阅读说明：本技术 普鲁士蓝纳米颗粒在制备预防、延缓或治疗神经系统退行性疾病药物中的应用 (Application of prussian blue nano-particles in preparation of medicine for preventing, delaying or treating nervous system degenerative diseases ) 是由 常津 窦妍 赵冬菊 唐雨晴 于 2020-06-09 设计创作，主要内容包括：本发明涉及普鲁士蓝纳米颗粒在制备预防、延缓或治疗神经系统退行性疾病药物中的应用。本发明研究发现普鲁士蓝纳米颗粒在预防、延缓或治疗神经系统退行性疾病方面均具有显著的功效。细胞水平试验结果表明,普鲁士蓝纳米颗粒能够降低过氧化氢刺激的神经细胞内的ROS水平,提高过氧化氢刺激的神经细胞中的活细胞比例；动物水平试验结果表明,普鲁士蓝纳米颗粒能够显著降低神经系统退行性疾病模型小鼠海马中氧化应激标志物的表达水平,显著提高神经系统退行性疾病模型小鼠的学习和记忆能力、改善运功功能障碍。且普鲁士蓝纳米颗粒的制备过程简单,易宏量生产,反应条件温和,易表面修饰。(The invention relates to application of prussian blue nano-particles in preparation of a medicine for preventing, delaying or treating nervous system degenerative diseases. The research of the invention finds that the prussian blue nano-particles have obvious effects on preventing, delaying or treating the degenerative diseases of the nervous system. Cell level test results show that the prussian blue nanoparticles can reduce the ROS level in nerve cells stimulated by hydrogen peroxide and improve the proportion of living cells in the nerve cells stimulated by hydrogen peroxide; animal level test results show that the prussian blue nanoparticles can obviously reduce the expression level of an oxidative stress marker in hippocampus of a nervous system degenerative disease model mouse, obviously improve the learning and memory capacity of the nervous system degenerative disease model mouse, and improve dysfunction of motor function. And the preparation process of the prussian blue nano-particles is simple, mass production is easy, the reaction condition is mild, and surface modification is easy.)

1. The prussian blue nano-particles are applied to the preparation of medicines for preventing, delaying or treating nervous system degenerative diseases.

2. The use of claim 1, wherein the neurodegenerative disease comprises alzheimer's disease, parkinson's disease, amyotrophic lateral sclerosis, huntington's disease, spinocerebellar ataxia, spinal muscular atrophy, multiple sclerosis, or epilepsy.

3. The use of claim 1 or 2, wherein the prussian blue nanoparticles are functionally modified or non-functionally modified prussian blue nanoparticles;

preferably, the prussian blue nanoparticles are prussian blue nanoparticles modified by a functional molecule that crosses the blood brain barrier and/or a specifically targeted a β deposition molecule.

4. The use of claim 3, wherein the functional molecule that crosses the blood-brain barrier comprises any one of transferrin, lactoferrin, Apo E, Angiopep-2, RVG29, or a TAT peptide, or a combination of at least two thereof;

preferably, the specifically targeted a β deposition molecule comprises any one of congo red, thioflavin S or anti-a β antibodies or a combination of at least two thereof.

5. The use according to any one of claims 1 to 4, wherein the prussian blue nanoparticles have a particle size of from 80 to 200 nm.

6. The use of any one of claims 1 to 5, wherein the Prussian blue nanoparticles are Prussian blue nanoparticles supported on a pharmaceutically acceptable carrier;

preferably, the prussian blue nanoparticles are prussian blue nanoparticles contained in a pharmaceutical composition.

7. The use of any one of claims 1 to 6, wherein the medicament is in the form of a tablet, powder, suspension, granule, capsule, injection, spray, solution, enema, emulsion, film, suppository, patch, nasal drop or drop pill.

8. The use of any one of claims 1 to 7, wherein the route of administration of the medicament comprises intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration or transdermal administration.

9. Use of prussian blue nanoparticles for the preparation of an inhibitor of the expression of an oxidative stress marker, wherein the oxidative stress marker comprises 4-hydroxynonenal, malondialdehyde, or 8-hydroxyguanosine.

10. A medicament for preventing, delaying or treating a nervous system degenerative disease, which comprises prussian blue nanoparticles.

Technical Field

The invention belongs to the technical field of biological medicines, relates to a new application of Prussian blue nano-particles, and particularly relates to an application of Prussian blue nano-particles in preparation of a medicine for preventing, delaying or treating a nervous system degenerative disease.

Background

Neurodegenerative diseases are a group of irreversible neurological diseases caused by the loss of neuronal cells in the brain and spinal cord, and mainly include alzheimer's disease, parkinson's disease, amyotrophic lateral sclerosis, huntington's disease, etc. With the aging of society, the incidence of degenerative diseases of the nervous system is increasing, which causes huge social medical treatment consumption and family pressure. Most degenerative diseases of the nervous system still lack effective treatment methods, so finding effective methods for preventing, delaying and treating the diseases is a problem to be solved urgently.

Oxidative stress plays a key role in the development of degenerative diseases of the nervous system. Oxidative stress refers to imbalance between oxidation and antioxidation in vivo, and accumulation of Reactive Oxygen Species (ROS) and reactive nitrogen radicals in large quantities causes molecular oxidation and tissue damage, which ultimately leads to disease occurrence. Compared with other tissues, the brain tissue is more easily attacked by ROS, the function of an antioxidant system in the brain, including superoxide dismutase, catalase, peroxidase and the like, is weakened by the aging factors, the ROS generation is obviously increased and cannot be effectively eliminated, and the oxidative stress is aggravated. Studies have shown that excessive ROS disrupt intracellular calcium ion balance, causing synaptic damage by modulating the release of neurotransmitters at the presynaptic terminal and postsynaptic neuronal responses, affecting neuronal signaling in the brain. Meanwhile, oxidative stress can mediate apoptosis of neuron cells by regulating expression of apoptosis-related proteins such as Bcl-2 and the like. In addition, oxidative stress can activate glial cells, further leading to the release of pro-inflammatory factors, thereby causing damage and loss of neurons through neuroinflammation. Therefore, oxidative stress plays an important role in the development of neurodegenerative diseases such as alzheimer's disease, parkinson's disease, amyotrophic lateral sclerosis, huntington's disease, and the like.

The Prussian blue nano-particles are simple in preparation process, mild in reaction conditions and easy to modify on the surface, and have abundant oxidation-reduction potentials and unique electron spin characteristics, so that the application of the Prussian blue nano-particles in the field of biomedicine becomes a research hotspot in recent years. For example, CN105288665A discloses a prussian blue nanoparticle contrast agent, which comprises an inner core of prussian blue nanoparticles and a polyethylene glycol shell layer coated on the surface of the prussian blue nanoparticles, and the prussian blue nanoparticle contrast agent has good water solubility and biocompatibility, and is beneficial to application in organisms. For example, CN105477648A discloses a lymph-targeting prussian blue-like nanoparticle and a preparation method thereof, in which hyaluronic acid is crosslinked on diethylenetriaminepentaacetic acid and chelated on gadolinium ions to form a stable prussian blue-like nanoparticle with hyaluronic acid on the surface, and the inner core prussian blue-like nanoparticle is a position using gadolinium to replace ferric iron, has more unpaired electrons, and has stronger magnetic resonance signal; the surface coating is hyaluronic acid which is one of the components of human tissues, and the biocompatibility is very good.

However, no relevant report exists on the research of applying the prussian blue nanoparticles to the prevention, delay or treatment of the nervous system degenerative diseases.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a new application of prussian blue nanoparticles, and particularly provides an application of prussian blue nanoparticles in preparation of a medicine for preventing, delaying or treating nervous system degenerative diseases.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the invention provides an application of prussian blue nanoparticles in preparation of a medicament for preventing, delaying or treating a nervous system degenerative disease.

The invention relates to a new application of Prussian blue nano-particles, which comprises three aspects, namely the application of the Prussian blue nano-particles in preparing a medicament for preventing nervous system degenerative diseases; secondly, the application of the Prussian blue nano-particles in preparing the medicine for delaying the degenerative diseases of the nervous system; thirdly, the application of the prussian blue nano-particles in preparing the medicine for treating the nervous system degenerative diseases. The cell level test result shows that the prussian blue nano-particles can reduce the ROS level in the nerve cells stimulated by hydrogen peroxide and improve the proportion of living cells in the nerve cells stimulated by hydrogen peroxide; animal level test results show that the prussian blue nanoparticles can obviously reduce the expression level of hippocampal oxidative stress markers of a nervous system degenerative disease model mouse, obviously reduce the expression level of hippocampal inflammatory related molecules of the nervous system degenerative disease model mouse, and obviously improve the learning and memory abilities of the nervous system degenerative disease model mouse.

The prussian blue nanoparticles related to the present invention can be prepared according to conventional methods disclosed in the prior art by those skilled in the art, and the present invention does not specifically limit the preparation method of prussian blue nanoparticles. Illustratively, the prussian blue nanoparticles can be prepared by a one-step method, and the specific preparation steps comprise the following steps:

(1) respectively dissolving potassium ferrocyanide and carboxylated polyethylene glycol in deionized water, and fully and uniformly mixing to obtain a clear solution A; dissolving ferric chloride in deionized water, and fully dissolving to obtain a clear solution B; dropwise adding the solution B into the solution A, wherein the molar ratio of potassium ferrocyanide to ferric chloride is 1:1, and reacting for 0.5-2h at 40-80 ℃;

(2) and when the reaction system is cooled to 20-30 ℃, reacting for 0.5-2h, and centrifugally washing to obtain the Prussian blue nano-particles without functional modification.

Preferably, the neurodegenerative disease includes alzheimer's disease, parkinson's disease, amyotrophic lateral sclerosis, huntington's disease, spinocerebellar ataxia, spinal muscular atrophy, multiple sclerosis or epilepsy.

The pathogenesis of the nervous system degenerative diseases is related to oxidative stress, namely, the oxidation and the antioxidation in vivo are unbalanced, and a large amount of reactive oxygen Radicals (ROS) and reactive nitrogen radicals are accumulated to cause molecular oxidation and tissue damage; excessive ROS can destroy the calcium ion balance in cells, and synapse damage is caused by regulating the release of neurotransmitters at the presynaptic terminal and the reaction of postsynaptic neurons, so that the signal conduction of neurons in brain is influenced; at the same time, the glial cells are activated, leading to the release of proinflammatory factors, causing the damage and loss of neurons, and finally leading to the occurrence of the above-mentioned diseases.

Preferably, the prussian blue nanoparticles are prussian blue nanoparticles which are functionalized or not functionalized.

The preparation process of the Prussian blue nano-particles is simple, the reaction conditions are mild, the surface modification is easy, and the functional modification can be carried out on the surface of the Prussian blue nano-particles by a person skilled in the art according to the actual application requirement.

Preferably, the prussian blue nanoparticles are prussian blue nanoparticles modified by a functional molecule that crosses the blood brain barrier and/or a specifically targeted a β deposition molecule.

Preferably, the blood brain barrier spanning functional molecule comprises any one of transferrin, lactoferrin, Apo E, Angiopep-2, RVG29 or TAT peptide or a combination of at least two thereof. The combination of at least two of the above-mentioned compounds, such as the combination of transferrin and lactoferrin, the combination of Angiopep-2 and RVG29, the combination of RVG29 and TAT peptide, etc., can be selected in any combination manner, and thus, the description thereof is omitted.

Preferably, the specifically targeted a β deposition molecule comprises any one of congo red, thioflavin S or anti-a β antibodies or a combination of at least two thereof. The combination of at least two of the above-mentioned compounds, such as the combination of congo red and thioflavin S, the combination of thioflavin S and anti-a β antibody, etc., can be selected in any other combination manner, and will not be described in detail herein.

Preferably, the particle size of the prussian blue nanoparticle is 80-200nm, for example, 80nm, 100nm, 120nm, 140nm, 150nm, 160nm, 180nm or 200nm, and other specific values within the above range can be selected, and are not described in detail herein.

Preferably, the prussian blue nanoparticles are prussian blue nanoparticles supported on a pharmaceutically acceptable carrier.

Such as liposomes, micelles, dendrimers, microspheres or microcapsules, etc.

Preferably, the prussian blue nanoparticles are prussian blue nanoparticles contained in a pharmaceutical composition.

The Prussian blue nano-particles related by the invention can also be matched with other bioactive components with the functions of preventing, delaying or treating nervous system degenerative diseases according to different proportions to form a pharmaceutical composition, and the pharmaceutical composition plays a role together.

Preferably, the dosage form of the medicine comprises tablets, powder, suspension, granules, capsules, injections, sprays, solutions, enemas, emulsions, films, suppositories, patches, nasal drops or dropping pills.

The prussian blue nano-particles can be independently administered or can be matched with auxiliary materials to be prepared into a proper dosage form for administration, and the auxiliary materials comprise any one or the combination of at least two of diluent, excipient, filler, adhesive, wetting agent, disintegrating agent, emulsifier, cosolvent, solubilizer, osmotic pressure regulator, surfactant, pH regulator, antioxidant, bacteriostatic agent or buffering agent. Combinations of the at least two such as diluents and excipients, emulsifiers and co-solvents, fillers and binders and wetting agents, and the like.

When the dosage form is a tablet, excipients such as microcrystalline cellulose, starch, or calcium carbonate, and the like; disintegrants may also be included, such as croscarmellose sodium and the like. When the preparation is a capsule, the preparation can be prepared into a hard capsule or a soft capsule, and the Prussian blue nano-particles and the auxiliary materials can be prepared into powder or granules to be filled into the capsule. When the preparation is suspension, flavoring agent, suspending agent, etc. can be added to adjust taste and mouthfeel. When the dosage form is emulsion, emulsifier and cosolvent can be added to adjust solubility and emulsifying degree for administration.

Preferably, the route of administration of the medicament includes intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration or transdermal administration.

Generally, the oral administration is carried out in the form of tablets or capsules, and in addition, when the oral administration is carried out in the form of tablets or capsules, the oral administration can be prepared into controlled release preparations or sustained release preparations, and controlled release auxiliary materials or sustained release auxiliary materials with proper dosage are selected according to the required drug effect and action time.

In a second aspect, the present invention provides use of prussian blue nanoparticles for the preparation of an inhibitor of expression of an oxidative stress marker comprising 4-hydroxynonenal, malondialdehyde, or 8-hydroxyguanosine.

The invention also provides application of the Prussian blue nano-particles in preparation of expression inhibitors of astrocyte markers GFAP, microglia markers Iba-1, inflammatory factors TNF-alpha, inflammatory factors IL-1 beta, apoptosis protein P53 or apoptosis protein Caspase-3.

The invention also provides application of the Prussian blue nano-particles in preparation of an expression promoter of a synaptic injury marker SYN1, a synaptic injury marker PSD95 or an apoptosis protein Bcl-2.

In a third aspect, the invention provides a medicament for preventing, delaying or treating a nervous system degenerative disease, wherein the medicament for preventing, delaying or treating a nervous system degenerative disease comprises prussian blue nanoparticles.

In a fourth aspect, the present invention provides the use of prussian blue nanoparticles for the prevention, delay or treatment of degenerative diseases of the nervous system.

Compared with the prior art, the invention has the following beneficial effects:

the Prussian blue nano-particles have remarkable effects in the aspects of preparing, delaying or treating nervous system degenerative diseases. The cell level test result shows that the prussian blue nano-particles can reduce the ROS level in the nerve cells stimulated by hydrogen peroxide and improve the proportion of living cells in the nerve cells stimulated by hydrogen peroxide; animal level test results show that the prussian blue nanoparticles can obviously reduce the expression level of hippocampal oxidative stress markers of a nervous system degenerative disease model mouse, obviously reduce the expression level of hippocampal inflammatory related molecules of the nervous system degenerative disease model mouse, and obviously improve the learning and memory abilities of the nervous system degenerative disease model mouse. And the preparation process of the prussian blue nano-particles is simple, mass production is easy, the reaction condition is mild, and surface modification is easy.

Drawings

Fig. 1 is a transmission electron micrograph of double-targeted prussian blue nanoparticles;

fig. 2 is a graph of particle size characterization of dual-targeted prussian blue nanoparticles;

fig. 3 is a graph of potential characterization of dual-targeted prussian blue nanoparticles;

FIG. 4 is a graph showing the results of reactive oxygen species measurements of ROS levels in various groups of cells;

FIG. 5 is a graph showing the results of detecting the level of apoptosis using Annexin V-FITC/PI kit;

FIG. 6 is a graph showing the results of detecting the expression levels of pathological feature markers by Western blotting;

FIG. 7 is a graph showing the results of the water maze test.

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

The reagents or starting materials mentioned in the following examples are commercially available or may be prepared according to the common general knowledge of those skilled in the art, unless otherwise specified.

The neural cell strain PC12 cells were donated by the general Hospital of Tianjin medical university, and both APP/PS1 transgenic mice and C57BL/6 mice were purchased from Beijing Huafukang Biotech GmbH.