A chemically stimulated oxidative Aβ 1-42 Protein nanoparticles and their preparation method and application

Abstract

The present invention discloses a method for oxidizing Aβ based on chemical stimulation 1‑42 Protein nanoparticles, preparation methods and applications thereof, the structural formula of the photosensitizer is shown in formula (I). Compared with other substances in the brain, the fluorescence of the photosensitizer is significantly enhanced after binding to Aβ aggregates, and it has good imaging capabilities for Aβ amyloid plaques. Compared with dark conditions, it has a good singlet oxygen generation capacity under light, which can photooxidize Aβ aggregates. However, the limitation of external light sources on brain tissue penetration and the toxic side effects on normal tissues limit the development of photooxidized Aβ aggregates. Furthermore, after the present invention prepares nanoparticles with photosensitizers and polymer materials, the nanoparticles are characterized by particle size, stability, and transmission electron microscopy. After incubation with hydrogen peroxide, the nanoparticles can also generate singlet oxygen through chemical excitation, inhibiting Aβ protein aggregation, and can be used to prepare drugs for the treatment of Alzheimer's disease.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a preparation method and application of a nanosystem capable of chemically stimulating and reducing abnormal aggregation of Aβ proteins. Background Art

[0002] Alzheimer's disease is a neurodegenerative disease characterized by neurotoxic pathology manifested by oxidative stress, abnormal protein aggregation, mitochondrial dysfunction, decreased neurotransmitter production, and inflammation. Theories of Alzheimer's disease pathogenesis currently receiving significant attention include the Aβ protein cascade hypothesis, the Tau protein hypothesis, the immune-inflammatory response hypothesis, the mitochondrial dysfunction hypothesis, and the oxidative stress hypothesis. The aggregation of amyloid proteins into extracellular amyloid plaques is a primary hallmark of Alzheimer's disease. Many scientists believe that reducing the abnormal Aβ aggregation at the pathological sites of Alzheimer's disease is a potential therapeutic approach. Therefore, due to its controllable and minimally invasive nature, photooxidation of Aβ aggregates has emerged as a new therapeutic approach.

[0003] Currently, the main agents for photooxidizing Aβ include small molecule photosensitizers (Rose Bengal, methylene blue, ThT, CRANAD, BODIPY, curcumin), metal complexes (Ru, M, Re, Ir, etc.), nanomaterials, and photoelectrode materials. In 2014, researchers discovered that Rose Bengal (RB) can significantly reduce Aβ amyloid aggregation under green light stimulation and can also reduce the neurotoxicity induced by abnormal Aβ protein aggregation. However, limited external laser penetration and adverse effects on normal brain tissue have hindered progress in Alzheimer's disease treatment.

[0004] Chemical excitation is to transfer energy through chemical reactions to activate prodrugs, thereby solving the limitation of external light sources. Classic peroxalate reacts with hydrogen peroxide to generate a high-energy intermediate (1,2-dioxyethanedione intermediate DOD), which transfers chemical energy to the photosensitizer through resonance energy transfer (CRET). The high concentration of H2O2 in the pathological microenvironment of Alzheimer's disease (AD) can be used to chemically excite photosensitizers to produce singlet oxygen ( 1 O2), inhibiting Aβ protein aggregation. For Alzheimer's disease (AD), nano-drug delivery system with in situ chemical excitation of prodrugs is a very promising therapeutic strategy. Summary of the Invention

[0005] The main purpose of the present invention is to break through the obstacles of the existing technology and design and synthesize a type of photosensitizer that can target Aβ aggregates.

[0006] Yet another object of the present invention is to develop a nano-delivery system for chemically stimulated prodrugs.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] The first aspect of the present invention is the synthesis of two photosensitizers.

[0009] A type of protein that can target Aβ amyloid protein and produce singlet oxygen ( 1 O2), a photosensitizer that inhibits the aggregation of Aβ aggregates. The S atom is replaced with a Se atom and cyclohexane, and the side chain 1-methyl-6-dimethylaminoquinoline is connected with a styrene bridge, and the structure is shown in Formula (I).

[0010] A photosensitizer targeting β-amyloid protein, wherein the photosensitizer has any one of the following structures:

[0011]

[0012] The preparation method of the photosensitizer comprises the following steps:

[0013]

[0014] (1) 3,6-dibromo-1,2-phenylenediamine and cyclohexanone undergo condensation reaction in a toluene solution to obtain a crude product. Manganese dioxide and ultra-dry dichloromethane are added to the crude product, and the reaction is stirred under inert gas protection. The mother core intermediate 3 (BD-6) is obtained by filtration and purification.

[0015] (2) The core intermediate 3 (BD-6) reacts with 4-formylphenylboronic acid pinacol ester under the catalysis of tetrakis(triphenylphosphine)palladium (Pd(PPh3)4) to undergo nucleophilic substitution reaction to obtain the core BD-6-CHO;

[0016] (3) 1-Methyl-6-dimethylaminoquinoline and the parent nucleus BD-6-CHO are catalytically dehydrogenated under alkaline conditions of piperidine to generate the final product, the photosensitizer BD-6-QM.

[0017] The preparation method of the photosensitizer comprises the following steps:

[0018]

[0019] (1) Add selenium dioxide solution to the ethanol solution of 3,6-dibromobenzene-1,2-diamine, and heat to 60±10°C to react to obtain intermediate 5 (BD-Se);

[0020] (2) Intermediate 5 (BD-Se) and 4-formylphenylboronic acid pinacol ester undergo a nucleophilic substitution reaction in an alkaline environment of toluene solution, catalyzed by tetrakis(triphenylphosphine)palladium, to obtain the intermediate 6 (BD-Se-CHO);

[0021] (3) 1-Methyl-6-dimethylaminoquinoline and intermediate 6 (BD-Se-CHO) were dehydrogenated by the strong base piperidine to obtain the final product, the photosensitizer BD-Se-QM.

[0022] A chemically stimulated oxidative Aβ 1-42 The protein nanoparticles are prepared by encapsulating the photosensitizer and bisoxalate or its derivatives in claim 1 with a polymer that can respond to hydrogen peroxide as a carrier to obtain the nanoparticles.

[0023] Preferably, the bisoxalate is CPPO, and the carrier is polyoxalate or polythioacetal, with the following structural formula:

[0024]

[0025] Preferably, the photosensitizer BD-Se-QM, CPPO, and polyoxalate are dissolved in an organic solvent, added dropwise to an aqueous solution of polyvinyl alcohol, and an oil / water emulsion is prepared by ultrasound, and the organic solvent is removed by dialysis to obtain nanoparticles BD-Se-QM / NPs; or

[0026] The photosensitizer BD-6-QM, CPPO, and polythioacetal are dissolved in an organic solvent, added dropwise to an aqueous solution, stirred for reaction, and then the organic solvent is removed by dialysis to prepare nanoparticles BD-6-QM / NPs.

[0027] Preferably, the preparation conditions of the nanoparticles BD-Se-QM / NPs are as follows: the organic solvent is dimethyl sulfoxide (DMSO), the photosensitizer BD-Se-QM: polyoxalate: CPPO = 1:10±2:2±1; the concentration of the aqueous solution of polyvinyl alcohol is 5±3%, polyvinyl alcohol: DMSO (volume ratio) = 6±3:1; the ultrasonic time is 30±10 min, the dialysis bag has a molecular weight of 3500±500, and the dialysis time is 48±12 hours.

[0028] Preferably, the preparation conditions of the nanoparticles BD-6-QM / NPs are as follows: the organic solvent is dimethyl sulfoxide, the photosensitizer BD-6-QM: polythioacetal: CPPO = 1:10±2:2±1, the reaction time is 30±10 min; the water: DMSO (volume ratio) = 9±3:1.

[0029] Application of the photosensitizer and nanoparticles in the preparation of beta-amyloid protein fluorescent imaging agents.

[0030] Preferably, the amyloid beta protein is Aβ 1-42 protein.

[0031] The use of the photosensitizer and nanoparticles in the preparation of drugs for treating Alzheimer's disease.

[0032] The preparation method of polyoxalate comprises the following steps:

[0033]

[0034] (1) In order to protect the hydroxyl groups on glucose, D-glucose and acetone were reacted to form the intermediate 7-isopropylidene glucose.

[0035] (2) Intermediate 7 is subjected to esterification reaction with 2,4-dihydroxybenzoic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 4-dimethylaminopyridine in dichloromethane, and intermediate 8 is obtained after purification.

[0036] (3) Intermediate 8, 1,8-octanediol, and oxalyl chloride undergo a nucleophilic addition reaction under alkaline conditions to produce a crude product. The crude product is then deprotected in trifluoroacetic acid solution to obtain the final polyoxalate product.

[0037] The preparation method of polythioacetal is characterized by comprising the following steps:

[0038]

[0039] (1) Carbon disulfide (CS2), Na2S·9H2O, and 48 ml of water were added to a round-bottom flask and heated with stirring to react. Excess carbon disulfide was removed by concentration under reduced pressure, and 70 ml of water was added to obtain a 33% sodium trithiocarbonate solution. 1,3-Dichloropropanol was then added in an ice bath and heated to 60°C. After reacting for 5 hours, the product, 1,3-dimercapto-2-propanol, was obtained by extraction and acidification.

[0040] (2) 1,3-Dimercapto-2-propanol and cinnamaldehyde were stirred and reacted for 30 minutes under the catalysis of a small amount of hydrochloric acid, and intermediate 10 was purified. Intermediate 10 and 2,2-dithiodipyridine were dissolved in ultra-dry tetrahydrofuran, and glacial acetic acid was slowly added dropwise. The reaction was stirred at room temperature for 24 hours. The thiol group was protected to obtain intermediate 11.

[0041] (3) Intermediate 11 and N,N'-disuccinimidyl carbonate were subjected to hydroxyl activation reaction in DMF (5 ml) solution and purified to obtain intermediate 12. Intermediate 12, intermediate 9 and methoxypolyethylene glycol amine were dissolved in DMF (5 mL), vacuumed, filled with inert gas, and stirred for 24 hours. After the reaction was completed, the final product was obtained by concentration under reduced pressure and purification.

[0042] The synthesis steps of the photosensitizer are as follows:

[0043] (1) Crotonaldehyde, toluene, and N,N-dimethyl-1,4-p-phenylenediamine (p-dimethylaminoaniline) are added to a hydrochloric acid solution, evacuated and filled with inert gas, and the reaction system is heated to 115±5°C for reflux reaction. After the reaction is completed, the reaction is cooled to room temperature and post-treated to obtain intermediate 1, whose structure is shown in Formula (II).

[0044]

[0045] (2) The intermediate 1 and the organic solvent are added to a round-bottom flask, and iodomethane is added. The mixture is evacuated and filled with inert gas for protection. The mixture is heated to 80±5°C for reflux reaction. After the reaction is completed, the mixture is cooled to room temperature and post-treated to obtain the intermediate 2, whose structure is shown in Formula (III).

[0046]

[0047] The ratio of crotonaldehyde to N,N-dimethyl-1,4-p-phenylenediamine (p-dimethylaminoaniline) in step (1) is 1:1.86. The amount of toluene used is 8-10 ml per gram of N,N-dimethyl-1,4-p-phenylenediamine (p-dimethylaminoaniline). The concentration of the hydrochloric acid solution is 6 mol / L. The inert gas is nitrogen or argon. The reaction time is 4-6 hours. The post-treatment is to first extract and remove the toluene, then adjust the pH to neutral in an ice bath, and then extract with dichloromethane and spin-dry to make sand. Purification is performed by column chromatography, using a 200-mesh silica gel column and a mobile phase of petroleum ether:ethyl acetate = 20:1.

[0048] The organic solvent in step (2) is an ethanol solution. The ratio of intermediate 1 to iodomethane is 1:2.45. The reaction time is 8 to 12 hours (optimized to 10 hours). The post-processing step is to spin-dry the reaction solution and make sand. The reaction solution is purified by column chromatography using 200-mesh silica gel and a mobile phase of dichloromethane:methanol = 40:1.

[0049] (3) 3,6-dibromo-1,2-phenylenediamine and cyclohexanone were added to a toluene solution, the solution was evacuated and filled with inert gas, and the reaction system was heated to 120°C for reflux reaction for 6 hours. After the reaction, a crude product was obtained. 85% active manganese dioxide was added to the organic solution of the crude product and stirred at room temperature for 2 hours under inert gas protection. The reaction mixture was then filtered, extracted, and purified to obtain the intermediate 3 mother core 4,7-dibromo-2H-benzimidazole-2-spirocyclohexane (BD-6).

[0050]

[0051] In step (3), the ratio of 3,6-dibromo-1,2-phenylenediamine to cyclohexanone is 1:2.15. The ratio of 85% active manganese dioxide to 3,6-dibromo-1,2-phenylenediamine is 1:1.3. The organic solvent is dichloromethane (ultra-dry). The purification step is column chromatography, the filler is SiO2, and the mobile phase is petroleum ether to ethyl acetate in a ratio of 15:1.

[0052] (4) A K2CO3 aqueous solution was added dropwise to a toluene solution of 4,7-dibromo-2H-benzimidazole-2-spirocyclohexane, 4-formylphenylboronic acid pinacol ester, and tetrakis(triphenylphosphine)palladium (Pd(PPh3)4). The mixture was evacuated and filled with inert gas. The reaction system was then heated to 100°C and refluxed for 12 hours. After the reaction was completed, the mixture was cooled to room temperature and post-treated to obtain the intermediate 4, the core BD-6-CHO shown in formula (IV).

[0053]

[0054] The BD-6 in step (4) comprises 4-formylphenylboronic acid pinacol ester and Pd(PPh3)4 in a ratio of 1:4:0.05. The K2CO3 aqueous solution is 6 mol / L. The post-processing step comprises drying the solvent, re-extracting and sanding. The product is purified by column chromatography using 200-mesh silica gel and a mobile phase of dichloromethane:petroleum ether in a ratio of 10:3.

[0055] (5) Selenium dioxide solution was added to the ethanol solution of 3,6-dibromobenzene-1,2-diamine and heated to 60°C for 24 hours. The reaction was then concentrated to obtain the intermediate 5, 4,7-dibromobenzo[c][1,2,5]selenadiazole (BD-Se), as shown in formula (V).

[0056]

[0057] The ratio of 3,6-dibromobenzene-1,2-diamine described in step (5) to selenium dioxide is 1:1.2.

[0058] (6) Toluene is added to BD-Se, 4-formylphenylboronic acid pinacol ester, and tetrakis(triphenylphosphine)palladium, and then sodium carbonate solution is added. The mixture is evacuated and filled with inert gas for protection. The reaction system is then heated to 100°C for 24 hours. The reaction solution is then concentrated under reduced pressure, washed, and dried to obtain the intermediate 6, the mother core BD-Se-CHO shown in formula (VI).

[0059]

[0060] The BD-Se in step (6) is 1:4:0.05, 4-formylphenylboronic acid pinacol ester benzaldehyde, and the sodium carbonate solution has a concentration of 2M.

[0061] (7) Piperidine is added dropwise to the ethanol solution of 1-methyl-6-dimethylaminoquinoline and BD-6-CHO / BD-Se-CHO, heated to 80°C in a pressure tube for 2-5 hours, and then cooled to room temperature. The solvent is removed and the product is purified to obtain the final product BD-6-QM or BD-Se-QM as shown in Formula (VII).

[0062]

[0063] The ratio of 1-methyl-6-dimethylaminoquinoline to BD-6-CHO / BD-Se-CHO in step (7) is 2.5:1. The piperidine solution is 200 μl and the ethanol solution is 8 ml. The purification step is to filter the solution and repeatedly wash the solid with ethanol until the filtrate is colorless.

[0064] A second aspect of the present invention provides methods for synthesizing two polymers that are responsive to hydrogen peroxide degradation. The first is a polyoxalate containing D-glucose in its side chain, and the second is a polythioacetal.

[0065] (8) Synthesis of polyoxalate intermediate 7: D-glucose was added to acetone, and concentrated sulfuric acid was slowly added dropwise in an ice bath. The mixture was stirred at room temperature for 4 hours. The pH was adjusted to neutral in an ice bath, and the mixture was extracted, dried, and concentrated to obtain polyoxalate intermediate 7 as shown in Formula (VIII).

[0066]

[0067] For every mol of D-glucose described in step (8), 6.6 ml of acetone and 0.2 ml of concentrated sulfuric acid were added.

[0068] (9) Intermediate 7, 2,4-dihydroxybenzoic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine, and dichloromethane were added to a round-bottom flask and stirred at room temperature for 12 hours. After the reaction, the reaction solution was concentrated under reduced pressure to obtain sand and purified to obtain polyoxalate intermediate 8 as shown in Formula (IX).

[0069]

[0070] The intermediate 7 in step (9) comprises: 2,4-dihydroxybenzoic acid: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride: 4-dimethylaminopyridine = 1: 1.2: 1.5: 0.2. The purification step is performed by column chromatography using 200-mesh silica gel and a mobile phase of petroleum ether: ethyl acetate = 12:1.

[0071] (10) The intermediate 8, 1,8-octanediol was added to a three-necked round-bottom flask, and then ultra-dry dichloromethane was added to dissolve it. The mixture was evacuated and filled with inert gas. Pyridine was added dropwise in an ice bath, and then oxalyl chloride was slowly added dropwise. The mixture was stirred and reacted at room temperature for 12 hours. After the reaction was completed, the mixture was extracted with saturated brine, dried and concentrated. The crude product and trifluoroacetic acid solution were then added to the round-bottom flask and reacted at room temperature for 2 hours. After the reaction was completed, the reaction solution was concentrated and dried to obtain the final polyoxalate product as shown in Formula (X).

[0072]

[0073] The intermediate 8 described in step (10) is prepared by mixing 1,8-octanediol, pyridine, and oxalyl chloride in a ratio of 1:0.15:16:1.1. The trifluoroacetic acid solution is a mixture of trifluoroacetic acid and water in a ratio of 1:1. For every 90 mg of the crude product added, 1.5 ml of the trifluoroacetic acid solution is used.

[0074] (11) Intermediate 7 and 5-amino-2-methylbenzoic acid were weighed and added to a round-bottom flask. Dicyclohexylcarbodiimide and 4-dimethylaminopyridine were added and dissolved in an organic solvent. The mixture was stirred at room temperature for 12 h. After the reaction, the solution was spin-dried and sanded. After purification, it was added to a trifluoroacetic acid solution to remove the isopropylidene group, thereby obtaining intermediate 9 as shown in Formula (XI).

[0075]

[0076] The intermediate 7 in step (11) is prepared by mixing 5-amino-2-methylbenzoic acid, dicyclohexylcarbodiimide, and 4-dimethylaminopyridine in a ratio of 1:2:1:0.2. The organic solvent is DCM:DMF in a ratio of 1:1, with 0.8 ml of the organic solvent added per mole of intermediate 7. The purification step is performed by column chromatography using 200-mesh silica gel and a mobile phase of petroleum ether:ethyl acetate in a ratio of 8:1. The trifluoroacetic acid and water are in a ratio of 1:1.

[0077] (12) Synthesis steps of 1,3-dimercapto-2-propanol of polythioacetal: Carbon disulfide CS2, Na2S·9H2O and 48 ml of water were added to a round-bottom flask, heated to 40°C and stirred for 5 hours. The excess carbon disulfide was removed by concentration under reduced pressure, and 70 mL of water was added to obtain a 33% sodium trithiocarbonate solution. Subsequently, 1,3-dichloropropanol was added in an ice bath and heated to 60°C for 5 hours. After the reaction was completed, the solution was cooled to room temperature. It was then washed with ethyl acetate (5×100 mL), the aqueous phase was collected and slowly acidified with concentrated sulfuric acid, and then extracted with ether until the oil phase was colorless. The crude product obtained by concentration and drying was a light brown oil, which was further purified by distillation under reduced pressure, heated to 160°C, and the light yellow liquid was collected as the obtained product 1,3-dimercapto-2-propanol.

[0078]

[0079] In step (12), the ratio of carbon disulfide to sodium sulfide nonahydrate is 1:0.9. The ratio of carbon disulfide to 1,3-dichloropropanol is 0.3:1.

[0080] (13) 1,3-Dimercapto-2-propanol and cinnamaldehyde were added to a round-bottom flask, and a drop of hydrochloric acid was added. The mixture was evacuated, filled with inert gas, and stirred at room temperature for 30 min. The viscous solution was washed with water to remove most of the hydrochloric acid, dissolved in tetrahydrofuran, and precipitated in excess cold n-hexane to obtain intermediate 10. Intermediate 10 and 2,2-dithiodipyridine were added to a round-bottom flask and dissolved in ultra-dry tetrahydrofuran. Glacial acetic acid was then slowly added dropwise and stirred at room temperature for 24 h. The mixture was concentrated under reduced pressure and purified to obtain intermediate 11.

[0081]

[0082] In step (13), the ratio of 1,3-dimercapto-2-propanol to cinnamaldehyde is 1:1.05. The ratio of the intermediate 10 to 2,2-disulfide dipyridine is 1:2. The drop of hydrochloric acid is 8 μl, and the glacial acetic acid is 100 μl. The purification is performed by gel column chromatography. The gel used is 1% cross-linked: it separates lipophilic polymers with a molecular weight of 600-14,000, and the eluent is tetrahydrofuran.

[0083] (14) Intermediate 11 and N,N'-disuccinimidyl carbonate were dissolved in DMF (5 ml) solution, evacuated, filled with inert gas, and stirred to react for 24 hours. Then, the reaction solution was concentrated under reduced pressure and purified to obtain intermediate 12. Intermediate 12, intermediate 9, and methoxypolyethylene glycolamine were dissolved in DMF (5 mL), evacuated, filled with inert gas, and stirred to react for 24 hours. After the reaction was completed, the solution was concentrated under reduced pressure and purified to obtain the final product.

[0084]

[0085] The intermediate 11 described in step (14): N,N'-disuccinimidyl carbonate = 1:20.

[0086] The intermediate 12 described in step (14): intermediate 9: methoxypolyethylene glycol amine = 1:15:1.5.

[0087] The purification in step (14) is performed by gel column chromatography. The gel used is 1% cross-linked: separation molecular weight lipophilic polymers of 600-14000, and the eluent is N,N-dimethylformamide.

[0088] The third aspect of the present invention is to provide chemically activated nanoparticles. The photosensitizer and bis(oxalate) CPPO of Invention 1 are encapsulated with the polymer capable of responding to hydrogen peroxide of Invention 2 as a carrier to obtain the nanoparticles BD-Se-QM / NPs.

[0089] (15) The photosensitizers BD-Se-QM, CPPO, and polyoxalate were dissolved in an organic solvent and dropped into an aqueous solution of polyvinyl alcohol. Ultrasonication was used to prepare an excellent oil / water emulsion, and then the organic solvent was removed by dialysis.

[0090] The organic solvent in step (15) was dimethyl sulfoxide (DMSO), used in an amount of 0.5 ml. The BD-Se-QM:polyoxalate:CPPO ratio was 1:10:2. The polyvinyl alcohol solution had a concentration of 5%, and the polyvinyl alcohol:DMSO (volume ratio) was 6:1. The ultrasound was performed using an ultrasonic disruptor for 30 minutes. The dialysis bag had a molecular weight of 3500, and the dialysis time was 48 hours.

[0091] (16) The photosensitizer BD-6-QM, CPPO, and polythioacetal were dissolved in an organic solvent, added dropwise to the aqueous solution, stirred for 30 min, and then the organic solvent was removed by dialysis to obtain the nanoparticles BD-6-QM / NPs.

[0092] The organic solvent described in step (16) is dimethyl sulfoxide, and the amount used is 1 ml.

[0093] BD-6-QM described in step (16): polythioacetal: CPPO = 1:10:2.

[0094] The water described in step (16) : DMSO (volume) = 9:1.

[0095] The use of the photosensitizer targeting Aβ amyloid protein in the preparation of a product for diagnosing Alzheimer's disease (AD). The Aβ amyloid protein is Aβ 1-42 Aggregate.

[0096] The products include fluorescent probes, detection (diagnosis) reagents and kits, etc. The photosensitizer targeting Aβ amyloid protein is used in the preparation of drugs for diagnosing and treating Alzheimer's disease (AD).

[0097] The nanoparticles are used in the preparation of drugs for diagnosing and treating Alzheimer's disease (AD).

[0098] The photooxidation treatment preferably utilizes 520 nm, 1000 mW white light. The chemical excitation treatment preferably utilizes 500 μM H 2 O 2 .

[0099] Compared with the prior art, the present invention has the following advantages and effects:

[0100] (1) The present invention includes two unreported photosensitizers targeting Aβ amyloid protein, which can be used for imaging and photooxidation therapy of Aβ protein and for the diagnosis and treatment of early Alzheimer's disease.

[0101] (2) The photosensitizer of the present invention reacts with Aβ in PBS solution 1-42 After the aggregates bind, the fluorescence is significantly enhanced, and it has obvious selectivity for other ions, amino acids, Aβ oligomers, and monomers in the brain of AD mice. At the same time, it has good imaging of the cortex and brain of APPswe / PSEN1 transgenic AD mice, and can be used to prepare drugs for diagnosing Alzheimer's disease (AD). Under white light irradiation and excitation, singlet oxygen ( 1 O2), used to inhibit Aβ 1-42 Aggregates, reducing the neurotoxicity caused by Aβ aggregation, and have the potential to treat Alzheimer's disease (AD).

[0102] (3) The nanoparticles described in the present invention have good biocompatibility. Without the need for white light irradiation and excitation, hydrogen peroxide (H2O2) can also produce singlet oxygen through chemical excitation, reducing the aggregation of Aβ amyloid protein and reducing neurotoxicity. This solves the limitations of white light's limited penetration ability and toxicity to normal animal tissues, and is an excellent strategy for treating Alzheimer's disease (AD). BRIEF DESCRIPTION OF THE DRAWINGS

[0103] Figure 1 For Aβ 1-42 Aggregate-specific binding fluorescence and viscosity response graphs. Figure 1 (a) BD-6-QM and Aβ 1-42 Combining the fluorescence spectra before and after, Figure 1 (b) BD-6-QM and Aβ 1-42 Combined with the quantitative comparison of fluorescence intensity before and after Figure 1 (c) BD-6-QM and Aβ 1-42 , comparison of fluorescence intensity of common amino acids and ion binding, Figure 1 (d) The fluorescence intensity change of BD-6-QM with increasing viscosity. Figure 1 (e) BD-Se-QM and Aβ 1-42 Combining the fluorescence spectra before and after, Figure 1 (f) BD-Se-QM and Aβ 1-42 Combined with the quantitative comparison of fluorescence intensity before and after Figure 1 (g) BD-6-QM and Aβ 1-42 , comparison of fluorescence intensity of common amino acids and ion binding, Figure 1(h) Changes in fluorescence intensity of BD-Se-QM with increasing viscosity.

[0104] Figure 2 Singlet oxygen generation ability of photosensitizer and photooxidation of Aβ 1-42 Aggregate. Figure 2 (a) is the UV / Vis change of ABDA, BD-6-QM and RB at 378nm under white light irradiation. Figure 2 (b) Circular dichroism (CD) spectroscopy monitoring of the changes in the Aβ secondary structure of BD-6-QM under different conditions. Figure 2 (c) is the UV / Vis change of ABDA, BD-Se-QM and RB at 378nm under white light irradiation. Figure 2 (d) Circular dichroism (CD) spectroscopy monitoring of the changes in the secondary structure of Aβ under different conditions using BD-Se-QM.

[0105] Figure 3 Images of the cortex and brain of APPswe / PSEN1 transgenic AD mice treated with photosensitizers. Figure 3 (a) Histological staining of the brain and cortex of APPswe / PSEN1 transgenic AD mice using Thio-S and BD-Se-QM. Figure 3 (b) Histological staining of the brain and cortex of APPswe / PSEN1 transgenic AD mice using Thio-S and BD-6-QM.

[0106] Figure 4 Nanoparticle characterization diagram. Figure 4 (a) The hydrodynamic diameter of BD-6-QM / NPs was measured by dynamic light scattering (DLS). Figure 4 (b) Dynamic light scattering (DLS) test of the stability of BD-6-QM / NPs. Figure 4 (c) is the morphology characterization of BD-6-QM / NPs by transmission electron microscopy (TEM). Figure 4 (d) The hydrodynamic diameter of BD-Se-QM / NPs was measured by dynamic light scattering (DLS). Figure 4 (e) Dynamic light scattering (DLS) was used to test the stability of BD-Se-QM / NPs. Figure 4 (f) Transmission electron microscopy (TEM) morphology characterization of BD-Se-QM / NPs.

[0107] Figure 5 shows the ability of nanoparticles to generate singlet oxygen by chemical excitation and their ability to inhibit Aβ aggregation. Figure 5(a) shows the UV / Vis changes of DPBF with BD-6-QM / NPs and H2O2 at 378 nm, Figure 5(b) shows the UV / Vis changes of DPBF with BD-Se-QM / NPs and H2O2 at 378 nm, Figure 5(c) shows the morphology of BD-6-QM / NPs incubated with Aβ under different conditions (scale bar: 200 nm), Figure 5(d) shows the morphology of BD-Se-QM / NPs incubated with Aβ under different conditions (scale bar: 200 nm), Figure 5(e) shows the morphology of BD-6-QM / NPs incubated with Aβ under different conditions (scale bar: 500 nm), and Figure 5(f) shows the morphology of BD-Se-QM / NPs incubated with Aβ under different conditions (scale bar: 500 nm).

[0108] Figure 6 Polyoxalate 1 HNMR.

[0109] Figure 7 For polythioacetal intermediate 11 1 HNMR.

[0110] Figure 8 The final product of polythioacetal 1 HNMR. DETAILED DESCRIPTION

[0111] The present invention will be further explained in detail below with reference to examples, but the embodiments of the present invention are not limited thereto. The reagents, equipment, and consumables used in the present invention are all conventional reagents, equipment, and consumables purchased from commercial companies. Unless otherwise specified, the experimental methods used in the present invention are all conventional experimental methods or experimental methods recommended by the manufacturer's instructions.

[0112] Example 1:

[0113] (1) Dissolve 11.6 g, 85.2 mmol) of N,N-dimethyl-1,4-p-phenylenediamine (p-dimethylaminoaniline) in a hydrochloric acid solution (6 M, 500 ml), add crotonaldehyde (13 ml), evacuate and fill with inert gas, and inject 80 ml of ultra-dry toluene. Then heat the reaction system to 115±5°C for reflux reaction. Monitor the reaction progress by thin layer chromatography (TLC) every 1 h until the reaction is completed. After the reaction is completed, cool to room temperature, extract and remove toluene, adjust the pH to neutral with water in an ice bath, extract with dichloromethane, and spin-dry to make sand. Purify by column chromatography, silica gel column 200 mesh, mobile phase petroleum ether: ethyl acetate = 20:1, and purify to obtain a yellow solid 1.

[0114]

[0115] N,N,2-trimethylquinolin-6-amine: yellow solid: yield: 35.00%; 1 H-NMR (400MHz, Chloroform-d): δ = 7.91 (dd, J = 14.3, 8.9 Hz, 2H), 7.36 (dd, J = 9.3, 2 .8Hz,1H),7.18(d,J=8.4Hz,1H),6.81(d,J=2.9Hz,1H),3.07(s,6H),2.70(s,3H).

[0116] (2) The obtained compound 1 (5.2 g, 27.92 mmol) was fully dissolved in 80 ml of ethanol solution, and then iodomethane (9.72 g, 68.48 mmol) was added. The mixture was evacuated and filled with inert gas for protection. The mixture was heated to 80±5°C and refluxed for 10 hours. After the reaction was completed, the mixture was cooled to room temperature and the reaction solution was spin-dried and sanded. The mixture was purified by column chromatography using 200 mesh silica gel and a mobile phase of dichloromethane:methanol = 40:1 to obtain an orange solid 2.

[0117]

[0118] 6-(dimethylamino)-1,2-dimethylquinolin-1-ium: orange solid; yield: 26.08%; 1 HNMR (400MHz, DMSO-d6): δ = 8.73 (d, J = 8.6Hz, 1H), 8.35 (d, J = 9.7Hz, 1H), 7.86 (d, J = 8.6Hz, 1H), 7. 77(dd,J=9.9,3.0Hz,1H),7.27(d,J=3.0Hz,1H),4.36(s,3H),3.13(d,J=1.2Hz,6H),2.95(s,3H).

[0119] (3) Add (413.4 mg, 1.56 mM) 3,6-dibromo-1,2-phenylenediamine and (325 μl, 3.35 mM) cyclohexanone to 75 ml toluene solution and dissolve thoroughly. Vacuum and fill with inert gas, then heat the reaction system to 120 ° C and reflux for 6 hours. After the reaction, a crude product is obtained. 85% active manganese dioxide (1.3 g, 1.2 mM) and 8 ml of ultra-dry dichloromethane are added to the crude product and stirred at room temperature for 2 hours under inert gas protection. The reaction mixture is then filtered, 40 ml of dichloromethane is added to the filtrate, extracted with 40 ml of water, and dried with anhydrous sodium sulfate. Purify by column chromatography (filler: SiO2, mobile phase: petroleum ether: ethyl acetate = 15:1) to obtain the core intermediate 3 (BD-6).

[0120]

[0121] 4,7-dibromospiro[benzo[d]imidazole-2,1'-cyclohexane](BD-6): yellow solid; yield: 83.0%; 1 H-NMR (400MHz, CDCl3-d) δ7.21 (s, 2H), 1.99 (d, J = 6.0Hz, 2H), 1.78 (s, 4H), 1.74 (s, 4H).

[0122] (4) K2CO3 aqueous solution (5.2 ml, 2M) was added dropwise to a toluene solution (12 ml) of 4,7-dibromo-2H-benzimidazole-2-spirocyclohexane (549.8 mg, 1.6 mmol), 4-formylphenylboronic acid pinacol ester (1.5 mg, 6.4 mmol) and tetrakis(triphenylphosphine)palladium (Pd(PPh3)4) (96.6 mg, 0.084 mmol), and the mixture was evacuated and filled with inert gas for protection. The reaction system was then heated to 100°C for reflux reaction for 12 hours. After the reaction was completed, the mixture was cooled to room temperature, the solvent was dried, and the mixture was extracted with dichloromethane and water again, dried and sanded. The mixture was purified by column chromatography using 200 mesh silica gel and a mobile phase of dichloromethane: petroleum ether = 10:3 to obtain the intermediate 4 mother core BD-6-CHO.

[0123]

[0124] 4,4'-(spiro[benzo[d]imidazole-2,1'-cyclohexane]-4,diyl)dibenzaldehyde (BD-6-CHO): yellow solid; yield: 65.86%; 1 H NMR (400MHz, CDCl3-d) δ 10.10 (s, 2H), 8.16 (s, 4H), 8.00 (d, J = 8.4Hz, 4H), 7.45 (s, 2H), 1.91 (m, 4H), 1.69 (m, 2H), 1.64 (s, 4H).

[0125] (5) Selenium dioxide solution (1.34 g, 12 mmol) was added to an ethanol solution of 3,6-dibromobenzene-1,2-diamine (2.66 g, 10 mmol) and heated to 60°C for 24 hours. After the reaction was completed, the mixture was cooled to room temperature and extracted with ethyl acetate. The organic phase was dried and concentrated to obtain the intermediate 5-dibromobenzo[c][1,2,5]selenadiazole (BD-Se).

[0126]

[0127] 4,7-dibromobenzo[c][1,2,5]selenadiazole (BD-Se): yellow solid; yield: 61.05%; 1 H NMR (400MHz, CDCl3) δ7.63 (s, 2H, phenylene ring).

[0128] (6) 15 ml of toluene was added to 4,7-dibromo-[c][1,2,5]benzoselenadiazole (0.41 g, 1.2 mmol), 4-boronic acid benzaldehyde (0.55 g, 2.4 mmol), and tetrakis(triphenylphosphine)palladium (Pd(PPh3)4) (0.072, 0.063 mmol), and then sodium carbonate (2 M) solution was added. The mixture was evacuated and filled with inert gas for protection. The reaction system was heated to 100 ° C for 24 h, and the reaction solution was concentrated under reduced pressure, washed, and dried to obtain the intermediate 6 (BD-Se-CHO).

[0129]

[0130] 4,4'-(benzo[c][1,2,5]selenadiazole-4,7-diyl)dibenzaldehyde (BD-Se-CHO): orange solid; yield: 64.50%; 1 H NMR (400MHz, CDCl3) δ10.16(s,2H),8.21-8.19(d,4H),8.11-8.09(d,4H),7.94(s,2H).

[0131] (7) Add 200 μl of piperidine dropwise to an ethanol solution of 1-methyl-6-dimethylaminoquinoline (0.148 g, 0.48 mmol) and the core BD-6-CHO (0.067 g, 0.19 mmol). Heat to 80°C and reflux for 12 h. Cool to room temperature. Filter the precipitated red solid and wash with ethanol until the filtrate becomes colorless. Dry the mixture to obtain the final product, BD-6-QM.

[0132]

[0133] 2,2'-((1E,1'E)-(spiro[benzo[d]imidazole-2,1'-cyclohexane]-4,7-diylbis(4,1-phenylene))bis(ethene-2,1-diyl))bis(6-(dimethylamino)-1-methylquinolin-1-ium)(BD-6-QM): red solid; yield: 47.80%; 1 H NMR (400MHz, DMSO-d6) δ8.71(d,J=9.9Hz,2H),8.33(d,J=8.7Hz,4H),8.20(s,4H),8.01(t,J=8.8Hz,8H),7.7 5–7.65(m,4H),7.26(d,J=11.4Hz,2H),4.53(s,6H),3.13(d,J=11.5Hz,12H),1.96(s,4H),1.81–1.54(m,6H).

[0134] (8) Add 200 μl of piperidine dropwise to an ethanol solution of 1-methyl-6-dimethylaminoquinoline (0.090 g, 0.45 mmol) and the core BD-Se-CHO (0.069 g, 0.18 mmol). Heat to 80°C and reflux for 5 h. Cool to room temperature. Filter the precipitated red solid and wash with ethanol until the filtrate becomes colorless. Dry the mixture to obtain the final product, BD-Se-QM.

[0135]

[0136] 2,2'-((1E,1'E)-(benzo[c][1,2,5]selenadiazole-4,7-diylbis(4,1phenylene))bis(ethene-2,1-diyl))bis(6-(dimethylamino)-1-methylquinolin-1-ium)(BD-Se-QM): red solid; yield: 49.85%; 1 H NMR(400MHz, DMSO-d6)δ8.76(d,J=8.6Hz,2H),8.39(t,J=9.2Hz,4H),8.08(d,J=6.9Hz, 10H),7.99–7.90(m,4H),7.76(d,J=7.5Hz,2H),7.29(s,2H),4.55(s,6H),3.16(s,12H).

[0137] (9) D-glucose (90 mg, 0.5 mmol) and 3.3 ml of acetone were added to a round-bottom flask. Concentrated sulfuric acid (0.1 ml) was slowly added dropwise using a syringe in an ice bath. The mixture was stirred at room temperature for 4 h. The pH of the reaction solution was adjusted to neutral in an ice bath, and then extracted with ethyl acetate and water. The organic phase was retained and concentrated under reduced pressure to obtain intermediate 7.

[0138]

[0139] ((3aS,5R,5aR,8aS,8bR)-2,2,7,7-tetramethyltetrahydro-5H-bis([1,3]dioxolo)

[0140] [4,5-b:4',5'-d]pyran-5-yl)methanol: white solid; yield: 95.30% 1 H NMR(δ[ppm],400MHz,CDCl3):1.33(s,6H,2×CH3),1.47(s,3H,CH3),1.52(s,3H,CH3),2.35(br s,1H,OH),3.83–3.89(m,3H,CH2+CH),4.26(dd,J=7.9Hz,J=1.7Hz,1H,CH),4.33(dd,J=5 .0Hz,J=2.4Hz,1H,CH),4.60(dd,J=7.9Hz,2.4Hz,1H,CH),5.60(d,J=5.0Hz,1H,anomeric CH).

[0141] (10) Intermediate 7 (0.087 g, 0.33 mmol), 2,4-dihydroxybenzoic acid (0.062 g, 0.4 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.096 g, 0.5 mmol), 4-dimethylaminopyridine (0.008 g, 0.066 mmol), and 1.5 ml of dichloromethane were added to a round-bottom flask and stirred at room temperature for 12 hours. After the reaction was completed, the reaction solution was evaporated under reduced pressure to remove the solvent and sanded. The product was purified by column chromatography using 200-mesh silica gel and a mobile phase of petroleum ether:ethyl acetate = 12:1 to obtain intermediate 8.

[0142]

[0143] ((3aS,5R,5aR,8aS,8bR)-2,2,7,7-tetramethyltetrahydro-5H-bis([1,3]dioxolo)

[0144] [4,5-b:4',5'-d]pyran-5-yl)methyl 2,4-dihydroxybenzoate: pale yellow oil; yield: 52.30%; 1 H NMR(400MHz,Chloroform-d)δ7.75(d,J=8.6Hz,1H),6.42(s,1H),6.06(d,J=3.7Hz,1H),5.63(s,1H),4.64(d,J=3.7Hz,1H),4.54(dd ,J=11.8,3.2Hz,1H),4.43(s,2H),4.30(d,J=3.8Hz,1H),3.94(t,J=6.1Hz,1H),1.53(s,3H),1.41(s,3H),1.37(s,3H),1.28(s,3H).

[0145] (10) Intermediate 8 (0.25 g, 0.6 mmol) and 1,8-octanediol (0.013 g, 0.09 mmol) were added to a three-necked round-bottom flask and dissolved with ultra-dry dichloromethane. The mixture was evacuated and filled with inert gas. Pyridine was added dropwise in an ice bath, and then oxalyl chloride was slowly added dropwise. The mixture was stirred and reacted at room temperature for 12 hours. After the reaction was completed, the mixture was extracted with saturated brine, and the organic phase was dried and concentrated. The crude product (0.09 mg) and 1.5 ml of trifluoroacetic acid solution (trifluoroacetic acid: water = 1:1) were then added to the round-bottom flask and reacted at room temperature for 2 hours. After the reaction was completed, the reaction solution was concentrated and dried to obtain the final polyoxalate product.

[0146]

[0147] (11) Intermediate 7 (0.05 g, 0.2 mmol) and 5-amino-2-methylbenzoic acid (0.061 g, 0.4 mmol) were weighed and added to a round-bottom flask. Dicyclohexylcarbodiimide (0.041 g, 0.2 mmol) and 4-dimethylaminopyridine (0.004 g, 0.04 mol) were added and dissolved in 0.8 ml of DCM and 0.8 ml of DMF. The mixture was stirred at room temperature for 12 h. After the reaction was completed, the reaction solution was concentrated and sanded, purified by column chromatography, and then purified with petroleum ether:ethyl acetate = 1:8. After that, the mixture was reacted with trifluoroacetic acid:water = 1:1 for 2 h to remove the isopropylidene group to obtain intermediate 9.

[0148]

[0149] ((2R,3S,4S,5R,6R)-3,4,5,6-tetrahydroxytetrahydro-2H-pyran-2-yl)methyl5-amino-2-methylbenzoate: colorless oil; yield: 43.80%; 1 H NMR(400MHz,DMSO-d6)δ7.09(d,J=2.5Hz,1H),6.98(d,J=8.2Hz,1H),6.71(d d,J=8.1,2.6Hz,1H),5.95(d,J=3.8Hz,1H),5.22(d,J=3.0Hz,1H),5.18(s,2 H),4.67(d,J=3.7Hz,1H),4.33(dt,J=7.7,5.5Hz,1H),4.20(dd,J=7.7,3.0H z,1H),4.05(dd,J=8.5,6.1Hz,1H),3.92(dd,J=8.5,5.1Hz,1H),2.34(s,3H).

[0150] (11) Synthesis steps of polythioacetal 1,3-dimercapto-2-propanol: Dissolve carbon disulfide CS2 (13.2 ml, 220 mmol) and Na2S·9H2O (48 g, 200 mmol) in water (48 ml), heat to 40 °C and stir for 5 hours. Concentrate under reduced pressure to remove excess carbon disulfide, and then add 70 mL of water to obtain a 33% sodium trithiocarbonate solution. Then add 1,3-dichloropropanol (6.72 ml, 70 mmol) in an ice bath and heat to 60 °C for 5 hours. After the reaction is completed, the solution is cooled to room temperature and then washed with ethyl acetate (5×100 mL). The aqueous phase is collected and slowly acidified with concentrated sulfuric acid in an ice bath, and then extracted with ether until the oil phase is colorless. The crude product obtained by concentration and drying is a light brown oil. It is further purified by vacuum distillation and heated to 160 °C. The light yellow liquid is collected as the obtained product 1,3-dimercapto-2-propanol.

[0151]

[0152] 1,3-dimercaptopropan-2-ol: pale yellow oil; yield: 30.46%; 1 H NMR (400MHz, Chloroform-d): δ3.72 (tq, J=7.5, 4.3Hz, 1H), 2.85–2.63 (m, 5H), 1.55–1.42 (m, 2H).

[0153] (12) 1,3-Dimercapto-2-propanol (0.26 g, 2.09 mmol) and cinnamaldehyde (0.26 g, 2 mmol) were added to a round-bottom flask, 8 μl of hydrochloric acid was added dropwise, the mixture was evacuated and filled with inert gas, and the mixture was stirred at room temperature for 30 min. The viscous solution was washed with water to remove most of the hydrochloric acid, and the mixture was dissolved in 2 ml of tetrahydrofuran and precipitated in an excess of cold n-hexane. The n-hexane was removed, and the precipitate was dissolved in a very small amount of tetrahydrofuran and purified using a gel column with a molecular weight of 600-14000, using tetrahydrofuran as the eluent, to obtain intermediate 10 (0.2 g, 38.36%). Intermediate 2 (0.4 g, 0.09 mmol) and 2,2-dithiodipyridine (0.39 g, 0.18 mmol) were added to a round-bottom flask, evacuated, and filled with inert gas. Dissolved in ultra-dry tetrahydrofuran, 100 μl of glacial acetic acid was slowly added dropwise, and the mixture was stirred at room temperature for 24 hours. The mixture was concentrated and purified under reduced pressure to yield Intermediate 11 (0.18 g, 22.78%).

[0154]

[0155] (13) Intermediate 11 (0.18 g, 0.04 mmol) and N,N'-succinimidyl carbonate (DSC) (0.19, 1.2 mmol) were dissolved in 5 ml of DMF solution, evacuated, filled with inert gas, and stirred for 24 hours. The reaction solution was then concentrated under reduced pressure and purified using a gel with a 1% cross-linking: molecular weight separation of 600-14000 lipophilic polymer, and N,N-dimethylformamide as the eluent to obtain intermediate 12 (0.2 g, 54.05%). Intermediate 12 (0.2 g, 0.033 mmol), intermediate 9 (0.04 g, 0.12 mmol) and methoxypolyethylene glycol amine (0.09 g, 0.04 mmol) were dissolved in DMF (5 mL), evacuated, filled with inert gas, and stirred for 24 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure and purified using a 1% cross-linked gel with a separation molecular weight of 600-14000 lipophilic polymer. The eluent was N,N-dimethylformamide. After removing DMF, the final product (0.1 g, yield: 30.30%) was obtained.

[0156]

[0157] 1. The excitation wavelength and emission wavelength of the compounds BD-6-QM and BD-Se-QM synthesized above are shown in Table 1:

[0158] Table 1 Excitation wavelength and emission wavelength of photosensitizer

[0159] name λex(nm) λem(nm) BD-6-QM 518 740 BD-Se-QM 518 755

[0160] λex: excitation wavelength of the probe; λem: maximum emission wavelength of the probe.

[0161] 2. Determine the effect of BD-6-QM and BD-Se-QM on Aβ 1-42 The mother solutions of BD-6-QM and BD-Se-QM were diluted to 100 μM and used separately. The mother solution concentrations of different proteins were all 100 μM. 6 μM photosensitizer and 60 μM A0 1-42 The protein was diluted to 1 μM and 10 μM with PBS. The photosensitizer was measured by fluorescence spectrophotometer. 1-42 Fluorescence spectra of proteins before and after incubation. 1-42 After the addition of Aβ 1-42 The fluorescence intensity increased 18 times.

[0162] 3. Common metal ions Zn 2+ 、Al 3+ 、Fe 2+ 、Fe 3+ 、Na + , K + 、Cu + Mg 2+ 、Ni + 、Cd 2+ 、Pd 2+ , Gla, Tle arginine (Arg), glutamic acid (Glu), glutathione (GSH), alanine (Ala), aspartic acid (Asp), cysteine ​​(Cys), lysine (Lys), isoleucine (Ile), methionine (Met), serine (Ser), Aβ 1-42 After the aggregates were diluted to 10 μM and incubated with photosensitizers BD-6-QM and BD-Se-QM (1 μM) for 10 minutes, the fluorescence intensity of the photosensitizers to common amino acids, metal ions, and different proteins was measured using a 96-well plate and a multifunctional microplate assay system. The excitation wavelength was the maximum absorption wavelength of the photosensitizer, and the receiving emission wavelength was the emission wavelength of the photosensitizer. 1-42 The fluorescence intensity after protein addition was significantly higher than that of BD-6-QM and other metal ions and amino acids. 1-42 The fluorescence intensity after protein was significantly higher than that of BD-6-QM and other metal ions and amino acids.

[0163] 4. The photosensitizers (BD-6-QM and BD-Se-QM) were diluted to 1 μM in water / glycerol systems of varying viscosities (0:100 to 99:1). Fluorescence intensity was then measured using a fluorescence spectrophotometer under different conditions. The fluorescence intensity of BD-6-QM increased with increasing viscosity. The fluorescence intensity of BD-Se-QM also increased with increasing viscosity.

[0164] 5. The singlet oxygen production of photosensitizers BD-Se-QM and BD-6-QM was measured. Prepare the mother solution (10mM) of 9,10-anthracenediyl-bis(methylene)dicarboxylic acid (ABDA) indicator under light-proof conditions, and use Rose Bengal RB (100μM) as the control group for photosensitizers BD-Se-QM and BD-6-QM. Take 6μl of indicator and 60μl of photosensitizer and dilute them to 100μM and 10μM with PBS respectively. Under white light (10mW / cm 2 ) was irradiated with an ELISA to test the time-dependent ultraviolet absorption of ABDA (378 nm). Within 240 s, the absorbance of the photosensitizer BD-6-QM decreased significantly over time and almost reached a plateau within 60 s. Compared with Rose Bengal RB, the rate of decrease of BD-6-QM was faster, indicating that it may have a better efficiency in producing singlet oxygen. Within 300 s, the absorbance of the photosensitizer BD-Se-QM decreased significantly over time. Compared with Rose Bengal RB, the rate of decrease of BD-Se-QM was faster, indicating that it may have a better efficiency in producing singlet oxygen.

[0165] 6. Aβ 1-42 (40 μM) and / or photosensitizers (BD-6-QM, BD-Se-QM) (10 μM) were tested in 300 μl of distilled water using circular dichroism (CD). 1-42 (40μM)+Dark(2)Aβ 1-42 (40μM) + photosensitizer (BD-6-QM, BD-Se-QM) + darkness (3) Aβ 1-42 (40μM) + photosensitizer (BD-6-QM, BD-Se-QM) + light. All samples were kept at 37°C and the light exposure time was 24 hours. Aβ in natural state 1-42 There is an obvious positive peak at 190nm and an obvious positive peak at 220nm. 1-42 After 24 hours of light exposure, the negative peak at 220 nm and the positive peak at 190 nm decreased significantly, while the peak shapes under other conditions remained basically unchanged. 1-42 After 24 hours of illumination, the negative peak at 220 nm and the positive peak at 190 nm decreased significantly, while the peak shapes under other conditions remained basically unchanged.

[0166] 7. Brain and cortical sections from APPswe / PSEN1 double transgenic AD model mice (C57BL6, 13-month-old, male) were purchased for in vitro fluorescence imaging. Paraffin sections were first dewaxed and soaked in m-xylene for 5 minutes, followed by 5 minutes in anhydrous ethanol (repeated twice); 5 minutes in 95% ethanol (repeated twice); 5 minutes in 85% ethanol (repeated twice); and finally 5 minutes in PBS (repeated twice). For staining, a PBS solution containing a photosensitizer (5 μM) was added dropwise to the sections, incubated for 30 minutes, and then washed three times with PBS. A PBS solution containing the positive control compound Thio-S was then added dropwise to the sections, incubated for 30 minutes, washed three times with PBS, and coverslipped. Sections were imaged using a laser confocal microscope. The commercial probe Thio-S stains Aβ plaques in the cortex and brain of AD mice in the green channel, while BD-6-QM can well image Aβ plaques in the red channel. Comparison of the two revealed a high degree of overlap. The commercial probe Thio-S stained Aβ plaques in the cortex and brain of AD mice in the green channel, and BD-Se-QM can well image Aβ plaques in the red channel. Comparing the two, it was found that they had a high degree of overlap.

[0167] The third aspect of the present invention provides chemically activated nanoparticles. The photosensitizer and bis(oxalate) ester CPPO of Invention 1 are encapsulated using the polymer capable of responding to hydrogen peroxide of Invention 2 as a carrier to obtain the nanoparticles BD-Se-QM / NPs and BD-6-QM / NPs.

[0168] 8. The prepared nanoparticles (BD-6-QM / NPs, BD-Se-QM / NPs) were diluted to 5 μM with distilled water, and the particle size was measured by dynamic light scattering (DLS). The morphology of the nanoparticles was observed by transmission electron microscopy (TEM). The hydrodynamic diameter of the nanoparticles was measured by dynamic light scattering (DLS) over a period of seven days. The average particle size of BD-6-QM / NPs was 125.8 nm, and the morphology was that of prototype nanospheres. The hydrodynamic diameter maintained good stability over a period of seven days. The average particle size of BD-Se-QM / NPs was 216.32 nm, and the morphology was round under transmission electron microscopy, showing good stability over a period of seven days.

[0169] 9. Singlet oxygen production by the nanoparticles under hydrogen peroxide excitation was measured. A 10 mM stock solution of 1,3-diphenylisobenzofuran (DPBF) indicator and a 100 μM stock solution of the nanoparticles were prepared in the dark. 6 μl of the indicator and 60 μl of the photosensitizer were diluted to 100 μM and 10 μM, respectively, in PBS. Under hydrogen peroxide excitation (H₂O₂), the UV absorbance of DPBF (410 nm) was measured over time using a microplate reader. Compared to the DPBF and DPBF+H₂O₂ groups, the UV absorbance of BD-6-QM / NPs under hydrogen peroxide excitation decreased significantly. The addition of hydrogen peroxide to BD-Se-QM / NPs also significantly decreased the UV absorbance of DPBF. Both BD-6-QM / NPs and BD-Se-QM / NPs can generate singlet oxygen under hydrogen peroxide excitation.

[0170] 10. Aβ 1-42 (40 μM) and / or nanoparticles (BD-6-QM / NPs, BD-Se-QM / NPs) (10 μM) were incubated in 300 μl of distilled water for 24 hours. The experimental groups were (1) Aβ 1-42 (40μM)(2)Aβ 1-42 (40μM)+H2O2(3)Aβ 1-42 (40μM)+Nanoparticles (BD-6-QM / NPs, BD-Se-QM / NPs)(4)Aβ 1-42 (40 μM) + nanoparticles (BD-6-QM / NPs, BD-Se-QM / NPs) + H2O2. After incubation, each sample solution was diluted threefold and 10 μL was applied to a carbon-coated copper grid. After drying, it was negatively stained with 1.5% phosphotungstic acid (w / v) and allowed to dry. Aβ under different conditions was observed using a Talos L120c transmission electron microscope at an accelerating voltage of 120 kV. 1-42 The morphology of Aβ 1-42 , Aβ 1-42 +H2O2、Aβ 1-42 +Nanoparticles (BD-6-QM / NPs, BD-Se-QM / NPs) showed fibrous shape under electron microscopy without changing the basic morphology. 1-42 The Aβ morphology of the nanoparticles (BD-6-QM / NPs, BD-Se-QM / NPs) + H2O2 was significantly changed to a dot-like shape. BD-6-QM / NPs and BD-Se-QM / NPs, stimulated by hydrogen peroxide, produced singlet oxygen that oxidized Aβ into monomers.

[0171] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A photosensitizer targeting β-amyloid protein, characterized in that The photosensitizer has any of the following structures:

2. The method for preparing the photosensitizer according to claim 1, wherein The steps include: (1) 3,6-dibromo-1,2-phenylenediamine and cyclohexanone undergo condensation reaction in toluene solution. After the reaction is completed, a crude product is obtained. Manganese dioxide and ultra-dry dichloromethane are added to the crude product. Under the protection of inert gas, the reaction is stirred and filtered to purify the product. (2) With 4-formylphenylboronic acid pinacol ester, a nucleophilic substitution reaction occurs under the catalysis of tetrakis(triphenylphosphine)palladium (Pd(PPh3)4) to obtain (3) 1-methyl-6-dimethylaminoquinoline and Under alkaline conditions, piperidine is catalytically dehydrogenated to generate the final product, photosensitizer BD-6-QM.

3. The method for preparing the photosensitizer according to claim 1, characterized in that: The steps include: (1) Add selenium dioxide solution to the ethanol solution of 3,6-dibromobenzene-1,2-diamine and heat to 60±10℃ to react to obtain (2) 4-Formylphenylboronic acid pinacol ester undergoes nucleophilic substitution reaction in the alkaline environment of toluene solution under the catalysis of tetrakis(triphenylphosphine)palladium to obtain (3) 1-methyl-6-dimethylaminoquinoline, The product was dehydrogenated by the strong base piperidine to obtain the final product, the photosensitizer BD-Se-QM.

4. A method based on chemically stimulated oxidation of Aβ 1-42 Protein nanoparticles, characterized in that The photosensitizer and bisoxalate of claim 1 are encapsulated with a polymer that can respond to hydrogen peroxide as a carrier to obtain the nanoparticles; the bisoxalate is CPPO, and the carrier is polyoxalate or polythioacetal; The structure of polyoxalate is as follows: The structure of polythioacetal is as follows:

5. The method for preparing nanoparticles according to claim 4, characterized in that: The photosensitizer BD-Se-QM, CPPO, and polyoxalate were dissolved in an organic solvent and dropped into an aqueous solution of polyvinyl alcohol. The oil / water emulsion was prepared by ultrasonication, and the organic solvent was removed by dialysis to obtain nanoparticles BD-Se-QM / NPs. Alternatively, the photosensitizer BD-6-QM, CPPO, and polythioacetal are dissolved in an organic solvent, added dropwise to an aqueous solution, stirred for reaction, and then the organic solvent is removed by dialysis to prepare nanoparticles BD-6-QM / NPs.

6. The method for preparing nanoparticles according to claim 5, characterized in that: Preparation conditions for nanoparticles BD-Se-QM / NPs: the organic solvent is dimethyl sulfoxide (DMSO), the photosensitizer BD-Se-QM:polyoxalate:CPPO = 1:10±2:2±1; the concentration of the polyvinyl alcohol aqueous solution is 5±3%, and the polyvinyl alcohol:DMSO = 6±3:1; the ultrasonication time is 30±10 min, the dialysis bag has a molecular weight of 3500±500, and the dialysis time is 48±12 hours. Preparation conditions of nanoparticles BD-6-QM / NPs: the organic solvent is dimethyl sulfoxide, the photosensitizer BD-6-QM: polythioacetal: CPPO = 1:10±2:2±1, the reaction time is 30±10 min; the water: DMSO volume ratio = 9±3:1, the dialysis bag has a molecular weight of 3500±500, and the dialysis time is 48±12 hours.

7. Use of the photosensitizer according to claim 1 or the nanoparticles according to claim 4 in the preparation of a β-amyloid fluorescent imaging agent.

8. The use according to claim 7, characterized in that The beta amyloid protein is Aβ 1-42 protein.

9. Use of the photosensitizer according to claim 1 or the nanoparticles according to claim 4 in the preparation of a drug for treating Alzheimer's disease.

Images