish, after cardiac injection has been performed, an apparent green fluorescence appears, indicating that the drug can pass through the BBB to smoothly enter the center.

Detailed description of the embodiment

The embodiments of the present invention will be described below in detail; However, it should be understood by those skilled in the art that the following examples are intended to illustrate the invention and are not intended to limit the scope of the invention. The conventional conditions or conditions recommended by the manufacturer are included in the examples unless otherwise specified. The reagents or instruments not mentioned are manufacturers of commercial products.

Embodiment 1: Preparation of Ach-Loaded Nanoparticles In this embodiment, human serum albumin (HSA) was used as a nanocarrier material for encapsulating and loading acetylcholine. The active ingredient used was acetylcholine chloride, the selected good solvent was 10 mmol / L sodium chloride solution and the bad solvent used was ethanol, the crosslinking agent used was 10% glutaraldehyde, the targeting molecule used was Tween-80 and human serum albumin (HSA) is considered to be Carrier material used. The manufacturing process was as follows.

Step ( 1 Preparation of drug-loaded HSA nanoparticles by anti-solvent method: 20 mg of HSA and 10 mg of ACh were dissolved in 1 ml of sodium chloride solution (10 mmol / l), filtered through a 0.22 μm aqueous solution filter membrane, and the solution was added dropwise to 10 ml of ethanol at a rate of 50 μl / min. at a rotation speed of 600 rpm; after completion of the dropwise addition, the stirring speed was increased to 800 rpm, 20 μl of 10% glutaraldehyde was added and reacted in the dark for 24 hours. The reaction sample was centrifuged at 13,600 rpm for 10 minutes, then the supernatant was removed and the precipitate was collected and the sample became Centrifuged three times with an equal amount of physiological saline, then the precipitate was collected to obtain acetylcholine (Ach) -loaded HSA nanoparticles maintained at 4 ° C.

The mass of product was determined by a differential method and the entrapment efficiency and drug loading rate of the nano-drug were calculated as follows.

$$\begin{array}{c}\text{Arzneimittelbeladungsrate}=\left({\text{m}}_{\text{Gesamtarzneimitte}}-{\text{m}}_{\text{nicht eingekapseltes Arzneimittel im Überstand}}\right)/\text{m}\\ {}_{\text{Produktarzneimitte}}\times 100\%.\end{array}$$

The solvent in step ( 1 ) was removed by rotary evaporation, the solid powder was redispersed in 8 ml of physiological saline and centrifuged for 10 min at 13600 rpm; 1 ml of the supernatant was drawn into 8 ml of physiological saline and shaken well; The amount of unencapsulated drug in the supernatant was determined by a basic colorimetric hydroxylamine method (or ultraviolet spectrophotometry). The entrapment efficiency and drug loading rate of the nano-drug were calculated by the difference between the total drug amount and the unencapsulated drug amount according to the following formula: $$\begin{array}{c}\text{entrapment efficiency}=\left({\text{m}}_{\text{Total drug center}}-{\text{m}}_{\text{unencapsulated drug in the supernatant}}\right)/\text{m}\\ {}_{\text{total drug}}\times 100\%;\end{array}$$

$$\begin{array}{c}\text{Drug loading rate}=\left({\text{m}}_{\text{Total drug center}}-{\text{m}}_{\text{unencapsulated drug in the supernatant}}\right)/\text{m}\\ {}_{\text{Product drug center}}\times 100\%,\end{array}$$

The amount of unencapsulated drug in the supernatant over a period of time was measured. After 24 hours, the amount of unencapsulated drug in the supernatant reached a stable level. By calculation, the inclusion efficiency of the Ach-loaded HSA nanoparticle was 46.14 ± 1.13% and the drug loading rate was 21.95 ± 1.26%.

The drug-loaded HSA nanoparticle was observed with a SEM. The results were in 3 shown. The unmodified nanoparticle was spherical with a diameter of about 200 nm.

Step ( 2 ): Surface modification of drug-loaded HSA nanoparticles: those in step ( 1 Nanoparticles prepared were dispersed in 1 ml of physiological saline and 1 ml of 0.1% Tween-80 physiological saline was added, stirred for 30 min at 800 rpm and sonicated for 15 min; finally, an HSA nanoparticle modified with Tween-80 and loaded with Ach (HSA-ACh NPs) was obtained.

The modified drug-loaded HSA nanoparticle was observed using a SEM. The results were in 4 and 5 shown. The modified drug-loaded nanoparticle was a cube with a side length of about 200 nm.

The Tween-80 modified drug-loaded HSA nanoparticles were determined with a dynamic light scattering particle size analyzer (with zeta potential test function). The average particle size was about 198.5 nm and the particle size was uniform and stable. The zeta potential was about - 28.4 mV. The test results were shown in Table 1. Table 1 Characterization parameters Sample Batch □ x ± s 1 2 3 HSA ACh nanoparticles Particle size (nm) 193.5 178.6 224.6 198.5 ± 18.28 Zeta potential (mv) -27.4 -28.6 -27.7 - (28.38 ± 1.213) HSA RT nanoparticles Particle size (nm) 203.3 189.7 202.7 198.6 ± 7,685 Zeta potential (mv) +18.2 +16.6 +17.4 + (17.4 ± 0.8)

Embodiment 2: Preparation of rivastigmine (RT) -loaded nanoparticles

In this embodiment, a human particle albumin (HSA) nanoparticle was used as a drug carrier to produce a rivastigmine (RT) -loaded nanoparticle. The active ingredient used was rivastigmine hydrogen tartrate, the good solvent used was 10 mmol / l sodium chloride solution, the bad solvent used was anhydrous ethanol (greater than 99.7% purity), the crosslinker used was 10% glutaraldehyde, and the targeting molecule used was tween 80th The manufacturing process was as follows.

(1) Preparation of drug-loaded HSA nanoparticles by an anti-solvent method: 20 mg of HSA and 10 mg of RT were dissolved in 1 ml of sodium chloride solution (10 mmol / l) and the pH of the solution was adjusted to 8.3 adjusted with 0.1 mol / l sodium hydroxide solution; after being filtered through a 0.22 μm filter membrane for an aqueous solution, the solution was added dropwise to 10 ml of anhydrous ethanol at a rate of 50 μl / min. at a rotation speed of 600 rpm; after completion of the dropwise addition, the stirring rate was increased to 800 rpm, and 20 μl of 10% glutaraldehyde was added to react in the dark for 24 hours. The reaction sample was centrifuged at 13,600 rpm for 10 minutes, then the supernatant was removed and the precipitate was collected and the sample was centrifuged three times with an equal amount of physiological saline, then the precipitate was collected to obtain spherical drug-loaded HSA nanoparticles which were kept at 4 ° C.

(2) Surface modification of drug-loaded HSA nanoparticles: those in step ( 1 Nanoparticles prepared were dispersed in 1 ml of physiological saline and 1 ml of 0.1% Tween-80 physiological saline was added, stirred for 30 min at 800 rpm and sonicated for 15 min; finally, RT-loaded HSA nanoparticle (HSA-RT-NPs) modified with Tween-80 was obtained, which was a cube.

The Tween-80 modified drug-loaded HSA nanoparticles were determined with a dynamic light scattering particle size analyzer (with zeta potential test function). The average particle size was about 198.6 nm and the particle size was uniform and stable. The zeta potential was about +17.4 mV. The test results are shown in Table 1.

Embodiment 3: Structural characterization of the nanoparticle

Ultraviolet absorption spectroscopy

The ultraviolet absorption spectroscopy of aqueous ACh solution, aqueous HSA solution, blind HSA nanoparticles without loading of active ingredient and without modification with Tween-80 (the preparation process may refer to step ( 1 ) in Example 1) and the HSA Ach nanoparticle without modification with Tween-80 prepared in step ( 1 ) of Example 1 were each conducted, and the UV absorption curves of samples were recorded in 6 shown. The HSA solution showed a distinct characteristic absorption peak at 280 nm (curve 1 ), while the Ach solution at 280 nm had no absorption peak (curve 2 ). After the formation of nanoparticles by HSA, the absorption peak at 280 nm was still clearly visible (curve 3 ); while the absorption peak at 280 nm was significantly reduced for the Ach-loaded HSA nanoparticle (curve 4 ). Therefore, after the drug molecule (ACh) was falsified and encapsulated, the HSA nanoparticles interacted with drug molecules. The UV characteristic absorption peaks of HSA would be reduced or disappeared. The results showed that the drug molecules were successfully falsified and encapsulated in the nanocarrier.

XRD analysis

The XRD test was performed for the following samples: 1. Blind-HSA nanoparticles without drug loading and without modification with Tween-80 (the preparation process may refer to step ( 1 ) in Example 1)); 2. HSA-ACh nanoparticles without modification with Tween-80 prepared in step ( 1 ) of Example 1; 3. Tween-80-modified HSA nanoparticle without drug loading ((the Manufacturing process may refer to the step ( 1 ) and step ( 2 ) in Example 1)); 4. Tween-80-modified HSA-Ach nanoparticle prepared in Example 1. The XRD spectra of all samples were recorded in 7 shown.

In the figure, the Tween-80 modified HSA nanoparticles showed an apparent sharp peak at 20 = 31.6 ° (curve 3 and curve 4 ), while the unmodified nanoparticles showed no peak (curve 1 and curve 2 ). The results showed that the targeting molecule Tween-80 was successfully modified on the surface of the nanoparticles.

Embodiment 4: Preparation and in vitro release evaluation of doxorubicin-loaded HSA nanoparticles

In this embodiment, ultraviolet spectroscopy was used to quantify the release amount of nanoparticles. Since acetylcholine had no UV absorption peak while rivastigmine had a significant UV absorption peak at 263 nm (the characteristic absorption peak of HSA itself was 280 nm, which coincided with that of rivastigmine), accurate quantitative analysis by ultraviolet spectroscopy could not be performed become. Therefore, a simulated alternative drug should be used to characterize the release effect of nanoparticles.

The simulated alternative drug in this embodiment was doxorubicin. On the one hand, doxorubicin has similar solubility to drugs such as acetylcholine and rivastigmine (both low molecular weight drugs with good water solubility). On the other hand, doxorubicin has a suitable UV absorption peak. According to Annex IV of the 2010 edition of Pharmacopoeia, doxorubicin has large absorption peaks at the wavelengths 233 nm, 252 nm, 288 nm, 480 nm, 495 nm and 530 nm, with the maximum absorption peak at 480 nm having the maximum intensity and a small contamination impairment. Therefore, in this example, a doxorubicin-loaded HSA nanoparticle was used as an in vitro release model to evaluate the in vitro release effect of the nanoparticle of the present application by ultraviolet spectrophotometry using the absorbance value at 480 nm as a quantitative index.

The preparation of the nanoparticle was performed by replacing the target drug with doxorubicin hydrochloride (Dox). The synthesis procedure was the same as that in Example 1.

Test Procedure: First, doxorubicin standard solutions of a variety of concentration gradients were prepared by a gradient dilution method, and the UV absorbance values were measured at a wavelength of 480 nm to generate a doxorubicin standard curve, as in 8th shown (y = 0.020x-0.000, R ² = 0.998).

The doxorubicin solution group and the doxorubicin-loaded nanoparticle group were determined. Calculated by the drug loading rate of nanoparticles of 10%, the concentration of the doxorubicin solution group was 0.5 mg / ml (4 ml total, corresponding to 2 mg doxorubicin) and the concentration of the drug-loaded nanoparticle group was 5 mg / ml (4 ml total, corresponding to 2 mg doxorubicin). The two solution groups were placed in a semipermeable membrane having a molecular weight of 3000 and each placed in a release system with 900 ml of physiological saline solution and released continuously at a rotation speed of 200 rpm. At regular intervals, 2 ml of the release solution was taken out and the UV absorbance value was measured at 480 nm and the same amount of normal saline was supplemented.

9 showed the release results. In the doxorubicin solution group, the maximum cumulative release rate was reached in about 10 hours with a recovery rate in excess of 90%, proving that doxorubicin was completely released. In the doxorubicin-loaded nanoparticle group, the cumulative release rate increased stably and continuously within 80 hours, and the cumulative release rate in neutral saline was only 30% of the drug loading rate. Therefore, it could be concluded that the drug-loaded nanoparticle could maintain structural stability during transport of body fluid, thereby ensuring that the nanoparticles entering the center could maintain a high drug loading rate.

Embodiment 5: Behavioral evaluation of nanoparticles on D-galactose-induced cognitive impairment in mice

Experimental grouping and rearing

Male Kunming mice were randomized into 6 groups by body weight, 10 animals in each group, including the blind control group (no modeling and no administration), model group (modeling but no administration), control group with aqueous ACh solution (aqueous acetylcholine solution) after modeling), HSA-ACh nanoparticle group (Tween-80 modified ACH-loaded HSA nanoparticles administered after modeling), RT solution control group (aqueous rivastigmine solution administered after modeling) and HSA-RT nanoparticle group (Tween-80 modified rivastigmine loaded HSA nanoparticles administered after modeling).

The rearing environment: temperature of (24 ± 2) ° C; Humidity of (60 ± 10)%; daily light / dark at intervals of 12 h; Diet and water drink at will.

Animal modeling and drug therapy

① Animal Modeling: With the exception of the blank control group, the mice in the other 5 groups were subcutaneously injected with D-galactose solution 100 mg / kg / d, and the aluminum chloride solution 20 mg / kg / d was intragastrically injected. In the normal control group, the same amount of water was added. The modeling cycle was 90 days.

② Therapeutic Scheme: In 60 days after modeling, mice in the ACh solution group were given an aqueous acetylcholine solution (1 mg / kg); mice in the HSA-ACh nanoparticle group were given Tween-80-modified ACH-loaded nanoparticles (10 mg / kg, wherein the content of loaded ACh was equivalent to 1 mg / kg), mice in the RT dissolution group were given aqueous rivastigmine solution (1.25 mg / kg), and mice in the HSA-RT nanoparticle group became Tween -80-modified rivastigmine-loaded nanoparticle (12.5 mg / kg, the loaded RT content being equivalent to 1.25 mg / kg); the dosing cycle was twice a week, for 4 weeks in a row.

Morris water maze behavioral assessment procedures

① The Morris Water Labyrinth (MWM) had a test duration of 5 days with the first 4 days being the training period and the 5th day being the test duration.

② The swimming pool had a diameter of 1 m and was divided into 4 quadrants and four entry points were fixed in each of the four quadrants. The water depth of the swimming pool was about 0.6 m and the water temperature was 22 ± 2 ° C.

③ During the training period, a black platform with a diameter of 0.1 m was placed in the third quadrant about 2 cm below the water surface. Each mouse received training four times a day as follows: First, the mouse was placed in the fixed entry point of the first quadrant in the direction of the pelvic wall so that it could swim freely for 1 min. When the mouse found the platform position within 1 min and stayed for at least 3 seconds, the time measurement was immediately stopped and data such as latency and swimming speed were recorded. If the mouse did not find the platform within 1 minute or did not stay for 3 seconds after finding the platform, the mouse was artificially supported to find the platform and stay for 15 seconds, and the latency was recorded as 60 seconds. The mouse was wiped dry after a single swim, and when all the mice were swimming, the training was performed sequentially in the second, third, and fourth quadrants.

④ During the trial period, the platform was removed and the entry point was fixed. The mouse was allowed to swim free for 1 min and the latency for first finding the platform position, the PPT (platform time crossed) and the swim time in the target quadrant were recorded.

Experimental results:

The time to find the platform for mice in each group was in 10 shown. During the training period, the time to find the platform on the first day was not significantly different between the groups; on the third day and the fourth day was the time to find the platform for mice in the blank control group 20 s, while the time to find the platform for mice in the model group, ACh-solution group and RT-solution group was significantly longer than that of the blank control group. The time to find the platform for mice in the HSA-ACh nanoparticle group and the HSA-RT nanoparticle group was not significantly different from the blind control group, but was significantly shorter than that of the model group. Experiments showed that the short-term spatial learning ability of mice was improved after HSA-ACh nanoparticles or HSA-RT nanoparticles were administered.

During the test period, the time for mice to find the original platform position was shortened, but there was a statistically significant difference between the groups. As in 11 showed the mice in the blind control group to have the shortest latency. The analysis of variance showed that the latency of the model group of the ACh dissolution group and the RT solution group was significantly longer than that of the dummy control group (P <0.05), but the latency of the HSA-ACh nanoparticle group and the HSA-RT nanoparticle group differed but not significantly different from that of the blind control group.

The PPT and swimming time in the target quadrant were another two indices for assessing the long-term ability of mice in spatial memory. The PPT represented the crossed platform times of experimental animals within 60 s, the higher the PPT, the better the animals' spatial learning and memory ability. As in 12 The PPT of mice in the blind control group was significantly higher than that in the model group, the ACh dissolution group and the RT solution group (P <0.01), but did not differ significantly from that of the HSA-ACh nanoparticle group or the HSA RT nanoparticles group.

The swim time in the target quadrant is the sum of the time spent by laboratory animals having activity on the original platform and in the environment. The longer the time, the better the spatial learning and memory ability. As in 13 showed that the swim time in the target quadrant of the model group and the ACh dissolution group showed a statistically significant difference (P <0.05) compared to the blind control group, while in other groups the no-statistically significant difference was shown in comparison with the blind control group.

The above experimental MWM data showed that the learning and memory ability of mice in the model group was significantly lower than that of the normal group and the mouse models for D-galactose-induced cognitive impairment were successfully constructed. After administration of aqueous acetylcholine solution or aqueous rivastigmine solution, the cognitive impairment of mice was not alleviated; After administration of HSA-ACh nanoparticles or HSA-RT nanoparticles, the short-term learning ability and long-term spatial memory of mice were significantly improved, demonstrating that HSA-ACh nanoparticles and HSA-RT nanoparticles alleviate the cognitive impairment of mice and can achieve a long release.

Embodiment 6: Effect of a nanoparticle on brain biochemical parameters in mice with D-galactose-induced cognitive impairment

Pretreatment of the samples

① The body weights of the 6 groups of mice of Example 5 were accurately weighed and recorded;

② The blood was taken from mouse eyeball and collected in a 5 ml anticoagulation collection tube with heparin sodium and then placed in an ice bath;

The mice were decapitated to remove the brainstem. Then the brain weight was accurately weighed and recorded. The left and right hemispheres were cut into an ice box and the left hemisphere was immersed in a 4% neutral formaldehyde solution. The right hemisphere was crushed by filter paper to remove blood and capillaries, and then a cholinesterase extraction solution of 20 times by weight was added for homogenization. The cholinesterase extraction solution was centrifuged for 10 minutes at 4 ° C at 5000 rpm and then the supernatant was collected and stored at 4 ° C.

Determination of the activity of brain acetylcholinesterase (AChE)

① 10 μl of brain supernatant was accurately taken out and diluted in 0.5 ml of PBS, after the tip was repeatedly rinsed, the solution was mixed by vortexing;

② The plates were prepared, and four replicate wells were prepared for each sample, 20 μl of sample was added to each well, 80 μl of PBS was added to the first wells, and 50 μl of PBS and 30 μl of acetylthiocholine iodide (ATCH) became two others Given depressions;

③ The solutions were centrifuged for 1 min to remove air bubbles and incubated for 30 min in a constant temperature water bath at 37 ° C;

④ After removing the plate, 20 μl of 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) was added to each well and the absorbance value was measured at 412 nm after shaking for 30 sec.

Determination of brain choline acetyltransferase (ChAT) activity

① ChAT was measured using a kit (Nanjing Jiancheng, Freight Number: A079-1). The reagents 1 to 6 were added sequentially, mixed well and preheated for 5 minutes in a 37 ° C water bath;

② 25 μl tissue homogenate supernatant was added to the test tube and the same amount of cooked homogenate supernatant was added to the appropriate control tube, mixed well, heated in a water bath at 37 ° C for 20 minutes and then boiled in a 100 ° C water bath Warmed water bath for 2 min, then the reaction was stopped;

③ 425 μl of distilled water was added to the reaction system in each tube, mixed well; and centrifuged for 10 min at 4000 rpm; 500 μl supernatant was removed and 10 μl reagent 7 (Color developer) was added; after mixing well, the tubes were allowed to stand for 15 minutes, then the absorbance value of each tube was measured with a quartz cuvette with 1 cm beam path and 2 mm inside diameter at 324 nm wavelength using distilled water for zero adjustment.

Determination of acetylcholine (ACh) content

① The content of ACh was determined by a colorimetric alkaline hydroxylamine method. The sample was first added with a cholinesterase inhibitor to prevent the cholinesterase from decomposing acetylcholine, and then immediately added with trichloroacetic acid to precipitate the protein;

② The sample supernatant and ACh standard solution were added to the test tube and the standard tube, respectively; After basic hydroxylamine solution was added, the tubes were heated in a water bath at 37 ° C for 30 minutes, and then hydrochloric acid was added to terminate the reaction. Finally, ferric chloride was added to develop. Sample supernatant and water were added to the control tube and the blank tube respectively; The tubes were heated in a water bath at 37 ° C for 30 min and then hydrochloric acid and ferric chloride were added, finally alkaline hydroxylamine was added;

③ The plates were prepared, 200 μl of the solution was added to each well on a plate, and the absorbance value was measured at 540 nm wavelength.

Determination of malondialdehyde (MDA) content

① The MDA content was determined by a kit (Nanjing Jiancheng, Freight No: A003-1). 0.1 ml of homogenate supernatant was added to the test tube, the same amount of absolute ethanol was added to the blank tube, and the same amount of 10 nmol / ml standard was added to the standard tube, and then 0.1 ml of reagent 1 . 3 ml of reagent 2 (Application solution) and 1 ml of reagent 3 (Application solution) added successively and mixed well by vortex;

② The mouth of the test tube was wrapped with a plastic wrap and a small hole was pierced with a needle. Then the tube was heated in a water bath at 95 ° C for 40 min, taken out, cooled by running water and centrifuged for 10 min at 4000 rpm; and then the supernatant was taken to measure the absorbance value of each tube at 532 nm wavelength with 1 cm beam path using double distilled water for zero adjustment.

Determination of the total activity of superoxide dismutase (T-SOD)

① T-SOD was measured with a kit (Nanjing Jiancheng, freight number A001-1). The reagents 1 to 4 and the samples were added sequentially, mixed well by vortex, and then placed in a constant temperature water bath at 37 ° C for 40 minutes;

② The color developer was added to the solution, mixed well, placed at room temperature for 10 minutes, and then the absorbance value of each tube was measured with a 1 cm beam at a wavelength of 550 nm using double-distilled water for zero adjustment.

Histopathological examination of the brain

The left hemisphere immobilized with 4% neutral formaldehyde solution was subjected to the steps of washing, dehydrating, waxing, embedding, cutting and sticking, dewaxing, hematoxylin-eosin staining, dehydration, transparency and foil sealing, and finally brain tissue sections with blue stained core and red stained To get cytoplasm.

Experimental results:

The cholinergic system-related enzyme activity and brain oxidative stress level were important biochemical indices of the brain for evaluating cognitive ability. The measurement results of all indices are shown in Table 2. Table 2 cholinergic system Oxidative stress ACh content AChE activity ChAT activity MDA content T-SOD activity (Ug / mgprot) (U / mgprot) (U / mgprot) (Nmol / mgprot) (U / mgprot) Blind control group 4.16 ± 0.25 1.69 ± 0.14 1.42 ± 0.33 1.06 ± 0.07 40.54 ± 2.34 model group 4.19 ± 0.56 1.71 ± 0.20 1.75 ± 0.20 1.49 ± 0.18 *** 43.23 ± 3.79 ACh solution group 3.89 ± 0.48 1.86 ± 0.12 1.59 ± 0.15 1.50 ± 0.18 *** 42.57 ± 2.88 HSA ACh nanoparticles group 4.51 ± 0.36 1.93 ± 0.14 ^{* / #} 1.55 ± 0.36 1.09 ± 0.14 ^### 42.76 ± 1.78 RT solution group 3.97 ± 0.36 1.70 ± 0.17 1.30 ± 0.17 ^## 1, 12: TO, 08 ^## 40.76 ± 2.30 HSA-RT nanoparticle group anoparticle group 4.55 ± 0.16 2.21 ± 0.15 ^{** / ##} 1.36 ± 0.14 1.06 ± 0.06 ^### 43.90 ± 1.83 * p <0.05, ** p <0.01, *** p <0.001, compared with the blank control group. # p <0.05, ## p <0.01, ### p <0.001, compared to the model group; n = 8.

As shown in Table 2, the activity and content of ChAT, ACh and AChE were maintained at a relatively stable level in normal brain tissues; In the model group, the ChAT activity was significantly increased (p <0.05), while in the HSA-ACh nanoparticle group, HSA-RT nanoparticle group and the RT solution group, the activity and content of ChAT, ACh and AChE are still at normal levels, indicating that the nanoparticles of the present invention effectively maintained the relative balance of activity and content of ChAT, ACh, and AChE in the cholinergic system of the brain. by virtue of From the results of the oxidative stress test, the content of MDA in the model group and the ACh solution group was significantly increased (p <0.001) in comparison with the blank control group, while the content of MDA in the HSA-ACh nanoparticle group, the HSA-RT nanoparticle group and the RT solution group was maintained at a normal level, demonstrating that the nanoparticles of the present invention were effective in reducing the level of oxidative stress in the brain and protecting neurons.

14 showed the results of histopathological examination of the brain. The normal CA2 region in the hippocampus and the frontotemporal cortical neurons were round or elliptical, with a large nucleoplasmic ratio. The nuclear membranes were obvious, round or elliptical and showed a uniform coloration of light blue-purple ( 14A . 14B) , The hippocampal neurons in the model group and the ACh dissolution group were loosely arranged and more neurons in the hippocampal and cortical areas showed shrinkage, cytoplasmic condensation, nuclear pyknosis, and the entire cell was deeply violet stained ( 14C . 14D . 14E . 14F) ; after administration of the HSA-ACh nanoparticle, HSA-RT nanoparticle or RT solution, the above phenomena were significantly improved and the nuclear pyknosis of the frontotemporal cortical neurons was significantly reduced ( 14G . 14H . 141 . 14J . 14K . 14L ). Experiments showed that the nanoparticle of the present invention had a significant protective effect on neuronal cells.

Embodiment 7: Central Targeting and Penetration of the Blood-Brain Barrier of Nanoparticles

The Tween-80-modified acetylcholine-loaded nanoparticles and the Tween-80-modified rivastigmine-loaded nanoparticles were prepared according to the procedures of Examples 1 and 2 by modifying fluorescein isothiocyanate (FITC) on human serum albumin to obtain nanoparticles with FITC. After the FITC-containing nanoparticles in each group were diluted to 0.1 mg / ml, 10 μl of solution from the heart was injected into the zebrafish body. The successfully injected and surviving zebrafish was selected and immobilized in the gel, and then the in vivo distribution of zebrafish fluorescence was observed by confocal laser microscopy.

The 15A - 15C The confocal laser microscope images showed the saline group (control group), the acetylcholine nanoparticle group and the rivastigmine nanoparticle group, respectively. FITC is a water-soluble fluorescent dye that is tightly bound to a nanoparticle through a chemical bond. Due to chemical bonding, the fluorescein can not be released freely from the nanoparticle to enter the system as a free small molecule, so that the fluorescence observed under the microscope comes from nanoparticles and there are no free small FITC molecules in the system. As can be clearly seen from the figure, there was no in vivo fluorescence distribution in normal zebrafish ( 15A) , In the acetylcholine nanoparticle group and the rivastigmine nanoparticle group, although only one heart injection was performed, apparent green fluorescence occurred in the brain of the zebrafish, indicating that the drug could easily enter the center through the blood-brain barrier.

The above embodiments comprehensively evaluated the effect of the nanoparticles of the present application on improving the cognitive ability of mice. The overall behavioral changes of mice were observed and their biochemical indices of the brain and pathological parameters were examined. Based on the experimental results, it can be concluded that the nanoparticles of the present application can transport acetylcholine, which can not enter the center by itself, to the center and slowly release it to achieve a long-term low-dose supplementation of exogenous neurotransmitters; In addition, the nanoparticles of the present application can transport rivastigmine, which tends to rapidly decompose, to the center to achieve long-acting administration.

Although specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes can be made in accordance with the teachings disclosed herein and such modifications and changes will fall within the scope of the present invention. The scope of the present invention is indicated by the appended claims and any equivalents thereof.

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Cited non-patent literature

• Helen F. Stanyon and John H. Viles, Human Serum Albumin Can Regulate Amyloid-β Peptides Fiber Growth in the Brain Interstitium, J. Biol. Chem. 2012, 287: 28163-28168 [0009]

Claims (9)

Nanoparticles comprising a carrier material, an active agent and a targeting molecule, wherein the carrier material is human serum albumin (HSA) and the active ingredient can prevent and / or treat a neurodegenerative disease; preferably, wherein the drug is loaded into the carrier material to form a drug-loaded HSA nanoparticle; in which the drug-loaded HSA nanoparticle is formed by a process comprising the following steps: Step (1-1): dissolving or dispersing the human serum albumin and the drug in a good solvent to form a solution (1); Step (1-2): adding dropwise the solution (1) to a poor solvent and adding a crosslinking agent to conduct a reaction to obtain a drug-loaded HSA nanoparticle; preferably, wherein the nanoparticle has a drug loading rate of 15% to 25%. Nanoparticles after Claim 1 wherein the active agent is selected from a group consisting of a cholinergic transmitter supplement and a cholinesterase inhibitor; preferably, wherein the cholinergic transmitter supplement is selected from a group consisting of acetylcholine, a prodrug of acetylcholine or a pharmaceutically acceptable salt of acetylcholine (for example, hydrochloride); preferably, wherein the cholinergic transmitter supplement is acetylcholine or acetylcholine chloride; preferably, wherein the cholinesterase inhibitor is selected from a group consisting of rivastigmine, galantamine or donepezil or a pharmaceutically acceptable salt thereof (for example tartrate); preferably, wherein the cholinesterase inhibitor is rivastigmine hydrogen tartrate. Nanoparticles after Claim 1 or 2 wherein the targeting molecule is selected from a group consisting of a surfactant, a polyethylene glycol polymer, and a blood brain barrier specific antibody; preferred, wherein the targeting molecule is a surfactant; preferred, wherein the targeting molecule is Tween-80; preferred wherein the targeting molecule is modified on the surface of the nanoparticle; preferred wherein the targeting molecule is modified on the surface of the drug-loaded HSA nanoparticle; preferably, wherein the targeting molecule is modified on the surface of the nanoparticle by adsorption. Nanoparticles after one of the Claims 1 to 3 wherein the nanoparticle has a diameter of 150 nm to 250 nm; preferred, wherein the nanoparticle has a zeta potential of -30 mV to +20 mV; preferred, wherein the nanoparticle has an entrapment efficiency of 40% to 50%. Process for the preparation of the nanoparticle according to one of Claims 1 to 4 comprising the following steps: Step (1): preparing a drug-loaded HSA nanoparticle; Step (2): Modifying a targeting molecule on the surface of the drug-loaded HSA nanoparticle. Method according to Claim 5 wherein, in step (1), the method of making a drug-loaded HSA nanoparticle is an anti-solvent method; preferably, wherein step (1) comprises the steps of: step (1-1): dissolving or dispersing the human serum albumin and active ingredient in a good solvent to form a solution (1); Step (1-2): dropwise adding the solution (1) in a poor solvent, and adding a crosslinking agent to carry out the reaction to form a drug-loaded HSA nanoparticle; preferably, wherein step (1) further comprises the step of (1-3): isolating the drug-loaded HSA nanoparticle. Method according to Claim 5 or 6 in which step (2) comprises the following steps: Step (2-1): dispersing the drug-loaded HSA nanoparticle in physiological saline to obtain a solution (2); Step (2-2): mixing the solution (2) with a targeting molecule or a solution thereof. Pharmaceutical composition comprising the nanoparticle according to one of Claims 1 to 4 ; optionally wherein the pharmaceutical composition further comprises a pharmaceutically acceptable adjuvant (eg, a carrier and / or carrier). Use of the nanoparticle according to one of Claims 1 to 4 or the pharmaceutical composition Claim 8 for the manufacture of a medicament for the prevention and / or treatment of a neurodegenerative disease in an individual; preferably, wherein the neurodegenerative disease is selected from a group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis and cerebrospinal multiple sclerosis; preferred, wherein the subject is a mammal, such as a bovine, equine, ovine, porcine, canine, feline, rodent, primate; for example, the individual is a human.

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