Abeta-targeted black phosphorus cinnamate nano composition as well as preparation method and application thereof

Abstract

The invention discloses an A beta-targeted black phosphorus cinnamate nano composition as well as a preparation method and application thereof. The composition takes 4-(dimethylamino) cinnamic acid as a group for targeting A beta, and is composed of a drug loading main body BP (black phosphorus nanosheet), a modification material C18-PEG-NH2 (amino polyethylene glycol stearic acid), a targeting material Tar (4-(dimethylamino) cinnamic acid) and a therapeutic material Cur (curcumin). The preparation method comprises the following steps: (1) preparation of PEG-Tar, (2) preparation of PEG-Tar-Cur, and (3) preparation of the BP-PEG-Tar-Cur composition. Black phosphorus nanosheets are used for loading Cur (curcumin), the drug loading capacity of the Cur is remarkably improved due to the good specific surface area, 4-(dimethylamino) cinnamic acid serves as a targeted A beta group, diffuse distribution of drugs in the brain is avoided, the drug utilization rate of the Cur is greatly improved, the synthesis process is simple, repeatability is high, and the preparation method is suitable for industrial production. The medicine is easy for industrial large-scale application, and has a positive effect in the field of Alzheimer's disease treatment.

Description

Technical Field

[0001] The invention relates to the technical field of biomedical materials, and in particular to a cinnamic acid black phosphorus nanocomposite targeting Aβ, and a preparation method and application thereof. Background Art

[0002] Studies have shown that the onset of Alzheimer's disease (AD) is related to the abnormal deposition of amyloid beta (Aβ) in the brain. Aβ is produced by the hydrolysis of its precursor protein (APP) by secretase, transported to the brain, and abnormally aggregated to produce Aβ oligomers, causing neurotoxic reactions such as inflammation, ROS oxidative stress, and calcium overload, destroying synaptic membranes, and ultimately causing neuronal cell death. Therefore, reducing Aβ production, promoting Aβ clearance, regulating Aβ transport, inhibiting Aβ aggregation, and counteracting its neurotoxicity can effectively treat AD clinically.

[0003] Black phosphorus (BP) is a new type of nanomaterial composed of a single phosphorus element. Black phosphorus nanomaterials have the following advantages: they produce singlet oxygen under near-infrared light irradiation and can be used as a photosensitizer for photodynamic therapy; they can have a wide range of light absorption in the longer wavelength region, and the thermal properties of this near-infrared light can be applied to photothermal therapy; black phosphorus nanosheets have a high specific surface area and a unique wrinkled structure, which gives them an extremely high drug loading capacity, and they have relatively high biological activity and biodegradability.

[0004] Curcumin is a hydrophobic polyphenol extracted from plants such as Curcumaceae and Araceae, and has been shown to have anti-inflammatory, antioxidant, and antiviral effects. Studies have shown that curcumin can play a neuroprotective role in Parkinson's disease through multiple pathways such as protecting dopaminergic neurons, anti-inflammatory, anti-oxidation, anti-apoptosis, anti-mitochondrial damage, and regulating autophagy. Compared with chemical drugs, traditional Chinese medicine has good tolerance and safety, few adverse reactions, and a wide range of applicability. However, due to the low water solubility of curcumin, its absorption rate in the body is low and its bioavailability is low, which greatly limits the use of curcumin in the medical field.

[0005] At present, the biocompatibility of curcumin is improved by forming a composition of curcumin with peptides, such as the curcumin-peptide complex proposed in Chinese patent CN105358179B (authorization publication date: 2018.12.07). Although the curcumin-peptide complex improves the solubility of curcumin, its main limitation is that it is easily cleaved and degraded by proteases, and lacks specificity, resulting in a part of it being degraded before it reaches the brain, thereby greatly reducing the utilization rate of the drug. Another problem is that the use of peptides to encapsulate drugs has a low loading capacity and cannot achieve large-capacity drug delivery. Moreover, simple drug delivery without targeting the target substance will cause the drug to be diffusely distributed in the brain, non-specifically binding to other parts, resulting in a decrease in drug utilization. Summary of the invention

[0006] In order to overcome the above-mentioned deficiencies of the prior art, the object of the present invention is to provide a cinnamic acid black phosphorus nanocomposition targeting Aβ and a preparation method and application thereof.

[0007] The first aspect of the present invention proposes a cinnamic acid black phosphorus nanocomposite for targeting Aβ, which is composed of a drug-carrying body black phosphorus nanosheet, a modifying material aminopolyethylene glycol stearic acid, a targeting material 4-(dimethylamino)cinnamic acid) and a therapeutic material Cur (curcumin).

[0008] The cinnamic acid black phosphorus nanocomposite targeting Aβ of the present invention uses 4-(dimethylamino)cinnamic acid as a group targeting Aβ.

[0009] The second aspect of the present invention provides a method for preparing a cinnamic acid black phosphorus nanocomposite targeting Aβ, comprising the following steps:

[0010] (1) 4-(dimethylamino)cinnamic acid, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide are mixed in a certain proportion, and a solvent is added to carry out a dissolving reaction, and then amino polyethylene glycol stearic acid is added to continue the reaction.

[0011] (2) After adding curcumin to the solution obtained in step (1) for reaction, the solution is then dropped into rapidly stirred water and heated until the odor is completely volatilized. The upper layer of the solution is collected after centrifugation.

[0012] (3) reacting the upper layer solution obtained in step (2) with black phosphorus nanosheets to generate a final product, a cinnamic acid black phosphorus nanocomposite targeting Aβ.

[0013] In the present invention, the molar ratio of the four materials of 4-(dimethylamino)cinnamic acid, aminopolyethylene glycol stearic acid, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide in step (1) is (1-2):1:(1-2):(1-2).

[0014] More preferably, the molar ratio of the four materials of 4-(dimethylamino)cinnamic acid, aminopolyethylene glycol stearic acid, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide in step (1) is 2:1:2:2.

[0015] In the present invention, the solvent in step (1) is tetrahydrofuran, and the reaction is carried out for 4 to 6 hours after the solvent is added.

[0016] In the present invention, the hydrophobic reaction is carried out for 20 to 25 hours after the amino polyethylene glycol stearic acid is added in step (1).

[0017] In the present invention, step (2) specifically comprises adding curcumin to the solution obtained in step (1) and reacting for 10 to 15 hours, dripping the obtained product into rapidly stirred water, heating in a water bath until the odor is completely volatilized, and taking the upper layer solution after centrifugation.

[0018] In the present invention, the step (3) specifically comprises reacting the solution obtained in step (2) with black phosphorus nanosheets through phosphorus-oxygen bonds for 10 to 15 hours, and then repeating centrifugation twice to obtain the final product, cinnamic acid black phosphorus nanocomposite particles targeting Aβ.

[0019] The third aspect of the present invention provides the use of a cinnamic acid black phosphorus nanocomposition targeting Aβ in the preparation of a drug for treating Alzheimer's disease, wherein the composition is prepared by any of the above methods.

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

[0021] 1. The present invention can target Aβ more accurately by adding cinnamic acid as a targeting substance, avoiding diffuse distribution of drugs in the brain, greatly improving drug utilization, and combining the many advantages of the new nanomaterial black phosphorus, which is non-toxic, has good biodegradability and biocompatibility, and a high surface-to-volume ratio, so that the drug loading capacity of black phosphorus nanosheets for curcumin is significantly increased, overcoming the problem that peptides are easily degraded during in vivo transportation and the loading rate is low. This cinnamic acid black phosphorus composite nanocomposite has the dual functions of photothermally opening the blood-brain barrier under NIR irradiation and being able to target Aβ, and has broad prospects in the field of drugs for treating Alzheimer's disease.

[0022] 2. The present invention does not require the use of complex synthesis procedures. By simply adding BP as a carrier and using cinnamic acid as a target substance, the drug loading rate and targeting ability of Cur can be greatly improved. The synthesis process is simple, highly repeatable, and easy to apply on an industrial scale. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] 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.

[0024] Figure 1 The UV spectrum results of Example 1 show that Cur and BP are not bound;

[0025] Figure 2 The UV spectrogram result of Example 2 is shown below:

[0026] Figure 3 The UV spectrogram result of Example 3 is shown below:

[0027] Figure 4A This is the BP microscope morphology image;

[0028] Figure 4B This is the microscope morphology of BP-PEG-Tar@Cur;

[0029] Figure 4C Potential diagrams for various materials;

[0030] Figure 4D It is the particle size distribution density diagram of various materials;

[0031] Figure 4E is the Fourier infrared spectrum of each material;

[0032] Figure 4F is the spectrophotometric value of each material solution;

[0033] Figure 4G is the Raman spectrum value of each material;

[0034] Figure 4H It is the dimensional stability diagram of various materials in PBS;

[0035] Fig. 4I It is the dimensional stability diagram of various materials in DMEM;

[0036] Figure 4J This is a diagram of the in vitro drug release model of curcumin;

[0037] Figure 5A Schematic diagram of cell viability test results;

[0038] Figure 5B This is the fluorescence experiment diagram of Aβ disaggregation;

[0039] Figure 5C This is a schematic diagram of the BCA protein quantification experiment results;

[0040] FIG5D is a schematic diagram of ThT fluorescence test results;

[0041] Figure 6 Schematic diagram of the cell targeted uptake experimental results;

[0042] Figure 7 Schematic diagram of the mitochondrial ROS clearance experiment results;

[0043] Figure 8 Schematic diagram of Transwell experiment results. DETAILED DESCRIPTION

[0044] 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.

[0045] In order to enable those skilled in the art to better understand the solution of the present application, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the accompanying drawings.

[0046] Example 1

[0047] This embodiment shows a method for preparing a black phosphorus nanocomposite material, which specifically includes the following steps:

[0048] Step 1: Weigh 2 mg of Cur and dissolve it in 1 ml of anhydrous ethanol. Use ultrasound to assist dissolution, and then slowly add 8 ml of anhydrous ethanol to dissolve;

[0049] Step 2: Divide the above solution into two 5 ml EP tubes, add 0.5 ml 1 mg / ml BP into one of the tubes, mix well, and shake on a shaker overnight;

[0050] Step 3: Take out the EP tube and place it in a high-speed centrifuge for centrifugation at a speed of 7830 r / min. Filter after centrifugation for 20 minutes;

[0051] Step 4: Determine the material synthesis result by detecting the UV spectrum, such as Figure 1 As shown, the UV spectra of the solution with BP added and the solution without BP added are not much different, proving that Cur and BP are not combined.

[0052] Example 2

[0053] This embodiment shows a method for preparing a cinnamic acid black phosphorus nanocomposite, which specifically includes the following steps:

[0054] Step 1: First, 4-(dimethylamino)cinnamic acid, N-hydroxysuccinimide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide were added to a flask, and 2 ml of tetrahydrofuran was added to dissolve the mixture to activate the hydroxyl group in the 4-(dimethylamino)cinnamic acid;

[0055] Step 2: After reacting for 5 hours, amino polyethylene glycol stearic acid is added to form a hydrophobic chain to encapsulate Cur, and the reaction is continued for 24 hours. The molar ratio of the four materials of 4-(dimethylamino)cinnamic acid, amino polyethylene glycol stearic acid, N-hydroxysuccinimide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 1:1:1:1;

[0056] Step 3: Add 10 mg of Cur to the flask for reaction. After 12 hours of reaction, slowly drip the reaction solution into 12 ml of rapidly stirred water and heat in a 40°C water bath until the odor is completely evaporated;

[0057] Step 4: After the odor has completely evaporated, place the solution in a high-speed centrifuge and centrifuge at a speed of 6000r / min for 5 minutes. After the centrifugation, extract the supernatant and adjust the volume to 14ml;

[0058] Step 5: Draw out half of the PEG-Tar@Cur supernatant to measure the UV spectrophotometric value, and put the other half of the supernatant in a flask. Centrifuge 4 ml of 1 mg / ml BP, resuspend it in 1 ml of water and add it to the flask. PEG-Tar@Cur reacts with BP through the phosphorus-oxygen bond.

[0060] Step 6: After 12 hours of reaction, the reaction solution in the flask was extracted and placed in a high-speed centrifuge for centrifugation at a speed of 7000 r / min for 5 minutes. After the centrifugation, the supernatant was extracted and the ultraviolet spectrophotometry value was determined. Then, 5 ml of water was added for resuspending. The above centrifugation steps were repeated to wash away the unbound PEG-Tar@Cur to obtain 5.3 ml of the final product suspension. The absorbance value was determined by ultraviolet spectrophotometer. The final suspension was measured by ultraviolet spectroscopy for Cur content, and the drug loading of curcumin was measured according to the steps of Example 4. Finally, the total amount of BP-PEG-Tar@Cur nanoparticles was dried to obtain 1 mg.

[0061] Step 7: The synthesis of the material was determined by detecting the UV spectra of different products and the encapsulation efficiency and loading rate were calculated by detecting the Cur content in the supernatant. As shown in Table 1, the encapsulation efficiency and loading rate were 11.72% and 45.84%, respectively, and both efficiencies were relatively low.

[0062] Table 1 Encapsulation efficiency and drug loading rate results of Example 2

[0063]

[0064] Example 3

[0065] This embodiment shows a method for preparing a cinnamic acid black phosphorus nanocomposite, which specifically includes the following steps:

[0066] Step 1: First, add 4-(dimethylamino)cinnamic acid, N-hydroxysuccinimide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide into a flask, and add 2 ml of tetrahydrofuran to dissolve, so as to activate the hydroxyl group in 4-(dimethylamino)cinnamic acid;

[0067] Step 2: After reacting for 5 hours, amino polyethylene glycol stearic acid is added to form a hydrophobic chain to encapsulate Cur, and the reaction is continued for 24 hours. The molar ratio of the four materials of 4-(dimethylamino)cinnamic acid, amino polyethylene glycol stearic acid, N-hydroxysuccinimide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 2:1:2:2;

[0068] Step 3: Add 10 mg of Cur to the flask for reaction. After 12 hours of reaction, slowly drip the reaction solution into 12 ml of rapidly stirred water and heat in a 40°C water bath until the odor is completely evaporated.

[0069] Step 4: After the odor has completely evaporated, place the solution in a high-speed centrifuge and centrifuge at a speed of 6000r / min for 5 minutes. After the centrifugation, extract the supernatant and adjust the volume to 14ml;

[0070] Step 5: Draw out half of the supernatant of the PEG-Tar@Cur reaction solution into a flask, centrifuge 5 ml of 1 mg / ml BP, resuspend it in 1 ml of water and add it to the flask, and PEG-Tar@Cur reacts with BP through a phosphorus-oxygen bond;

[0071] Step 6: After 12 hours of reaction, the reaction solution in the flask was extracted and placed in a high-speed centrifuge for centrifugation at a speed of 7000 r / min for 5 minutes. After the centrifugation, the supernatant was extracted and the ultraviolet spectrophotometric value was determined. 5 ml of water was added for resuspending, and the above centrifugation steps were repeated to wash away the unbound PEG-Tar@Cur to obtain 5.3 ml of the final product suspension. The Cur content of the final suspension was determined by ultraviolet spectroscopy, and the drug loading of curcumin was measured according to the steps of Example 4. Finally, the total amount of BP-PEG-Tar@Cur nanoparticles was obtained by drying.

[0072] Step 7: The synthesis of the materials was determined by detecting the UV spectra of different products and the encapsulation efficiency and loading rate were calculated by detecting the Cur content in the supernatant. As shown in Table 3, after adjusting the ratio between the materials, the encapsulation efficiency and drug loading rate were improved, which were 22.75% and 68.51%, respectively.

[0073] Table 2 Calculation results of encapsulation efficiency and drug loading rate of Example 3

[0074]

[0075] Characterization and analysis of BP, PEG-Tar, PEG-Tar@Cur and BP-PEG-Tar@Cur

[0076] 1. Microscope morphology of BP and BP-PEG-Tar@Cur. Part of BP and BP-PEG-Tar@Cur samples were dispersed in ethanol solution and ultrasonicated. Then a few drops of the dispersed liquid were dripped onto the copper mesh. After drying, the morphology was photographed (high resolution) using a TEM transmission electron microscope (FEI Talos F200X G2, USA) with an acceleration voltage of 200 kV and an energy spectrum model of EDS super-X. The results are shown in the figure. Figure 4A , 4B shown.

[0077] 2. The particle size and potential of each material were measured by a laser particle size analyzer (model: Vern Nanosizer Nano ZS). The results are as follows: Figure 4C , 4D , 4H, and 4I.

[0078] 3. The Fourier transform infrared spectra of each material were measured by Fourier transform infrared spectrometer (model: Thermo Science Nicolet iS20). In a dry environment, the ATR accessory was placed in the optical path of the spectrometer, the air background was scanned, a drop of liquid was dropped on the crystal surface of the ATR accessory with a dropper, and then the infrared spectrum of the sample was collected, with a resolution of 4cm-1, 32 scans, and a test wave number range of 400-4000cm-1. The results are as follows: Figure 4E shown.

[0079] 4. The spectrophotometric values ​​of each material were measured by an ultraviolet spectrophotometer (model: UV-2450). The results are as follows: Figure 4F shown.

[0080] 5. The Raman spectrum values ​​of each material were measured by using a high-resolution confocal laser Raman microscope (model: German WITec alpha300R, laser: 532nm). Single spectrum test conditions: laser energy 36mW, 1800g / mm grating, Olympus 20x objective (Olympus20x / 0.25), integration time 30s, accumulation times 10 times, the results are as follows Figure 4G shown.

[0081] Example 4

[0082] This embodiment proposes a method for measuring the drug loading of curcumin in BP-PEG-Tar@Cur nanoparticles in Examples 2 and 3 using an ultraviolet spectrophotometer. First, a standard curve of curcumin is drawn. 1 mg of curcumin is dissolved in anhydrous ethanol and diluted to different concentrations (0.625, 1.25, 2.5, 5, 10, 20 μg / ml). According to the absorbance value of each concentration solution measured by an ultraviolet spectrophotometer at 425 nm, a concentration-absorbance standard curve of curcumin is drawn. In order to calculate the loading of curcumin in the synthetic material, the BP-PEG-Tar@Cur solvent is centrifuged and resuspended with 1 ml of anhydrous ethanol, and the absorbance value is measured at 425 nm. The drug loading percentage is obtained by dividing the amount of curcumin in the nanoparticles by the amount of the total nanoparticles multiplied by the percentage. The test showed that the concentration of curcumin in the anhydrous ethanol solvent of the product BP-PEG-Tar@Cur of Example 2 was 458.45 μg / ml, and the concentration of curcumin in the anhydrous ethanol solvent of the product BP-PEG-Tar@Cur of Example 3 was 931.74 μg / ml.

[0083] Example 5

[0084] This example proposes a method for determining the in vitro cumulative release of curcumin in BP-PEG-Tar@Cur nanoparticles. 0.5 ml of a material solution loaded with 435 mg / ml curcumin is added to a dialysis bag with a molecular weight cutoff of 500 and sealed with a clamp. The dialysis bag is placed in a release medium of 50 ml of anhydrous ethanol (0.01 M, PH=7.4) and placed on a shaker at a constant temperature of 37°C. At predetermined time intervals (0, 2, 4, 6, 8, 10, 12, 24 hours), 3 ml of the release solution is taken out and an equal volume of anhydrous ethanol is added at the same time. The absorbance value of the release solution extracted each time is measured at 425 nm using an ultraviolet spectrophotometer, and the cumulative release curve of curcumin is calculated based on each result, as shown in FIG. Figure 4J As shown, about 20% Cur was initially released from BP-PEG-Tar@Cur nanoparticles within 2 hours, and then released continuously to about 50% within 24 hours.

[0085] Example 6

[0086] This example provides a method for preparing Aβ monomers and fibrils. 1 mg of frozen Aβ 1-42 Allow to equilibrate at room temperature for 30 minutes to prevent peptide condensation. Add 222 μl of HFIP to the Aβ 1-42 The resulting solution was sonicated in a water bath for 10 minutes and incubated at 4°C for 2 hours. The peptide solution was vortexed and 10 μl aliquots were taken. All aliquots were placed in a SpeedVac at 45°C for 30 minutes to remove any residual HFIP.1-42 The monomers were stored in small EP tubes and stored at -20°C until use. Before use, these dried aliquots were resuspended in anhydrous DMSO to a concentration of 1 mM (amount of substance 10 nmol), treated in an ultrasonic bath for about 10 minutes, and then diluted to the required concentration using PBS (pH 7.4, 10 mM). The prepared solution was used immediately for the experiment. Aβ 1-42 The monomers were incubated at 37°C for 72 hours to form fibrils.

[0087] Example 7

[0088] This example proposes a cytotoxicity test method, inoculating a cell suspension (100 μl) in a 96-well plate, about 5000 cells / well, and adding Aβ after 12 hours of cell culture. 1-42 The fibrils were incubated for 6 hours. Five solutions of Cur, PEG-Tar@Cur, and BP-PEG-Tar@Cur at concentrations of 0.1, 0.5, 1, 2, and 4 μg / ml were added and incubated for 30 minutes, followed by NIR irradiation for 3 minutes (0.75 W cm-2) to maximize the targeting effect, and then incubated for another 24 hours. 10 μl of CCK8 solution was added to each well, and the blank wells were set to add equal amounts of cell culture medium, drugs, and CCK8 solution but no cells. The results are shown in Figure 5A As shown, the material showed low toxicity.

[0089] Example 8

[0090] This example proposes an Aβ fiber disaggregation fluorescence experiment. A volume of 75 μl of Aβ with a concentration of 25 μM is taken. 1-42 The samples were divided into three parts and added to 100 μl PBS, Cur and BP-PEG-Tar@Cur (0.5 μg / ml) respectively and incubated for 24 h. After the incubation, 10 μl of the sample was drawn out and added to a glass slide to air dry. After staining with ThT (10 μM) for 10 min, the samples were gently rinsed with water and air dried. Finally, the images were taken with a fluorescence microscope (Olympus, Japan). The results are shown in Figure 2. Figure 5B As shown, BP-PEG-Tar@Cur can disaggregate the aggregated Aβ fibers.

[0091] Example 9

[0092] This example proposes a method for determining the effect of Cur, PEG-Tar@Cur, and BP-PEG-Tar@Cur in inhibiting Aβ fibril aggregation using the BCA protein quantification method. A volume of 100 μl of 25 μM Aβ 1-42Divide into four parts, one of which is used as a blank, and the other three are incubated with Cur, PEG-Tar@Cur, and BP-PEG-Tar@Cur respectively. Except for the blank, ensure that the Cur concentration in each solution is 5μg / ml. The four solutions are shaken at 100rpm for 72 hours at 37°C in PBS (PH=7.4, 1X) buffer, and then centrifuged at 20000rpm / min for 20 minutes. Then 75% of the supernatant is extracted and the concentration of soluble Aβ is determined by BCA protein quantification method. After resuspending the aliquots with 10μl DMSO, add PBS (PH=7.4, 1X) to dilute the total volume to 100μl. Take 25μl of each and add it to the EP tube, add 75μl PBS (PH=7.4, 1X) to the tube, and the total volume is 100μl. The results are as follows Figure 5C As shown, with the monomer content as 100%, the monomer content of Aβ fibrils in the untreated group was about 30%, while the monomer content of the treated groups with added materials increased successively, and the monomer content of the BP-PEG-Tar@Cur group even reached 80%, proving that BP-PEG-Tar@Cur can effectively inhibit the aggregation of Aβ fibrils.

[0093] Example 10

[0094] This example proposes a ThT fluorescence test method, taking an equal portion of the sample Aβ 1-42 A tube was placed in a tube and balanced at room temperature for 10 minutes. Then, 10 μl DMSO was added and ultrasonicated in an ice water bath for 10 minutes. Then, 50 μl PBS (16.7 μM) was added and mixed. Cur, PEG-Tar@Cur, and BP-PEG-Tar@Cur solutions with a Cur concentration of 0.5 μg / ml were added to the EP tube, placed in a 37°C constant temperature shaker, and incubated at 100 rpm for 72 hours. After the reaction, the sample was mixed with 400 μl ThT (25 μM). The fluorescence intensity was detected using a fluorimeter (model: Japan HITACHI F-4500) at 25°C, a slit of 5 nm, and excitation and emission wavelengths of 440.480 nm, respectively. The results are shown in Figure 5D. The sample does not contain Aβ. 1-42 The blank solution was used as the background, containing only Aβ 1-42 The solution has a fluorescence intensity of 100%. The higher the fluorescence intensity, the more fibrils are formed. The results showed that the fluorescence intensity of the BP-PEG-Tar@Cur treatment group was reduced to about 35%, indicating that the material can effectively inhibit the aggregation of Aβ fibrils.

[0095] Embodiment 11

[0096] This example proposes a method for cell-targeted uptake experiment. N2a cells were plated at 2×10 5 The cells were seeded into the confocal dish at a density of 100 cells. After the cells adhered to the wall, Aβ was added.1-42 After incubation for 12 hours, 10 μg / ml of Cur, PEG-Tar@Cur, and BP-PEG-Tar@Cur solutions were added, respectively, and incubation continued for 12 hours. After the incubation, the culture medium was replaced and washed three times with PBS (PH=7.4, 1X). The cells were fixed with 4% paraformaldehyde by weight for 10 minutes and then stained with DAPI for 5 minutes. The cells were observed using a laser scanning confocal microscope at an excitation wavelength of 488 nm. The results are shown in Figure 2. Figure 6 As shown, the images under the confocal microscope show that the cell uptake in the material group with the targeting substance Tar increased significantly and was able to target Aβ.

[0097] Example 12

[0098] This example proposes a method for removing mitochondrial ROS. N2a cells in the logarithmic growth phase were selected, and single-cell suspensions were prepared using 50 ml of culture medium supplemented with 100 U / ml double-antibody cell culture medium (500 μl), 10% FBS in DMEM (23 ml), and opti-MEM (23 ml). 5 Cells / dish were seeded in confocal dishes, 1 ml of culture medium was added to each dish, and the dish was placed in a cell culture incubator. After incubation for 12 hours, 25 μM Aβ fibrils were added and incubated for 12 hours, and then 5 μg / ml of Cur, PEG-Tar@Cur, and BP-PEG-Tar@Cur were added and incubated for 12 hours, respectively, and washed twice with PBS (PH=7.4, 1X), and 1 ml of working solution containing 5 μM superoxide indicator MitoSOX was added in the dark at 37°C, and the staining was incubated in a 37°C cell culture incubator for 10 minutes, and the cells were fixed with wt4% paraformaldehyde for 15 minutes, washed twice with PBS (PH=7.4, 1X), counterstained with 1 μM DAPI for 5 minutes, and the fluorescence image of ROS clearance was obtained using a laser scanning confocal microscope. The results are shown in Figure 7 As shown, the ROS content of cells treated with Aβ fibrils was greatly increased, while the ROS content was significantly reduced after drug treatment, indicating that the material can effectively reduce the ROS generated by Aβ fibril aggregation.

[0099] Embodiment 13

[0100] This example proposes a Transwell in vitro invasion assay method, using immortalized mouse brain microvascular endothelial cells (bEnd.3 cells) to simulate the in vitro environment of the BBB at the cellular level to study the BBB penetration ability of BP-PEG-Tar@Cur under the photothermal effect. 4Cells) were seeded on the front side of a gelatin-coated 6-well plate Transwell chamber and cultured in DMEM containing 10% FBS and 1% penicillin / streptomycin. Transendothelial electrical resistance (TEER) was measured every 3-4 days, and the integrity of the BBB monolayer was assessed using a volt-ohmmeter (EVOM) from World Precision Instruments (Sarasota, FL, USA). The values ​​of transendothelial electrical resistance (TEER) were measured before and after the experiment, and the experiment could be started when the TEER value was stable. To ensure the accuracy of the values, the entire process was carried out at a constant temperature of 37°C. Three points were taken in different directions for each cell insert, and the measurement was repeated 3 times. After a few days, the cells converged to form a reliable BBB monolayer, forming a tight monolayer (transendothelial electrical resistance (TEER) exceeded 200Ωcm). The same concentration of Cur, BP-PEG-Tar@Cur, and BP-PEG-Tar@Cur materials under NIR irradiation were added to the apical donor chamber and incubated for 2 hours at 37°C and 5% CO2. 200μl samples were collected from the basolateral side and the Cur content was detected by UV spectrophotometer. The initial Cur content of the three treatment materials was 43.5μg. After 2 hours, the Cur content of the BP-PEG-Tar@Cur material group was 11.30μg, and the transmittance was 25.9%; the Cur transmittance of the BP-PEG-Tar@Cur material group under NIR irradiation was 14.08μg, and the transmittance was 32.4%. The results are as follows: Figure 8 As shown in the figure, under NIR irradiation, BP-PEG-Tar@Cur can effectively open the blood-brain barrier and enhance the permeability of Cur. At the same time, the cells treated with Cur, BP-PEG-Tar@Cur, and BP-PEG-Tar@Cur under NIR irradiation were stained with Calcein-AM / PI to distinguish live cells from dead cells, as shown in the figure. Figure 8 As shown, the microscopic conditions of the three treatment groups were similar, and there were no excessive dead cells, indicating that NIR irradiation did not increase the toxicity of BP-PEG-Tar@Cur.

[0101] Definitions of Abbreviations and Key Terms

[0102] BP: Black phosphorus, a black semiconductor crystal with a metallic luster, its density is 2.70g / cm 3 , hardness is 2. Its lattice is composed of two atomic layers, each layer is composed of a tortuous chain of phosphorus atoms. In these chains, the PPP bond angle is 90° and the phosphorus-phosphorus bond distance is 2.17 angstroms. Black phosphorus is the least reactive among the allotropes of phosphorus and it does not spontaneously ignite in the air.

[0103] PEG: C18-PEG-NH2, amino polyethylene glycol stearate.

[0104] Cur: Curcumin is a plant-derived polyphenolic compound that occurs naturally in turmeric, a food and medicine widely used in India and China.

[0105] Tar: 4-(dimethylamino)cinnamic acid, used as a targeting material to target Aβ.

[0106] PEG-Tar: polyethylene glycol stearate cinnamic acid composition.

[0107] PEG-Tar@Cur: polyethylene glycol stearate cinnamate curcumin composition.

[0108] BP-PEG-Tar@Cur: Cinnamic acid black phosphorus nanocomposite targeting Aβ.

[0109] NHS: N-hydroxysuccinimide, white to off-white crystals, used for the synthesis of amino acid protective agents, semi-synthetic kanamycin and pharmaceutical intermediates.

[0110] EDC: 1-ethyl-(3-dimethylaminopropyl)carbodiimide, is an organic compound with the chemical formula C8H17N3.

[0111] AD: Alzheimer's disease is a progressive neurodegenerative disease with an insidious onset. It is clinically characterized by global dementia such as memory impairment, aphasia, apraxia, agnosia, visual-spatial skill impairment, executive dysfunction, and personality and behavioral changes. The cause of the disease is still unknown.

[0112] Aβ (amyloid β-protein): β-amyloid protein, with a molecular weight of about 4kDa, is hydrolyzed from β-amyloid precursor protein (APP), secreted by cells, and has a strong neurotoxic effect after precipitating and accumulating in the cell matrix.

[0113] FTIR: Fourier transform infrared.

[0114] DLS: dynamic light scattering.

[0115] TEM: transmission electron microscope.

[0116] HFIP: hexafluoroisopropanol.

[0117] SpeedVac: Vacuum concentrator.

[0118] DMSO: dimethyl sulfoxide.

[0119] CLSM: confocal laser scanning microscopy.

[0120] PBS: Phosphate buffer solution is the most widely used buffer in biochemical research. Its main components are Na2HPO4, KH2PO4, NaCl and KCl. It is generally used as a solvent to dissolve and protect reagents.

[0121] N2a cells: N2a cells stand for mouse neuroblastoma N2a cells, which are mouse-derived neuroblastoma cells.

[0122] DAPI: 4,6-diamidino-2-phenylindole, a fluorescent dye that can strongly bind to DNA and is commonly used in fluorescence microscopy observations.

[0123] NIR: Near infrared spectrometer.

[0124] CCK-8: Cell Counting Kit-8 cell counting reagent.

[0125] ROS: Reactive oxygen species, are chemically reactive species containing oxygen, including peroxides, superoxides, hydroxyl radicals, singlet oxygen, and α-oxygenase.

[0126] FBS: fetal bovine serum.

[0127] DMEM: is a culture medium containing various amino acids and glucose.

[0128] Opti-MEM: used to culture hematopoietic cells.

[0129] MitoSOX: Superoxide Indicator is a new fluorescent dye that specifically targets mitochondria in living cells.

[0130] BBB: Blood-brain barrier, refers to the barrier between plasma and brain cells formed by the walls of brain capillaries and glial cells, and the barrier between plasma and cerebrospinal fluid formed by the choroid plexus. These barriers can prevent certain substances (mostly harmful) from entering the brain tissue from the blood.

[0131] Calcein-AM / PI staining: Calcein AM is hydrophobic and can easily enter the cell membrane of living cells. Calcein AM (which does not fluoresce itself) is cut into membrane-impermeable polar molecules Calcein by esterases in the cells and emits green fluorescence, while dead cells lack esterases and cannot perform the cutting and thus do not emit light; PI cannot pass through the cell membrane of living cells, but can only pass through the disordered area of ​​the dead cell membrane to reach the cell nucleus and embed into the cell's DNA double helix to produce red fluorescence. Staining with the two dyes simultaneously can distinguish between living and dead cells.

[0132] 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 cinnamic acid black phosphorus nanocomposite targeting Aβ, characterized in that: It consists of black phosphorus nanosheets as the drug carrier, aminopolyethylene glycol stearic acid as the modification material, 4-(dimethylamino)cinnamic acid as the targeting material and curcumin as the therapeutic material.

2. The cinnamic acid black phosphorus nanocomposition targeting Aβ according to claim 1, characterized in that: 4-(Dimethylamino)cinnamic acid was used as the group targeting Aβ.

3. A method for preparing a cinnamic acid black phosphorus nanocomposite targeting Aβ, characterized in that: The preparation steps are as follows: (1) 4-(dimethylamino)cinnamic acid, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide are mixed in a certain proportion, a solvent is added to carry out dissolution reaction, and then aminopolyethylene glycol stearic acid is added to continue the reaction; (2) adding curcumin to the solution obtained in step (1) for reaction, then dropping it into rapidly stirred water, heating it until the odor is completely volatilized, and collecting the upper layer solution after centrifugation; (3) reacting the upper layer solution obtained in step (2) with black phosphorus nanosheets to generate a final product, a cinnamic acid black phosphorus nanocomposite targeting Aβ.

4. The method for preparing a cinnamic acid black phosphorus nanocomposite targeting Aβ according to claim 3, characterized in that: The molar ratio of the four materials of 4-(dimethylamino)cinnamic acid, aminopolyethylene glycol stearic acid, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide in step (1) is (1-2):1:(1-2):(1-2).

5. The method for preparing a cinnamic acid black phosphorus nanocomposite targeting Aβ according to claim 3, characterized in that: The molar ratio of the four materials of 4-(dimethylamino)cinnamic acid, aminopolyethylene glycol stearic acid, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide in step (1) is 2:1:2:

2.

6. The method for preparing a cinnamic acid black phosphorus nanocomposite targeting Aβ according to claim 3, characterized in that: The solvent in step (1) is tetrahydrofuran, and the reaction is carried out for 4 to 6 hours after the solvent is added.

7. The method for preparing a cinnamic acid black phosphorus nanocomposite targeting Aβ according to claim 3, characterized in that: After adding amino polyethylene glycol stearic acid in step (1), the hydrophobic reaction is carried out for 20 to 25 hours.

8. The method for preparing a cinnamic acid black phosphorus nanocomposite targeting Aβ according to claim 3, characterized in that: Step (2) specifically comprises adding curcumin to the solution obtained in step (1) and reacting for 10 to 15 hours, dripping the obtained product into rapidly stirred water, heating in a water bath until the odor is completely volatilized, and taking the upper layer solution after centrifugation.

9. The method for preparing a cinnamic acid black phosphorus nanocomposite targeting Aβ according to claim 3, characterized in that: Step (3) specifically comprises reacting the solution obtained in step (2) with black phosphorus nanosheets through phosphorus-oxygen bonds for 10 to 15 hours, and then repeating centrifugation twice to obtain the final product, cinnamic acid black phosphorus nanocomposite particles targeting Aβ.

10. Application of a cinnamic acid black phosphorus nanocomposite targeting Aβ in the preparation of drugs for treating Alzheimer's disease.

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