CN113057963B - Specific capture carrier for eliminating beta-amyloid and application thereof - Google Patents

Specific capture carrier for eliminating beta-amyloid and application thereof Download PDF

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CN113057963B
CN113057963B CN202110345476.9A CN202110345476A CN113057963B CN 113057963 B CN113057963 B CN 113057963B CN 202110345476 A CN202110345476 A CN 202110345476A CN 113057963 B CN113057963 B CN 113057963B
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beta
amyloid
msns
antibody
mesoporous silica
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CN113057963A (en
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骆海明
刘妮
胡顺
曹凯
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Huazhong University of Science and Technology
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/14—Peptides being immobilised on, or in, an inorganic carrier
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Abstract

The invention belongs to the technical field of biology, and particularly relates to a specific capture carrier for removing beta-amyloid and application thereof, wherein the specific capture carrier is specifically a functional nanoparticle, and comprises the following components: mesoporous silica nanoparticles, Ca-MOF, Ce-Mn MOF, mesoporous silica nanoparticles functionally modified by an anti hA beta 1-42 monoclonal antibody and hyaluronic acid capable of passing through a blood brain barrier. The functional nanoparticles can rapidly remove the A beta 1-42 in blood and even brain, can reshape the distribution of the A beta in intestinal tract by changing the metabolic pathway of the antibody, and relieve the disease process of neurodegenerative diseases such as Alzheimer disease, compared with a control group, the APP/PS1 mice injected with the antibody functional nanoparticles intravenously have the advantages of obviously reducing beta-amyloid in peripheral blood and brain, obviously increasing caecum and colon beta-amyloid, and having potential application value in the treatment of neurodegenerative diseases such as Alzheimer disease.

Description

Specific capture carrier for eliminating beta-amyloid and application thereof
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a specific capture carrier for removing beta-amyloid and application thereof.
Background
Amyloid-beta (Α β) deposits are the key pathological basis for Cerebral Amyloid Angiopathy (CAA) and Alzheimer's Disease (AD). Clinical data show that 80-98% of AD patients are associated with cerebrovascular dysfunction and CAA, and that the amount of a β deposited on the walls of the cerebral vessels is positively correlated with the severity and course of the disease. AD is the most common neurodegenerative disease that causes dementia. The deposition of a β in the extracellular space of the cerebral cortex and in the walls of cerebral blood vessels is a hallmark of AD neuropathology. Current studies indicate that defects in excessive a β production or a β clearance play a key or pathogenic role in the pathogenesis of AD. AD includes familial AD and sporadic AD. Familial AD accounts for approximately 1% of total AD patients, and is caused by mutations in the APP, PS1 or PS2 genes, resulting in overproduction of a β, whereas sporadic AD affects more than 99% of AD patients, where impaired clearance of a β from the brain is one of the major factors contributing to their pathogenesis. Therefore, increasing the clearance of a β in the brain and blood vessels is one of the most promising strategies for the prevention and treatment of CAA and AD.
It is currently believed that AD is primarily caused by excessive β -amyloid deposition in the brain due to overproduction or impaired clearance of β -amyloid, resulting in neurotoxicity, resulting in a decrease in the number of synapses and neuronal death. Phase III clinical verification of an anti-A beta immunotherapy AD patient based on an A beta antibody-Aducanumab proves the significance of the amyloid hypothesis, indicates that the anti-A beta immunotherapy eliminates the brain A beta deposition, and improves the AD pathological process, and is the most effective treatment means of clinical research so far. Currently, the a β clearance involves two pathways, central clearance and peripheral clearance, wherein the central clearance severely hampers the availability of the drug due to the presence of the blood-brain barrier, compared to the peripheral clearance pathway, which is more promising. In the peripheral clearance pathway, recent studies indicate that peripheral organ tissues have the ability to clear a β under physiological conditions, with the liver and kidney being important sites for peripheral clearance of a β. Research shows that intravenous injection of m266(A beta monoclonal antibody) on PD-APP transgenic mice can significantly reduce A beta deposition and increase central nervous system and plasma A beta clearance rate to reduce brain A beta burden; secondly, the heterogeneous symbiosis model of Wild Type (WT) and AD model mice finds that the accumulation of A beta plaques in the brains of the AD mice can be obviously reduced. Recent studies have shown that a β also has transmission activity similar to that of prions, and tail vein injection of peripheral-source a β can enter the brain of WT mice and deposit, inducing a β plaque aggregation and expression of proinflammatory factors (TNF-a and IL-6), resulting in neuronal function deficiency. The above studies indicate the importance and feasibility of peripheral clearance of a β, but pure peripheral clearance of a β cannot be achieved with simple antibodies to a β.
Disclosure of Invention
Aiming at the defects and the improvement needs of the prior art, the invention provides a specific capture carrier for clearing beta-amyloid and application thereof, and aims to flexibly, safely and efficiently realize the rapid clearing of the beta-amyloid in a human body.
To achieve the above objects, according to one aspect of the present invention, there is provided a specific capture carrier for clearing amyloid beta, which is a functional nanoparticle that adsorbs amyloid beta physically or performs capture enrichment of amyloid beta in a specific binding reaction; wherein the beta-amyloid is specifically beta-amyloid monomer and beta-amyloid oligomer.
The invention has the beneficial effects that: the functional nanoparticles are used as the specific capture carrier of the beta-amyloid for the first time, the beta-amyloid can be physically adsorbed by using the structural characteristics of the nanoparticles, the beta-amyloid can be captured and enriched by carrying out specific binding reaction on the beta-amyloid through functionalization of the nanoparticles, and the removal mode is selected according to actual needs or actual medical conditions, so that the flexibility of removing the human beta-amyloid can be greatly improved. In addition, the specific capture carrier provided by the invention can be used for intravenous injection by means of a nano drug delivery system, and can relieve the burden of beta-amyloid in the brain by removing beta-amyloid monomers and beta-amyloid oligomers in blood, is non-invasive, has the characteristics of simple and convenient operation, rapidness, no need of special equipment and the like, and is suitable for popularization and application in a basic layer. Therefore, the invention can utilize the characteristics of the body autonomous fluid circulation metabolism, and combines the unique advantages of non-invasiveness of a nano drug delivery system, high drug stability, high drug utilization rate and the like, and can relieve the beta-amyloid burden in the brain by quickly removing the beta-amyloid in peripheral blood, thereby having great promotion effect on the field of medical research.
In a further preferred embodiment of the present invention, the functional nanoparticles are mesoporous silica nanoparticles.
The invention has the further beneficial effects that: the functional nanoparticles are not limited to mesoporous silica, but may be ferroferric oxide-coated mesoporous silica, dendritic mesoporous silica, biosoluble mesoporous silica, biomaterial-modified (such as PLGA, platelet, dopamine, metal framework materials, and the like) mesoporous silica, composite mesoporous silica, silica microspheres with different structures, and nanoparticles with similar biological functional properties, wherein mesoporous silica nanoparticles are approved in biomedicine and are biologically safe, and therefore, mesoporous silica nanoparticles are preferably used in the invention.
As a further preferred aspect of the present invention, the functional nanoparticles are MOF scaffolds.
As a further preference of the invention, the MOF scaffold is Ca-MOF or Ce-Mn MOF.
The invention has the further beneficial effects that: in the biomedical field, the characteristics of the MOFs such as regular pore channel structure, high porosity, biodegradability, adjustable structural composition and function and the like enable the MOFs to have wide application value. Wherein, Ca-MOF can adsorb and enrich pathological proteins, polypeptides and toxic small molecular substances by using the acting force of pi bonds between benzene rings; and the MOF material based on Ce and Mn can simultaneously realize free radical removal and MRI (nuclear magnetic resonance) functional imaging, and realize diagnosis and treatment integration. Therefore, Ca-MOF or Ce-Mn MOF functional nanoparticles are preferably used in the present invention.
In a further preferred embodiment of the present invention, the functional nanoparticles are antibody-functionalized mesoporous silica nanoparticles.
The invention has the further beneficial effects that: first, as described above, mesoporous silica nanoparticles are approved on forest beds and have biosafety, and the present invention improves specificity by performing antibody functional modification on the nanoparticles. Therefore, according to the scheme, on one hand, the antibody functionalized mesoporous silica nanoparticles are prepared by combining the immunotherapy and the nanotechnology, and the advantages of non-invasiveness of a nano drug delivery system and high specificity of the immunotherapy are comprehensively integrated; on the other hand, based on the advantages of specificity, high efficiency, rapidness, safety and the like of the antibody functionalized nanoprobe, the clearance speed of the beta-amyloid in peripheral blood and brain of the patients with the Alzheimer's disease is improved to a certain extent, and a new technical source is provided for clinical rapid clearance of the beta-amyloid and reduction of the toxicity of the beta-amyloid of the Alzheimer's disease. The method has better clearing effect than common immunotherapy.
In a further preferred embodiment of the present invention, the antibody-functionalized mesoporous silica nanoparticle is a mesoporous silica nanoparticle functionalized and modified by an anti-hA β 1-42 monoclonal antibody.
In a further preferred embodiment of the present invention, the antibody-functionalized mesoporous silica nanoparticle is a mesoporous silica nanoparticle that is functionalized by the anti-hA β 1-42 monoclonal antibody and a ligand that can cross the blood brain barrier.
The invention has the further beneficial effects that: the anti hAbeta 1-42 monoclonal antibody is combined with a ligand capable of penetrating through a blood brain barrier to jointly functionalize the nano particles, so that the nano particles can penetrate through the blood brain barrier and enter the brain, and can directly remove beta-amyloid in the brain, the reduction speed of the beta-amyloid content in the brain is greatly accelerated, and a new technical source is provided for clinical rapid removal of the beta-amyloid in Alzheimer's syndrome and reduction of the toxicity of the beta-amyloid.
As a further preferred aspect of the present invention, the ligand is hyaluronic acid, an antibody, a protein or a small molecule polypeptide.
In a further preferred embodiment of the present invention, in the mesoporous silica nanoparticles functionalized and modified by the antibody, if the size of the mesoporous silica nanoparticles is 400-500nm, the metabolic pathway of the antibody is intestinal metabolism.
The invention has the further beneficial effects that: the size of the mesoporous silica nanoparticles is reasonably designed, and the mesoporous silica nanoparticles are functionally modified, so that the metabolic pathway and metabolic time of an antibody in vivo can be changed, specifically, the liver and kidney metabolic pathways of the antibody can be changed into the metabolic pathways mainly through intestinal tracts by the antibody functionally modified mesoporous silica nanoparticles, the removal of blood Abeta is accelerated, the antibody functionally modified mesoporous silica nanoparticles can form a complex with beta-amyloid, and the content of the beta-amyloid in blood is reduced through the intestinal tract metabolism; in addition, the mesoporous silica nanoparticles modified by the antibody function remodel the microenvironment of the intestinal tract by removing beta-amyloid protein monomers and beta-amyloid protein oligomers from the intestinal tract.
The invention also provides application of the specific capture carrier for removing the beta-amyloid in preparation of a medicine for relieving the disease process of the neurodegenerative disease.
Drawings
FIG. 1 is a schematic diagram of the capture of amyloid beta by a specific capture carrier according to an embodiment of the present invention;
FIG. 2 is a graph of the adsorption of different forms of beta-amyloid by MSNs, Ca-MOF and Ce-Mn MOF provided in an example of the present invention; wherein, A picture is MSNs scanning electron microscope picture and adsorption detection picture of different forms of beta-amyloid; b is a Ce-Mn MOF transmission electron microscope image and an adsorption detection image of Ce-Mn MOF/Ca-MOF on A beta 1-42 Ms; the C picture is a Ca-MOF XRD detection picture and an adsorption detection picture of Ce-Mn MOF/Ca-MOF on A beta 1-42 Os;
FIG. 3 is a representation of MSNs-1F12 functional nanoparticles provided by an embodiment of the present invention; wherein, A is a diagram of MSNs-NHS particle size with large particle size and a corresponding scanning electron microscope diagram; b is a particle size diagram of the antibody functionalized nanoparticles MSNs-1F12 and a corresponding scanning electron microscope diagram; panel C is an affinity map of the antibody functionalized nanoparticles MSNs-1F12 prepared by the invention to different forms of beta-amyloid; panel D is a specific detection map of MSNs-1F12 prepared by the invention; e is SDS-PAGE of MSNs-NHS-conjugated 1F12 antibody of the invention;
FIG. 4 is a graph showing the biological distribution of the antibody-functionalized nanoparticles MSNs-1F12 and HA-MSNs-1F12 in mice after intravenous injection; wherein, A is a diagram of the metabolic distribution map and the quantitative analysis diagram of liver and intestinal tract of the antibody functionalized nano particles MSNs-1F12, 1F12 and MSNs in male 7-month-old APP/PS1 mice; b is a distribution graph of brain tissues of APP/PS1 mice after the antibody functionalized nanoparticles MSNs-1F12, 1F12 and HA-MSNs-1F12 are injected intravenously;
FIG. 5 is a graph of the remodeling of beta-amyloid in intestinal tracts by intravenous antibody functionalized nanoparticles MSNs-1F12 and HA-MSNs-1F12 provided by the embodiment of the invention; wherein, A picture is a picture of ELISA detection result of the antibody functionalized nano particle MSNs-1F12 of the invention on the clearance of human beta-amyloid in the peripheral blood of APP/PS1 mice; b is a graph of the result of ELISA detection of the antibody functionalized nanoparticles HA-MSNs-1F12 on the clearance of human beta-amyloid in the peripheral blood of APP/PS1 mice; FIG. C is an ELISA detection graph of beta-amyloid in intestinal tracts of APP/PS1 mice in an antibody functionalized nanoparticle HA-MSNs-1F12 treatment group and a control group of the invention; FIG. D is a graph showing the statistical results of the relative areas of the beta-amyloid in the intestinal tracts of APP/PS1 mice in the treated group and the control group of antibody functionalized nanoparticles HA-MSNs-1F12 according to the present invention;
FIG. 6 is a graph showing the effect of intravenous antibody functionalized nanoparticles HA-MSNs-1F12 on real-time MRI monitoring and clearance of beta-amyloid in APP/PS1 mouse brain; wherein, the A picture is a real-time MRI monitoring picture of the antibody functionalized nano particle HA-MSNs-1F12 on beta-amyloid in the brain of APP/PS1 mice; b is a fluorescence image of brain tissue slices after thioflavin counter-staining in the brain tissues of the antibody functionalized nano particle HA-MSNs-1F12 treatment group and the control group APP/PS1 mice; FIG. C is a graph showing the statistical results of the number of beta-amyloid plaques in the brain tissues of APP/PS1 mice in the antibody functionalized nanoparticle HA-MSNs-1F12 treatment group and the control group of the invention; and D is a graph of statistical results of relative areas of beta-amyloid plaques in brain tissues of the antibody functionalized nanoparticle HA-MSNs-1F12 treated group and the APP/PS1 mouse control group.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
For ease of reference hereinafter, the meanings of the major abbreviations are now explained as follows:
beta-amyloid monomer (Abeta)1–42Monomers): abbreviated as Abeta1–42Ms；
Beta-amyloid oligomers (Abeta)1-42Oligomers): abbreviated as Abeta1–42Os；
Mesoporous Silica Nanoparticles (mesoporus Silica Nanoparticles): abbreviated MSNs;
anti hAbeta1-42Monoclonal antibody functionalized modified mesoporous silica nanoparticles: abbreviated MSNs-1F 12;
hyaluronic acid (hyaluronic acid): abbreviated as HA;
1F12 and hyaluronic acid functionalized mesoporous silica nanoparticles capable of passing through the blood brain barrier: abbreviated as HA-MSNs-1F 12.
The embodiment of the invention provides a specific capture carrier for removing beta-amyloid, wherein the specific capture carrier is a functional nanoparticle which adsorbs the beta-amyloid in a physical mode or captures and enriches the beta-amyloid in a specific binding reaction; among them, the beta-amyloid is specifically a beta-amyloid monomer and a beta-amyloid oligomer. Preferably, the functional nanoparticles can be mesoporous silica nanoparticles, Ca-MOF, Ce-Mn MOF or antibody functionalized mesoporous silica nanoparticles. The mesoporous silica nanoparticles functionally modified by the antibody are mesoporous silica nanoparticles functionally modified by an anti-hAbeta 1-42 monoclonal antibody or mesoporous silica nanoparticles functionally modified by the anti-hAbeta 1-42 monoclonal antibody and a ligand capable of penetrating a blood brain barrier.
In the embodiment, the functional nanoparticles are used as the specific capture carrier of the beta-amyloid, the beta-amyloid can be physically adsorbed by using the structural characteristics of the nanoparticles, the beta-amyloid can be captured and enriched by performing specific binding reaction on the beta-amyloid through functionalization of the nanoparticles, and the removal mode is selected according to actual needs or actual medical conditions, so that the flexibility of removing the human beta-amyloid can be greatly improved. In addition, the specific capture carrier provided by the embodiment can be used for intravenous injection by means of a nano drug delivery system, and can relieve the burden of beta-amyloid in the brain by removing beta-amyloid monomers and beta-amyloid oligomers in blood, is non-invasive, has the characteristics of simple and convenient operation, rapidness, no need of special equipment and the like, and is suitable for basic popularization and application. Therefore, the embodiment can utilize the characteristics of the body autonomous fluid circulation metabolism, and combines the unique advantages of non-invasiveness of a nano drug delivery system, high drug stability, high drug utilization rate and the like, and the beta-amyloid burden in the brain is relieved by quickly removing the beta-amyloid in peripheral blood, thereby having great promotion effect on the field of medical research.
In order to better explain the effects of the embodiments of the present invention, the following examples are given.
Example 1: adsorption of beta-amyloid by MSNs and Ca-MOF/Ce-Mn MOF
1.1 Synthesis of MSNs and Ca-MOF/Ce-Mn MOF
Synthesis of MSNs:
5mL hexadecyl-trimethyl-amine chloride (CTAC 0.274mmol) and 5mL Triethanolamine (Triethaneamine, TEA 51.1mmol) were mixed and heated to 95 deg.C with magnetic stirring for 1h, 0.5mL of Tetraethylorthosilicate (TEOS 2.23mmol) was added dropwise and the temperature was maintained and magnetic stirring was continued for 1 h. Then, MSNs were obtained by centrifugation and 3-time ethanol washing. Stirring the obtained MSNs in 1% hydrochloric acid ethanol solution at 60 ℃ for 3h, repeating for 3 times, and centrifuging to obtain the template-removing agent CTAC-MSNs. The transmission electron micrograph is shown as A in FIG. 2.
Synthesis of Ce-Mn MOF:
103.89g of MnCl are respectively taken2 4H2O and 83.92g CeCl3 7H2Dissolving O in 25mL of absolute ethanol, performing ultrasonic treatment for 10min, adding 189.126g of Trimesic Acid (TA) (dissolved in 15mL of absolute ethanol in advance), stirring the mixed solution at room temperature for 10min, and reacting at 160 ℃ for 10 h. After the reaction, the sample was cooled to room temperature and then dried under vacuum at 50 ℃. Transmission electron microscopeAs shown in diagram B of fig. 2.
Synthesis of curcumin Ca-MOF:
taking 20g of curcumin, dissolving the curcumin in 1000mL of ethanol-acetone (volume ratio of 4:1) mixed solution, and carrying out heat preservation treatment at 40-50 ℃ for 2h for later use. Dissolving 5g of anhydrous calcium chloride in 100mL of ethanol for later use; adding a calcium chloride ethanol solution into a curcumin mixed solution, carrying out heat preservation reaction for 2-3 h, adjusting the pH of the solution to 7-8 with ammonia water, and standing overnight; filtering and separating the curcumin Ca-MOF precipitate, washing with ethanol for 3 times, and drying at 70-80 ℃ to obtain the curcumin Ca-MOF. The XRD detection is shown as C in figure 2.
1.2 adsorption of beta-amyloid by MSNs, Ce-Mn MOF and Ca-MOF
To examine the adsorption of MSNs to beta-amyloid, we chose to use human Abeta1–42Ms、Aβ1–42Os and Abeta1–40Ms is substrate and MSNs are incubated for 1h at room temperature by shaking, and then the substrate is centrifuged at 5000rpm for 5min to remove unadsorbed Abeta1–42Ms、Aβ1–42Os and Abeta1–40Ms, washed 3 times with PBS and ELISA detected. The detection result shows that the MSNs can adsorb and combine different forms of beta-amyloid. The specific detection result is shown in graph A in FIG. 2.
To examine the adsorption of Ce-Mn MOF and Ca-MOF to beta-amyloid, we chose to use human Abeta1– 42Ms、Aβ1–42Incubating Os with Ce-Mn MOF and Ca-MOF at room temperature for 1h, centrifuging at 5000rpm for 5min to remove unadsorbed Abeta1–42Ms and Abeta1–42Os, washed 3 times with PBS and tested by ELISA. The detection result shows that Ce-Mn MOF and Ca-MOF can adsorb A beta1–42Ms and Abeta1–42And (7) Os. The specific detection results are shown in the B and C diagrams in FIG. 2, respectively.
Example 2: MSNS-1F12 nanoparticle probe for clearance of APP/PS1 mouse peripheral blood beta-amyloid
2.1、MSNSPreparation and detection of-1F 12 nanoparticle probe
NHS-modified MSNs: 5.4X 10-6mol NHS (NHS, N-hydroxysuccinimide, click-reacted with amino group)Chemical reaction for biological coupling and labeling) is added into a 3mL CTAB-MSNs sample for reaction for 3 h; centrifuged, washed 3 times with PBS, and dispersed in 200. mu.L DI and stored in the dark.
Conjugation of 1F12 antibody: MSNs-NHS and 1F12 antibodies were mixed at a ratio of 10:3 and 0.1M Na2CO3Adjusting the pH value to be 8.5-9.0, fully mixing the two under the vortex state, and incubating for 3-5 h at 4 ℃; obtaining MSNS-1F12 nanoparticle probe for MSNS-1F12 was dissolved in PBS at a concentration of 1 μ g/μ L for use; dissolving 1F12 in PBS at a concentration of 1 μ g/μ L for use; MSNs-NHS was dissolved in PBS at a concentration of 1. mu.g/. mu.L for use. Mixing MSNs and MSNSDiluting the 1F12 nano particle probe to 0.5 mu g/mu L, then taking 20 mu L of sample copper net, and observing the size of the nano particles by a transmission electron microscope; the particle size was measured by taking 100. mu.L. The results are shown in FIGS. 3A and B.
MSNS-1F12 validation: separately, 20. mu.L of MSN in PBS was addedSmu.L of-1F 12, 1F 1220. mu.L and MSNs-NHS 20. mu.L were mixed with 3.5. mu.L of 6 × loading buffer. Decocting at 95 deg.C for 10 min. Electrophoresis was carried out at a voltage of 110V and a current of 60mA for 80min, followed by staining with Coomassie Brilliant blue for 30min and destaining for 12 h. The results are shown in graph E of FIG. 3. As can be seen from the figure, the 1F12 antibody has been successfully linked to MSNs-NHS.
MSNSDetection of affinity of the 1F12 antibody: in order to detect the affinity of 1F12 in the MSNs-1F12 nanoparticle probe, A beta is selected to be used1–42Os is used as a substrate, and ELISA detection is carried out with 1F12 with different concentrations, and the detection is specifically divided into a simple 1F12 antibody and MSNs-1F 12. The detection result shows that after 1F12 is coupled to MSNs-NHS, the immune affinity is not obviously changed. The specific detection result is shown in the graph C in FIG. 3.
Specific detection of MSNs-1F 12: to test the specificity of the MSNs-1F12 probe, human A.beta.was chosen for this example1–42Ms、Aβ1–42Os and Abeta1–40Ms is substrate and MSNs and MSNS1F12 was incubated at 4 ℃ for 3h, and then unbound 1F12 was separated from the supernatant using a magnetic frame and the probes were washed 3 times with PBS for ELISA detection. The detection result shows that the MSNS1F12 specifically binds to A beta 1-42. Specific test resultsSee fig. 3D.
2.2 metabolic distribution path of antibody functionalized nano-particles MSNs-1F12 in APP/PS1 mice
Synthetic targeting of Abeta1-42A fluorescent probe: with 0.1M Na2CO3Adjusting the pH value of 1F12 to 8.5-9.0. NHS-Cy3(Cy3 belongs to amino reactive dye, is a bright orange fluorescent dye, and can use 532nm laser line) was added under vortex state and mixed for 3h at room temperature. After mixing, the synthesized fluorescent probe is passed through a PD-10 column, and unbound NHS-Cy3 is removed; then mixing the extract with MSNs-NHS (pH 8.5-9.0) with the concentration of 1 mug/muL for 3 hours at room temperature, then centrifugally separating the unbound 1F12-Cy3 in the supernatant at 5000rpm for 5 minutes, and washing the probe for 3 times by using PBS (phosphate buffer solution), thus obtaining the target Abeta1-42Fluorescent probe MSNs-1F12-Cy 3.
MSNs-1F12 metabolic pathway: in order to examine the metabolic pathway of MSNs-1F12 in AD mice, 100. mu.L of MSNs-1F12-Cy3, 1F12-Cy3, MSNs-Cy3 and PBS, which have been dissolved in PBS and had a concentration of 1. mu.g/. mu.L, were administered intravenously to male 7-month-old APP/PS1 mice, heart perfusion was performed 3h and 6h after the injection, hearts, livers, spleens, lungs, kidneys, brains and small intestines were extracted, ground thoroughly, chloroform was used to extract Cy3 from each tissue, and the amount of MSNs-1F12-Cy3 in each tissue was converted from a standard curve of MSNs-1F12-Cy 3. From the results, 1F12 mainly passes through the liver and then is metabolized by the small intestine, MSNs-1F12-Cy3 are directly metabolized by the intestinal tract, and the specific detection result is shown in a graph A in figure 4; HA-MSNs-1F12 could cross the blood brain barrier and enter the brain tissue, and after 6h, it was metabolized mainly through the small intestine in APP/PS1 mice. The specific detection result is shown in B diagram in FIG. 4.
2.3 clearance of APP/PS1 mouse peripheral blood beta-amyloid by MSNs-1F12
In order to detect the effect of MSNs-1F12 on the clearance of beta-amyloid in the peripheral blood of APP/PS1 mice, 100 mu L of MSNs-1F12 which is dissolved in PBS and has the concentration of 1 mu g/mu L is injected into tail veins of male 7-month-old APP/PS1 mice, tail blood collection is carried out before injection, 24h after injection, 3d after injection and 9d after injection respectively, ELISA detection is carried out after the blood collection amount of 50 mu L, and the detection result shows that the MSNs-1F12 rapidly reduces the beta-amyloid in the peripheral blood after 24h of tail vein injection, the beta-amyloid in the subsequent blood is increased again, and the effects of 1F12 and MSNs are not as good as the clearance effect of MSNs-1F 12. The specific detection result is shown in graph A in FIG. 5.
Example 3: HA-MSNs-1F12 nanoparticle probe for eliminating beta-amyloid in peripheral blood and brain of APP/PS1 mouse
3.1 preparation of HA-MSNs-1F12 functionalized nanoparticles
Fe3O4Preparation of @ OA nanoparticles: monodisperse Fe3O4The @ OA nanoparticles were prepared by chemical co-precipitation with 6.56g FeCl in a stream of nitrogen2·4H2O (33.5mmol) and 11.30g FeCl3 6H2O (41.8mmol) was dissolved in 80mL Deionized (DI) water. The solution was heated to 80 ℃ and stirred vigorously for 0.5 h. 45mL of ammonium hydroxide was added slowly and the resulting suspension was stirred vigorously for 5 min. 2mL of OA was added and the mixture was held at 80 ℃ for 25 min. The mixture was allowed to cool to room temperature. The black product was collected using a magnet and washed thoroughly with methanol and DI water to remove excess OA. The obtained oleic acid-stabilized monodisperse magnetite nanoparticles (Fe)3O4@ OA) was dried under vacuum at 50 ℃ for 24 h.
Synthesis of magnetic mesoporous silica nano (CTAB-MSNs): mixing 7.5mg Fe3O4@ OA was dispersed in 0.5mL of chloroform, and added to 5mL of an aqueous solution containing 0.1g of cetyltrimethylammonium bromide (CTAB (0.274 mmol)). After vigorous stirring of the resulting solution, a homogeneous oil-in-water microemulsion is obtained. Heating at 60 ℃ for 10min induces evaporation of chloroform from the solution, thereby producing water-phase dispersed nanoparticles. The resulting aqueous solution was then diluted with 100mL of DI water. The mixture was stirred rapidly at 40 ℃ for 2 h. Then 3mL of ammonium hydroxide (22.7mmol), 0.5mL of TEOS (2.23mmol)) and 5mL of EtOAc (51.1mmol) were added successively to the dilute aqueous solution containing the magnetonanoparticles. The resulting mixture was stirred for 30s and then held at 80rpm for an additional 3h at 40 ℃ to age the product. The precipitate was collected by centrifugation and washed 4 times with ethanol and DI. And (4) freeze-drying the product to obtain CTAB-MSNs.
HA-MSNs-NHS synthesis:take 10.8 x 10-6mol HA dissolved in 500. mu.L DMSO and 10.8X 10 added-6mol 1- (3-Dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (1- (3-methylenepropyl) -3-ethylcarbodiimide hydrochloride, EDC) and 5.4X 10-6Activating mol NHS for 0.5 h; adding the activated HA into a 3mL CTAB-MSNs sample for reaction for 3 h; centrifuged, washed 3 times with water, and dispersed in 200. mu.L DI and stored in the dark.
Synthesis of HA-MSNs-1F 12: mixing HA-MSNs-NHS and 1F12 antibody according to the proportion of 10:3, and incubating for 3-5 h at 4 ℃; obtaining an HA-MSNs-1F12 nanoparticle probe, and dissolving HA-MSNs-1F12 in PBS at the concentration of 1 mug/muL for later use. 1F12 was dissolved in PBS at a concentration of 1. mu.g/. mu.L for use. HA-MSNs-NHS was dissolved in PBS at a concentration of 1. mu.g/. mu.L for use.
3.2 removal of APP/PS1 mouse peripheral blood beta-amyloid by HA-MSNs-1F12
In order to test the clearance effect of HA-MSNs-1F12 on beta-amyloid in peripheral blood of APP/PS1 mice, 100 mu L of HA-MSNs-1F12 which is dissolved in PBS and HAs the concentration of 1 mu g/mu L is injected into tail veins of male APP/PS1 mice with the age of 7 months, tail blood collection is carried out at 24h, 3d and 9d before and after injection respectively, ELISA test is carried out on the blood collection quantity of 50 mu L, and the test result shows that HA-MSNs-1F12 rapidly reduces the beta-amyloid in the peripheral blood after 24h of tail vein injection, while the clearance effect of 1F12 and MSNs is not as good as the clearance effect of HA-MSNs-1F 12. The specific detection result is shown in the B diagram in FIG. 5.
3.3 remodeling of beta-amyloid in the gut of APP/PS1 mice by HA-MSNs-1F12
100 μ L of the HA-MSNs-1F12 probe, a synthetic functionalized nanoparticle probe, was injected intravenously into APP/PS1 mice once a week for three weeks. Simultaneously setting unfunctionalized nano particles MSNs and PBS as a control, after treatment, respectively obtaining 50mg gastrointestinal tracts of a treatment group and a control mouse, grinding in 1mL TBS buffer solution, obtaining tissue homogenate, and respectively taking 100 mu L to carry out beta-amyloid relative area statistics. As a result, HA-MSNs-1F12 was found to decrease beta-amyloid in the intestine and increase beta-amyloid in the caecum and colon in the treatment group 12. The specific detection results are shown in the C and D diagrams in FIG. 5.
3.4 real-time monitoring of beta-amyloid in APP/PS1 mouse brain by HA-MSNs-1F12
To detect HA-MSNs-1F12 in AD mice to monitor beta-amyloid in brain in real time, 100. mu.L of HA-MSNs-1F12 dissolved in PBS at a concentration of 1. mu.g/. mu.L was administered intravenously to male 7-month-old APP/PS1 mice, an equal volume of PBS was injected as a control group, and Magnetic Resonance Imaging (MRI) was performed at 3h and 9h before and after injection. From the results, HA-MSNs-1F12 can enter brain tissues to realize real-time monitoring of changes of beta-amyloid in the brain. The specific detection result is shown in graph A in FIG. 6.
3.4 clearance of beta-amyloid in APP/PS1 mouse brain by HA-MSNs-1F12
100 μ L of the HA-MSNs-1F12 probe, a synthetic functionalized nanoparticle probe, was injected intravenously into APP/PS1 mice once a week for three weeks. Meanwhile, unfunctionalized nano particles MSNs and PBS are set as controls, and after treatment is finished, the brain coronal sections of mice of a treatment group and the control group are obtained to carry out statistical analysis of beta-amyloid after thioflavin counterstaining. As a result: compared with the control group, after APP/PS1 mice are treated by the functionalized HA-MSNs-1F12 probe through intravenous injection, the HA-MSNs-1F12 functionalized nanoparticle probe is proved to have better effect of relieving the burden of beta-amyloid in the brains of the APP/PS1 mice than 1F12 alone. The specific detection results are shown in the B, C and D diagrams in FIG. 6.
In conclusion, the antibody functionalized nanoparticles provided by the invention can be applied to rapidly reduce peripheral blood beta-amyloid, further reduce the burden of the beta-amyloid in brain, and have an obvious effect. The prepared nano-particle for rapidly removing the Alzheimer's disease beta-amyloid has great significance for targeted and accurate treatment.
Examples 1, 2 and 3 have described in detail the preparation of nanoparticles for the rapid clearance of beta-amyloid from peripheral blood and their use, but the antibody functionalized nanoparticles of the present invention are not limited to those described in the examples of this patent. The invention describes a specific capture carrier (functional nano-scale) for rapidly removing beta-amyloid in bloodParticles) of the protein, wherein the monoclonal antibody used by the protein is 1F12, and can also be monoclonal antibodies 6E10 and Aducanumab, as well as polyclonal antibodies, single-chain antibodies and other proteins and small molecular polypeptides with similar functions, wherein the anti-hA beta is obtained1-42Antibody 1F12 was secreted against hA β1-42The hybridoma cell strain 1F12 of the protein monoclonal antibody is prepared, and the hybridoma cell strain 1F12 has the preservation number: CCTCC NO: C2020131. the ligands that cross the blood-brain barrier described in the present invention are not limited to hyaluronic acid, but may be other antibodies, proteins or small molecule polypeptides that can assist in crossing the blood-brain barrier. The nano-particles described in the invention are not limited to mesoporous silica and ferroferric oxide-coated mesoporous silica, and can also be dendritic mesoporous silica, biosoluble mesoporous silica, biomaterial-modified (such as PLGA, platelets, dopamine, metal framework materials and the like) mesoporous silica, composite mesoporous silica, silica microspheres with different structures and nano-particles with similar biological functional properties. The functional nano-particle described by the invention is not limited to clearing beta-amyloid, and can also be applied to the aspects of antibiosis, antioxidation, degradation and clearing of in vivo harmful protein or small molecular polypeptide and the like. All of which are within the scope of the present invention.
It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims (3)

1. A specific capture carrier for clearing beta-amyloid, which is characterized in that the specific capture carrier is a functional nanoparticle for capturing and enriching the beta-amyloid by a specific binding reaction; wherein the beta-amyloid is specifically beta-amyloid monomer and beta-amyloid oligomer;
the functional nano particle is a functional mesoporous silica nano particle which is an anti hA beta 1-42 monoclonal antibody and can penetrate throughThe mesoporous silica nanoparticle which is subjected to surface modification by the common functionalization of hyaluronic acid ligands passing through a blood brain barrier and carries ferroferric oxide magnetic nano materials on the core has the size of 400-500nm, can pass through the blood brain barrier and can be used as a high-specificity scavenger and a contrast agent of beta-amyloid; the anti hA beta 1-42 antibody 1F12 is secreted by anti hA beta1-42The hybridoma cell strain 1F12 of the protein monoclonal antibody is prepared, and the hybridoma cell strain 1F12 has the preservation number: CCTCC NO: C2020131.
2. the specific capture carrier for clearing beta-amyloid according to claim 1, wherein the metabolic pathway of the antibody in the antibody-functionalized modified mesoporous silica nanoparticle is intestinal metabolism.
3. Use of a specific capture vector according to claim 1 or 2 for clearance of β -amyloid in the manufacture of a medicament for alleviating the disease progression of a neurodegenerative disease.
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