Protein degradation agent-loaded host-guest nanoparticle as well as preparation method and application thereof

Abstract

The invention provides subject-object nanoparticles loaded with a protein degradation agent as well as a preparation method and application thereof, and the nanoparticles comprise: 1) functionalized polysialic acid, one part of which is covalently grafted with cyclodextrin, crown ether, pillararene or cucurbituril and other macrocyclic subjects, and the other part of which is covalently grafted with a targeting ligand and is used for crossing a blood brain barrier; 2) a heterodimer composed of a responsive protein degradation agent and a therapeutic drug; and 3) a fluorescent dye or other therapeutic small molecules. The heterodimer, the fluorescent dye and the like are combined with a macrocyclic host in the functionalized polysialic acid through host-guest interaction, and the nanoparticles are formed through self-assembly. The targeting ligand guides the nanoparticles to cross the blood brain barrier, and responds to the specific stimulation of the pathological environment through the responsive bond in the heterodimer, so that the controllable release of the protein degradation agent, the therapeutic drug and the fluorescent dye or other therapeutic small molecules is realized; finally, precise delivery and selective intervention of the medicine in the pathological area of the Alzheimer's disease are realized.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and relates to a target macrocyclic functionalized polysialic acid and a responsive protein degrader-heterodimer therapeutic drug, a host-guest nanoparticle formed by self-assembly of the two through host-guest interaction, a preparation method and application thereof in preparation of a neurodegenerative disease treatment drug. BACKGROUND

[0002] A protein degrader is a kind of molecular tool that selectively degrades specific pathological proteins by binding to an E3 ubiquitin ligase, and has shown great application potential in the research of various neurodegenerative diseases in recent years. However, the protein degrader often faces the problem of "off-target effect" in the application process, that is, it cannot accurately function in the lesion tissue, but degrades the target protein in the non-lesion area, thereby reducing the treatment effect and causing unnecessary side effects. For central nervous system diseases such as Alzheimer's disease, the effective delivery of the protein degrader is particularly challenging. This is because the protein degrader not only needs to achieve selective degradation of pathological proteins, but also must pass through the blood-brain barrier (BBB) to reach the brain lesion area. However, the blood-brain barrier has very low permeability to most drugs, especially macromolecules, which significantly limits the application of protein degraders in the treatment of central nervous system diseases. In addition, even if the protein degrader can pass through the blood-brain barrier, if it is not strong in selectivity in the lesion area, it will still exacerbate the off-target effect and affect its treatment effect. Therefore, how to design a protein degrader delivery system that can not only efficiently pass through the blood-brain barrier, but also accurately function in a specific lesion area, is an important bottleneck in the clinical application of this technology. SUMMARY

[0003] In order to solve the problems of the prior art, one of the technical purposes of the present application is to provide a protein degrader-loaded host-guest nanoparticle with a lesion microenvironment response function for the treatment of Alzheimer's disease, which can achieve targeted protein degradation by responding to the microenvironment conditions of the lesion, and effectively deliver to the central nervous system to improve the treatment effect.

[0004] Another technical purpose of the present application is to provide a preparation method of the protein degrader-loaded host-guest nanoparticle.

[0005] Still another technical purpose of the present application is to provide the use of the protein degrader-loaded host-guest nanoparticle in the preparation of an Alzheimer's disease treatment drug.

[0006] To achieve the above purposes, the specific technical solutions of the present application are as follows:

[0007] A protein degrader-loaded host-guest nanoparticle, comprising the following components:

[0008] 1) a target macrocyclic functionalized polysialic acid represented by the following formula I:

[0009] 2) a protein degrader-therapeutic drug heterodimer represented by the following formula II;

[0010] 3) a fluorescent dye or a therapeutic small molecule;

[0011] wherein, in the nanoparticle, the functionalized polysialic acid 1) loads the heterodimer 2) with the fluorescent dye or the therapeutic small molecule 3) through host-guest interaction;

[0012]

[0013] Formula I

[0014] wherein, in Formula I, x, y, z respectively represent the molar percentage of the three structural units in the polymer, and satisfy x + y + z = 100%; under this constraint, the value range of x, y, z is: x = 10-80 mol%, y = 10-80 mol%, z = 0-80 mol%; preferably, x = 20 mol%, y = 40 mol%, z = 40 mol%;

[0015] The macrocyclic host is a macrocyclic derivative used as a host in host-guest chemistry;

[0016] The target head is a ligand capable of crossing the blood-brain barrier;

[0017] The macrocyclic host and the target head are respectively covalently linked to the polysialic acid;

[0018]

[0019] Formula II

[0020] wherein, in Formula II, the protein degrader is a targeted protein degrader capable of binding to an E3 ubiquitin enzyme containing Von Hippel-Lindau receptor or thalidomide receptor;

[0021] The linking group is a small molecule or a polypeptide that specifically responds to the pathological environment of neurodegenerative diseases;

[0022] The therapeutic drug is a drug with functional group characteristics;

[0023] One end of the linking group is covalently linked to the protein degrader, and the other end is covalently linked to the therapeutic drug;

[0024] The targeting macroring functional polysialic acid is used as a host, a protein degradation agent-therapeutic drug heterodimer is used as a main guest, and a fluorescent dye or a therapeutic small molecule is used as a functional guest, and a nanoparticle is formed by self-assembly through host-guest interaction; wherein the protein degradation agent-therapeutic drug heterodimer can target degradation of abnormal proteins and release of therapeutic drugs; the fluorescent dye is used for in vivo fluorescence imaging; the therapeutic small molecule can produce synergistic or sensitizing effect with the therapeutic drug, and the therapeutic effect is improved together. The mass ratio of the host, the main guest and the functional guest is 1:0.1-0.5:0.01-0.5; in the self-assembly process, the concentration of the aqueous phase system is controlled at 1-2 mg / mL, and the hydrodynamic radius of the obtained host-guest nanoparticle is 60-200 nm.

[0025] Further, the macroring derivative is selected from any one of dextrin, crown ether, pillararene or cucurbituril and derivatives thereof, and specifically is any one of the following:

[0026] .

[0027] Further, the target head is a ligand capable of crossing the blood-brain barrier, and specifically is at least one of a peptide, an antibody, a fusion protein or a small molecule;

[0028] The peptide is at least one of a transport peptide, a transferrin receptor targeting peptide, a glucose transporter targeting peptide and an antibody-derived peptide;

[0029] The antibody is at least one of an antibody capable of binding to a transferrin receptor, a low-density lipoprotein receptor-related protein, an insulin receptor or a glucose transporter;

[0030] The fusion protein is at least one of a multifunctional fusion protein carrying a blood-brain barrier penetrating fragment or a variant thereof;

[0031] The small molecule is at least one of a small molecule capable of interacting with a blood-brain barrier receptor and promoting crossing of the blood-brain barrier:

[0032] The target head is specifically selected from at least one of the following structural formulae:

[0033] .

[0034] Further, the targeting protein degradation agent is selected from a targeting protein degradation agent for degrading tau protein or phosphorylated tau protein or other proteins related to the pathology of neurodegenerative diseases; the other proteins include beta-amyloid protein (Aβ), TDP-43, alpha-synuclein, ApoE4 and FYN kinase;

[0035] Specifically, it is selected from the following molecules:

[0036] .

[0037] Further, the linker group is selected from small molecules or polypeptides capable of responding to oxidative stress, high expression of matrix metalloproteinase-3, matrix metalloproteinase-9, myeloperoxidase and cytokines IL-1β or IL-6 in the pathological environment of neurodegenerative diseases;

[0038] In particular, at least one of the following molecules is selected:

[0039] .

[0040] Further, the therapeutic drug is a drug with functional group characteristics, capable of forming a responsive protein degradation agent-therapeutic drug heterodimer with the protein degradation agent, including memantine, galantamine, pramipexole, minocycline, deferasirox, acetylglutamate, curcumin, methylene blue, rapamycin or rutin;

[0041] In particular, at least one of the following molecules is selected:

[0042] .

[0043] Further, the host-guest nanoparticles can bind fluorescent dyes through host-guest interaction for fluorescence imaging (such as in vivo tracking, lesion positioning or drug distribution monitoring, etc.);

[0044] The fluorescent dye is any one of rhodamine, coumarin, cyanine (Cy) series dye, fluorescein and porphyrin:

[0045] .

[0046] Further, the therapeutic small molecule includes cholinesterase inhibitors such as donepezil (for improving cognitive function in Alzheimer's disease patients), monoamine oxidase inhibitors such as rasagiline (for treating Parkinson's disease to delay dopamine degradation), alkaloid compounds such as berberine (with anti-inflammatory, antibacterial and glucose-lipid metabolism regulating effects), anticonvulsants such as pregabalin (for seizure control and neuropathic pain relief), antioxidants such as acetylcysteine (to reduce oxidative stress damage by scavenging free radicals), and CSF1R inhibitors; such as PLX5622 (for inhibiting microglial cell activation to improve the microenvironment of neurodegenerative diseases) are particularly selected from any one of the following:

[0047] .

[0048] A method for preparing the above-mentioned host-guest nanoparticles, the targeting macrocyclic functionalized polysialic acid, the preparation thereof comprising the following steps:

[0049] The functionalized polysialic acid is prepared by connecting the polysialic acid with the functionalized macrocycle and the functionalized ligand in proportion, wherein the mole percentages of the three structural units in the functionalized polysialic acid are represented by x, y and z respectively, and x + y + z = 100%; under the constraint, the value ranges of x, y and z are respectively: x = 10-80 mol%, y = 10-80 mol%, and z = 0-80 mol%.

[0050] In particular, the method comprises the following steps:

[0051] When the reaction group of the functionalized macrocycle or the ligand is a carboxyl group, the carboxyl group is activated by dissolving the functionalized macrocycle or the ligand in N,N-dimethylformamide (DMF) or dimethyl sulfoxide (DMSO) and sequentially adding 1.2-3.0 times of the molar equivalent of the carboxyl group of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI), 1.2-3.0 equivalents of 4-dimethylaminopyridine (DMAP), 1.2-3.0 equivalents of 1-hydroxybenzotriazole (HOBT) and 1.2-3.0 equivalents of an organic base (DIEA or TEA) under the condition of an ice-water bath for 1-2 hours; then, 1.0-2.0 equivalents of ethylenediamine relative to the carboxyl group is added, and the reaction is carried out at room temperature for 12-24 hours; the reaction product is precipitated by ice-ether / acetone to obtain an aminated macrocycle or ligand; then, the polysialic acid is dissolved in DMF or DMSO, and the above-mentioned activation steps of EDCI, DMAP, HOBT and the organic base are repeated, and after the carboxyl group is activated for 1-2 hours, the aminated macrocycle or ligand is added, and the stirring is carried out at room temperature for 12-24 hours; after the reaction is completed, the product is dialyzed by using an organic solvent and water, and finally, the targeted macrocycle functionalized polysialic acid is obtained by freeze-drying;

[0052] When the reaction group of the functionalized macrocycle or the ligand is a carboxyl group, the carboxyl group is activated by dissolving the functionalized macrocycle or the ligand in N,N-dimethylformamide (DMF) or dimethyl sulfoxide (DMSO) and sequentially adding 1.2-3.0 times of the molar equivalent of the carboxyl group of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI), 1.2-3.0 equivalents of 4-dimethylaminopyridine (DMAP), 1.2-3.0 equivalents of 1-hydroxybenzotriazole (HOBT) and 1.2-3.0 equivalents of an organic base (DIEA or TEA) under the condition of an ice-water bath for 1-2 hours; then, 1.0-2.0 equivalents of ethylenediamine relative to the carboxyl group is added, and the reaction is carried out at room temperature for 12-24 hours; the reaction product is precipitated by ice-ether / acetone to obtain an aminated macrocycle or ligand; then, the polysialic acid is dissolved in DMF or DMSO, and the above-mentioned activation steps of EDCI, DMAP, HOBT and the organic base are repeated, and after the carboxyl group is activated for 1-2 hours, the aminated macrocycle or ligand is added, and the stirring is carried out at room temperature for 12-24 hours; after the reaction is completed, the product is dialyzed by using an organic solvent and water, and finally, the targeted macrocycle functionalized polysialic acid is obtained by freeze-drying;

[0053] The organic base is N,N-diisopropyl ethylamine (DIEA) or triethylamine (TEA), and the organic solvent used in dialysis is N,N-dimethyl formamide (DMF), N,N-dimethyl acetamide (DMA) or dimethyl sulfoxide (DMSO);

[0054] The preparation of the protein degradation agent-therapeutic drug heterodimer comprises the following steps:

[0055] The other end of the linker is covalently connected to the therapeutic drug to obtain the responsive protein degradation agent-therapeutic drug heterodimer;

[0056] In particular, the preparation comprises the following steps:

[0057] When a thio-ketone derivative is used as a linker to couple the protein degradation agent and the therapeutic drug together, first, 1.0 equivalent of methyl mercaptan and 1.1-1.5 equivalents of acetone are reacted to generate a thiol-acetone ketal derivative, which is then reduced with 1.2-2.0 equivalents of lithium aluminum hydride in tetrahydrofuran (THF) to generate a bis-thiol ethyl chain with a hydroxyl group; then, the hydroxyl group is protected by 1.1-1.5 equivalents of a silylating agent (such as TBDMS-Cl) under basic conditions (such as the presence of pyridine), and then coupled with a therapeutic drug containing an active hydroxyl or amino group for the first time by a carbonate reaction, using 1.2-2.0 equivalents of p-nitrophenyl carbonate or NHS carbonate as an activating agent, and the reaction is carried out at room temperature; after the coupling is completed, 1.5-2.5 equivalents of tetrafluoroborates TBAF are used to remove the silyl protecting group in THF to expose the free hydroxyl group, and the carbonate reagent is used again to activate and coupled with 1.0-1.5 equivalents of a protein degradation agent containing an amino or hydroxyl group for the second time; the final crude product is purified by column chromatography to obtain the target responsive protein degradation agent-therapeutic drug heterodimer, and the structure is verified by high performance liquid chromatography (HPLC), mass spectrometry (MS) and nuclear magnetic resonance hydrogen spectrum (1H-NMR);

[0058] The preparation of the host-guest nanoparticle comprises:

[0059] 1) coupling a functionalized macrocycle or ligand with polysialic acid to prepare the functionalized polysialic acid targeting macrocycle;

[0060] 2) covalently coupling a protein degradation agent and a therapeutic drug through a linker to prepare the protein degradation agent-therapeutic drug heterodimer;

[0061] 3) 0.01-0.5 mg of the fluorescent dye or therapeutic small molecule, 0.1-0.5 mg of the protein-degrading agent-therapeutic drug heterodimer is dissolved in 100-200 μL of organic solvent (such as DMF and DMSO); under the condition of ultrasonic frequency of 40-60 kHz and power of 40-100 W, the organic phase is slowly dropped or injected into the prepared aqueous solution of the targeting macrocyclic functionalized polysialic acid with a concentration of 0.5-5 mg / mL and pH of 6.5-7.5, to form an emulsified dispersion system; the dispersion system is subjected to dialysis with MWCO of 0.8-14 kDa or centrifugal ultrafiltration at a speed of 3000-8000 rpm to remove the organic solvent and small molecule impurities, so as to obtain the host-guest nanoparticles;

[0062] The organic solvent is selected from N,N-dimethylformamide, N,N-dimethylacetamide or dimethyl sulfoxide.

[0063] The volume ratio of the aqueous phase to the organic solvent is 100-10:1, preferably 10:1.

[0064] The ultrafiltration process is performed by centrifugal ultrafiltration or ultrafiltration in an ultrafiltration machine, and the molecular weight cut-off of the ultrafiltration tube or ultrafiltration membrane used in the process is 1500D, 3kD, 5kD or 10KD.

[0065] The molecular weight cut-off of the dialysis bag used in the dialysis process is 800, 1000, 3500 or 14000.

[0066] The host-guest nanoparticles are used for preparing a drug for treating neurodegenerative diseases, such as any one of Alzheimer's disease, Parkinson's disease and Huntington's disease.

[0067] The technical scheme of the present application has at least the following technical effects:

[0068] The present application provides a host-guest particle formed by a targeting macrocyclic functionalized polysialic acid and a responsive protein-degrading agent-therapeutic drug heterodimer and self-assembly thereof. The polysialic acid is covalently grafted with a macrocyclic host and a specific target head, and the target head can recognize and bind to a specific receptor or molecule on the blood-brain barrier, so as to realize the targeted crossing of the blood-brain barrier. At the same time, the heterodimer is coupled by a protein-degrading agent and a therapeutic drug through a responsive linker, and dissociates under the stimulation of a lesion microenvironment (such as oxidative stress or high expression of a specific enzyme), so as to realize the controlled release of the drug and ensure that the drug can be accurately enriched in the pathological area of the neurodegenerative disease. The release of the protein-degrading agent can accurately degrade the pathological related protein, and the therapeutic drug can further regulate the pathological process.

[0069] The nanoparticles formed by self-assembly of the host-guest interaction after combination can significantly improve the targeted delivery of drugs in the central nervous system. The polysialic acid provides the targeting ability, so that the nanoparticles can non-invasively cross the blood-brain barrier, and the heterodimer responds to the microenvironment stimulus in the lesion area to control the release of the protein degradation agent and the therapeutic drug, so that the drug is selectively accumulated in the pathological area. The nanoparticle system significantly improves the distribution of the drug in the pathological area in the brain and reduces the systemic side effects. The targeting function of polysialic acid and the responsive release function of the heterodimer together ensure the high efficiency and selectivity of the host-guest nanoparticles in the treatment of neurodegenerative diseases such as Alzheimer's disease, and both are indispensable. BRIEF DESCRIPTION OF DRAWINGS

[0070] Figure 1 NMR hydrogen spectrum data and mass spectrum data of C6 amino-modified D-galactose are shown; wherein a graph is an NMR hydrogen spectrum, and b graph is a mass spectrum;

[0071] Figure 2 NMR hydrogen spectrum data of functional polysialic acid modified at different galactose and cyclodextrin ratios are shown; wherein a graph is a modification ratio graph, and b to d graphs are NMR hydrogen spectrum graphs of different modification ratios;

[0072] Figure 3 NMR hydrogen spectrum data and mass spectrum data of the target phosphated tau protein degradation agent are shown; wherein a graph is an NMR hydrogen spectrum, and b graph is a mass spectrum;

[0073] Figure 4 NMR hydrogen spectrum data and mass spectrum data of the protein degradation agent-rivastigmine heterodimer are shown; wherein a graph is an NMR hydrogen spectrum, and b graph is a mass spectrum;

[0074] Figure 5 Hydrodynamic particle size and transmission electron microscope graph of the nanoparticles prepared in Example 5 of the application are shown; wherein the left graph is a particle size distribution histogram, and the upper right corner graph is a transmission electron microscope graph;

[0075] Figure 6 Nanoparticle active oxygen response performance graph in Example 6 of the application is shown; wherein a graph is a protein degradation agent cumulative release amount graph in the presence of different concentrations of active oxygen, and b graph is a particle size distribution histogram in the presence of active oxygen;

[0076] Figure 7 Protein blotting graph of the nanoparticle degradation ability for phosphated tau protein at the cell level in Example 7 of the application is shown;

[0077] Figure 8 Flow quantification positive rate (B) schematic diagram of the nanoparticles crossing the in vitro blood-brain barrier model in Example 8 of the application is shown;

[0078] Figure 9 Figure 9 shows the western blotting diagram of the ability of phosphorylated tau protein degradation in wild type mice, 3xTg AD mice, and 3xTg AD mice treated with nanoparticles in the embodiment 9 of the present application;

[0079] Figure 10 Figure 4 is a schematic diagram of the nanoparticle structure of the present application. DETAILED DESCRIPTION

[0080] The present application will be further described in conjunction with the following specific examples and drawings. The process, conditions, experimental methods, etc. for implementing the present application are the general knowledge and common sense in the art, and the present application does not have special limitations.

[0081] In order to solve the problems of off-target degradation of protein degradation agents in non-lesion tissues, low efficiency of crossing the blood-brain barrier, and uneven distribution of drugs in pathological areas, the host-guest nanoparticles formed by the self-assembly of a targeting macrocyclic functionalized polysialic acid and a responsive protein degradation agent-therapeutic drug heterodimer are proposed in the embodiments of the present application. The polysialic acid covalently grafts the macrocyclic host and the target head, guiding the nanoparticles to cross the blood-brain barrier. At the same time, the heterodimer controls the release of the protein degradation agent and the therapeutic drug and makes them precisely enriched in the pathological area by responding to specific stimuli (such as oxidative stress or expression of specific enzymes) in the lesion microenvironment, achieving efficient degradation of pathological-related proteins. Through host-guest interaction, the nanoparticles formed by the self-assembly of polysialic acid and heterodimer not only have good biocompatibility, but also can effectively improve the enrichment of drugs in the brain and reduce systemic side effects. The nanoparticle system further optimizes the distribution of drugs in pathological areas, significantly improves the treatment effect of neurodegenerative diseases such as Alzheimer's disease, and overcomes the problems of poor penetration and off-target effects in traditional drug delivery systems.

[0082] The present application provides a protein degradation agent-loaded host-guest nanoparticle (see Figure 10 ), which comprises the following components:

[0083] 1) a targeting macrocyclic functionalized polysialic acid represented by the following formula I;

[0084] 2) a protein degradation agent-therapeutic drug heterodimer represented by the following formula II;

[0085] 3) a fluorescent dye or a therapeutic small molecule;

[0086] In the nanoparticle, the functionalized polysialic acid 1) loads the heterodimer 2) and the fluorescent dye 3) through host-guest interaction,

[0087]

[0088] Formula I

[0089] In Formula I, x, y, z respectively represent the molar percentage of three kinds of structural units in the polymer, and satisfy x + y + z = 100%. Under this constraint condition, the value range of x, y, z is: x = 10-80 mol%, y = 10-80 mol%, z = 0-80 mol%; preferably, x = 20 mol%, y = 40 mol%, z = 40 mol%;

[0090] The macrocyclic host is a macrocyclic derivative used as a host in host-guest chemistry;

[0091] The target head is a ligand capable of crossing the blood-brain barrier;

[0092] The macrocyclic host and the target head are respectively connected to polysialic acid by a covalent bond.

[0093]

[0094] Formula II

[0095] In Formula II, the protein degradation agent is a targeted protein degradation agent capable of binding to an E3 ubiquitinase containing a Von Hippel-Lindau receptor or a thalidomide receptor, the linking group is a small molecule or a polypeptide that specifically responds to the pathological environment of a neurodegenerative disease such as Alzheimer's disease, and the therapeutic drug is a drug with functional group characteristics; one end of the linking group is covalently connected to the protein degradation agent, and the other end is covalently connected to the therapeutic drug.

[0096] In a specific embodiment, the macrocyclic host is selected from macrocyclic derivatives used as a host in host-guest chemistry; wherein the host refers to a macromolecular structure capable of selectively binding to a guest molecule through non-covalent interactions (such as hydrogen bonding, hydrophobic interaction or π-π stacking). The macrocyclic host can form a stable host-guest complex with the guest such as protein degradation, effectively loading and releasing the protein degradation agent;

[0097] Preferably, the macrocyclic host is selected from at least one of cyclodextrin, crown ether, pillararene or cucurbituril and derivatives thereof:

[0098] .

[0099] In the present application, the target head is selected from at least one of peptides, antibodies, fusion proteins or small molecules capable of crossing the blood-brain barrier;

[0100] Preferably, the peptide-based targeting head is selected from at least one of the following: a transport peptide that crosses the blood-brain barrier, a transferrin receptor-targeting peptide, a glucose transporter-targeting peptide, an antibody-derived polypeptide;

[0101] Preferably, the antibody targeting head is selected from at least one of the following: an antibody that binds to a transferrin receptor, a low-density lipoprotein receptor-related protein, an insulin receptor, or a glucose transporter;

[0102] Preferably, the fusion protein targeting head is selected from at least one of the following: a multifunctional fusion protein carrying a blood-brain barrier-penetrating fragment or a variant thereof;

[0103] Preferably, the small molecule targeting head is selected from at least one of the following: a small molecule that can interact with a blood-brain barrier receptor and facilitate crossing of the blood-brain barrier:

[0104] .

[0105] In the present application, the protein degrader is selected from a targeted protein degrader that can bind to an E3 ubiquitin ligase containing a Von Hippel-Lindau receptor or a thalidomide receptor;

[0106] Preferably, the protein degrader is selected from a targeted protein degrader for degrading tau protein or phosphorylated tau protein or other proteins associated with the pathology of neurodegenerative diseases such as Alzheimer's disease, including but not limited to beta-amyloid protein (Aβ), TDP-43, alpha-synuclein, ApoE4, and FYN kinase;

[0107] More preferably, the protein degrader is selected from the following molecules:

[0108] .

[0109] In the present application, the linker group is selected from a specific small molecule or polypeptide that can respond to oxidative stress, highly expressed matrix metalloproteinase-3, matrix metalloproteinase-9, myeloperoxidase, and cytokines IL-1β or IL-6, etc. in the pathological environment of neurodegenerative diseases such as Alzheimer's disease;

[0110] Preferably, the linker group is selected from at least one of the following molecules:

[0111] .

[0112] In the present application, the therapeutic drug is a drug with functional group characteristics, which can form a responsive protein degrader-therapeutic drug heterodimer with the protein degrader, including but not limited to memantine, galantamine, pramipexole, minocycline, deferasirox, acetyl glutamate, curcumin, methylene blue, rapamycin, rutin;

[0113] Preferably, the therapeutic drug is selected from at least one of the following molecules:

[0114] .

[0115] In another aspect, the present application also provides a preparation method of the targeted macrocycle functionalized polysialic acid, comprising the following steps:

[0116] The polysialic acid is combined with the functionalized macrocycle and the functionalized ligand in a suitable ratio to obtain the targeted macrocycle functionalized polysialic acid;

[0117] In some embodiments, the preparation method comprises the following steps:

[0118] When the reaction group of the functionalized macrocycle or the ligand is a carboxyl group, it is dissolved in a DMF or DMSO solvent, and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI), 4-dimethylaminopyridine (DMAP), 1-hydroxybenzotriazole (HOBT) and an organic base are sequentially added under ice water bath conditions to activate the carboxyl group for 1-2 hours. Then, ethylenediamine is added, and the reaction is carried out at room temperature for 12-24 hours. The reaction product is precipitated by ice ether / acetone to obtain the aminated macrocycle or ligand. Subsequently, the polysialic acid is dissolved in a DMF or DMSO solvent, and the above-mentioned activation steps of EDCI, DMAP, HOBT and the organic base are repeated. After the carboxyl group is activated for 1-2 hours, different proportions of the aminated macrocycle or ligand are added, and the mixture is stirred at room temperature for 12-24 hours. After the reaction is completed, the product is dialyzed (using organic solvents and water), and finally freeze-dried to obtain the targeted macrocycle functionalized polysialic acid.

[0119] When the reaction group of the functionalized macrocycle or the ligand is a hydroxyl group or an amino group, the polysialic acid is dissolved in a DMF or DMSO solvent, and EDCI, DMAP, HOBT and an organic base are added to activate the carboxyl group for 1-2 hours. Then, an appropriate amount of the macrocycle or ligand with a reaction group of a hydroxyl group or an amino group is added, and the mixture is stirred at room temperature for 12-24 hours. The reaction product is dialyzed three times (using organic solvents and water), and then freeze-dried to obtain the targeted macrocycle functionalized polysialic acid.

[0120] The organic base in the preparation method can be N,N-diisopropylethylamine (DIEA) or triethylamine (TEA), and the organic solvent used in dialysis can be selected from N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA) or dimethyl sulfoxide (DMSO).

[0121] The present application also provides a preparation method of the responsive protein degradation agent-therapeutic drug heterodimer, comprising the following steps:

[0122] When one end of the linker is first covalently connected to the protein degrader molecule, the other end is covalently connected to the therapeutic drug to obtain a responsive protein degrader-therapeutic drug heterodimer; when one end of the linker is first covalently connected to the therapeutic drug, the other end is covalently connected to the protein degrader to obtain a responsive protein degrader-therapeutic drug heterodimer;

[0123] In particular, the method comprises the following steps:

[0124] When a thio-ketone derivative is used as a linker to couple the protein degrader and the therapeutic drug together. First, a thiol-acetone ketal derivative is generated by reacting methyl mercaptan and acetone, and then reduced with lithium aluminum hydride to generate a bis-thiol ethyl chain with a hydroxyl group. Subsequently, the therapeutic drug / protein degrader is coupled with the linker through silicon-based protection, introduction of the drug molecule, and multiple carbonate activations. Then, the hydroxyl group is exposed by deprotection, and then reacted with the activated carbonate. Finally, the protein degrader / therapeutic drug is introduced and reacted with the linker to generate a responsive protein degrader-therapeutic drug heterodimer.

[0125] In another aspect, the application also provides a host-guest nanoparticle self-assembled by the above-mentioned targeted macrocyclic functionalized polysialic acid and the above-mentioned responsive protein degrader-therapeutic drug heterodimer through host-guest interaction;

[0126] Preferably, the hydrodynamic radius of the host-guest nanoparticle is 60-200 nm, more preferably 100 nm.

[0127] The host-guest nanoparticle provided by the application can combine fluorescent dyes through host-guest interaction;

[0128] The fluorescent dye is any one of rhodamine, coumarin, cyanine (Cy) series dye, fluorescein or porphyrin;

[0129] Preferably, the fluorescent dye is at least one of the following molecules:

[0130] .

[0131] The host-guest nanoparticle provided by the application can load therapeutic small molecules through host-guest interaction, including but not limited to cholinesterase inhibitors such as donepezil, monoamine oxidase inhibitors such as rasagiline, alkaloid compounds such as berberine, anticonvulsants such as pregabalin, antioxidants such as acetylcysteine, and CSF1R inhibitors such as PLX5622;

[0132] Preferably, the therapeutic small molecule is selected from any one of the following:

[0133] .

[0134] In yet another aspect, the present application also provides a method for preparing the host-guest nanoparticle, comprising the following steps:

[0135] The responsive protein degrader-therapeutic drug heterodimer is dissolved in an organic solvent, and is dispersed in a targeting macrocyclic functionalized polysialic acid aqueous solution under ultrasonication, and the host-guest nanoparticle is obtained by dialysis or ultrafiltration.

[0136] In some embodiments, the organic solvent is selected from N,N-dimethylformamide, N,N-dimethylacetamide or dimethyl sulfoxide.

[0137] In some embodiments, the volume ratio of the aqueous phase to the organic solvent is 100:1-10:1, preferably 10:1.

[0138] In some embodiments, the ultrafiltration process is performed by centrifugal ultrafiltration using an ultrafiltration tube or ultrafiltration using an ultrafiltration machine, and the molecular weight cut-off of the ultrafiltration tube or ultrafiltration membrane used in the process is 1500D, 3kD, 5kD or 10KD.

[0139] In some embodiments, the molecular weight cut-off of the dialysis bag used in the dialysis process is 800, 1000, 3500 or 14000.

[0140] In yet another aspect, the present application also provides the use of the host-guest nanoparticle in the preparation of a medicament for treating a neurodegenerative disease.

[0141] Preferably, the neurodegenerative disease is selected from any one of Alzheimer's disease, Parkinson's disease and Huntington's disease.

[0142] The present application proposes a host-guest particle formed by self-assembly of a targeting macrocyclic functionalized polysialic acid and a responsive protein degrading agent-therapeutic drug heterodimer. The main function of the targeting macrocyclic functionalized polysialic acid is to realize efficient delivery of drugs across the blood-brain barrier by binding with specific targeting ligands, and to ensure that the drugs can enter the central nervous system. On the other hand, the responsive protein degrading agent-therapeutic drug heterodimer can realize precise enrichment and controlled release of drugs in the lesion area by accurately responding to the pathological environment signals (such as oxidative stress or high expression of specific enzymes) of neurodegenerative diseases such as Alzheimer's disease through specific linking groups. The heterodimer can target the pathologically related proteins and inhibit the progression of the disease by precisely degrading these proteins in combination with therapeutic drugs. The combination of the two can form a host-guest nanoparticle by self-assembly through host-guest interaction, which can not only cross the blood-brain barrier, but also realize the responsive release of drugs in the lesion microenvironment. The system realizes the precise control of drug release in the lesion area, ensures the precise action of protein degrading agent and therapeutic drug in the lesion area, optimizes the pharmacokinetic properties of the drug, reduces the systemic side effects, and significantly improves the treatment effect of neurodegenerative diseases such as Alzheimer's disease.

[0143] The reagents and instruments used in the following examples are as follows:

[0144] Methyl thioacetate, lithium aluminum hydride, 1-(3-dimethylaminopropyl), 4- dimethylaminopyridine (DMAP), triphenylphosphine, 1-hydroxybenzotriazole (HOBT), acetic acid, pyridine, [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (Pd(dppf)Cl2), tert-butyldiphenylsilyl chloride (TBDPSCl), and phthalimide potassium were purchased from Bide Pharmaceutical Co., Ltd. (Shanghai, China). Bis(diethyleneglycol)boron, 3-(4-bromophenyl)propanol, potassium acetate (KOAc), tetrakis(triphenylphosphine)palladium (Pd(PPh3)4), and 10% palladium on carbon (Pd / C) were purchased from Shanghai Aosun Chemical Co., Ltd. Sulfuric acid, tert-butyldimethylsilyl chloride, imidazole, bis(p-nitrophenyl)carbonate, tetrabutylammonium fluoride, diacetylgalactoside, p-toluenesulfonyl chloride, and memantine were purchased from J&K Chemical (Shanghai, China). 3-Bromo-4-nitropyridine, di-tert-butyl dicarbonate (Boc20), and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) were purchased from Haohong Biomedical Technology Co., Ltd. Sodium azide, 3-ethylcarbodiimide hydrochloride (EDCI), triethylamine (TEA), N,N-diisopropylethylamine (DIEA), trifluoroacetic acid (TFA), hydrazine hydrate, methanesulfonyl chloride, sodium carbonate (Na2CO3), sodium sulfate (Na2SO4), 1,4-dioxane, ethyl acetate, dichloromethane (DCM), N,N-dimethylformamide (DMF), tetrahydrofuran (THF), ethanol (EtOH) were purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd. Triethylphosphine (P(OEt)3), 4-methylmorpholine, sodium hydride (NaH), and iodomethane (CH3I) were purchased from Angene Chemicals. Pyropheophytin-a (PPa) and mono(6-amino-6-deoxy)-β-cyclodextrin (NH2-β-CD) were purchased from Dibao Biotech Co., Ltd. (Shanghai, China), and polysialic acid (PSA) was purchased from Zhongke Hongji Biotech Co., Ltd. (Shanxi, China). The remaining reagents and solvents were purchased from National Pharmaceutical Group (Shanghai) Chemical Reagent Co., Ltd. unless otherwise specified.

[0145] PC12 cells and SH-SY5Y cells were purchased from Shanghai Cell Bank of Chinese Academy of Sciences. Female C57 (6 ~ 8 weeks, 18 ~ 20 g) were purchased from Shanghai Experimental Animal Center.

[0146] The hydrodynamic size of the nanoparticles was measured by a Malvern laser particle size analyzer (ZEN3690, Malven, USA); the morphology of the nanoparticles was investigated by a 120 kV transmission electron microscope (Talos L120C, FEI, USA). Electronic balance (Quintix 224-1, Sartorius Germany); rotary evaporator (B-40, Buchi, Switzerland); constant temperature magnetic stirrer (DF-101S, Shanghai Yihua Instrument Co., Ltd.); nuclear magnetic resonance spectrometer (Mercury Plus 400, Varian, USA); Waters 2695 high performance liquid chromatograph (waters e2695 chromatographic pump, Xbridge C18 5μm 19*250mm chromatographic column, Waters 2998 ultraviolet detector). Flow cytometry data was measured by a BD FACS Fortessa flow cytometer. Unless otherwise specified, the equipment and test methods used are conventional equipment and methods in the art.

[0147] Example 1: Synthesis of aminoated D-galactose at C6 position

[0148]

[0149] Diethyketone galactose (3.55 g, 13.65 mmol) was mixed with anhydrous pyridine (30 mL) and p-toluenesulfonyl chloride (TsCl, 3.90 g, 20.48 mmol) and stirred at 0 °C under nitrogen protection for 8 hours. After the reaction was completed, the mixture was poured into ice-cold 1M hydrochloric acid solution, extracted with ethyl acetate, and dried over anhydrous sodium sulfate. Silica gel column chromatography was performed using petroleum ether / ethyl acetate (4:1, v / v) as the eluent to obtain yellow liquid 1 (yield: 99%, 5.59 g).

[0150] Compound 1 (5.37 g, 12.97 mmol) was dissolved in anhydrous N,N-dimethylformamide (DMF, 30 mL), sodium azide (NaN3, 1.69 g, 25.94 mmol) was added, heated to 90 °C under nitrogen protection and stirred overnight. After the reaction was completed, the mixture was poured into a cooled saturated sodium bicarbonate solution, extracted with ethyl acetate, washed with brine, and dried over anhydrous sodium sulfate. Purification was performed by silica gel column chromatography using petroleum ether / ethyl acetate (4:1, v / v) as the eluent to obtain compound 2 with a yield of 70% (2.59 g).

[0151] Compound 2 (285 mg, 1 mmol) was dissolved in 8 mL of THF / H2O mixed solvent, and triphenylphosphine (786 mg, 3 mmol) was added. It was refluxed at 50 °C for about 3 hours. After the reaction was completed, it was extracted, dried, and purified by silica gel column chromatography with petroleum ether / dichloromethane-methanol (10:1, v / v) as the eluent to obtain compound 3 (yield: 85%, 220 mg).

[0152] Compound 3 (259 mg, 1 mmol) was dissolved in a DCM / TFA / H2O mixed solvent (volume ratio 10:1:1) and stirred at 0 °C for 4 hours under nitrogen protection. After the reaction was completed, the product was precipitated with ether and purified by preparative high-performance liquid chromatography to finally obtain C6-amino D-galactose (yield: 40%, 71 mg).

[0153] The nuclear magnetic resonance hydrogen spectrum and mass spectrum data of C6-amino D-galactose are shown in Figure 1 , indicating that it was successfully synthesized.

[0154] Example 2: Synthesis of Galactose and Cyclodextrin Functionalized Polysialic Acid

[0155]

[0156] Polysialic acid (PSA, 50 mg, 0.161 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 87 mg, 0.484 mmol), and 1-hydroxybenzotriazole (HOBT, 65 mg, 0.484 mmol) were dissolved in dimethyl sulfoxide (DMSO) and transferred to a three-necked flask. Then triethylamine (TEA, 49 mg, 0.484 mmol) was added, and the mixture was activated for 2 hours. Then DMSO solutions containing different amounts of mono(6-amino-6-deoxy)-β-cyclodextrin (NH2-β-CD) were added, with the amount being 77 mg (0.067 mmol), 127 mg (0.113 mmol), and 178 mg (0.158 mmol), respectively. After 24 hours of reaction, the white solid PSA-CD polymer generated was purified by water dialysis through a dialysis bag with a molecular weight cutoff of 3.5 kDa, and then freeze-dried. Then, the PSA-CD polymers with different grafting ratios were further functionalized by adding different amounts of C6-amino D-galactose through an amidation reaction to form PSA-CD / Gal polymers, i.e., galactose and cyclodextrin functionalized polysialic acid.

[0157] The nuclear magnetic resonance hydrogen spectrum of the functionalized polysialic acid modified by different galactose and cyclodextrin ratios is shown in Figure 2 , indicating that it was successfully synthesized.

[0158] Example 3: Synthesis of a degradation agent targeting phosphorylated tau protein

[0159]

[0160] NaH (48 mg, 1.19 mmol, purity: 60%) was added to a solution of compound 4 (500 mg, 1.08 mmol) in dimethylformamide (DMF, 6 mL) and stirred at 0 °C for 20 min. Then, methyl iodide (CH3I, 169 mg, 1.19 mmol) was added dropwise to the mixture and stirring was continued at 0 °C for 40 min. After the reaction, the mixture was diluted with water and extracted with petroleum ether / ethyl acetate (2:1, v / v). The organic layer was washed with saturated brine, then dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by flash column chromatography (silica gel, 1% to 5% methanol / DCM as eluent) to give the desired compound 5 (344 mg, yield: 66.5%).

[0161] To a stirred solution of compound 5 (320 mg, 0.67 mmol) in tetrahydrofuran (THF, 5 mL), tetrabutylammonium fluoride (TBAF, 1.0 M solution, 1.01 mL, 1.01 mmol) was added at 0 °C. The mixture was stirred at this temperature for 2 h, then concentrated under reduced pressure. The crude product was purified by flash column chromatography (silica gel, 2% to 10% DCM / methanol as eluent) to give compound 6 (113 mg, yield: 70.2%).

[0162] To a stirred solution of compound 6 (95 mg, 0.40 mmol) in DCM (3 mL), triethylamine (Et3N, 121 mg, 166 μL, 1.20 mmol) and methanesulfonyl chloride (MsCl, 69 mg, 0.60 mmol) were added at 0 °C. After the mixture was stirred at room temperature for 2 h, it was concentrated under reduced pressure. The crude product was purified by flash column chromatography (silica gel, 2% to 5% DCM / methanol as eluent) to give compound 7 (106 mg, yield: 83.5%).

[0163] Compound 7 (80 mg, 0.25 mmol) and sodium azide (NaN3, 33 mg, 0.50 mmol) were mixed in DMF (3 mL) and stirred at 80 °C under nitrogen atmosphere for 5 h. After cooling to room temperature, the resulting compound 8 was used directly in the next step. To the above resulting mixture, 10% palladium on carbon (Pd / C, 16 mg) and methanol (1 mL) were added and stirred at room temperature under 1 atm of hydrogen atmosphere for 2 h. Subsequently, the mixture was filtered through Celite and the filtrate was concentrated under vacuum to give crude 9. The crude 9 was used directly in the next step without further purification. To a solution of crude 9 and compound 10 (120 mg, 0.20 mmol), DIPEA (78 mg, 105 μL, 0.60 mmol) and HATU (106 mg, 0.28 mmol) were added and reacted in DMF (2 mL) at room temperature for 2 h. After the reaction, the mixture was concentrated under reduced pressure and the resulting crude was purified by preparative high-performance liquid chromatography to give compound 11, a targeted phospho-tau protein degrader (46 mg, overall yield: 22.4% for three steps).

[0164] The nuclear magnetic resonance hydrogen spectrum and mass spectrum data of the targeted phospho-tau protein degrader are shown in Figure 3 , indicating the successful synthesis thereof.

[0165] Example 4: Synthesis of a responsive protein degrader - amphetamine heterodimer

[0166]

[0167] Methyl thioacetate (21.2 g, 0.2 mol) and acetone (5.8 g, 0.1 mol) were dissolved in methanol (MeOH) under ice bath, followed by the slow dropwise addition of 4 mL of concentrated sulfuric acid. The mixture was reacted at room temperature for 48 h. After the reaction, the reaction was diluted with 20 mL of dichloromethane (DCM), washed with saturated sodium bicarbonate solution in sequence, and dried over anhydrous sodium sulfate. Compound 12 (20.1 g, 80% yield) was obtained as a colorless oily liquid. Lithium aluminum hydride (1.5 g, 40 mmol) was dispersed in 10 mL of tetrahydrofuran (THF) under nitrogen atmosphere and ice water bath conditions, and 50 mL of the previously obtained oily liquid (5 g, 79 mmol) was added dropwise. After the addition, the reaction was continued at room temperature for 4 h. Subsequently, 1.5 mL of water, 1.5 mL of 15% sodium hydroxide solution, and an additional 4.5 mL of water were added to the reaction mixture cooled in an ice water bath in sequence. The mixture was reacted at room temperature for 15 min, then filtered, washed with water, and extracted with ethyl acetate. The extract was dried over anhydrous sodium sulfate and purified by silica gel column chromatography to obtain compound 13 (70% yield) as a light yellow oily liquid.

[0168] Compound 13 (200 mg, 1.018 mmol) was dissolved in DCM, imidazole (69 mg, 1.018 mmol) was added, then tert-butyldimethylsilyl chloride (TBDPSCl, 138 mg, 0.916 mmol) in DCM was added dropwise under ice bath conditions, and finally the reaction was carried out at room temperature for 24 hours. After the reaction was completed, the mixture was washed with water and saturated NaCl solution, dried over anhydrous sodium sulfate, and purified by column chromatography on silica gel with petroleum ether / ethyl acetate (10 / 1, v / v) as the eluent to obtain compound 14 (41% yield, 130.7 mg) as a yellow oily liquid.

[0169] Compound 14 (100 mg, 0.322 mmol), bis(p-nitrophenyl) carbonate (45 mg, 0.386 mmol), and triethylamine (TEA, 48 mg, 0.483 mmol) were dissolved in DCM and reacted at room temperature overnight. The reaction mixture was diluted with DCM, then washed with water, dried over anhydrous sodium sulfate, and evaporated under reduced pressure to obtain compound 15 (95% yield, 145 mg) as a light yellow oily liquid.

[0170] Compound 15 (415 mg, 0.872 mmol) and memantine (156 mg, 0.726 mmol) were dissolved in DCM, 4-dimethylaminopyridine (DMAP, 106 mg, 0.872 mmol) and N,N-diisopropylethylamine (DIEA, 112 mg, 0.872 mmol) were added, and the mixture was reacted at room temperature overnight. The reaction mixture was diluted with DCM, washed, dried over anhydrous sodium sulfate, and purified by column chromatography on silica gel with petroleum ether / ethyl acetate (6 / 1, v / v) as the eluent to obtain compound 16 (75% yield, 281 mg) as a colorless oily liquid.

[0171] Compound 16 (100 mg, 0.194 mmol) was dissolved in 2 mL of THF, acetic acid (HOAC, 7.5 mg, 0.291 mmol) and tetrabutylammonium fluoride (TBAF, 76 mg, 0.291 mmol) were added, and the reaction was carried out at room temperature overnight. The reaction mixture was washed, extracted, dried over anhydrous sodium sulfate, and purified by column chromatography on silica gel with petroleum ether / ethyl acetate (6 / 1, v / v) as the eluent to obtain compound 17 (85% yield, 66.2 mg) as a light yellow solid.

[0172] Compound 17 (100 mg, 0.249 mmol), bis(p-nitrophenyl) carbonate (91 mg, 0.299 mmol), and TEA (38 mg, 0.374 mmol) were dissolved in DCM and allowed to react at room temperature overnight. The reaction mixture was diluted with DCM, washed with water, dried over anhydrous sodium sulfate, and then evaporated under reduced pressure to afford Compound 18 as a yellow solid (95% yield, 134 mg).

[0173] A targeted phosphorylated tau protein degrader (54 mg, 0.074 mmol) and compound 18 (45 mg, 0.088 mmol) were dissolved in DCM, followed by the addition of 4-dimethylaminopyridine (DMAP, 12 mg, 0.111 mmol) and N,N-diisopropylethylamine (DIEA, 19 mg, 0.148 mmol). The mixture was allowed to react overnight at room temperature. After completion of the reaction, the mixture was diluted with DCM, washed sequentially with water, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography using DCM / methanol (20 / 1, v / v) as the eluent to afford heterodimeric compound 19 as a white solid (yield: 56%, 48 mg).

[0174] The nuclear magnetic resonance hydrogen spectrum and mass spectrometry data of the responsive protein degrader-memantine heterodimer are as follows Figure 4 As shown, it was successfully synthesized.

[0175] Example 5: Preparation of host-guest nanoparticles

[0176] The heterodimer and PLX5622 (0.2 mg and 0.03 mg, respectively) were dissolved in 150 μL of the organic phase and added to a 1 mL aqueous solution containing 1 mg of galactose and cyclodextrin-functionalized polysialic acid under ultrasonication (85W, 5 seconds on, 2 seconds off). The organic solvent was then removed through dialysis tubing with a molecular weight cutoff of 3.5 kDa, and the resulting GPtMP nanoparticles were concentrated to 100 μL by ultrafiltration.

[0177] like Figure 5 As shown, dynamic light scattering (DLS) showed that the hydrated particle size of the nanoparticles was 108.13 ± 1.47 nm, the PDI was 0.12 ± 0.02, and the potential was -24.4 ± 2.0 mV; transmission electron microscopy (TEM) observation showed that it was spherical and about 70 nm.

[0178] Example 6: Active oxygen response performance of host-guest nanoparticles

[0179] The nanoparticles were placed in 10 mL of release medium (PBS buffer at pH 7.4 containing 0.5% Tween 80 and different concentrations of H2O2) and the experiment was carried out at 37 °C. The release of the protein degrader was monitored periodically by HPLC, while the particle size and morphology were evaluated by dynamic light scattering (DLS) and transmission electron microscopy (TEM), respectively.

[0180] As shown in Fig. a, the release rate of the protein degrader in the responsive nanoparticles was significantly accelerated with the increase of the concentration of H2O2, and the responsive nanoparticles showed obvious release behavior only in the presence of H2O2. Figure 6 As shown in Fig. b, the particle size of the responsive nanoparticles gradually increased and decomposed in the presence of H2O2, confirming that the host-guest nanoparticles had ROS-responsive drug release ability.

[0181] Figure 6 As shown in Fig. b, the particle size of the responsive nanoparticles gradually increased and decomposed in the presence of H2O2, confirming that the host-guest nanoparticles had ROS-responsive drug release ability.

[0182] Example 7: In vitro determination of the degradation ability of host-guest nanoparticles for phosphorylated tau protein

[0183] PC12 or SH-SY5Y cells (3 x 10 5 ) were seeded in 6-well plates and cultured for 10 hours. Subsequently, the cells were treated with different concentrations of okadaic acid (OA) for 12 or 24 hours. Then, the host-guest nanoparticles containing the protein degrader-rivastigmine heterodimer (100 nM) were used to treat the cells for 24 hours. As a control, the cells were pretreated with 100 μM N-acetyl cysteine (NAC) for 1 hour. After that, the cells were lysed with RIPA lysis buffer and the proteins were collected by centrifugation at 12,000 x g for 20 minutes. The proteins were separated by 7.5% SDS-PAGE and then transferred to a membrane, which was subjected to Western Blot detection using antibodies against phosphorylated Tau (Ser396), β-actin, PP2Aα (Leu309), and β-tubulin. After incubation with the corresponding secondary antibodies and luminescence development, the signals were detected using a gel imager (Bio-Rad Laboratories, USA).

[0184] As shown in Fig. a, the release rate of the protein degrader in the responsive nanoparticles was significantly accelerated with the increase of the concentration of H2O2, and the responsive nanoparticles showed obvious release behavior only in the presence of H2O2. Figure 7 As shown in Fig. b, the particle size of the responsive nanoparticles gradually increased and decomposed in the presence of H2O2, confirming that the host-guest nanoparticles had ROS-responsive drug release ability.

[0185] ​Example 8: In vitro evaluation of host-guest nanoparticles' ability to cross the blood-brain barrier

[0186] bEnd.3 cells were seeded at a density of 5 x 105cells per well on the upper chamber of a Transwell (No. 3401 Costar, pore size 0.4 μm, growth area 1.13 cm2) and cultured for one week. When the transendothelial electrical resistance (TEER) value exceeded 200 Ω-cm2, it was confirmed that the blood-brain barrier (BBB) monolayer had been formed. Subsequently, 1 x 105PC12 cells were seeded in the lower chamber and cultured for 24 hours. Then, 5 μM of fluorescent dye (PPa)-loaded galactose-modified nanoparticles (GPtM@PPa) were added to the upper chamber and incubated for 12 hours. As a control group, galactose-free PtM@PPa nanoparticles containing the same fluorescent dye concentration were used, as well as a group that was pre-treated with 100 μM phloretin for 1 hour before being treated with the nanoparticle drug. Finally, the PC12 cells in the lower chamber were collected and subjected to flow cytometry (FCM) analysis. 5 4

[0187] As shown in FIG. 8, the fluorescence intensity of the galactose-modified (GPtM@PPa) nanoparticle group was 5.5 times that of the non-galactose-modified (PtM@PPa) nanoparticle group. However, this enhanced effect was inhibited after treatment with the GLUT1 inhibitor phloretin, indicating the important role of galactose in promoting the crossing of nanoparticles through the BBB and their uptake by PC12 cells. Figure 8

[0188] Example 9: Evaluation of host-guest nanoparticles' ability to degrade phosphorylated tau protein in 3xTg AD mice

[0189] Nine-month-old three-transgenic (3xTg) Alzheimer's disease mice were randomly divided into different experimental groups (6-7 per group), and were injected with nanoparticles or PBS through the tail vein every 2-3 days for a total of 28 days of treatment, with a total of 12 injections. The dose of the protein degradation agent was 6.5 mg / kg, that of memantine was 1.4 mg / kg, and that of the CSF1R inhibitor was 1.6 mg / kg. Wild-type C57BL6 / 129sv mice (WT) were used as a control group and were administered the same dose, with each injection being 100 μL. After the treatment ended, the mice were euthanized by aortic perfusion with 100 mL of 0.9% saline, and the brain tissue was removed and the hippocampal region was isolated. The hippocampal tissue was homogenized with RIPA lysis buffer, then centrifuged at 12,000 x g for 20 minutes, and the supernatant was collected and subjected to Western Blot (WB) analysis of the p-tau level and PP2A activity in each treatment group.

[0190] As shown in FIG. 9, the phosphorylated tau protein level in the galactose-modified (GPtM@PPa) nanoparticle group was 0.5 times that of the non-galactose-modified (PtM@PPa) nanoparticle group. However, this enhanced effect was inhibited after treatment with the GLUT1 inhibitor phloretin, indicating the important role of galactose in promoting the crossing of nanoparticles through the BBB and their uptake by PC12 cells. Figure 9 ​​​As shown, the p-tau and its aggregation in the hippocampus of 3xTg AD mice were significantly reduced, close to the level of wild type control group, while the PP2A activity was significantly improved. The results showed that the host-guest nanoparticles could effectively inhibit the production and accumulation of p-tau.

[0191] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, but not limited to them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand: it can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A host-guest nanoparticle loaded with a protein degradation agent, comprising: 1) a targeted macrocyclic functionalized polysialic acid represented by the following formula I; 2) a protein degrader-therapeutic agent heterodimer represented by the following formula II; 3) Fluorescent dyes or therapeutic small molecules; Wherein, in the nanoparticles, the functionalized polysialic acid 1) loads the heterodimer 2) and the fluorescent dye or therapeutic small molecule 3) through host-guest interaction; ； Formula I Wherein, in Formula I, x, y, and z represent the molar percentages of the three structural units in the polymer, respectively, and satisfy x + y + z = 100%. Under this restriction, the value ranges of x, y, and z are: x = 10-80 mol%, y = 10-80 mol%, and z = 0-80 mol%. Macrocyclic hosts are macrocyclic derivatives used as hosts in host-guest chemistry; The target head is a ligand that can cross the blood-brain barrier; The macrocyclic body and the target head are respectively connected to polysialic acid by covalent bonds; ； Formula II Wherein, in Formula II, the protein degrader is a targeted protein degrader capable of binding to an E3 ubiquitin enzyme containing a Von Hippel-Lindau receptor or a thalidomide receptor; The linker is a small molecule or peptide that specifically responds to the pathological environment of neurodegenerative diseases; The therapeutic drug is a drug with functional group characteristics; One end of the linker is covalently linked to the protein degrader, and the other end is covalently linked to the therapeutic drug; The targeted macrocyclic functionalized polysialic acid serves as the host, the protein degrader-therapeutic drug heterodimer serves as the main guest, and the fluorescent dye or therapeutic small molecule serves as the functional guest, and the nanoparticles are self-assembled through host-guest interactions. The protein degrader-therapeutic drug heterodimer can target and degrade abnormal proteins and release therapeutic drugs. The fluorescent dye is used for in vivo fluorescence imaging. The therapeutic small molecule can produce a synergistic or sensitizing effect with the therapeutic drug. The mass ratio of the host, main guest, and functional guest is 1:0.1-0.5:0.01-0.

5. During the self-assembly process, the concentration of the aqueous phase system is controlled at 1-2 mg / mL, and the resulting host-guest nanoparticles have a hydrodynamic radius of 60-200 nm.

2. The host-guest nanoparticles according to claim 1, characterized in that The macrocyclic derivative is selected from any one of dextrin, crown ether, pillar aromatic hydrocarbon or cucurbituril and its derivatives, specifically any one of the following: 。 3. The host-guest nanoparticles according to claim 1, characterized in that The target head is a ligand capable of crossing the blood-brain barrier, specifically at least one of a peptide, an antibody, a fusion protein or a small molecule; The peptide is at least one of a transport peptide, a transferrin receptor targeting peptide, a glucose transporter targeting peptide, and an antibody-derived peptide; The antibody is at least one of an antibody capable of binding to transferrin receptor, low-density lipoprotein receptor-related protein, insulin receptor or glucose transporter; The fusion protein is at least one multifunctional fusion protein carrying a blood-brain barrier penetrating fragment or a variant thereof; The small molecule is at least one of the small molecules that can interact with the blood-brain barrier receptor and promote crossing the blood-brain barrier: The target head is specifically selected from at least one of the following structural formulas: 。 4. The host-guest nanoparticles according to claim 1, characterized in that The targeted protein degrader is selected from a group consisting of targeted protein degraders for degrading tau protein or phosphorylated tau protein or other proteins associated with the pathology of neurodegenerative diseases; the other proteins include β-amyloid protein (Aβ), TDP-43, α-synuclein, ApoE4 and FYN kinase; Specifically, selected from the following molecules: 。 5. The host-guest nanoparticles according to claim 1, characterized in that The linking group is selected from small molecules or polypeptides that can respond to oxidative stress in the pathological environment of neurodegenerative diseases, highly expressed matrix metalloproteinase-3, matrix metalloproteinase-9, myeloperoxidase, and cytokines IL-1β or IL-6; Specifically, at least one selected from the following molecules: 。 6. The host-guest nanoparticles according to claim 1, characterized in that The therapeutic drug is a drug with functional group characteristics, which can form a responsive protein degrader-therapeutic drug heterodimer with a protein degrader, including memantine, galantamine, pramipexole, minocycline, deferasirox, acetylglutamate, curcumin, methylene blue, rapamycin or rutin; Specifically, at least one selected from the following molecules: 。 7. The host-guest nanoparticles according to claim 1, characterized in that The fluorescent dye is any one of rhodamine, coumarin, cyanine (Cy) series dyes, fluorescein and porphyrin: 。 8. The host-guest nanoparticles according to claim 1, characterized in that The therapeutic small molecules include cholinesterase inhibitors, monoamine oxidase inhibitors, alkaloid compounds, anticonvulsants, antioxidants and CSF1R inhibitors; specifically selected from any one of the following: 。 9. A method for preparing the host-guest nanoparticles according to claim 1, characterized in that: The preparation of the targeted macrocyclic functionalized polysialic acid comprises the following steps: Polysialic acid is linked to a functionalized macrocycle and a functionalized ligand in a proportional manner to prepare a targeted macrocyclic functionalized polysialic acid. The molar percentages of the three structural units in the functionalized polysialic acid are denoted as x, y, and z, respectively, and x + y + z = 100%. Under this constraint, the values ​​of x, y, and z are in the ranges of x = 10-80 mol%, y = 10-80 mol%, and z = 0-80 mol%. When the reactive group of the functionalized macrocycle or ligand is a carboxyl group, it is dissolved in N,N-dimethylformamide (DMF) or dimethyl sulfoxide (DMSO), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) in an amount 1.2 to 3.0 times the molar equivalent of the carboxyl group, 1.2 to 3.0 equivalents of 4-dimethylaminopyridine (DMAP), 1.2 to 3.0 equivalents of 1-hydroxybenzotriazole (HOBT), and 1.2 to 3.0 equivalents of an organic base (DIEA or TEA) are added in sequence under ice-water bath conditions to activate the carboxyl group for 1 to 2 hours; then, the reaction mixture is added. 1.0 to 2.0 equivalents of ethylenediamine relative to the carboxyl group are reacted at room temperature for 12 to 24 hours; the reaction product is precipitated with glacial ether / acetone to obtain an amino macrocycle or ligand; then, the polysialic acid is dissolved in DMF or DMSO solvent, and the activation steps of EDCI, DMAP, HOBT, and an organic base are repeated. After activating the carboxyl group for 1 to 2 hours, the amino macrocycle or ligand is added, and the mixture is stirred at room temperature for 12 to 24 hours; after the reaction is completed, the product is dialyzed using an organic solvent and water, and finally lyophilized to obtain a targeted macrocyclic functionalized polysialic acid; When the reactive group of the functionalized macrocycle or ligand is a hydroxyl group or an amino group, polysialic acid is dissolved in N,N-dimethylformamide (DMF) or dimethyl sulfoxide (DMSO) solvent, and 1.2 to 3.0 times the molar equivalent of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) relative to the hydroxyl group or amino group, 1.2 to 3.0 equivalents of 4-dimethylaminopyridine (DMAP), 1.2 to 3.0 equivalents of 1-hydroxybenzotriazole (HOBT), and 1.2 to 3.0 equivalents of an organic base (DIEA or TEA) are added to activate the carboxyl group at 0°C for 1 to 2 hours. Subsequently, the macrocycle or ligand having a hydroxyl group or amino group is added, and the mixture is stirred at room temperature for 12 to 24 hours. The reaction product is dialyzed three times with an organic solvent and water, and then lyophilized to obtain a targeted macrocycle-functionalized polysialic acid. The organic solvents used in dialysis were N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), or dimethyl sulfoxide (DMSO); The preparation of the protein degrader-therapeutic drug heterodimer comprises the following steps: When one end of the linker is covalently linked to the protein degrader molecule, the other end is covalently linked to the therapeutic drug to obtain a responsive protein degrader-therapeutic drug heterodimer; When a thioketal derivative is used as a linker, a protein degrader and a therapeutic drug are coupled together; first, 1.0 equivalents of methyl mercaptan and 1.1-1.5 equivalents of acetone are reacted to generate a thiol-acetone ketal derivative, which is then reduced with 1.2-2.0 equivalents of lithium aluminum hydride in tetrahydrofuran (THF) to generate a dithiol ethyl chain with a hydroxyl group; subsequently, the hydroxyl group is protected by 1.1-1.5 equivalents of a silylating agent under alkaline conditions, and then the hydroxyl group is first coupled with a therapeutic drug containing an active hydroxyl group or an amino group through a carbonation reaction, and the activation reagent used is 1.2-2.0 equivalents of acetone. The reaction is carried out at room temperature with p-nitrophenyl carbonate or NHS carbonate. After the coupling is completed, 1.5-2.5 equivalents of ammonium tetrafluoride (TBAF) are used in THF to remove the silicon-based protecting group, exposing the free hydroxyl group. The free hydroxyl group is activated again with a carbonate reagent and coupled a second time with 1.0-1.5 equivalents of a protein degrader containing an amino or hydroxyl group. The resulting crude product is purified by column chromatography to obtain the target responsive protein degrader-therapeutic drug heterodimer, and its structure is verified by high-performance liquid chromatography (HPLC), mass spectrometry (MS), and hydrogen nuclear magnetic resonance (H-NMR). The preparation of the host-guest nanoparticles comprises the following steps: 0.01-0.5 mg of the fluorescent dye or therapeutic small molecule and 0.1-0.5 mg of the protein degrader-therapeutic drug heterodimer are dissolved in 100-200 μL of an organic solvent; under ultrasonic conditions of a frequency of 40-60 kHz and a power of 40-100 W, the organic phase is slowly added dropwise or injected into the prepared aqueous phase with a concentration of 0.5-5 mg / mL and a pH of 6.5-7.5 of a targeted macrocyclic functionalized polysialic acid aqueous solution to form an emulsified dispersion system; the dispersion system is dialyzed with a MWCO of 0.8-14 kDa or ultrafiltered at a centrifugal speed of 3000-8000 rpm to remove the organic solvent and small molecule impurities, thereby obtaining the host-guest nanoparticles; Wherein, the organic solvent is selected from N,N-dimethylformamide, N,N-dimethylacetamide or dimethyl sulfoxide; Wherein, the volume ratio of the aqueous phase to the organic solvent is 100~10:1; The ultrafiltration process is carried out by ultrafiltration tube centrifugal ultrafiltration or ultrafiltration machine ultrafiltration, and the ultrafiltration tube or ultrafiltration membrane package used in the process has a molecular weight cutoff of 1500D, 3kD, 5kD or 10KD; The molecular weight cut-off of the dialysis bag during the dialysis process is 800, 1000, 3500 or 14000.

10. Use of the host-guest nanoparticles according to claim 1 in preparing a drug for treating neurodegenerative diseases, characterized in that: The neurodegenerative disease is any one of Alzheimer's disease, Parkinson's disease and Huntington's disease.

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