WO2018137483A1 - Sustained-release nano-drug for targeting neurodegenerative disease - Google Patents

Abstract

A sustained-release nanoparticle for targeting a neurodegenerative disease, a preparation method thereof, a pharmaceutical composition comprising the same, and a use thereof in preparing a drug for preventing and/or treating a neurodegenerative disease in a subject. The nanoparticle comprises a carrier material, an active ingredient, and a targeting molecule. The carrier material is human serum albumin, and the active ingredient can prevent and/or treat the neurodegenerative disease.

Description

A nano drug capable of sustained release and targeted application to neurodegenerative diseases Technical field

The present invention relates to the field of medical technology, and in particular to a nanoparticle comprising a carrier material, an active ingredient and a targeting molecule, the carrier material being human serum albumin, the active ingredient being capable of preventing and/or treating a neurodegenerative disease. Active ingredient. The present invention also relates to a method of preparing the nanoparticles, a pharmaceutical composition comprising the nanoparticles, and a medicament for preparing a neurodegenerative disease for preventing and/or treating a subject in the nanoparticle or pharmaceutical composition. Use in.

Background technique

1, Alzheimer's disease cholinergic hypothesis

Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive decline in memory and cognitive ability. At present, there is still no clear conclusion about the etiology of AD, mainly in the cholinergic theory and β- Amyloid (Aβ) theory.

The central cholinergic system is closely related to learning and memory. Acetylcholine is one of the important neurotransmitters in the central cholinergic system. Its main function is to maintain consciousness and play an important role in learning and memory. Although the exact cause of AD is still unknown, the massive loss of neurons in specific brain regions is a direct cause of progressive decline in cognitive and memory capabilities, especially the loss of cholinergic neurons in the hippocampus. At present, there are five drugs approved by the US FDA for the treatment of AD, namely tacrine, neostigmine, galantamine, donepezil and memantine. The first four are reversible acetylcholinesterase inhibitors. The acetylcholinesterase inhibitor can firmly interact with the acetylcholinesterase in the body, occupying the enzyme site, and the enzyme loses the function of decomposing acetylcholine, thereby indirectly increasing the concentration of acetylcholine in the synaptic cleft, and achieving the effect of relieving AD.

2. Limitations of existing drug applications

At present, the drugs used to treat AD are still limited, not only because the mechanism of origin is unclear, but also because the brain's complex defense system prevents the drug from penetrating the blood-brain barrier (BBB). In addition, even if some of the drug passes through the BBB, it will be pumped out under the action of an extracellular transporter (eg, p-glycoprotein). Although these limitations of common drugs can be overcome by chemical modification, problems such as decreased efficacy after modification, introduction of toxic by-products during modification, and complicated modification processes will also emerge. For protein and peptide drugs, they are rapidly decomposed and inactivated by the action of peripheral proteases, limiting the application of a large number of effector molecules.

A neurodegenerative disease represented by AD is a type of chronic disease. After the diagnosis, the patient needs to take the drug for a long time without interruption or even take it for life. The existing AD treatment drugs have short plasma half-life, poor blood-brain barrier penetration, and rapid metabolism, so that patients need to take the medicine on time and in time, which is very difficult for patients whose memory is diminishing. Therefore, it is urgent to prepare a drug for the treatment of AD with high efficiency, sustained release and high targeting.

3. Progress in nanomedicine research

Most of the existing nanomedicines targeting the central nervous system use synthetic polymer materials as carriers, that is, the carrier material is coated on the outer layer of the target drug, and the core drug is degraded when the carrier material is degraded at the target site. It is released. Such nanomedicines are simple to prepare, have high stability, are non-toxic to some extent, and are easily degraded.

However, the neurodegenerative disease represented by AD belongs to chronic diseases of the elderly, and the brain metabolic capacity of patients is reduced. These exogenous synthetic polymer materials and their degradation products will undoubtedly increase the burden on the brain, so when preparing AD nanomedicines More attention should be paid to the biocompatibility of materials and the toxicity of material metabolites.

For the above reasons, India's Shrinidh A. Joshi research group used a biocompatible polylactic acid-glycolic acid copolymer (PLGA) as a carrier, loaded with the AD treatment drug Lismin, and prepared a packaged nanoparticle by a sedimentation method. From the perspective of material safety, although PLGA enhances biocompatibility to a certain extent, poor central targeting and rapid drug release still fail to meet the treatment needs of chronic diseases (Shrinidh A. Joshi, Sandip S. Chavhan) 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 Helen F. Stanyon team in the UK found that at physiological levels, human serum albumin (HSA) binds not only to 95% of Aβ in peripheral plasma, but also to nearly half of Aβ monomers in cerebrospinal fluid, effectively inhibiting β- The formation of amyloid fiber and significantly reduce the total amount of fibrils; in addition, HSA can also reduce the catalytic effect of metal ions by depriving Cu(II) in Aβ-Cu(II) complex, a dual mechanism Inhibition of fibril formation (Helen F. Stanyon and John H. Viles, Human Serum Albumin Can Regulate Amyloid-β Peptide Fiber Growth in the Brain Interstitium, J. Biol. Chem. 2012, 287: 28163-28168).

In summary, at present, neither molecular drugs nor existing nano-drugs can meet the therapeutic needs of high-efficiency, sustained-release and targeted treatment of neurodegenerative diseases. Human serum albumin, as a natural protein, exhibits good material properties and targeted properties for Aβ. Therefore, there is an urgent need to develop nanomedicines using human serum albumin nanoparticles as carrier materials to meet the needs of high-efficiency, sustained-release, targeted therapy.

Summary of the invention

In the present invention, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art, unless otherwise stated. Moreover, the laboratory procedures involved herein are routine steps that are widely used in the corresponding art. Also, for a better understanding of the present invention, definitions and explanations of related terms are provided below.

As used herein, the term "nanoparticle" (NP) refers to particles having a particle size on the order of nanometers, such as particles having a particle size of not more than 1000 nm, for example, having a particle diameter of 10 nm to 100 nm, 100 nm to 200 nm, and 200 nm to 300 nm. Particles between 300 nm and 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.

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

As used herein, the term "average particle size" refers to an actual population of particles consisting of particles of different sizes and shapes, compared to a group of hypothetical particles consisting of uniform spherical particles. When the total length of the diameter is the same, the diameter of the spherical particles is referred to as the average particle diameter of the actual particle group. Methods of measuring the average particle size are known to those skilled in the art, such as light scattering; and measuring instruments of average particle size include, but are not limited to, light scattering particle size analyzers.

As used herein, the term "choline energy transmitter supplement" refers to an active ingredient (eg, a drug) that exogenously supplements a cholinergic neurotransmitter (a cholinergic transmitter).

As used herein, the term "prodrug" refers to a compound obtained by modifying a compound which is inactive or less active in vitro and which exerts an efficacious effect by enzymatic or non-enzymatic conversion in vivo to release the active drug. The design principles and preparation methods of prodrugs are known to those skilled in the art.

As used herein, the term "pharmaceutically acceptable salts" refers to an acidic functional group (e.g. -COOH, -OH, -SO ₃ H and the like) compound (1) in the presence of a suitable inorganic or organic cation (base) a salt formed, for example, a salt of a compound with an alkali metal or an alkaline earth metal, an ammonium salt of a compound, and a salt of a compound with a nitrogen-containing organic base; and (2) a basic functional group (for example, -NH _{2 ,} etc.) present in the compound Salts formed with suitable inorganic or organic anions (acids), for example salts of the compounds with inorganic or organic carboxylic acids.

Therefore, in the present application, "pharmaceutically acceptable salts" include, but are not limited to, alkali metal salts such as sodium salts, potassium salts, lithium salts and the like; alkaline earth metal salts such as calcium salts, magnesium salts and the like; 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, Portuguese Glycosylamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, sulfonium salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N, N'- Dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, piperazine salt, tetramethylamine salt, tris (hydroxyl) Aminomethane salt; a hydrohalide salt such as a hydrofluoric acid salt, a hydrochloride salt, a hydrobromide salt, a hydroiodide salt, etc.; a mineral acid salt such as a nitrate salt, a perchlorate salt, a sulfate salt, a phosphate salt And lower; alkanesulfonates such as methanesulfonate, triflate, ethanesulfonate, etc.; arylsulfonates such as besylate, p-benzenesulfonate, etc.; Such as acetate, malate, fumaric acid , succinate, citrate, tartrate, oxalate, maleate, etc.; amino acid salts such as glycinate, trimethylglycine, arginine, ornithine, glutamate, Aspartate and the like.

As used herein, the term "neurodegenerative disease" refers to a disease caused by progressive disease of the nervous system, including but not limited to Alzheimer's disease (AD), Parkinson's disease, Huntington's disease, muscular atrophy. Lateral sclerosis and multiple sclerosis of the brain. As used herein, the term "good solvent" refers to a solvent capable of solubilizing human serum albumin, including but not limited to water and sodium chloride solutions.

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

As used herein, the term "room temperature" means 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 use of the term is not clear to the person skilled in the art, then "about" means no more than plus or minus 10% of the particular 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 labor, which can be used to deliver poor blood-brain barrier permeability and/or plasma. A drug with a short half-life effectively achieves the central targeting and sustained release targets of the drug, thereby providing the following inventions:

In one aspect, the present invention provides a nanoparticle comprising a carrier material, an active ingredient, and a targeting molecule, the carrier material being human serum albumin, the active ingredient being capable of preventing and/or treating a neurodegenerative disease ingredient.

The inventors have found that the natural protein drug carrier human serum albumin nanoparticles can effectively meet the requirements of central targeted administration in the selection of carrier materials. First of all, human serum albumin has a high affinity with Aβ monomer, and has a targeting property against Aβ. While ensuring a large amount of drug molecules, it can rapidly target the lesion site and achieve excellent targeting performance of the material itself. .

In addition, natural protein materials have high biosafety, meet the requirements of use in the pharmaceutical field, are non-immunogenic, and the decomposition products are amino acids, which can be reused or converted into other nitrogen-containing substances as the body's own nutrients, minimizing The damage and burden of the central nervous system by exogenous organic or inorganic polymer materials.

In certain embodiments, the active ingredient is loaded into a carrier material to form human serum albumin-loaded nanoparticles.

In certain embodiments, the human serum albumin drug-loaded nanoparticles are formed by a method 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);

Step (1-2): The solution (1) is added dropwise to a poor solvent, and a crosslinking agent is added to carry out a reaction to obtain human serum albumin-loaded nanoparticles.

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

In certain embodiments, the active ingredient is selected from the group consisting of a cholinergic neurotransmitter supplement and a cholinesterase inhibitor.

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

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

In certain embodiments, the cholinesterase inhibitor is selected from the group consisting of lismin, galantamine or donepezil, or a pharmaceutically acceptable salt of such compounds (eg, tartrate).

In certain embodiments, the cholinesterase inhibitor is Lismin heavy tartrate.

In order to ensure sufficient central input dose and enhance the central targeting of nanoparticles, the surface modification of nanoparticles has targeting molecules to increase the permeability of nanoparticles to the blood-brain barrier and achieve efficient central targeted transport.

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

In certain embodiments, the targeting molecule is a surfactant.

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

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

In certain embodiments, the targeting molecule is modified on the surface of a human serum albumin-loaded nanoparticle.

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

In certain embodiments, the nanoparticles have a diameter of from 150 nm to 250 nm; for example, 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 certain embodiments, the nanoparticles have a zeta potential of -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, -30 mV, -25 mV, -20 mV, -15 mV, -10 mV, -5 mV, 0, +5 mV, +10 mV, +15 mV or +20 mV.

In certain embodiments, the encapsulation efficiency of the nanoparticles is from 40% to 50%, such as 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49% or 50%.

Fig. 1 exemplarily shows a structure of the nanoparticles of the present application. Among the nanoparticles shown in the figure, HSA with Aβ targeting property is used as a nanocarrier, and a drug capable of treating AD is coated, and a surface molecule is modified on the surface of the HSA nanoparticle to enhance the HSA nanoparticle. The blood-brain barrier (BBB) is transmissive and prolongs circulation time in the body.

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

Step (1): preparing human serum albumin-loaded nanoparticles;

Step (2): modifying the targeting molecule on the surface of the human serum albumin-loaded nanoparticles.

In certain embodiments, in the step (1), the method of preparing human serum albumin-loaded nanoparticles is an anti-solvent method. .

In certain embodiments, the step (1) comprises the steps of:

Step (1-1): dissolving or dispersing human serum albumin and active ingredient in a good solvent to form a solution (1);

Step (1-2): The solution (1) is added dropwise to a poor solvent, and a crosslinking agent is added to carry out a reaction to obtain human serum albumin-loaded nanoparticles.

In certain embodiments, in step (1-1), the mass ratio of human serum albumin to active ingredient is from 1:1 to 10:1, such as 1:1, 2:1, 3:1, 4 1: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, or 10: 1.

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

In certain embodiments, in the step (1-1), the concentration of the sodium chloride solution is from 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 10 mmol. /L to 15 mmol / L or 15 mmol / L to 20 mmol / L.

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

In certain embodiments, the poor solvent is ethanol.

In certain embodiments, the step (1-2) is carried out under agitation.

In certain embodiments, the crosslinking agent in step (1-2) is glutaraldehyde.

In the present invention, the addition of the crosslinking agent can cross-link and solidify the human serum albumin particles so that they do not immediately swell and disperse in the aqueous solution, thereby avoiding the release of the drug encapsulated in the particles too quickly.

In certain embodiments, the step (1) further comprises the step (1-3) of isolating the human serum albumin-loaded nanoparticles.

In certain embodiments, in the step (1-3), the human serum albumin-loaded nanoparticles are separated by centrifugation.

In certain embodiments, the step (2) comprises the steps of:

Step (2-1): dispersing human serum albumin-loaded nanoparticles in physiological saline; obtaining a solution (2);

Step (2-2): Mixing the solution (2) with a targeting molecule or a solution thereof.

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

In certain embodiments, the concentration of the targeting molecule in the solution of the targeting molecule is from 0.1% to 10%, such as from 0.1% to 0.5%, from 0.5% to 1%, from 1% to 5%, or 5% to 10%.

In certain embodiments, in step (2-2), mixing is performed by agitation and/or ultrasound.

In certain embodiments, the drug loading rate and encapsulation efficiency of the drug loaded human serum albumin nanoparticles are tested after step (1).

Figure 2 exemplarily shows a method of preparing the nanoparticles of the present application. The method described in the figure comprises: mixing an active ingredient (such as acetylcholine or lissamine) with HSA, under the action of glutaraldehyde, HSA is cross-linked to form HSA-loaded nanoparticles; The surface-modifying target molecule (for example, Tween-80) is surface-modified to obtain a modified drug-loaded nanoparticle.

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

In the present invention, optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable adjuvant (e.g., a carrier and/or an excipient). In certain embodiments, the carrier and/or excipient is selected from the group consisting of: an ion exchanger, alumina, aluminum stearate, lecithin, serum protein (eg, human serum albumin), glycerin, sorbic acid, sorbic acid Potassium, a partial glyceride mixture of saturated plant fatty acids, water, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose Substance, polyethylene glycol, sodium carboxymethylcellulose, polyacrylate, beeswax, polyethylene-polyoxypropylene block polymer and lanolin.

In the present invention, the pharmaceutical composition can be formulated into any of pharmaceutically acceptable dosage forms. 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, Inhalant or spray.

Furthermore, the pharmaceutical composition of the present invention can also be administered to a subject by any suitable mode of administration, such as oral, parenteral, rectal, pulmonary or topical administration. In certain preferred embodiments, the pharmaceutical compositions are suitable for oral, parenteral (intravenous, intramuscular or subcutaneous), transdermal, translingual or respiratory administration.

When used for oral administration, the pharmaceutical composition can be formulated into an oral preparation, such as an oral solid preparation such as a tablet, a capsule, a pill, a granule, or the like; or an oral liquid preparation such as an oral solution or an oral mixture. Suspension, syrup, and the like. When formulated into an oral preparation, the pharmaceutical composition may further comprise a suitable filler, binder, disintegrant, lubricant, and the like. When used for parenteral administration, the pharmaceutical composition can be formulated into an injection, including a liquid injection, an injectable powder or an injectable tablet. When formulated as an injection, the pharmaceutical composition can be produced by a conventional method in the existing pharmaceutical field. When the injection is formulated, no additional agent may be added to the pharmaceutical composition, and a suitable additional agent may be added depending on the nature of the drug. When used for rectal administration, the pharmaceutical composition can be formulated into a suppository or the like. For pulmonary administration, the pharmaceutical composition can be formulated as an inhalant or a spray.

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

In certain preferred embodiments, the subject is administered an effective amount of the pharmaceutical composition. As used herein, the term "effective amount" 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 the disease; treating an effective amount of the disease means an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. Determination of such an effective amount is well within the capabilities of those skilled in the art. For example, the amount effective for therapeutic use will depend on the severity of the condition to be treated, the overall condition of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments for simultaneous administration. and many more.

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

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

In one aspect, the application provides a nanoparticle as described above for use in preventing and/or treating a neurodegenerative disease in a subject.

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

In certain embodiments, the subject is a mammal, such as a bovine, equine, ovine, porcine, canine, feline, rodent, primate; For example, the subject is a human.

The nanoparticles of the present application are particularly suitable for the 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 are unable to meet long-term sustained release requirements. Thus, in another aspect, the application provides the use of a nanoparticle as described above for enhancing the ability of an active ingredient to pass through a blood-brain barrier of a subject, and/or prolonging the release of the active ingredient in a subject The active ingredient is an active ingredient capable of preventing and/or treating a neurodegenerative disease in a subject.

In certain embodiments, the active ingredient is selected from the group consisting of a cholinergic neurotransmitter supplement and a cholinesterase inhibitor.

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

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

In certain embodiments, the cholinesterase inhibitor is selected from the group consisting of lismin, galantamine or donepezil, or a pharmaceutically acceptable salt of such compounds (eg, tartrate).

In certain embodiments, the cholinesterase inhibitor is Lismin heavy tartrate.

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

In certain embodiments, the subject is a mammal, such as a bovine, equine, ovine, porcine, canine, feline, rodent, primate; For example, the subject is a human.

Beneficial effect

The nanoparticles of the present application have one or more of the following benefits:

1. The nanoparticle of the present application solves the problem that a drug having high water solubility is difficult to pass the blood-brain barrier, and/or a drug having a short plasma half-life cannot achieve a long-term slow drug release problem.

2. The nanoparticles of the present application are highly safe. The nanocarrier used in the nanoparticle of the present application is a natural protein material, has no immunogenicity, high biocompatibility, and the degradation product is a plurality of essential amino acids, and has no toxic and side effects.

3. Optimization of the drug loading mode of the nanoparticles of the present application. The drug-loading methods reported in the literature mostly focus on the adsorption of drug molecules on the outer layer of nanoparticles, and the drug-loading rate is low and easy to desorb. However, the present invention uses nano-particles to encapsulate drug molecules, and the drug molecules are wrapped and wrapped in the nano-carriers, along with the nanoparticles. Gradually disintegrating, the drug molecules are slowly released.

4. The nanoparticles of the present application can effectively improve the spatial learning and memory ability of mice, maintain the activity of the enzymes related to the cholinergic system and the dynamic balance of acetylcholine content, and reduce the level of oxidative stress, especially for the frontotemporal cortex. effect.

The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and embodiments. The various objects and advantageous aspects of the invention will be apparent to those skilled in the <

DRAWINGS

Fig. 1 exemplarily shows a structure of the nanoparticles of the present application. Among the nanoparticles shown in the figure, HSA with Aβ targeting property is used as a carrier material, and a drug capable of treating AD is coated, and a surface molecule is modified on the surface of the HSA nanoparticle to enhance the HSA nanoparticle. The BBB is transmissive and extends the circulation time in the body.

Figure 2 exemplarily shows a method of preparing the nanoparticles of the present application. The method described in the figure comprises: mixing an active ingredient (such as acetylcholine or lissamine) with HSA, under the action of glutaraldehyde, HSA is cross-linked to form HSA-loaded nanoparticles; The surface-modifying target molecule (for example, Tween-80) is surface-modified to obtain a modified drug-loaded nanoparticle.

Figure 3 is a scanning electron micrograph of HSA nanoparticles loaded with acetylcholine unmodified Tween-80 in Example 1. As shown, the unmodified HSA drug-loaded nanoparticles are spherical and have a diameter of about 200 nm.

4 is a scanning electron micrograph of HSA nanoparticles loaded with acetylcholine modified with Tween-80 in Example 1. As shown in the figure, the HSA drug-loaded nanoparticles modified with Tween-80 are regular cubes with a side length of about 200 nm.

Figure 5 is a scanning electron micrograph of HSA nanoparticles loaded with acetylcholine modified with Tween-80 in Example 1. As shown in the figure, the HSA drug-loaded nanoparticles modified with Tween-80 are regular cubes with a side length of about 200 nm.

6 shows the UV absorption curves of the ACh aqueous solution, the HSA aqueous solution, the unloaded drug and unmodified Tween-80 blank HSA nanoparticles, and the unmodified Tween-80 HSA-ACh nanoparticles in Example 3. In the figure, the HSA solution has a distinct characteristic absorption peak around 280 nm (curve 1), while the Ach solution has no absorption near 280 nm (curve 2). After the nanoparticles were formed by HSA, the absorption peak near 280 nm was still clearly visible (curve 3); while the HSA nanoparticles loaded with Ach showed a significant decrease in the absorption peak near 280 nm (curve 4). The results show that the drug molecules are successfully doped in the nanocarriers.

Figure 7 shows the blank HSA nanoparticles of unloaded drug and unmodified Tween-80 in Example 3, HSA-Ach nanoparticles without Tween-80 modified, Tween-80 modified unloaded drug HSA nanoparticles XRD patterns of granules and Tween-80 modified HSA-Ach nanoparticles. In the figure, HSA nanoparticles modified by Tween-80 showed sharp peaks at 2θ = 31.6° (curves 3 and 4), while unmodified nanoparticles had no peaks here (curves 1 and 2). The results demonstrate that the targeting molecule Tween-80 has been successfully modified on the surface of nanoparticles.

Figure 8 is a standard curve of doxorubicin as determined by ultraviolet spectrophotometry in Example 4.

Figure 9 is a graph showing the in vitro cumulative release rate of the doxorubicin sample in Example 4. As shown in the figure, the doxorubicin solution group reached the maximum cumulative release rate at around 10 h, and the recovery rate was over 90%, which proved that doxorubicin was completely released; while the doxorubicin-loaded drug nanoparticle group was within 80 h. The cumulative release rate is steadily increasing, 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 body fluid transportation, ensuring that the nanoparticles entering the central center can still maintain a high drug loading rate.

Fig. 10 is a trend diagram showing the time of finding the platform time of each group of mice within 4 days of the training period of the water maze behavior evaluation in the fifth embodiment. During the training period, the mice in each group found no significant difference in platform time on the first day; on the third and fourth days, the blank control group found the platform time was 20s, and the model group, ACh solution group and RT solution group were found. The platform time was significantly longer than that of the blank control group, and the platform time of the HSA-ACh nanoparticle group and the HSA-RT nanoparticle group was not significantly different from that of the blank control group, and was significantly lower than that of the model group. Experiments have shown that the short-term spatial learning ability of mice is improved after HSA-ACh nanoparticles and HSA-RT nanoparticles are separately administered.

Figure 11 is a graph showing the latency of the first time a mouse was found in each group of mice during the water maze behavioral evaluation test in Example 5. As shown in the figure, the latency of the blank control group was the shortest. The analysis of variance showed that the latency of the model group, ACh solution group and RT solution group was significantly longer than that of the blank control group (P<0.05), while the HSA-ACh nanoparticle group and HSA. There was no statistically significant difference between the -RT nanoparticle group and the blank control group.

Figure 12 shows the number of times each group of mice crossed the platform within 60 s during the test period of the water maze behavioral evaluation in Example 5. As shown in the figure, the number of penetrating mice in the blank control group was significantly higher than that in the model group, ACh solution group and RT solution group (P<0.01), but no significant statistics were found with HSA-ACh nanoparticle group and HSA-RT nanoparticle group. Learn the difference.

Figure 13 is a graph showing the time in which the mice of each group swim in the target quadrant during the test period of the water maze behavior evaluation in Example 5. As shown in the figure, there was a statistically significant difference between the model group and the ACh solution group compared with the blank control group (P<0.05), while there was no statistically significant difference between the other groups and the blank control group.

Fig. 14 is a photograph showing the HE staining pathology of the left hemisphere of each group of mice in Example 6, and Fig. AL corresponds to the blank control group hippocampus (A), cortex (B), model group hippocampus (C), cortex (D), ACh. Solution group hippocampus (E), cortex (F), HSA-ACh nanoparticle group hippocampus (G), cortex (H), RT solution group hippocampus (I), cortex (J), HSA-RT nanoparticle group hippocampus (K ), cortex (L).

Figure 15 is a photograph of a laser confocal microscope in Example 7. Panels A-C correspond to the heart-injected saline group (control group), the acetylcholine nanoparticle group, and the Lissian nanoparticle group, respectively. It can be clearly observed from the figure that there is no fluorescence distribution in normal zebrafish; for the acetylcholine nanoparticle group and the Lissian nanoparticle group, although the heart is injected, there is obvious green fluorescence in the brain of the zebrafish. Prove that the drug can smoothly enter the center through the blood-brain barrier.

detailed description

The embodiments of the present invention will be described in detail below with reference to the accompanying drawings, however, the following examples are intended to illustrate the invention and are not intended to limit the scope of the invention. Those who do not specify the specific conditions in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are conventional products that can be obtained commercially.

Example 1: Preparation of nanoparticles loaded with acetylcholine (Ach)

In this embodiment, human serum albumin (HSA) is used as a nanocarrier material, and acetylcholine is encapsulated. The active ingredient used is acetylcholine chloride, the preferred solvent is 10mmol/L sodium chloride solution, the poor solvent selected is ethanol, the selected cross-linking agent is 10% glutaraldehyde, and the selected targeting molecule is spit. Wen-80, using human serum albumin (HSA) nanoparticles as a carrier material. The preparation process is as follows.

Step (1): Preparation of HSA drug-loaded nanoparticles by an anti-solvent method: 20 mg of HSA and 10 mg of ACh are dissolved in 1 mL of sodium chloride solution (10 mmol/L), filtered through a 0.22 μm water-based filter, and then rotated at 600 rpm. This solution was added dropwise to 10 mL of ethanol at a dropping rate of 50 μL/min; after the completion of the dropwise addition, the stirring speed was increased to 800 rpm, 20 μL of 10% glutaraldehyde was added, and the reaction was protected from light for 24 hours. The reaction sample was centrifuged at 13600 rpm for 10 min, the supernatant was removed, the precipitate was collected, and the sample was centrifuged three times with an equal amount of physiological saline, and the precipitate was collected to obtain acetylcholine (Ach)-loaded HSA nanoparticles, which were stored at 4 °C.

The mass method was used to determine the product quality, and the nano drug encapsulation rate and drug loading rate were calculated as follows:

The solvent in the sample prepared in the step (1) was spin-dried, the solid powder was redispersed in 8 mL of physiological saline, centrifuged at 13600 rpm for 10 min, 1 mL of the supernatant was aspirated in 8 mL of physiological saline, shaken, and the basic hydroxylamine colorimetric method was used. Or ultraviolet spectrophotometry) to determine the amount of unencapsulated drug in the supernatant, calculate the nano drug encapsulation rate and drug loading rate by the difference between the total drug amount and the unencapsulated drug amount in the supernatant, and the formula is as follows:

Encapsulation efficiency = (m -m _{total dose supernatant dose)} / m _{total dose} × 100%;

Drug loading rate = (m _{total dose -} m _{supernatant amount} ) / m _product × 100%.

The amount of unencapsulated drug in the supernatant was measured for a certain period of time. After 24 hours, the amount of unencapsulated drug in the supernatant reached a stable level. The encapsulation efficiency of the HSA nanoparticles loaded with Ach was calculated to be 46.14±1.13%, and the drug loading rate was 21.95±1.26%.

The HSA drug-loaded nanoparticles were observed using a scanning electron microscope, and the results are shown in Fig. 3. The unmodified nanoparticles are spherical and have a diameter of about 200 nm.

Step (2): surface modification of the HSA-loaded nanoparticles: the nanoparticles prepared in the step (1) are dispersed in 1 mL of physiological saline, and 0.1 mL of a Tween-80 physiological saline solution is added, and the mixture is stirred at 800 rpm for 30 minutes. After 15 min of sonication, HSA nanoparticles (HSA-ACh NPs) modified with Tween-80 and loaded with Ach were finally obtained.

The modified HSA drug-loaded nanoparticles were observed using a scanning electron microscope, and the results are shown in Figs. 4 and 5. The modified drug-loaded nanoparticles are regular cubes with a side length of about 200 nm.

The HSA drug-loaded nanoparticles modified with Tween-80 were measured using a dynamic light scattering particle size analyzer (with zeta potential test function), the average particle size was about 198.5 nm, the particle size was uniform, and the zeta potential was about -28.4. mV. The test results are shown in Table 1.

Table 1

Example 2: Preparation of Nanoparticles loaded with Lissamine (RT)

In this example, human serum albumin (HSA) nanoparticles were used as a drug carrier, and nanoparticles loaded with Lissamine (RT) were prepared. The active ingredient used is Lismin heavy tartaric acid, the preferred solvent is 10mmol/L sodium chloride solution, the poor solvent selected is anhydrous ethanol (purity greater than 99.7%), and the selected cross-linking agent is 10°. % glutaraldehyde, the selected targeting molecule is Tween-80. The preparation process is as follows.

(1) Preparation of HSA drug-loaded nanoparticles by 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 with 0.1 mol/L sodium hydroxide solution. After filtering through a 0.22 μm water-based filter, the solution was dropwise added to 10 mL of absolute ethanol at a flow rate of 50 μL/min at a rotation speed of 600 rpm. After the completion of the dropwise addition, the stirring speed was increased to 800 rpm, and 10% by weight of glutaraldehyde was added. 20 μL, protected from light for 24 h. The reaction sample was centrifuged at 13600 rpm for 10 min, the supernatant was removed, the precipitate was collected, and the sample was centrifuged three times with an equal amount of physiological saline, and the precipitate was collected to obtain spherical HSA-loaded nanoparticles, which were stored at 4 °C.

(2) Surface modification of HSA-loaded nanoparticles: Disperse the nanoparticles prepared in step (1) in 1 mL of physiological saline, add 1 mL of 0.1% Tween-80 physiological saline solution, stir at 800 rpm for 30 min, and sonicate. At 15 min, finally, RT-loaded HSA nanoparticles (HSA-RT NPs) modified with Tween-80 were obtained, which were tetragonal.

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

Example 3: Structural characterization of nanoparticles

(1) Ultraviolet absorption spectrum analysis

The AHS aqueous solution, the HSA aqueous solution, the blank HSA nanoparticles not loaded with the active ingredient and not modified Tween-80 (preparation process refers to step (1) of Example 1), and the preparation of step (1) of Example 1 The HSA-ACh nanoparticles modified with Tween-80 were analyzed by UV absorption spectroscopy. The UV absorption curve of the sample is shown in Fig. 6. The HSA solution has a distinct characteristic absorption peak around 280 nm (curve 1), while the Ach solution has no absorption near 280 nm (curve 2). After HSA formed blank nanoparticles, the absorption peak near 280 nm was still visible (curve 3); while the AHS-loaded HSA nanoparticles showed a significant decrease in the absorption peak near 280 nm (curve 4). Therefore, when the HSA nanoparticles are doped with drug molecules (ACh) and interact with the drug molecules, the HSA's own UV absorption peak will decrease or disappear. The results show that the drug molecules are successfully doped in the nanocarriers.

(2) XRD analysis

The following samples were subjected to XRD test: 1. Blank HSA nanoparticles without active ingredient and without modification of Tween-80 (preparation process refers to step (1) of Example 1), 2. Preparation of step (1) of Example 1. Unmodified Tween-80 HSA-Ach nanoparticles, 3. Tween-80 modified unloaded active HSA nanoparticles (process reference to step (1) and step (2) of Example 1), 4. Tween-80 modified HSA-Ach nanoparticles prepared in Example 1. The XRD spectrum of each sample is shown in Fig. 7.

In the figure, HSA nanoparticles modified by Tween-80 showed sharp peaks at 2θ = 31.6° (curves 3 and 4), while unmodified nanoparticles had no peaks here (curves 1 and 2). The results demonstrate that the targeting molecule Tween-80 has been successfully modified on the surface of nanoparticles.

Example 4: Preparation of human serum albumin nanoparticles loaded with doxorubicin and evaluation of in vitro release

In this embodiment, ultraviolet spectroscopy is used to quantitatively determine the release amount of nanoparticles. Since acetylcholine has no ultraviolet absorption peak, and Lismin has a distinct ultraviolet absorption peak at 263 nm (the characteristic absorption peak of HSA itself is around 280 nm, which coincides with each other), accurate quantitative analysis cannot be performed by ultraviolet spectroscopy. Therefore, a simulated alternative drug is needed to characterize the release effect of the nanoparticles.

The analog replacement drug selected in this embodiment is doxorubicin. On the one hand, doxorubicin has similar solubility to acetylcholine and lysine, and is a small molecule drug with good water solubility. On the one hand, it is due to the fact that doxorubicin has a suitable UV absorption peak. According to Appendix IV of the 2010 edition of the Pharmacopoeia, doxorubicin has large absorption peaks at wavelengths of 233 nm, 252 nm, 288 nm, 480 nm, 495 nm and 530 nm, with the absorption peak intensity at 480 nm being the largest and the impurity interference being small. Therefore, in this example, the human serum albumin nanoparticles loaded with doxorubicin were used as an in vitro release model, and the in vitro release effect of the nanoparticles of the present application was evaluated by ultraviolet spectrophotometry with the absorption value at 480 nm as a quantitative index. .

The preparation of the nanoparticles was carried out by replacing the target drug with doxorubicin hydrochloride (Dox), and the synthesis procedure was the same as in Example 1.

Test procedure: First, a series of concentration gradients of doxorubicin standard solution was prepared by gradient dilution method, and the UV absorption value was measured at 480 nm to prepare a doxorubicin standard curve, as shown in Fig. 8 (y=0.020x- 0.000, R ² =0.998).

A doxorubicin solution group and a drug-loaded nanoparticle group loaded with doxorubicin were set. According to the nanoparticle loading rate of 10%, the concentration of doxorubicin solution group was 0.5mg/mL (4mL total, equivalent to 2mg of doxorubicin), and the concentration of drug-loaded nanoparticles was 5mg/mL (4mL total, Equivalent to doxorubicin 2mg). The two groups were fixed in a semi-permeable membrane with a molecular weight of 3000, and placed in a 900 mL physiological saline release system, respectively, and continuously released at 200 rpm. Take 2 mL of the release solution at regular intervals, measure the UV absorption at 480 nm, and replenish the same amount of normal saline.

Figure 9 shows the release results. The doxorubicin solution group reached the maximum cumulative release rate in about 10h, and the recovery rate was over 90%, which proved that doxorubicin was completely released. The cumulative release rate of doxorubicin-loaded nanoparticles was stable within 80h. It continues to rise, 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 body fluid transportation, ensuring that the nanoparticles entering the central center can still maintain a high drug loading rate.

Example 5: Behavioral Evaluation of Nanoparticles on D-galactose-induced Cognitive Impaired Mice

5.1 Experimental grouping and breeding

按体重随机分为6组，每组各10只动物，包括空白对照组(不建模不给药)、模型组(只建模不给药)、ACh水溶液对照组(建模后给予乙酰胆碱水溶液)、HSA-ACh纳米粒组(建模后给予吐温-80修饰的装载乙酰胆碱的人血清白蛋白纳米粒)、RT溶液组对照组(建模后给予利斯的明水溶液)、HSA-RT纳米粒组(建模后给予吐温-80修饰的装载利斯的明的人血清白蛋白纳米粒)。 Kunming mice,

They were randomly divided into 6 groups according to their body weight, including 10 animals in each group, including blank control group (not modeled and not administered), model group (modeled only), ACh aqueous solution control group (administered acetylcholine aqueous solution after modeling) ), HSA-ACh nanoparticle group (Tween-80 modified human serum albumin nanoparticles loaded with acetylcholine after modeling), RT solution group control group (applied to Lisz's aqueous solution after modeling), HSA-RT Nanoparticle group (modeled after Tween-80 modified Lismine's human serum albumin nanoparticles).

The feeding environment was: temperature (24 ± 2) ° C; humidity (60 ± 10)%; daily light / dark for 12 h; free feeding and drinking.

5.2 Animal Modeling and Drug Delivery Therapy

1 Animal model: In addition to the blank control group, the other 5 groups were injected subcutaneously with D-galactose solution 100 mg/kg/d, and the aluminum chloride solution was administered with 20 mg/kg/d; the normal control group was given the same amount of water. The modeling cycle is 90 days.

2 drug treatment protocol: 60 days after modeling, ACh solution group was given acetylcholine aqueous solution (1mg/kg), HSA-ACh nanoparticle group was given Tween-80 modified acetylcholine-loaded nanoparticles (10mg/kg, loaded ACh content) Equivalent to 1 mg/kg), the RT solution group was given Liss's clear aqueous solution (1.25 mg/kg), and the HSA-RT nanoparticle group was given Tween-80 modified Lissian Ming nanoparticles (12.5 mg/kg, loaded The content of RT is equivalent to 1.25 mg/kg); the dosing period is 2 times a week for 4 weeks.

5.3 Morris water maze behavior evaluation method

The 1Morris water maze (MWM) test period is 5 days, the first 4 days are the training period, and the 5th day is the test period;

2 The diameter of the swimming pool is 1m, divided into 4 quadrants, and 4 water inlet points are fixed in 4 quadrants respectively; the swimming pool water depth is about 0.6m, and the water temperature is 22±2°C;

During the training period, the black platform with a diameter of 0.1 m was placed in the third quadrant and was about 2 cm below the water surface; each mouse received four trainings per day, as follows: First, the mice were facing the pool wall. Put it into the first quadrant and fix it into the water point and swim for 1 minute. If within 1 min, the mouse finds the platform position and stays for at least 3 s, the time stops immediately, and the data such as latency and swimming speed are recorded; if within 1 min, the mouse fails to find the platform or finds the platform and fails to stay for 3 s, the artificial assisted mouse finds The platform stayed for 15 s. At this time, the recorded latency was 60 s. The mice were dried immediately after a swim, and after all the mice were swimming, the training was performed in the 2, 3, and 4 quadrants.

During the test period, the platform was removed and fixed into the water point. The mice were free to swim for 1 min, and the data of the incubation period, the number of passages and the swimming time in the target quadrant were recorded for the first time.

Experimental results:

The platform time for each group of mice was as shown in FIG. During the training period, the mice in each group found no significant difference in platform time on the first day; on the third and fourth days, the blank control group found the platform time was 20s, and the model group, ACh solution group and RT solution group were found. The platform time was significantly longer than that of the blank control group, and the platform time of the HSA-ACh nanoparticle group and the HSA-RT nanoparticle group was not significantly different from that of the blank control group, and was significantly lower than that of the model group. Experiments have shown that the short-term spatial learning ability of mice is improved after HSA-ACh nanoparticles and HSA-RT nanoparticles are separately administered.

During the test period, the time for the mouse to find the original platform position was further shortened, but there was a statistically significant difference between the groups. As shown in Figure 11, the blank control group had the shortest incubation period, and the variance analysis showed that the model group, The latency of ACh solution group and RT solution group was significantly longer than that of the blank control group (P<0.05), but there was no significant difference between HSA-ACh nanoparticle group and HSA-RT nanoparticle group and blank control group.

The number of times of wearing and the target quadrant swimming time are two other indicators for judging the long-term spatial memory ability of mice. The number of times of wearing is the number of times the experimental animal crosses the original platform within 60s. The more times the animal wears, the better the spatial learning and memory ability. As shown in Figure 12, the number of mice in the blank control group was significantly higher than that in the model group, ACh solution group and RT solution group (P<0.01), but not in the HSA-ACh nanoparticle group and HSA-RT nanoparticle group. Statistical differences.

The target quadrant swimming time is the sum of the time of the experimental animals in the original platform and the surrounding area. The longer the time, the better the spatial learning and memory ability, as shown in Figure 13, the model group and the ACh solution group have compared with the blank control group. There was significant statistical difference (P<0.05), while there was no statistically significant difference between the other groups and the blank control group.

The above MWM experimental data showed that the learning and memory ability of the model group mice was significantly lower than that of the normal group mice, and the D-galactose-induced cognitive impairment mouse model was successfully modeled; after administration of acetylcholine aqueous solution or Liss's clear aqueous solution, cognitive impairment No improvement was achieved; while HSA-ACh nanoparticles or HSA-RT nanoparticles were given, the short-term learning ability and long-term spatial memory ability of the mice were significantly improved, demonstrating that HSA-ACh nanoparticles and HSA-RT nanoparticles achieved remission. The purpose of cognitive impairment and long-term release.

Example 6: Effect of nanoparticles on brain biochemical parameters in mice with cognitive impairment induced by D-galactose

6.1 Sample preparation

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

2 The eyeball was taken for blood collection, collected in 5 mL of heparin sodium anticoagulated blood collection tube, and stored in an ice bath;

3 Decapitate the brain, remove the brain stem, accurately weigh the brain and record; cut the left and right hemispheres on the ice box, the left hemisphere is immersed in 4% neutral formaldehyde solution; the right hemisphere is crushed by filter paper Blood and microvessels were added to a 20-fold weight cholinesterase extract homogenate; the cholinesterase extract was centrifuged at 5000 rpm for 10 min at 4 ° C, and the supernatant was collected and stored at 4 ° C.

6.2 Determination of brain acetylcholinesterase (AChE) activity

1 accurately absorb 10 μL of brain supernatant diluted in 0.5mL PBS, repeatedly rinse the tip and vortex to mix;

2 plates, 4 replicate wells per sample, 20 μL sample per well, 80 μL PBS in the first two wells, 50 μL PBS and 30 μL thioacetylcholine (ATCH) in the latter two wells;

3 centrifuge for 1 min to remove air bubbles, and incubate in a constant temperature water bath at 37 ° C for 30 min;

4 After taking out, 20 μL of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) was added to each well, and the absorption value was measured at 412 nm after shaking for 30 s.

6.3 Determination of brain acetylcholine transferase (ChAT) activity

1ChAT was measured using a kit (Nanjing built, article number: A079-1), adding reagent 1 to reagent 6 in sequence, mixing, pre-warming for 5 min at 37 ° C water bath;

2 Add 25 μl of tissue homogenate supernatant to the measuring tube, add the same amount of boiled homogenate supernatant to the corresponding control tube, mix well, water bath at 37 ° C for 20 min, and terminate the reaction at 100 ° C boiling water bath for 2 min;

3 Add 425 μl of distilled water to each reaction system, mix well, centrifuge at 4000 rpm for 10 min, then take 500 μL of supernatant, add 10 μL of reagent 7 color developer, mix and let stand for 15 min, at 324 nm, 1 cm optical path, 2 mm inner diameter quartz The cuvette and the distilled water were zeroed, and the absorbance values of the respective tubes were measured.

6.4 Determination of acetylcholine (ACh) content

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

2 Add the sample supernatant and ACh standard solution to the measuring tube and the standard tube respectively, add the basic hydroxylamine solution and then heat the water bath at 37 °C for 30 min, add hydrochloric acid to terminate the reaction, and finally add ferric chloride; add the control tube and the blank tube respectively. The sample supernatant and water, 37 ° C constant temperature water bath for 30 min, adding hydrochloric acid and ferric chloride, and finally adding basic hydroxylamine;

3 plates were added, 200 μL was added to each well, and the absorbance value was measured at 540 nm.

6.5 Determination of malondialdehyde (MDA) content

The determination of 1MDA was carried out by kit (Nanjing completed, item No. A003-1), 0.1 mL homogenate supernatant was added to the measuring tube, the same amount of absolute ethanol was added to the blank tube, and the standard tube was added with the equivalent amount of 10 nmol/mL standard, and then added. 0.1mL reagent 1, 3mL reagent 2 application solution, 1mL reagent 3 application solution, vortex and mix;

2 test tube mouth with plastic wrap, puncture a small hole with a needle, 95 ° C water bath for 40min, remove water, cool, centrifuge at 4000rpm for 10min, take the supernatant at 532nm, 1cm light path, double distilled water zero, determine the absorbance of each tube value.

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

1T-SOD was measured by kit (Nanjing built, item number A001-1), reagent 1 to reagent 4 and sample were added in sequence, vortexed and mixed, placed in a constant temperature water bath at 37 ° C for 40 min;

2 Add the color developer, mix, place at room temperature for 10 min, at a wavelength of 550 nm, 1 cm optical diameter than the color cup, double distilled water to zero, determine the absorbance value of each tube.

6.7 Brain histopathology test

4% neutral formaldehyde solution fixed left hemisphere after washing, dehydration, waxing, embedding, sectioning and sticking, dewaxing, hematoxylin-eosin staining, dehydration, transparency, sealing and other steps, finally obtain the nucleus Blue stained, cytoplasmic red stained brain tissue sections.

Experimental results:

The cholinergic system-related enzyme activity and brain oxidative stress level are important brain biochemical indicators for evaluating cognitive ability. The measurement results of each index are shown in Table 2.

Table 2

^* p < 0.05, ^** p < 0.01, ^*** p < 0.001, compared with the blank control group. ^{# P <0.05, ## p <} 0.01, comparing ^### p <0.001, with model group; n = 8.

As can be seen from Table 2, in normal brain tissue, the activity and content of ChAT, ACh and AChE were maintained at a relatively stable level; in the model group, ChAT activity was significantly increased (p<0.05), while the HSA-ACh nanoparticle group, The HSA-RT nanoparticle group and the RT solution group were still maintained at normal levels, demonstrating that the nanoparticles of the present invention effectively maintain the relative balance of the activity and content of ChAT, ACh and AChE in the brain cholinergic system. From the results of oxidative stress test, compared with the blank control group, the MDA content in the model group and the ACh solution group was significantly increased (p<0.001), while the HSA-ACh nanoparticle group, the HSA-RT nanoparticle group and the RT solution group. It is still maintained at a normal level, demonstrating that the nanoparticles of the present invention are effective in reducing the level of oxidative stress in the brain and protecting neurons.

Figure 14 shows the results of brain histopathological examination. The normal hippocampal CA2 area and the frontotemporal cortex neurons are round or elliptical, with a large nucleoplasmic ratio, a clear nuclear or nuclear membrane, and a uniform color of light blue-violet (Fig. 14A, Fig. 14B). The hippocampal neurons in the model group and the ACh solution group were loosely arranged. The hippocampus and cortex showed more neuronal cell shrinkage, cytoplasm condensation, nuclear condensation, and the whole cell was deeply stained purple (Fig. 14C, Fig. 14D, Fig. 14E, Fig. 14F); after the administration of HSA-ACh nanoparticles, HSA-RT nanoparticles or RT solution, the above phenomenon was significantly improved, and the nuclear pyknosis of the frontotemporal cortex neurons was significantly reduced (Fig. 14G, Fig. 14H, Fig. 14I). Figure 14J, Figure 14K, Figure 14L). Experiments have shown that the nanoparticle of the present invention has a significant protective effect on neuronal cells.

Example 7: Central targeting of nanoparticles and penetration of blood-brain barrier

The fluorescein isothiocyanate (FITC) was modified onto human serum albumin, and the Tween-80 modified acetylcholine-loaded nanoparticles were prepared according to the methods of Examples 1 and 2, and the Tween-80 modified Liss was loaded. The nanoparticles of the bright, thereby obtaining nanoparticles with FITC. After diluting each group of FITC-containing nanoparticles to 0.1 mg/mL, 10 nL of the heart was injected into the zebrafish, and the successfully injected zebrafish was selected and fixed in the gel, and the zebrafish was observed by laser confocal microscopy. The distribution of fluorescence.

15A-15C are laser confocal micrographs of a heart-injected saline group (control group), an acetylcholine nanoparticle group, and a lismin nanoparticle group, respectively. FITC is a water-soluble fluorescent dye that is firmly bonded to the nanoparticles by chemical bonds. Because of chemical bonding, the fluorescein cannot be freely detached from the nanoparticles into free small molecules into the system, so under the microscope The observed fluorescence was derived from nanoparticles, and there were no free FITC small molecules in the system. It can be clearly observed from the figure that there is no fluorescence distribution in normal zebrafish (Fig. 15A); for the acetylcholine nanoparticle group and the Lissian nanoparticle group, although the heart is injected, there is obvious in the brain of the zebrafish. Green fluorescence, thus demonstrating that the drug can smoothly enter the center through the blood-brain barrier.

The above examples comprehensively evaluated the effect of the nanoparticles of the present application on the cognitive ability of mice, and observed the changes of behavior of the mice at the overall level, and the determination of brain biochemical indicators and pathological examination. Based on the above experimental results, it can be concluded that the nanoparticles of the present application can slowly release the acetylcholine which could not enter the central nervous system and then release it to achieve the purpose of long-term low-dose supplementation of exogenous neurotransmitters; Lissamine is slowly released after being transported to the center, achieving the purpose of long-acting administration.

While the invention has been described in detail, the embodiments of the invention . The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims (9)

A nanoparticle comprising a carrier material, an active ingredient and a targeting molecule, the carrier material being human serum albumin, the active ingredient being an active ingredient capable of preventing and/or treating a neurodegenerative disease; Preferably, the active ingredient is loaded in a carrier material to form human serum albumin-loaded nanoparticles; Preferably, the human serum albumin drug-loaded nanoparticles are formed by a method 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); Step (1-2): adding the solution (1) to a poor solvent, and adding a crosslinking agent to carry out a reaction to obtain human serum albumin-loaded nanoparticles; Preferably, the nanoparticle has a drug loading rate of 15% to 25%. The nanoparticle of claim 1 wherein said active ingredient is selected from the group consisting of a cholinergic neurotransmitter supplement and a cholinesterase inhibitor; Preferably, the cholinergic transmitter supplement is selected from the group consisting of acetylcholine, a prodrug of acetylcholine or a pharmaceutically acceptable salt of acetylcholine (eg hydrochloride); Preferably, the cholinergic transmitter supplement is acetylcholine or acetylcholine chloride; Preferably, the cholinesterase inhibitor is selected from the group consisting of lismin, galantamine or donepezil, or a pharmaceutically acceptable salt of such a compound (eg tartrate); Preferably, the cholinesterase inhibitor is Lismin heavy tartrate. The nanoparticle according to claim 1 or 2, wherein the targeting molecule is selected from the group consisting of a surfactant, a polyethylene glycol polymer, and a blood brain barrier specific antibody; Preferably, the targeting molecule is a surfactant; Preferably, the targeting molecule is Tween-80; Preferably, the targeting molecule is modified on the surface of the nanoparticle; Preferably, the targeting molecule is modified on the surface of the human serum albumin-loaded nanoparticles; Preferably, the targeting molecule is modified on the surface of the nanoparticles by adsorption. The nanoparticle according to any one of claims 1 to 3, wherein the nanoparticle has a diameter of 150 nm to 250 nm; Preferably, the nanoparticle has a zeta potential of -30 mV to +20 mV; Preferably, the nanoparticle has an encapsulation ratio of 40% to 50%. A method of preparing the nanoparticles of any of claims 1-4, the method comprising the steps of: Step (1): preparing human serum albumin-loaded nanoparticles; Step (2): modifying the targeting molecule on the surface of the human serum albumin-loaded nanoparticles. The method of claim 5, wherein in the step (1), the method for preparing human serum albumin-loaded nanoparticles is an anti-solvent method; Preferably, the step (1) comprises the following steps: Step (1-1): dissolving or dispersing human serum albumin and active ingredient in a good solvent to form a solution (1); Step (1-2): adding the solution (1) to a poor solvent, and adding a crosslinking agent to carry out a reaction to obtain human serum albumin-loaded nanoparticles; Preferably, the step (1) further comprises the step (1-3) of separating the human serum albumin-loaded nanoparticles. The method of claim 5 or 6, wherein said step (2) comprises the steps of: Step (2-1): dispersing human serum albumin-loaded nanoparticles in physiological saline to obtain a solution (2); Step (2-2): Mixing the solution (2) with a targeting molecule or a solution thereof. A pharmaceutical composition comprising the nanoparticles of any of claims 1-4; Optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable adjuvant (eg, a carrier and/or an excipient). Use of the nanoparticle of any one of claims 1 to 4 or the pharmaceutical composition of claim 8 for the preparation of a medicament for preventing and/or treating a neurodegenerative disease in a subject; Preferably, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and multiple sclerosis of the brain; Preferably, the subject is a mammal, such as a bovine, equine, ovine, porcine, canine, feline, rodent, primate; for example, said The subject is a human.

Images