A black phosphorus-methylene blue nanocomposite for precise targeted treatment of Tau phosphorylation and its synthesis method

Abstract

The present invention discloses a black phosphorus-methylene blue nanocomposition for precise targeted treatment of Tau phosphorylation and a synthesis method thereof, wherein the synthesis comprises the following steps: step 1, mixing black phosphorus nanosheets with polyethylene glycol for reaction; step 2, centrifuging and cleaning the reaction solution in step 1, and collecting the lower layer reactant; step 3, adding amination superparamagnetic nanoparticles and methylene blue to the lower layer reactant obtained in step 2 for reaction; step 4, centrifuging and cleaning the reaction solution obtained in step 3, and collecting the lower layer reactant. The present invention also discloses a black phosphorus-methylene blue nanocomposition synthesized by the above-mentioned synthesis method, wherein the drug loading rate of methylene blue in the composition exceeds 60%, the composition has precise targeted treatment capability, good biocompatibility, is nontoxic to cells, and the manufacturing process is very simple and convenient, and has broad application prospects in the treatment of Alzheimer's disease.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and more specifically, to a black phosphorus-methylene blue nanocomposite for precise targeted treatment of Tau phosphorylation and a synthesis method thereof. Background Art

[0002] Alzheimer's disease, also known as senile dementia, is a neurodegenerative disease and the most common type of dementia. Clinical manifestations include memory loss, persistent cognitive decline, and movement disorders, accompanied by a series of psychiatric disorders. The main cause of the disease is the excessive phosphorylation of Tau protein in the brain, which leads to excessive deposition of Aβ amyloid protein in brain tissue, thereby forming specific senile plaques, causing some neurons to become entangled, leading to neuronal damage, and further affecting the patient's brain neuronal system function. Therefore, inhibiting Tau protein aggregation and reducing Tau protein phosphorylation are important strategies for treating Alzheimer's disease.

[0003] Black phosphorus (BP) is the most thermodynamically stable allotrope of phosphorus. As an emerging two-dimensional semiconductor material, black phosphorus nanosheets have a high specific surface area and a unique wrinkled structure. They also have strong reactive oxygen scavenging ability and high biocompatibility, and have broad application prospects in the medical field.

[0004] Studies have shown that methylene blue (MB) can regulate the cell cycle by promoting cell autophagy, causing cells to enter a resting phase and inhibiting cell proliferation. In addition, methylene blue can also inhibit Tau protein aggregation and reduce Tau protein phosphorylation by promoting the autophagy-lysosome pathway in PC12-tau cells, and can be used to treat Alzheimer's disease. For example, a Chinese patent (CN107233631B) discloses a fiber scaffold material, which includes a matrix and methylene blue loaded on the surface of the matrix, wherein the matrix includes a fiber scaffold and graphene oxide coated on the surface of the fiber scaffold. At the same time, a method for preparing a fiber scaffold material and a method for adjusting the methylene blue loading amount are also disclosed, and a composite nanofiber scaffold material with controllable slow release of methylene blue is obtained by using the strong adsorption performance of graphene oxide on methylene blue. The material is used to treat Alzheimer's disease at different stages. However, the preparation of this material is complicated, the utilization rate of methylene blue drugs is not high, and it is not convenient to promote and use. Summary of the invention

[0005] In order to overcome the above-mentioned deficiencies of the prior art, the object of the present invention is to provide a black phosphorus-methylene blue nanocomposition and a synthesis method thereof, which has a high drug loading rate, a simple preparation method and can accurately target and treat Tau phosphorylation.

[0006] The first aspect of the present invention provides a method for synthesizing a black phosphorus-methylene blue nanocomposite for precise targeted treatment of Tau phosphorylation, comprising the following steps:

[0007] Step 1, mixing black phosphorus nanosheets and polyethylene glycol for reaction;

[0008] Step 2: Centrifuge and wash the reaction solution in step 1 to collect the lower layer of reactants;

[0009] Step 3, adding amino superparamagnetic nanoparticles and methylene blue to the lower layer reactant obtained in step 2 to carry out reaction;

[0010] Step 4: Centrifuge and wash the reaction solution obtained in step 3, and collect the lower layer of reactants.

[0011] Preferably, in the present invention, the molecular weight of polyethylene glycol in step 1 of the synthesis method of a black phosphorus-methylene blue nanocomposite for precise targeted treatment of Tau phosphorylation is 2000.

[0012] In the present invention, in step 3 of the synthesis method of a black phosphorus-methylene blue nanocomposite for precise targeted treatment of Tau phosphorylation, the volume molar ratio of the aminated superparamagnetic nanoparticles and the methylene blue is 1-2:1-2.

[0013] Preferably, in step 3 of the method for synthesizing a black phosphorus-methylene blue nanocomposite for precise targeted treatment of Tau phosphorylation described in the present invention, the volume molar ratio of the aminated superparamagnetic nanoparticles and methylene blue is 1:1.

[0014] In the present invention, the synthesis method of a black phosphorus-methylene blue nanocomposite for precise targeted treatment of Tau phosphorylation, in step 1, the black phosphorus nanosheet dispersant needs to be ultrasonicated for 20 to 40 minutes before the reaction.

[0015] In the present invention, the synthesis method of a black phosphorus-methylene blue nanocomposite for precise targeted treatment of Tau phosphorylation, the reaction in step 1 is magnetically stirred in a 4° C. water bath for 8 to 12 hours.

[0016] In the present invention, in the method for synthesizing a black phosphorus-methylene blue nanocomposite for precise targeted treatment of Tau phosphorylation, the reaction time of step three is 8 to 12 hours.

[0017] In the present invention, the synthesis method of a black phosphorus-methylene blue nanocomposite for precise targeted treatment of Tau phosphorylation, the centrifugation conditions in step 2 are a temperature of 4° C., a rotation speed of 9000-10000 r / min, and each centrifugation for 10 minutes.

[0018] In the present invention, the synthesis method of the black phosphorus-methylene blue nanocomposite for precise targeted treatment of Tau phosphorylation, the centrifugation conditions in step 4 are a temperature of 4° C., a rotation speed of 10,000-12,000 r / min, and each centrifugation for 10 minutes.

[0019] The second aspect of the present invention provides a black phosphorus-methylene blue nanocomposite for precise targeted treatment of Tau phosphorylation, which is prepared by any of the above methods.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention creatively adds amino superparamagnetic nanoparticles, utilizes the targeting property of the amino superparamagnetic nanoparticles under the action of an external magnetic field, can be magnetized and move in a directional manner, and when the external magnetic field is removed, the amino superparamagnetic nanoparticles can be suspended again, and has the advantages of good dispersibility and operability. After the amino superparamagnetic nanoparticles are added, methylene blue, a drug for treating Tau protein phosphorylation, can be successfully loaded into the black phosphorus nanosheets, and the multi-fold structure of the black phosphorus nanosheets is utilized to increase the drug loading rate of methylene blue to more than 60%. The composition has precise targeted treatment ability, good biocompatibility, is non-toxic to cells, and the preparation process is very simple and convenient, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the solutions in the present application or the prior art, a brief introduction is given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 The UV absorption spectrum analysis results of BP, MB, BP-PEG, and BP-PEG@MB in Example 1;

[0024] Figure 2 Zeta potential analysis results of BP (-30.47 mV), PEG (-1.52 mV), MB (-5.56 mV), BP-PEG (-33.91 mV), and BP-PEG@MB (-32.79 mV) in Example 1;

[0025] Figure 3The Zeta potential analysis results of BP (-30.47 mV), PEG (-1.52 mV), MB (-5.56 mV), SPION (24.44 mV), BP-PEG (-33.91 mV), and BP-PEG-SPION@MB (-11.19 mV) in Example 2. The change in potential confirmed the successful solidification of the components between the materials.

[0026] Figure 4 Transmission electron microscopy (TEM) analysis results of BP, BP-PEG and BP-PEG-SPION@MB. The average lateral size of pure black phosphorus nanosheets (BP) is about 130 nm (scale: 20 nm), and the size of the synthesized nanocomposites BP-PEG and BP-PEG-SPION@MB is about 200 nm (scale: 200 nm). The particles are small and suitable for in vivo application in Alzheimer's disease.

[0027] Figure 5 The results of energy dispersive x-ray spectroscopy (EDS) analysis of BP, SPION and the final reaction product in Example 2. The results show that BP and SPION are successfully combined with MB to form BP-PEG-SPION@MB;

[0028] Figure 6 The FTIR spectra of the substances in Example 2 show that BP and SPION are successfully combined with MB to form BP-PEG-SPION@MB.

[0029] Figure 7 The UV-visible spectrum analysis results of MB, SPION, BP and the final reaction product BP-PEG-SPION@MB in Example 2 show that MB has obvious absorption peaks at a wavelength of 664 nm and SPION has obvious absorption peaks at a wavelength of 400 nm, and BP-PEG-SPION@MB has visible absorption peaks at wavelengths of 664 nm and 400 nm, confirming the successful combination of MB and SPION;

[0030] Figure 8 The figure is the Western Blot protein blotting result of Example 4;

[0031] Fig. 9 This is the flow analysis result diagram of Example 5;

[0032] Fig.10 This is a graph showing the results of CCK8 biocompatibility analysis of BP-PEG-SPION@MB in Example 6. DETAILED DESCRIPTION

[0033] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0034] Example 1

[0035] This example proposes a method for synthesizing a black phosphorus-methylene blue nanocomposite for targeted treatment of Tau phosphorylation, which specifically includes the following steps:

[0036] Step 1: Take 2 mL of black phosphorus nanosheet dispersant (BPNSs, 1 mg / mL) and perform ultrasonic treatment for 30 minutes. Then weigh 20 mg of polyethylene glycol 2000 (HO(CH2CH2O)nH) and dissolve it in 2 mL of pure water (dH2O). Slowly add the ultrasonicated black phosphorus nanosheet dispersant and place the mixture in a 4°C water bath with magnetic stirring for 8-12 hours.

[0037] Step 2: Place the mixture in step 1 in a high-speed centrifuge and centrifuge at 4°C and 10,000 r / min. Centrifuge for 10 minutes and repeat the washing and centrifugation three times. Collect the supernatant and quantitatively divide the lower layer of reactants and store them at 4°C in the dark.

[0038] Step 3: Slowly add methylene blue (MB) to the lower layer of the reaction collected in step 2, and incubate it in a shaker at room temperature for 8 to 12 hours after it is fully dissolved;

[0039] Step 4: Place the mixture of step 3 in a high-speed centrifuge and centrifuge at 4°C and 12,000 r / min. Centrifuge for 10 minutes and repeat the washing and centrifugation three times. Collect the supernatant and quantitatively divide the lower layer of reactants and store them at 4°C in the dark.

[0040] Step 5: Detect the synthesis of the material using an ultraviolet-visible spectrometer and a Zeta potential analyzer.

[0041] like Figure 1 As shown in FIG. 1 , the UV-visible spectra of the supernatants collected in step 2 and step 4 are almost the same. After adding MB, the supernatant obtained in step 4 does not show a corresponding absorption peak at the characteristic wavelength of MB, 664 nm. Figure 2 As shown, the Zeta potential results of the reaction products obtained in step 2 and step 4 did not change significantly, which shows that methylene blue (MB) and black phosphorus nanosheet dispersant (BPNSs) did not combine successfully.

[0042] Example 2

[0043] This example proposes a method for synthesizing a black phosphorus-methylene blue nanocomposite for precise targeted treatment of Tau phosphorylation, which specifically includes the following steps:

[0044] Step 1: Take 2 mL of black phosphorus nanosheet dispersant (BPNSs, 1 mg / mL) and perform ultrasonic treatment for 30 minutes. Then weigh 20 mg of polyethylene glycol 2000 (HO(CH2CH2O)nH) and dissolve it in 2 mL of distilled water (dH2O). Slowly add the ultrasonicated black phosphorus nanosheet dispersant and place the mixture in a 4°C water bath with magnetic stirring overnight.

[0045] Step 2: Place the mixture in step 1 in a high-speed centrifuge and centrifuge at 4°C and 10,000 r / min. Centrifuge for 10 minutes and then wash and centrifuge three times. Collect the supernatant and store the lower layer of the reactant in aliquots at 4°C in the dark.

[0046] Step 3, slowly adding the amino superparamagnetic nanoparticles and methylene blue (MB) to the lower layer of the reaction product obtained in step 2, and incubating them on a shaker at room temperature for 8 to 12 hours after they are fully dissolved, wherein the molar volume ratio of the amino superparamagnetic nanoparticles to the methylene blue (MB) is 1:1;

[0047] Step 4: Place the mixture of step 3 in a high-speed centrifuge and centrifuge at 4°C and 12,000 r / min. Centrifuge for 10 minutes and then wash and centrifuge three times. Collect the supernatant and quantitatively divide the lower layer of reactants and store them at 4°C in the dark.

[0048] Step 5. As shown in Table 1, the MB content in the supernatant was detected by UV-visible spectrometer to calculate the encapsulation efficiency. The results showed that after adding amino superparamagnetic nanoparticles, the encapsulation efficiency of BP-PEG-SPION@MB reached 63.49%.

[0049] Table 1 Encapsulation efficiency calculation of Example 2

[0050]

[0051] The results were shown in the Zeta potential instrument. Figure 4 ), transmission electron microscopy TEM (results as shown Figure 5 as shown), energy dispersive x-ray spectrometer (results as shown Figure 6 As shown), FTIR Fourier transform infrared spectrometer (results as shown Figure 7 As shown), UV spectrometer (results as shown Figure 8 The immobilization status of each component in BP-PEG-SPION@MB was detected (as shown in Figure 5), and the results showed that after the addition of amino-modified superparamagnetic nanoparticles, MB and SPION could be successfully combined and solidified with BP.

[0052] In this embodiment, the oxidation of black phosphorus is effectively reduced by modifying it with polyethylene glycol through electrostatic adsorption. After adding amino superparamagnetic nanoparticles, the biological polymers can be easily embedded on the crystal surface by utilizing its specific surface area effect and magnetic effect, becoming a binding bridge between BP and MB, and effectively solidifying BP and MB together.

[0053] Example 3

[0054] This example proposes a method for measuring the MB concentration in the BP-PEG-SPION@MB solution using an ultraviolet spectrophotometer, which specifically comprises the following steps:

[0055] Step 1. Draw a standard curve of MB ultraviolet spectrophotometric absorbance: fully dissolve 1 mg MB in dH2O and dilute to different concentrations (1, 2, 2.5, 3, 4, 5, 6, 10 μg / mL). Then use a UV-visible spectrometer to measure the absorbance of MB at a characteristic wavelength of 664 nm to obtain a concentration-absorbance standard curve of MB.

[0056] Step 2: Measure and calculate the MB concentration in BP-PEG-SPION@MB. The BP-PEG-SPION@MB supernatant collected in step 4 of Example 2 was quantified to 20 mL, and then diluted 100 times with dH2O. The absorbance at a wavelength of 664 nm was measured using a UV-visible spectrometer, and the MB concentration was calculated based on the absorbance. The results are shown in Table 1.

[0057] Example 4.

[0058] This example proposes a method for in vitro verification of the therapeutic effect of inhibiting Tau protein phosphorylation, which specifically comprises the following steps:

[0059] Step 1: 293 cells and 293 cells stably transfected with human phosphorylated Tau (2.5×10 5 mL-1) were inoculated into 2 mL of culture medium in a 6-well plate and co-cultured with MB, the final reaction product BP-PEG@MB obtained in Example 1, and the final reaction product BP-PEG-SPION@MB obtained in Example 2 for 12 hours;

[0060] Step 2: Collect cells and lyse them with RIPA buffer containing 1% PMSF protease inhibitor and 1% phosphatase inhibitor;

[0061] Step 3: Place the samples in a high-speed centrifuge, set the temperature to 4°C, and centrifuge at 12000 rpm / min for 20 minutes, then collect the supernatant.

[0062] Step 4. Use the BCA kit to measure the protein content in the supernatant. Dilute the supernatant with PBS 5x buffer, heat to 95~100℃ for 3~5 minutes, and then separate by 10% SDS-PAGE. After electrophoresis, the protein is transferred to a polyvinylidene fluoride membrane and the membrane is blocked with a protein-free rapid blocking buffer at room temperature for 20-30 minutes. Then incubate with the primary antibody overnight at 4℃, and then incubate with the secondary antibody for another 1 hour at room temperature;

[0063] Step 5: Detection was performed using an enhanced chemiluminescence ECL kit. After each liquid change, the immunoblot results were washed with TBST buffered saline containing Tween-20. Finally, the immunoreactive bands were analyzed using Quantity One software and GAPDH was normalized using ImageJ software.

[0064] The results of this example are shown in Figure 8 By incubating highly phosphorylated Tau protein with different materials, MB and BP-PEG-SPION@MB have a significant inhibitory effect on the expression of highly phosphorylated Tau protein in cells. Compared with the control group incubated without adding materials, BP-PEG@MB showed no significant difference in the inhibitory effect on highly phosphorylated Tau protein in cells, which further illustrates that MB in Example 1 did not successfully bind to BPNSs. Therefore, during the synthesis process, adding amino superparamagnetic nanoparticles can effectively increase the MB loading rate, enhance the therapeutic effect, and inhibit Tau protein aggregation.

[0065] Example 5

[0066] This example shows an in vitro experimental method for verifying the therapeutic effect of ROS removal in mitochondria, which specifically includes the following steps: selecting 293 cells in the logarithmic growth phase and 293 cells stably transfected with human phosphorylated Tau, using DMEM (44.5 mL) culture medium supplemented with 100 U / mL dual antibody (500 μL) and 10% FBS per 50 mL to prepare a single cell suspension, and 2×10 5 The cells / dish were inoculated in a confocal dish, with 1 mL of culture medium per dish, placed in a cell culture incubator, and incubated for 24 hours. MB, the final reactant BP-PEG @MB obtained in Example 1, and the final reactant BP-PEG-SPION@MB obtained in Example 2 were then added, respectively. After incubation for 12 hours, the cells were collected and washed twice with PBS, and then 500 μL of working solution containing MitoSOX (5 μM) was added in the dark at 37°C, and finally incubated in a cell culture incubator at 37°C for 10 minutes and detected using a flow cytometer.

[0067] The results are as follows Fig. 9As shown in the figure, by incubating cells with highly phosphorylated Tau protein with different materials, BP-PEG-SPION@MB has a more significant inhibitory effect on the expression of ROS in cells overexpressing highly phosphorylated Tau protein compared with simple MB treatment. Compared with the control group incubated without adding materials, there is no significant difference in the inhibitory effect of BP-PEG@MB on ROS, which further illustrates that MB in Example 1 did not successfully bind to BPNSs. Therefore, during the synthesis process, the addition of amino superparamagnetic nanoparticles can effectively increase the MB loading rate and reduce the oxidative stress induced by highly phosphorylated Tau protein in cells.

[0068] Example 6

[0069] This example shows an in vitro experimental method for verifying the biocompatibility of BP-PEG-SPION@MB, which specifically includes the following steps:

[0070] A 293 cell suspension (100 μL) in the logarithmic growth phase was inoculated in a 96-well plate, with about 5,000 cells / well. After 24 hours of cell culture, different amounts of BP-PEG-SPION@MB were added to make the final BP-PEG-SPION@MB concentrations 1, 5, 10, 20, 40, and 60 μg / ml. After incubation for 12 hours, 10 μL of CCK8 solution was added to each well, and the blank wells were set with equal amounts of cell culture medium, BP-PEG-SPION@MB without cells, and CCK8 solution. Two hours after adding CCK8 solution, the absorbance of each well at 450 nm was measured using an ELISA reader.

[0071] The results are as follows Fig.10 As shown, after incubation with different concentrations of BP-PEG-SPION@MB for 12 hours, the viability of 293 cells still remained above 0.95, indicating that BP-PEG-SPION@MB has good biocompatibility.

[0072] Definitions of Abbreviations and Key Terms

[0073] Tau: Tau protein is a microtubule-associated protein that is widely distributed in the nerve cells of the nervous system and is an important component used to stabilize microtubules that serve as the skeleton of nerve cells.

[0074] AD: Alzheimer's disease;

[0075] MB: methylene blue;

[0076] BP: Black phosphorus, a black semiconductor crystal with a metallic luster, its density is 2.70g / cm 3, with a hardness of 2, its lattice is composed of two atomic layers, each of which is composed of a zigzag chain of phosphorus atoms;

[0077] PEG: polyethylene glycol;

[0078] BP-PEG@MB: black phosphorus polyethylene glycol methylene blue composition;

[0079] BP-PEG-SPION@MB: black phosphorus polyethylene glycol superparamagnetic iron oxide methylene blue composition;

[0080] BPNSs: black phosphorus nanodispersants;

[0081] SPION: aminated ferroferric oxide magnetic nanoparticles;

[0082] dH2O:Distilled water;

[0083] 293 cells: HEK293 cells, also known as human embryonic kidney cells 293, are a cell line derived from human embryonic kidney cells, with the characteristics of high transfection efficiency and easy culture;

[0084] PMSF: Methylsulfonyl fluoride, an irreversible serine protease inhibitor used in a variety of research and laboratory procedures;

[0085] Double antibody: Double antibody culture medium is a kind of culture medium, in which the double antibody is a mixture of penicillin and streptomycin;

[0086] ROS: Reactive oxygen species, which are chemically reactive species containing oxygen, including peroxides, superoxides, hydroxyl radicals, singlet oxygen, and α-oxygen;

[0087] FBS: fetal bovine serum;

[0088] DMEM: A medium containing various amino acids and glucose;

[0089] MitoSOX: Superoxide indicator is a new type of fluorescent dye that specifically targets mitochondria in living cells;

[0090] PBS: Phosphate buffer solution, the most widely used buffer in biochemical research, the main components of which are Na2HPO4, KH2PO4, NaCl and KCl. It is generally used as a solvent to dissolve protective reagents;

[0091] BCA kit: a protein concentration determination product developed based on the BCA method (bicinchoninic acid, diquinoline formic acid method) protein detection principle. Under alkaline conditions, the blue Cu 2+ The Cu reduced to purple by protein +, the model used in the embodiments of the present application is BL521A-Biosharp;

[0092] SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis, a discontinuous electrophoresis system, usually used to separate proteins with molecular weights between 5-250 kDa;

[0093] Phosphatase inhibitors: used for extracting proteins from animal cells or tissues, maintaining protein phosphorylation, and commonly used in the study of protein kinase activity and signal transduction in animal cells or tissues. The model used in the examples of this application is BL630B-Biosharp-China;

[0094] RIPA buffer: can lyse cultured mammalian cells, and can be used to lyse both adherent cells and suspended cells. The model used in the examples of this application is R0020-Solarbio-China;

[0095] Quantity One: Quantity One software, a member of The Discovery Series software family, is a powerful, flexible software package for imaging and analyzing one-dimensional electrophoresis gels, dot blots, slot blots, and colony counts;

[0096] ImageJ: is a Java-based image processing software developed by the National Institutes of Health (NIH);

[0097] GAPDH: glyceraldehyde-3-phosphate dehydrogenase, widely distributed in cells of various tissues, can be used to visualize cells in microscopic examinations and to study cell metabolism and functional activity;

[0098] PDVF: polyvinylidene fluoride membrane, the model used in the examples of this application is Millipore-MA-USA;

[0099] Protein-free rapid blocking buffer: a new type of blocking buffer that does not contain protein components, thus avoiding protein contamination and cross-reaction, and is suitable for various biological experiments and clinical tests. The model used in the examples of this application is PS108P-EpiZyme-China;

[0100] Primary antibody: refers to a protein that can specifically bind to a non-antibody antigen. The primary antibody used in the examples of this application is a mixture of Anti-pTau Thr231 antibody (1:1000, #71429, Cell Signaling Technology) and Anti-GAPDH antibody (1:10000, #2118, Cell Signaling Technology);

[0101] Secondary antibody: refers to a protein that can bind to an antibody and is an antibody of the primary antibody. The secondary antibody used in the present application example is goat anti-rabbit IgG H&L (1:20000, CSB-PA489724, CUSABIO, USA);

[0102] ECL kit: an ultra-sensitive ECL chemiluminescent kit that chemically reacts with horseradish peroxidase coupled to the secondary antibody to emit fluorescence, so that the sample can be detected by X-ray film or other appropriate chemiluminescent imaging equipment. The model used in the examples of this application is P0018M-Beyotime-China.

[0103] Obviously, the embodiments described above are only some embodiments of the present application, rather than all embodiments. The preferred embodiments of the present application are given in the accompanying drawings, but they do not limit the patent scope of the present application. The present application can be implemented in many different forms. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive. Although the present application is described in detail with reference to the aforementioned embodiments, for those skilled in the art, it is still possible to modify the technical solutions recorded in the aforementioned specific implementation methods, or to perform equivalent replacement of some of the technical features therein. Any equivalent structure made using the contents of the specification and drawings of this application, directly or indirectly used in other related technical fields, is similarly within the scope of patent protection of this application.

Claims

1. A method for synthesizing a black phosphorus-methylene blue nanocomposite for precise targeted treatment of Tau phosphorylation, characterized in that: The following steps are involved: Step 1, mixing black phosphorus nanosheets and polyethylene glycol for reaction; Step 2: Centrifuge and wash the reaction solution in step 1 to collect the lower layer of reactants; Step 3, adding amino superparamagnetic nanoparticles and methylene blue to the lower layer reactant obtained in step 2 to carry out reaction; Step 4: Centrifuge and wash the reaction solution obtained in step 3, and collect the lower layer of the reaction product; The molecular weight of the polyethylene glycol in step 1 is 2000; The volume molar ratio of the amino superparamagnetic nanoparticles and methylene blue in step 3 is 1-2:1-2.

2. The method for synthesizing a black phosphorus-methylene blue nanocomposite for precise targeted treatment of Tau phosphorylation according to claim 1, characterized in that: The volume molar ratio of the amino superparamagnetic nanoparticles and methylene blue in step 3 is 1:

1.

3. The method for synthesizing a black phosphorus-methylene blue nanocomposite for precise targeted treatment of Tau phosphorylation according to claim 2, characterized in that: In step 1, the black phosphorus nanosheets are subjected to ultrasound for 20 to 40 minutes before the reaction.

4. The method for synthesizing a black phosphorus-methylene blue nanocomposite for precise targeted treatment of Tau phosphorylation according to claim 3, characterized in that: The reaction in step 1 was magnetically stirred in a 4° C. water bath for 8 to 12 hours.

5. The method for synthesizing a black phosphorus-methylene blue nanocomposite for precise targeted treatment of Tau phosphorylation according to claim 1, characterized in that: The reaction time of step 3 is 8 to 12 hours.

6. The method for synthesizing a black phosphorus-methylene blue nanocomposite for precise targeted treatment of Tau phosphorylation according to claim 1, characterized in that: The centrifugation conditions in step 2 are as follows: temperature 4°C, speed 9000-10000 r / min, and each centrifugation for 10 minutes.

7. The method for synthesizing a black phosphorus-methylene blue nanocomposite for precise targeted treatment of Tau phosphorylation according to claim 1, characterized in that: The centrifugation conditions in step 4 are as follows: temperature 4°C, speed 10000-12000 r / min, and each centrifugation for 10 minutes.

8. A black phosphorus-methylene blue nanocomposite for precise targeted treatment of Tau phosphorylation, characterized in that: The composition is prepared by the method according to any one of claims 1 to 7.

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