Preparation method and performance research of BPQD-coated PDA-F fluorescent probe for detecting and regulating A beta aggregation

Abstract

The invention discloses and relates to a BPQD (at) PDA-F fluorescent probe for A beta detection and regulation. The BPQD (at) PDA-F fluorescent probe is synthesized from BPQD, PDA and a fluorescent probe F. According to the invention, surface modification is carried out on BPQD by using physicochemical properties of PDA, and then a fluorescent probe F is functionally modified to the surface of BPQD coated PDA. Fluorescence detection of A beta is realized by using a fluorescence probe F; bPQD, PDA and a fluorescent probe F are used for jointly realizing an inhibition effect on A beta; and the depolymerization effect on A beta is realized by utilizing the photothermal effects of the BPQD and the PDA. The BPQD (at) PDA-F fluorescent probe provided by the invention realizes the detection and regulation of A beta aggregate.

Description

Technical Field

[0001] The present invention relates to the field of medical material applications, and in particular to a preparation method and performance research of a BPQD@PDA-F fluorescent probe for detecting and regulating Aβ aggregates. Background Art

[0002] Alzheimer's disease (AD) is an insidious progressive neurodegenerative disease caused by damage and destruction of brain nerve cells. As more and more neurons in different parts of the brain are damaged and the degree of damage increases, AD patients' memory, abstract thinking, judgment, behavior and emotions will gradually change. According to statistics, about 50 million people suffer from AD worldwide, and it is estimated that there will be 130 million AD patients by 2050. However, so far, there is no effective drug that can cure AD or alleviate the progression of the disease. Abnormal aggregation of amyloid protein (Aβ) is one of the key links in the pathogenesis of AD. Aβ is a polypeptide with a molecular weight of about 4kDa and a length of 40-42 amino acids. It is produced by the proteolysis of amyloid beta precursor protein (APP) in neurons. Aβ has different lengths, the most abundant of which are 40 amino acids (Aβ40) and insoluble 42 amino acids (Aβ42). However, Aβ42 is dominant and has greater neurotoxicity than Aβ40, and is more likely to aggregate into insoluble oligomers. When Aβ clearance is impaired, Aβ accumulates in the brain and deposits into neurotoxic insoluble amyloid plaques. Therefore, the detection and regulation of Aβ aggregates is a new therapeutic strategy.

[0003] Aβ diagnosis, detection and treatment methods include magnetic resonance imaging (MRI), positron emission tomography (PET), single photon emission computed tomography (SPCET), optical imaging and other technologies. MRI has poor specificity, and the blood-brain barrier penetration of magnetic resonance imaging ligands with large molecular weight is poor. PET and SPCET can accurately improve the accuracy of diagnosis, but the process is cumbersome, costly and radioactive. Compared with PET, SPECT and MRI imaging techniques, near-infrared imaging technology (NIRF) has the advantages of excellent sensitivity, high resolution, non-invasiveness, and avoidance of interference from spontaneous fluorescence of biological tissues, and has been rapidly developed. There are currently two main types of NIRF probes: one is a molecule or nanoparticle (quantum dot) with spontaneous fluorescence emission characteristics, and the other is an organic fluorescent molecule, such as curcumin derivatives, phenothiazine derivatives, cyanine derivatives, etc. Fluorescent probe F is a compound with phenothiazine as the basic skeleton. After the fluorescent probe binds to Aβ42, a significant fluorescence enhancement phenomenon will occur.

[0004] There are two types of Aβ aggregation pathways: involving nucleation and elongation, accompanied by initial misfolding events, followed by self-assembly of polypeptide chains into soluble oligomer intermediates and then insoluble fibrils. The continuous deposition of Aβ will gradually affect the normal physiological function of neurons, and eventually lead to irreversible brain function damage. At present, a variety of inhibitors that regulate Aβ aggregation have been discovered, including small molecules, peptides, proteins, polymers, etc. They mainly interact with Aβ substances through hydrophobic interactions, hydrogen bonds, π-π stacking and electrostatic interactions, thereby inhibiting or interfering with Aβ fiber formation. Optics have been widely used in biomedical applications due to its remote controllability and minimal invasiveness. In order to achieve Aβ degradation, photothermal therapy (PTT) to regulate Aβ aggregation is considered to be an effective strategy. PTT can destroy the stability of Aβ aggregates, degrade mature Aβ aggregates, and significantly improve the inhibition efficiency of Aβ inhibitors. Nanomaterials have been widely used in the study of Aβ aggregation regulation due to their small particle size, large specific surface area, and easy modification. Various photothermal nanomaterials have been explored to regulate Aβ assembly.

[0005] Black phosphorus quantum dots (BPQDs) are a new type of zero-dimensional (OD) nanomaterial with excellent photocatalytic performance, high specific surface area, surface active sites, excellent biocompatibility and biosafety, and can be degraded into non-toxic phosphate and phosphite anions under physiological conditions. BPQDs have a wide absorption band spanning the ultraviolet to near-infrared region. Ultra-small BPQDs can convert near-infrared light into thermal energy and are ideal photothermal agents in PTT applications. However, there are currently few studies on BPQDs regulating Aβ aggregation, and photothermal depolymerization of Aβ aggregates is the first study.

[0006] Polydopamine (PDA) is usually prepared by polymerizing monomer dopamine under weak alkaline conditions and is an adhesive polymer. It has high biocompatibility, high biodegradability, high adhesive strength and stability. As one of the near-infrared light-responsive materials, PDA can effectively absorb near-infrared light energy and convert it into heat, making it an ideal photothermal therapeutic agent. The surface of PDA has a large number of chemically active sites, such as catechol and amine functional groups, which can interact with peptides and proteins and inhibit the formation of Aβ fibrils through hydrogen bonds and π-π stacking interactions. At the same time, PDA can act as a protective layer under alkaline conditions to prevent BPQD from being affected by oxygen and moisture, while enhancing the stability of BPQD. More importantly, PDA has many functional groups (such as amino groups) on the surface of the modified material, which can be used for functionalization and obtain the desired reactive groups.

[0007] The present invention is based on BPQD as the core. After surface modification with PDA under alkaline conditions, the fluorescent probe F is loaded on the BPQD@PDA surface through an amidation reaction to successfully prepare the BPQD@PDA-F fluorescent probe; the BPQD@PDA-F fluorescent probe can perform fluorescent imaging of AD mouse brain slices and regulate the formation of Aβ aggregates. Summary of the invention

[0008] The present invention is based on the research of fluorescent probes for detecting and regulating Aβ aggregation, using photothermal materials BPQD and PDA as the basic framework, and combining with fluorescent probe F to achieve fluorescent imaging and regulation of Aβ.

[0009] The purpose of the present invention is to achieve the inhibitory effect on Aβ aggregates by synthesizing the BPQD@PDA-F fluorescent probe. Under near-infrared light, BPQD@PDA-F can generate local heat to disaggregate mature Aβ aggregates; the loading of the fluorescent probe F further enhances the effect of inhibiting and disaggregating Aβ, and can also achieve fluorescent imaging of Aβ.

[0010] The present invention is achieved by the following technical solutions.

[0011] A BPQD@PDA-F fluorescent probe for detecting and regulating Aβ aggregation is based on BPQD, which is surface-modified with PDA and then coupled with fluorescent probe F.

[0012] The BPQD has a uniform particle size, with an average particle size of 5-7 nm, and is a nano-sized preparation.

[0013] The average particle size of the PDA-modified BPQDs is 85-100 nm.

[0014] The average particle size of the BPQD@PDA-F after coupling with the fluorescent probe F is 160-200 nm.

[0015] The present invention provides a method for preparing a BPQD@PDA-F fluorescent probe for detecting and regulating Aβ aggregation, comprising the following steps:

[0016] 1) Synthesis of fluorescent probe F: tri(3,6-dioxaheptyl)amine was dissolved in dichloromethane (DCM) solution, and then saturated potassium carbonate solution was added, and then (1,3-dioxolan-2-yl)methyltriphenylphosphonium bromide and 10-methyl-10H-phenothiazine-3-carboxaldehyde were added in sequence. The reaction system was heated, refluxed and protected from light, and nitrogen was protected. The reaction progress was monitored by TLC. After the reaction was completed, it was extracted twice with DCM and then extracted twice with saturated brine. The organic phase was dried with magnesium sulfate and concentrated by a rotary evaporator. Tetrahydrofuran (THF) and 10% hydrochloric acid (HCl) solution were added at 0°C, stirred at room temperature in the dark, and the reaction system was placed in an ice-water bath at 0°C, and the pH was adjusted to 7 with 10% sodium hydroxide (NaOH), and then extracted twice with DCM and twice with water, and finally dried with saturated brine and magnesium sulfate, concentrated and dried, and separated by silica gel column to obtain (E)-3-(10-methyl-10H-phenothiazine-3-yl) acrolein, the product of which was an orange solid. The product (E)-3-(10-methyl-10H-phenothiazine-3-yl) acrolein of the above reaction was dissolved in 1 mL of DCM, and rhodanine-3-acetic acid and piperidine were added in sequence, and stirred at room temperature in the dark. The pH was adjusted with acetic acid, and the organic phase was washed with saturated brine, purified by recrystallization, and the product was a red solid. The synthesized probe was named F.

[0017] 2) Synthesis of BPQDs: The BP crystals were dispersed in an organic solvent and ground at low temperature in the dark. The ground BP suspension was transferred to a beaker and sonicated in an ice-water bath in the dark. The suspension was then sonicated in a cell disruptor and centrifuged at high speed and low temperature. The supernatant was taken and then placed in a reactor for solvothermal reaction. After the reaction, the BPQD suspension was obtained by high-speed and low-temperature centrifugation.

[0018] 3) Synthesis of BPQD@PDA: BPQD and PDA were dissolved in an alkaline aqueous solution, reacted at room temperature in the dark, and then centrifuged at high speed and low temperature to remove the supernatant to obtain BPQD@PDA.

[0019] 4) Synthesis of BPQD@PDA-F: Add fluorescent probe F to BPQD@PDA, react at room temperature in the dark, centrifuge at high speed and low temperature, collect the precipitate, and obtain BPQD@PDA-F.

[0020] The reflux reaction time in step 1 is 30-36 hours.

[0021] In the step 1, the stirring time under room temperature and in the dark is 10-15 hours.

[0022] The grinding time in step 2 is 2-4h.

[0023] The ice bath ultrasonic time in step 2 is 2-4 hours.

[0024] The probe ultrasound time in step 2 is 4-6 hours.

[0025] The solvent thermal reaction time in step 2 is 10-15 hours.

[0026] The solvent thermal reaction temperature in step 2 is 120-150°C.

[0027] The high-speed low-temperature centrifugation conditions in step 2 are: temperature 4°C, speed 40000 rpm, and time 30 min.

[0028] The high-speed low-temperature centrifugation conditions in steps 3 and 4 are: temperature 4° C., rotation speed 12000-15000 rpm, and time 15 min.

[0029] The reaction time in steps 3 and 4 at room temperature and away from light is 24 hours.

[0030] The BPQD@PDA-F fluorescent probe for detecting and regulating Aβ aggregation provided by the present invention has the following advantages:

[0031] First, the BPQD@PDA-F fluorescent probe can realize fluorescent imaging of Aβ. In vitro AD mouse brain slice experiments proved that the BPQD@PDA-F fluorescent probe can specifically stain Aβ aggregates and distinguish between AD and WT mice.

[0032] Second, the BPQD@PDA-F fluorescent probe can effectively inhibit the transformation of Aβ42 from random coil conformation to β-fold conformation and reduce the formation of fiber structure. ThT fluorescence experiment and transmission electron microscopy experiment confirmed that BPQD@PDA-F can significantly inhibit the fibrillation of Aβ42 and change the aggregation morphology of Aβ42.

[0033] Third, the BPQD@PDA-F fluorescent probe can effectively disaggregate Aβ42 aggregates under near-infrared light and reduce the number of aggregates. ThT fluorescence experiments and transmission electron microscopy experiments confirmed that BPQD@PDA-F can significantly disaggregate Aβ42 aggregates.

[0034] The present invention synthesizes BPQD by mechanical stripping and solvothermal method, and loads fluorescent probe F molecules after PDA surface modification to synthesize BPQD@PDA-F fluorescent probe. BPQD@PDA-F fluorescent probe can perform fluorescent imaging of Aβ, regulate the formation of Aβ aggregates, and reduce the formation of fiber structures, providing a new strategy for the detection and treatment of AD. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0036] Figure 1 This is the synthesis route of the fluorescent probe F in the embodiment of the present invention.

[0037] Figure 2 These are the transmission electron microscopy images of BPQD (A), BPQD@PDA (B), and BPQD@PDA-F (C) in the embodiments of the present invention.

[0038] Figure 3 Graph showing the particle size distribution of BPQD (A), BPQD@PDA (B), and BPQD@PDA-F (C) in the embodiments of the present invention.

[0039] Figure 4 This is the Zeta potential distribution diagram of BPQD, BPQD@PDA, and BPQD@PDA-F in the embodiments of the present invention.

[0040] Figure 5 This is the fluorescence imaging image of brain slices of the fluorescent probe F and BPQD@PDA-F in the embodiments of the present invention.

[0041] Figure 6 This is the ThT fluorescence graph of BPQD@PDA, PDA-F, and BPQD@PDA-F inhibiting Aβ in the examples of the present invention.

[0042] Figure 7 Transmission electron microscopy images of BPQD@PDA, PDA-F, and BPQD@PDA-F inhibiting Aβ in the examples of the present invention.

[0043] Figure 8 Graphs showing the temperature changes of BPQD, BPQD@PDA, PDA-F, and BPQD@PDA-F under near-infrared laser irradiation of different powers in an embodiment of the present invention.

[0044] Fig. 9 ThT fluorescence images of BPQD@PDA, PDA-F, and BPQD@PDA-F depolymerizing Aβ in the embodiment of the present invention

[0045] Fig.10 Transmission electron microscopy images of BPQD@PDA, PDA-F, and BPQD@PDA-F depolymerizing Aβ in the examples of the present invention. DETAILED DESCRIPTION

[0046] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0047] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0048] The present invention synthesizes BPQD@PDA-F fluorescent probe with BPQD, PDA and fluorescent probe F. Experiments have shown that BPQD@PDA-F fluorescent probe can perform fluorescent imaging of AD mouse brain slices. BPQD@PDA-F fluorescent probe can destroy the formation of β-folding structure during the aggregation process of Aβ42, thereby effectively inhibiting the aggregation of Aβ42. In addition, BPQD@PDA-F fluorescent probe can disaggregate the aggregation of Aβ42 under near-infrared light.

[0049] Embodiment 1:

[0050] The specific preparation of the BPQD@PDA-F fluorescent probe in this embodiment is as follows:

[0051] 1) Preparation of fluorescent probe F

[0052] Dissolve tri(3,6-dioxaheptyl)amine (738mg, 2.28mmol, 1.1eq) in 10mL dichloromethane (DCM) solution, then add 15mL saturated potassium carbonate solution, then add (1,3-dioxolane-2-yl)methyltriphenylphosphonium bromide (1.43g, 3.32mmol, 1.6eq) and 10-methyl-10H-phenothiazine-3-carboxaldehyde (500mg, 2.07mmol, 1eq) in sequence. The reaction system is heated, and refluxed to avoid light for 30h, nitrogen protection, and the reaction progress is monitored by TLC. After the reaction is completed, extract twice with 50mL DCM, then extract twice with saturated brine, and the organic phase is dried with magnesium sulfate and concentrated by rotary evaporator. 20 mL of tetrahydrofuran (THF) and 20 mL of 10% hydrochloric acid (HCl) solution were added at 0°C, and the mixture was stirred in the dark for 1 h at room temperature. The reaction system was placed in an ice-water bath at 0°C, and the pH was adjusted to 7 with 10% sodium hydroxide (NaOH). The mixture was extracted twice with 50 mL of DCM and twice with water, respectively. The mixture was dried with saturated brine and magnesium sulfate, and then concentrated to dryness. The mixture was separated by silica gel column to obtain (E)-3-(10-methyl-10H-phenothiazine-3-yl)acrolein as an orange solid. The product (E)-3-(10-methyl-10H-phenothiazine-3-yl) acrolein (10 mg, 0.037 mmol, 1 eq) of the above reaction was dissolved in 1 mL of DCM, and rhodanine-3-acetic acid (7.78 mg, 0.0407 mmol, 1.1 eq) and piperidine (9.1 μL, 2.5 eq) were added in sequence, and stirred at room temperature in the dark for 10-15 h. The pH was adjusted with acetic acid, and the organic phase was washed with saturated brine and purified by recrystallization. The product was a red solid, and the synthesized probe was named F.

[0053] 2) Preparation of BPQDs

[0054] Weigh 50 mg of BP crystals, place them in an agate mortar, add 100-150 mL of N-methylpyrrolidone (NMP) solution, grind for 2-4 h, and transfer all the ground BP suspension to a beaker.

[0055] The BP dispersion was ultrasonicated in an ice-water bath in an ultrasonic machine for 2-4 h in the dark.

[0056] Then continue low-temperature ultrasound in a cell disruptor for 4-6 hours.

[0057] After completion, the BP suspension was taken out, allowed to stand at 4°C for 1-3 h, and the upper layer of the suspension was taken.

[0058] The product was then placed in a reaction vessel and subjected to solvent thermal reaction in an oven at 120-150°C for 10-15h.

[0059] The mixture was centrifuged at 40,000 rpm and 4°C for 30 min to obtain a BPQD precipitate, which was then washed with pure water and centrifuged 2-3 times to obtain a BPQD suspension, which was then stored in a 4°C refrigerator away from light.

[0060] 3) Preparation of BPQD@PDA

[0061] Take 1 mg of BPQD, weigh 10 times the weight of BPQD PDA, and adjust the pH to 7.5-8.5.

[0062] The reaction was carried out in the dark at room temperature for 24 h. After the reaction, the mixture was centrifuged at 12000 rpm and 4°C for 15 min. The supernatant was removed to obtain the PDA-modified BPQDs.

[0063] 4) Preparation of BPQD@PDA-F

[0064] The same amount of fluorescent probe F as BPQD@PDA was weighed, dissolved in DMSO, and added into MES buffer solution of EDC / NHS to activate the carboxyl group for 3-6 h.

[0065] BPQD@PDA was weighed and added to the activated fluorescent probe F solution. After reacting at room temperature in the dark for 24 h, the mixture was centrifuged at 4°C and 12,000 rpm for 15 min. The precipitate was BPQD@PDA-F after being washed several times with a mixture of pure water and DMSO. The product was freeze-dried and stored in a 4°C refrigerator in the dark.

[0066] Embodiment 2:

[0067] This example structurally characterizes the intermediates and products in the preparation process of the BPQD@PDA-F fluorescent probe.

[0068] 1) Synthesis route of fluorescent probe F ( Figure 1 ).

[0069] 1 H-NMR (400MHz, DMSO) δ7.62(d,J=3.5Hz,1H),7.56(d,J=8.0Hz,1H),7.51(dd,J=8.0,3.5Hz,1H),7.32(d,J=15.0Hz,1H),7.23(t ,J=7.0Hz,1H),7.18(dd,J=8.0,3.5Hz,1H),7.07(dd,J=15.0,15.0Hz,1H),7.05-6.95(m,3H),4.69(s,2H),3.35(s,3H); ESI-MS m / z:441.0[M+H] + .

[0070] 2) BPQD, BPQD@PDA and BPQD@PDA-F were observed using transmission electron microscopy.

[0071] from Figure 2 It can be seen that BPQDs are evenly dispersed and uniform in size; the morphology of BPQDs changes significantly after PDA surface modification, showing a spherical morphology; the fluorescent probe F is then loaded on BPQD@PDA, which still maintains a spherical shape and uniform size.

[0072] 3) The particle size distribution of BPQD, BPQD@PDA, and BPQD@PDA-F was measured and analyzed using a Malvern laser particle size analyzer.

[0073] from Figure 3 It can be seen that the average hydrated particle size of BPQD is about 6±0.23nm; after PDA modification, the average particle size of BPQD@PDA is 91±3.1nm; loading the fluorescent probe F on BPQD@PDA increases the average particle size of BPQD@PDA-F to 182±16nm, confirming the successful synthesis of BPQD@PDA-F.

[0074] 4) Zeta potential distribution analysis of BPQD, BPQD@PDA, and BPQD@PDA-F.

[0075] from Figure 4 It can be seen that the zeta potential of BPQD is -14.1mV, which is mainly because BP is composed of P elements, which makes its surface negatively charged; after PDA modification, the zeta potential of BPQD@PDA is -27.6mV; after modification with fluorescent probe F, the surface potential of BPQD@PDA-F becomes -36.5mV.

[0076] Embodiment 3:

[0077] In this example, brain slice fluorescence imaging was performed using the BPQD@PDA-F fluorescent probe.

[0078] APP / PSI double transgenic mice and C57 mice were anesthetized and killed. C57 mice served as the control group, and their brains were sliced.

[0079] AD mouse brain slices and C57 mouse brain slices were incubated with fluorescent probe F and BPQD@PDA-F at a constant temperature of 37°C for 30 minutes.

[0080] The brain slices were washed 2-3 times with 50% ethanol solution and PBS buffer respectively, and after wiping off the residual liquid with filter paper, the slices were observed under a fluorescence microscope, and adjacent slices were stained with 1% thioflavin T (30% ethanol solution).

[0081] from Figure 5 It can be seen that specific staining of Aβ plaques was observed in the brain tissue of APP / PS1 mice by ThT, fluorescent probe F, and BPQD@PDA-F fluorescent probe, while almost no spots were found in the brain sections of WT control mice, indicating that BPQD@PDA-F fluorescent probe can specifically stain Aβ aggregates and distinguish AD and WT mice.

[0082] Embodiment 4:

[0083] This example verifies the ability of BPQD@PDA-F to inhibit Aβ aggregation.

[0084] First, Aβ42 powder was fully dissolved in hexafluoroisopropanol (HFIP) solution to make the final concentration of Aβ42 1.0 mg / mL. The Aβ42 solution was then placed in an ice bath for ultrasonic treatment for 10-30 min, and then the Aβ42 solution was allowed to stand at 4°C for 2 h, and the HFIP solvent was removed using a vacuum dryer, and the treated Aβ was stored in a -20°C refrigerator.

[0085] Accurately weigh 16 mg of NaOH solid, dissolve it in 20 mL of pure water, prepare it to a concentration of 20 mM, and store it at 4°C. Dissolve the above-treated Aβ42 in a 20 mM NaOH solution to a final concentration of 275 μmol / L, and ultrasonically treat it in an ice bath until the polypeptide is completely dissolved. The Aβ42 solution was then centrifuged at 4°C and 16,000 g for 20 min, and the supernatant was collected as the Aβ42 monomer mother liquor. The Aβ42 monomer mother liquor was diluted to a concentration of 25 μmol / L with 100 mM PBS buffer (pH 7.4), and the sample was then placed in a shaker at 37°C and 150 rpm for 72 hours.

[0086] 1) ThT fluorescence detection of the inhibitory effect of BPQD@PDA, PDA-F, and BPQD@PDA-F on Aβ42 aggregation

[0087] BPQD@PDA, PDA-F, and BPQD@PDA-F solutions of different concentration gradients (25, 50, and 100 μg / mL) were added to a 25 μmol / L Aβ42 solution, and incubated at 37°C and 150 rpm in a shaker to obtain an Aβ sample polymer solution. At the time point, 200 μL of the Aβ sample polymer solution and 2 mL of ThT solution (25 μmol / L) were taken and mixed evenly. The ThT fluorescence intensity of the sample measured at 485 nm (Ex = 440 nm) was detected using an ELISA instrument. The sample without Aβ was subtracted as the background. The average value of three parallel experiments was taken, and the fluorescence value of the Aβ sample incubated alone was set to 100% for normalization.

[0088] from Figure 6 It can be seen that the ThT fluorescence value of Aβ42 incubated alone is the strongest at 100%, indicating the formation of Aβ42 fibrils. Compared with Aβ42 alone, BPQD@PDA, PDA-F, and BPQD@PDA-F all inhibited Aβ42 fibrillation in a dose-dependent manner. At a concentration of 100μg / mL, the ThT fluorescence value of the BPQD@PDA group decreased to 34.6%. The decrease in ThT fluorescence means a decrease in the binding sites of ThT in Aβ42 fibrils and a decrease in the number of Aβ42 fibrils. Similarly, the ThT fluorescence value of the 100μg / mL PDA-F group decreased to 23.6%, and the inhibitory effect of the BPQD@PDA-F group was more obvious, with the ThT fluorescence value reduced to 17.2%. The results show that BPQD@PDA-F can more effectively hinder the formation of Aβ42 aggregates.

[0089] 2) Transmission electron microscopy experiments were used to observe the effects of BPQD@PDA, PDA-F, and BPQD@PDA-F on the morphology of Aβ42 aggregates.

[0090] from Figure 7 It can be seen that after Aβ42 was incubated alone for 72 hours, a large number of dense and elongated fiber aggregates were produced, showing a typical Aβ morphology, indicating the formation of Aβ42 aggregates. Compared with the Aβ42 fibrils formed alone, after BPQD@PDA, PDA-F, BPQD@PDA-F and Aβ42 were co-incubated, the number of Aβ42 aggregates decreased significantly with the increase of BPQD@PDA, PDA-F and BPQD@PDA-F concentrations. When the concentration was 100μg / mL, it showed a better inhibitory effect than the low concentration of BPQD@PDA, and the number of Aβ42 aggregates was smaller. Under the same conditions, the inhibitory ability of PDA-F and BPQD@PDA-F on Aβ42 aggregates was tested. PDA-F and BPQD@PDA-F can inhibit Aβ42 aggregates, and there are almost no obvious Aβ plaques after 100μg / mL BPQD@PDA-F was co-cultured with Aβ42.

[0091] Embodiment 5:

[0092] This example verifies the ability of BPQD@PDA-F to disaggregate Aβ aggregates.

[0093] 1) Investigate the photothermal properties of BPQD, BPQD@PDA, PDA-F, and BPQD@PDA-F.

[0094] The aqueous dispersions of BPQD, BPQD@PDA, PDA-F, and BPQD@PDA-F (50 μg / mL) with the same concentration were prepared under different laser powers (1.0, 2.0 W / cm 2) for photothermal test, the sample volume was 200μL and placed in an EP tube, which was fixed on a tube rack. Control group: pure water. At room temperature of 25℃, the 808nm near-infrared illuminator laser probe was placed at the same height above the EP tube, and the laser completely covered the liquid surface in the EP tube. Each sample was irradiated for 10 minutes, and the temperature change was recorded every 1 minute and a photothermal curve was drawn.

[0095] from Figure 8 It can be seen that the BPQD group has a 2.0W / cm 2 Ratio 1.0W / cm 2 The temperature rises 5°C faster when the power is applied; the same is true for the BPQD@PDA, PDA-F and BPQD@PDA-F groups. The temperature increase is also proportional to the power, indicating that power is also one of the factors affecting the photothermal effect.

[0096] 2) ThT fluorescence detection of the disaggregation effect of BPQD@PDA, PDA-F, and BPQD@PDA-F on Aβ42 aggregates

[0097] Equal concentrations of BPQD@PDA, PDA-F, and BPQD@PDA-F (50 μg / mL) solutions were added to a 25 μmol / L Aβ42 aggregate solution, and the solution was exposed to NIR irradiation or not, and then incubated at 37°C and 150 rpm in a shaker.

[0098] from Fig. 9 It can be seen that the ThT fluorescence intensity of Aβ42 fibrils alone is 100%. At the same time, compared with Aβ42 fibrils alone, the ThT fluorescence intensity gradually decreased to 32.4% and 26.9% after Aβ42 fibrils were co-cultured with 50μg / mL BPQD@PDA and PDA-F, respectively. In addition, the ThT fluorescence intensity was further reduced to 21.6% after Aβ42 fibrils were co-cultured with 50μg / mL BPQD@PDA-F. However, in addition to the chemically induced disaggregation of Aβ42 fibrils, the photothermal effect of BPQD@PDA, PDA-F, and BPQD@PDA-F under NIR irradiation also produces local high temperatures, thereby disaggregating mature Aβ42 fibrils. The individual Aβ42 aggregates showed no obvious change in ThT fluorescence intensity before and after NIR laser irradiation, showing stable ThT fluorescence with a fluorescence intensity of about 100%. Compared with the individual Aβ42 fibrils, after the Aβ42 fibrils were co-cultured with 50 μg / mL BPQD@PDA, PDA-F, and BPQD@PDA-F and then irradiated with NIR, the ThT fluorescence decreased to 22.6%, 16.6%, and 11.0%, respectively, which was more obvious than the Aβ42 fibrils treated with BPQD@PDA, PDA-F, and BPQD@PDA-F without NIR irradiation.

[0099] 3) Transmission electron microscopy experiments were used to observe the morphological changes of BPQD@PDA, PDA-F, and BPQD@PDA-F after photothermal depolymerization of Aβ.

[0100] The results are as follows Fig.10 As shown, Aβ42 incubated alone showed a typical fiber aggregation state. After co-culture with BPQD@PDA, PDA-F, and BPQD@PDA-F, Aβ42 fibrils were broken into segments, indicating that BPQD@PDA, PDA-F, and BPQD@PDA-F had a depolymerization effect on Aβ42 fibrils without NIR irradiation, and few mature fibrils were found in the Aβ42 aggregate sample containing BPQD@PDA-F, and only small fibrils were found. TEM was also used to characterize the depolymerization effect of BPQD@PDA, PDA-F, and BPQD@PDA-F on Aβ42 fibrils under NIR irradiation. Aβ42 aggregates did not change significantly under NIR irradiation. After Aβ42 fibrils were co-incubated with BPQD@PDA, PDA-F, and BPQD@PDA-F and irradiated with NIR light, the BPQD@PDA-F group achieved more depolymerization of Aβ42 fibrils under NIR irradiation compared with BPQD@PDA and PDA-F, indicating that the excellent photothermal effect of BPQD@PDA-F can effectively depolymerize Aβ42 aggregation.

[0101] The present invention proposes a method for synthesizing a BPQD@PDA-F fluorescent probe and its application in Aβ fluorescence imaging and regulating Aβ aggregation. The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, all possible combinations of the technical features in the above-mentioned embodiments are not described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

Claims

1. A BPQD@PDA-F fluorescent probe for detecting and regulating Aβ aggregates, which is centered on BPQD and coupled with a fluorescent probe F molecule after surface modification with PDA.

2. The BPQDs as claimed in claim 1 have uniform particle size, an average particle size of 5-7 nm, and are a nano-size inhibitor. After PDA surface modification, the average particle size of BPQD@PDA is 85-100 nm, and after coupling with fluorescent probe F, the average particle size of BPQD@PDA-F is 160-200 nm.

3. The method for synthesizing the BPQD@PDA-F fluorescent probe for detecting and regulating Aβ aggregates according to claim 1, comprising the following steps: 1) BPQD synthesis: The BP crystals were dispersed in an organic solvent and ground at low temperature in the dark. The ground BP suspension was transferred to a beaker and sonicated in an ice-water bath in the dark in an ultrasonic machine. The mixture was then sonicated in a cell disruptor and centrifuged at high speed and low temperature. The supernatant was taken and then placed in a reactor for solvothermal reaction. After the reaction, the BPQD suspension was obtained by high-speed and low-temperature centrifugation. 2) Synthesis of BPQD@PDA-F: The fluorescent probe F was added to BPQD@PDA, and the reaction was carried out in the dark under room temperature. The reaction was centrifuged at high speed and low temperature, and the precipitate was collected to obtain BPQD@PDA-F. The grinding time in step 1 is 2-4h. The ice bath ultrasonic time in step 1 is 2-4 hours. The probe ultrasound time in step 1 is 4-6 hours. The solvent thermal reaction time in step 1 is 10-15 hours. The solvent thermal reaction temperature in step 1 is 120-150°C. The high-speed low-temperature centrifugation conditions in step 1 are: temperature 4° C., rotation speed 40,000 rpm, and time 30 min. The high-speed low-temperature centrifugation conditions in step 2 are: temperature 4° C., rotation speed 12000-15000 rpm, and time 15 min. The reaction time in step 2 at room temperature and away from light is 24 hours.

4. Application of the BPQD@PDA-F fluorescent probe described in claim 1 to Aβ fluorescence imaging.

5. Use of the BPQD@PDA-F fluorescent probe described in claim 1 to inhibit Aβ aggregation.

6. Use of the BPQD@PDA-F fluorescent probe described in claim 1 for photothermal disaggregation of Aβ aggregates.

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