A cinnamon acid black phosphorus nano composition targeting abeta and a preparation method and application thereof

Abstract

This invention discloses a cinnamic acid-black phosphorus nanocomposite targeting Aβ, its preparation method, and its application. The composition uses 4-(dimethylamino)cinnamic acid as the Aβ-targeting group and consists of a drug-loaded host (BP, black phosphorus nanosheets), a modifying 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 includes the following steps: (1) preparation of PEG-Tar, (2) preparation of PEG-Tar@Cur, and (3) preparation of the BP-PEG-Tar@Cur composition. This invention utilizes black phosphorus nanosheets to load Curcumin, whose excellent specific surface area significantly increases the drug loading capacity. Furthermore, by using 4-(dimethylamino)cinnamic acid as a group targeting Aβ, diffuse distribution of the drug in the brain is avoided, greatly improving the drug utilization rate of Curcumin. The synthesis process of this invention is simple, highly reproducible, and easy to scale up for industrial application, and it has a positive effect in the treatment of Alzheimer's disease.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a cinnamic acid black phosphorus nanocomposition targeting Aβ, its preparation method, and its application. Background Technology

[0002] Studies suggest that the pathogenesis of Alzheimer's disease (AD) is related to the abnormal deposition of β-amyloid protein (Aβ) in the brain. Aβ is produced from its precursor protein (APP) through hydrolysis by secretases, transported to the brain, and abnormally aggregated to produce Aβ oligomers. This leads to neurotoxic reactions such as inflammation, ROS oxidative stress, and calcium overload, damaging synaptic membranes and ultimately causing nerve cell death. Therefore, reducing Aβ production, promoting Aβ clearance, regulating Aβ transport, inhibiting Aβ aggregation, and counteracting its neurotoxicity can be effective in the clinical treatment of AD.

[0003] Black phosphorus (BP) is a novel nanomaterial composed of a single phosphorus element. Black phosphorus nanomaterials have the following advantages: they generate singlet oxygen under near-infrared light irradiation, which can be used as a photosensitizer for photodynamic therapy; they have a wide range of light absorption in a 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 extremely high drug loading capacity, as well as high bioactivity and biodegradability.

[0004] Curcumin, a hydrophobic polyphenol extracted from plants of the Curcumaceae and Araceae families, has been shown to possess anti-inflammatory, antioxidant, and antiviral properties. Studies have shown that curcumin exerts neuroprotective effects in Parkinson's disease through multiple pathways, including protecting dopaminergic neurons, anti-inflammation, antioxidant activity, anti-apoptosis, anti-mitochondrial damage, and regulation of autophagy. Compared to chemical drugs, traditional Chinese medicine (TCM) has better tolerability and safety, fewer adverse reactions, and is suitable for a wider range of populations. However, the low water solubility of curcumin results in low absorption and bioavailability in the body, significantly limiting its use in the pharmaceutical field.

[0005] Currently, improving the biocompatibility of curcumin often involves forming compositions of curcumin and peptides, such as the curcumin-peptide complex proposed in Chinese Patent CN105358179B (authorization publication date: 2018.12.07). While curcumin-peptide complexes improve the solubility of curcumin, their main limitations are susceptibility to protease cleavage and degradation, and a lack of specificity. This means that some of the drug may be degraded before reaching the brain, significantly reducing drug utilization. Another issue is that using peptides to encapsulate drugs results in low loading capacity, hindering large-volume drug delivery. Furthermore, simple drug delivery without a targeted substance leads to diffuse distribution of the drug in the brain, non-specific binding to other sites, and consequently, decreased drug utilization. Summary of the Invention

[0006] In order to overcome the shortcomings of the prior art, the present invention aims to provide a cinnamic acid black phosphorus nanocomposition targeting Aβ, its preparation method and application.

[0007] The first aspect of this invention proposes a cinnamic acid black phosphorus nanocomposition for targeting Aβ, comprising a drug-loaded black phosphorus nanosheet, a modifying material amino-polyethylene glycol stearic acid, a targeting material 4-(dimethylamino)cinnamic acid, and a therapeutic material Cur (curcumin).

[0008] The present invention discloses a cinnamic acid black phosphorus nanocomposition targeting Aβ, using 4-(dimethylamino)cinnamic acid as the Aβ-targeting group.

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

[0010] (1) Mix 4-(dimethylamino)cinnamic acid with N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide in a certain proportion, add solvent to dissolve and react, and then add amino polyethylene glycol stearic acid to continue the reaction.

[0011] (2) After adding curcumin to the solution obtained in step (1) and reacting, add it dropwise into water that is being stirred rapidly, and heat until the odor is completely volatilized. After centrifugation, take the upper layer solution.

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

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

[0015] In this 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 this invention, after adding amino polyethylene glycol stearic acid in step (1), a hydrophobic reaction is carried out for 20 to 25 hours.

[0017] In this invention, step (2) specifically includes adding curcumin to the solution obtained in step (1) and reacting for 10 to 15 hours, then dropping the obtained product into rapidly stirred water, heating in a water bath until the odor is completely volatilized, and then centrifuging to take the upper layer solution.

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

[0019] A third aspect of the present invention discloses 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 methods described above.

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

[0021] 1. This invention, by incorporating cinnamic acid as a targeting substance, can precisely target Aβ, avoiding diffuse drug distribution in the brain and greatly improving drug utilization. Combined with the numerous advantages of novel nanomaterial black phosphorus—non-toxic, with good biodegradability and biocompatibility, and a high surface-to-volume ratio—the black phosphorus nanosheets significantly increase the drug loading capacity of curcumin, overcoming the problems of easy degradation and low loading rates associated with peptide-encapsulated drugs during in vivo transport. This cinnamic acid-black phosphorus composite nanocomposition possesses the dual functions of photothermally opening the blood-brain barrier under NIR irradiation and targeting Aβ, showing broad prospects in the field of Alzheimer's disease treatment.

[0022] 2. This invention does not require a complex synthesis procedure. By simply adding BP as a carrier and 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 reproducible, and easy to scale up for industrial application. Attached Figure Description

[0023] To more clearly illustrate the solutions in this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0024] Figure 1 The UV spectrum results for Example 1 show that Cur and BP do not bind;

[0025] Figure 2 The results of the ultraviolet spectrum in Example 2;

[0026] Figure 3 The results of the ultraviolet spectrum in Example 3;

[0027] Figure 4A This is a BP microscope image of the morphology.

[0028] Figure 4B Microscopic morphology of BP-PEG-Tar@Cur;

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

[0030] Figure 4D Particle size distribution density diagrams for various materials;

[0031] Figure 4E Fourier transform infrared spectra of each material;

[0032] Figure 4F These are the spectrophotometric values ​​for each material solution;

[0033] Figure 4G These are the Raman spectral values ​​of each material;

[0034] Figure 4H Dimensional stability diagrams of various materials in PBS;

[0035] Figure 4I Dimensional stability diagrams of various materials in DMEM;

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

[0037] Figure 5A This is a schematic diagram of the cell viability test results;

[0038] Figure 5B This is a fluorescence experiment diagram of Aβ depolymerization;

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

[0040] Figure 5D is a schematic diagram of the ThT fluorescence test results;

[0041] Figure 6 This is a schematic diagram of the results of a cell-targeted uptake experiment.

[0042] Figure 7 This is a schematic diagram of the results of the mitochondrial ROS clearance experiment.

[0043] Figure 8 This is a schematic diagram of the Transwell experiment results. Detailed Implementation

[0044] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

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

[0046] Example 1

[0047] This embodiment illustrates a method for preparing black phosphorus nanocomposite materials, specifically including the following steps:

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

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

[0050] Step 3: Remove the EP tube and place it in a high-speed centrifuge at 7830 r / min for 20 minutes, then filter it.

[0051] Step 4: Determine the material synthesis results by detecting ultraviolet spectra, such as... Figure 1 As shown, the UV spectra of the solution with added BP and the solution without added BP are not significantly different, proving that Cur and BP do not bind.

[0052] Example 2

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

[0054] Step 1: First, add 4-(dimethylamino)cinnamic acid, N-hydroxysuccinimide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide to the flask, and add 2 ml of tetrahydrofuran to dissolve and activate the hydroxyl groups in 4-(dimethylamino)cinnamic acid.

[0055] Step 2: After reacting for 5 hours, add amino polyethylene glycol stearic acid to form hydrophobic chains to encapsulate Cur. React for 24 hours. The molar ratio of the four materials, 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 10mg of Cur to the flask and react. After reacting for 12 hours, slowly add the reaction solution dropwise to 12ml of rapidly stirred water and heat in a 40℃ water bath until the odor completely evaporates.

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

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

[0059] Step 6: After reacting for 12 hours, the reaction solution in the flask was extracted and centrifuged in a high-speed centrifuge at 7000 r / min for 5 minutes. After centrifugation, the supernatant was extracted and the UV spectrophotometric value was measured. Then, 5 ml of water was added to resuspend the solution, and the above centrifugation steps were repeated to wash away the unbound PEG-Tar@Cur, resulting in a suspension of 5.3 ml of the final product. The absorbance value was measured using a UV spectrophotometer. The Cur content of the final suspension was determined by UV spectroscopy, and the curcumin loading was measured according to the steps in Example 4. Finally, the total amount of BP-PEG-Tar@Cur nanoparticles was dried to obtain 1 mg.

[0060] Step 7: The synthesis of the material was determined by detecting the ultraviolet spectra of different products, and the encapsulation efficiency and loading rate were calculated by detecting the Cur content of the supernatant. As shown in Table 1, the encapsulation efficiency and drug loading rate were 11.72% and 45.84%, respectively, both of which were relatively low.

[0061] Table 1 shows the encapsulation efficiency and drug loading rate results for Example 2.

[0062]

[0063] Example 3

[0064] This embodiment illustrates a method for preparing a cinnamic acid black phosphorus nanocomposite, specifically including the following steps:

[0065] Step 1: First, add 4-(dimethylamino)cinnamic acid, N-hydroxysuccinimide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide to the flask, and add 2 ml of tetrahydrofuran to dissolve and activate the hydroxyl groups in 4-(dimethylamino)cinnamic acid.

[0066] Step 2: After reacting for 5 hours, add amino polyethylene glycol stearic acid to form a hydrophobic chain to encapsulate Cur. React for 24 hours. The molar ratio of the four materials, 4-(dimethylamino)cinnamic acid, amino polyethylene glycol stearic acid, N-hydroxysuccinimide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide, is 2:1:2:2.

[0067] Step 3: Add 10 mg of Cur to the flask and react. After reacting for 12 hours, slowly add the reaction solution dropwise to 12 ml of water with rapid stirring. Heat in a 40°C water bath until the odor completely evaporates.

[0068] Step 4: After the odor has completely evaporated, place the solution in a high-speed centrifuge and centrifuge at 6000 r / min for 5 minutes. After centrifugation, remove the supernatant and bring the volume up to 14 ml.

[0069] Step 5: Extract half of the supernatant of the PEG-Tar@Cur reaction solution into a flask, centrifuge 5 ml of BP with a concentration of 1 mg / ml, resuspend it in 1 ml of water and add it to the flask. PEG-Tar@Cur reacts with BP through phosphorus-oxygen bonds.

[0070] Step 6: After reacting for 12 hours, the reaction solution in the flask was extracted and centrifuged in a high-speed centrifuge at 7000 r / min for 5 minutes. After centrifugation, the supernatant was extracted and the UV spectrophotometric value was measured. 5 ml of water was added to resuspend the solution, and the above centrifugation steps were repeated to wash away unbound PEG-Tar@Cur, resulting in a suspension of 5.3 ml of the final product. The Cur content of the final suspension was determined by UV spectroscopy, and the curcumin loading was measured according to the steps in Example 4. Finally, the total amount of BP-PEG-Tar@Cur nanoparticles was 1.36 mg after drying.

[0071] Step 7: The synthesis of the material was determined by detecting the ultraviolet spectra of different products, and the encapsulation efficiency and loading rate were calculated by detecting the Cur content of the supernatant. As shown in Table 3, after adjusting the ratio between the materials, the encapsulation efficiency and drug loading rate were improved to 22.75% and 68.51%, respectively.

[0072] Table 2 shows the calculation results of encapsulation efficiency and drug loading rate in Example 3.

[0073]

[0074] Characterization analysis of BP, PEG-Tar, PEG-Tar@Cur and BP-PEG-Tar@Cur

[0075] 1. Microscopic morphology imaging of BP and BP-PEG-Tar@Cur: A portion of BP and BP-PEG-Tar@Cur samples were dispersed in an ethanol solution and sonicated. Several drops of the dispersed liquid were then added dropwise onto a copper grid. After drying, the morphology was imaged (high resolution) using a FEI Talos F200X G2 TEM microscope (USA), with an accelerating voltage of 200 kV and an energy dispersive spectroscopy (EDS super-X) mode. The results are as follows: Figure 4A , 4B As shown.

[0076] 2. The particle size and potential of each material were measured using a laser particle size analyzer (model: Vern Nanosizer Nano ZS), and the results are as follows: Figure 4C , 4D As shown in Figures 4H and 4I.

[0077] 3. The Fourier transform infrared (FTIR) spectra of each material were measured using a Thermo Science Nicolet iS20 Fourier transform infrared spectrometer. In a dry environment, the ATR attachment was placed in the optical path of the spectrometer, and the air background was scanned. One drop of liquid was placed on the crystal surface of the ATR attachment using a dropper, and then the infrared spectrum of the sample was acquired. The resolution was 4 cm⁻¹, the number of scans was 32, and the wavenumber range was 400-4000 cm⁻¹. The results are as follows: Figure 4E As shown.

[0078] 4. The spectrophotometric values ​​of each material were measured using a UV spectrophotometer (model: UV-2450), and the results are as follows: Figure 4F As shown.

[0079] 5. The Raman spectra of each material were determined using a high-resolution confocal laser-microscopic Raman spectroscopy instrument (model: WITec alpha300R, Germany; laser: 532nm). Single-spectrum testing conditions: laser energy 36mW, 1800g / mm grating, Olympus 20x objective lens (Olympus 20x / 0.25), integration time 30s, 10 iterations. Results are as follows: Figure 4G As shown.

[0080] Example 4

[0081] This embodiment presents a method for determining the curcumin loading in BP-PEG-Tar@Cur nanoparticles in Examples 2 and 3 using a UV spectrophotometer. First, a standard curve for curcumin is plotted. 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). Based on the absorbance values ​​of each concentration solution at 425 nm measured by the UV spectrophotometer, a concentration-absorbance standard curve for curcumin is obtained. To calculate the curcumin loading of the synthesized material, the BP-PEG-Tar@Cur solvent is centrifuged and resuspended in 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 total amount of nanoparticles and multiplying by the percentage. The curcumin concentration in anhydrous ethanol solvent of the product BP-PEG-Tar@Cur obtained in Example 2 was 458.45 μg / ml, and the curcumin concentration in anhydrous ethanol solvent of the product BP-PEG-Tar@Cur obtained in Example 3 was 931.74 μg / ml.

[0082] Example 5

[0083] This embodiment presents a method for determining the in vitro cumulative release of curcumin from BP-PEG-Tar@Cur nanoparticles. 0.5 ml of a material solution containing 435 mg / ml curcumin was added to a dialysis bag with a molecular weight cutoff of 500 and sealed with a clip. The dialysis bag was placed in 50 ml of anhydrous ethanol (0.01 M, pH 7.4) release medium and placed on a shaker at a constant temperature of 37°C. At predetermined time intervals (0, 2, 4, 6, 8, 10, 12, and 24 hours), 3 ml of the release solution was taken out, and the same volume of anhydrous ethanol was added simultaneously. The absorbance of each extracted release solution was measured at 425 nm using a UV spectrophotometer. The cumulative release curve of curcumin was calculated based on the results. Figure 4J As shown, approximately 20% Cur was initially released from BP-PEG-Tar@Cur nanoparticles within 2 hours, followed by a sustained release of approximately 50% over 24 hours.

[0084] Example 6

[0085] This embodiment presents a method for preparing Aβ monomer and fibrils, which involves using 1 mg of frozen Aβ... 1-42 Incubate at room temperature for 30 minutes to equilibrate and prevent peptide condensation. Add 222 μl of HFIP to Aβ. 1-42 A 1 mM solution was obtained. The resulting solution was sonicated in a water bath for 10 minutes, then incubated with shaking at 4°C for 2 hours. The peptide solution was then vortexed, and 10 μl aliquots were taken and placed in a SpeedVac at 45°C for 30 minutes to remove any residual HFIP. Finally, the pretreated solid Aβ was...1-42 The monomers were stored in small EP tubes at -20°C until use. Before use, these dried aliquots were resuspended in anhydrous DMSO to a concentration of 1 mM (10 nmol), sonicated for approximately 10 minutes, and then diluted with PBS (pH 7.4, 10 mM) to the desired concentration. The prepared solution was used immediately for experiments. Aβ 1-42 Monomers are incubated at 37°C for 72 hours to form fibrils.

[0086] Example 7

[0087] This embodiment proposes a cytotoxicity assay method. Cell suspension (100 μl) is seeded in a 96-well plate, approximately 5000 cells / well. After culturing the cells for 12 hours, Aβ is added. 1-42 Fibrous oxide was 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 each well was incubated for 30 minutes, followed by NIR irradiation for 3 minutes (0.75 W cm⁻²) to maximize targeting effect, and then incubation continued for 24 hours. 10 μl of CCK8 solution was added to each well. Blank wells were prepared by adding equal volumes of cell culture medium, drug, and CCK8 solution, but without cells. Results are as follows: Figure 5A As shown, the material exhibits low toxicity.

[0088] Example 8

[0089] This embodiment presents a fluorescence experiment on the depolymerization of Aβ fibers, using 75 μl of Aβ fiber at a concentration of 25 μM. 1-42 The sample was divided into three equal portions, and added to 100 μl of PBS, Cur, and BP-PEG-Tar@Cur (0.5 μg / ml), respectively. Each portion was incubated for 24 h. After incubation, 10 μl of each sample was transferred to a glass slide and air-dried. The slides were stained with ThT (10 μM) for 10 min, gently rinsed with water, and then air-dried again. Finally, the samples were imaged using a fluorescence microscope (Olympus, Japan). The results are shown below. Figure 5B As shown, BP-PEG-Tar@Cur can depolymerize Aβ fibers that have already aggregated.

[0090] Example 9

[0091] This embodiment proposes a method for determining the effects of Cur, PEG-Tar@Cur, and BP-PEG-Tar@Cur on inhibiting Aβ fibril aggregation using the BCA protein quantification method. 100 μl of Aβ at a concentration of 25 μM was taken. 1-42The sample was divided into four equal portions, one as a blank, and the other three incubated with Cur, PEG-Tar@Cur, and BP-PEG-Tar@Cur, respectively. Except for the blank, the Cur concentration in each solution was ensured to be 5 μg / ml. All four solutions were incubated at 37°C in PBS (pH 7.4, 1X) buffer, vortexed at 100 rpm for 72 hours, and then centrifuged at 20,000 rpm / min for 20 minutes. 75% of the supernatant was then collected for BCA protein quantification to determine the concentration of soluble Aβ. After resuspending the aliquots in 10 μl DMSO, PBS (pH 7.4, 1X) was added to dilute the mixture to a total volume of 100 μl. 25 μl of each aliquot was added to an EP tube, and 75 μl of PBS (pH 7.4, 1X) was added to the tube, bringing the total volume to 100 μl. Results are as follows: Figure 5C As shown, with a monomer content of 100%, the Aβ fibril monomer content in the untreated group was about 30%, while the monomer content in the treated groups with added materials increased sequentially, with the monomer content in the BP-PEG-Tar@Cur group even reaching 80%. This proves that BP-PEG-Tar@Cur can effectively inhibit the aggregation of Aβ fibrils.

[0092] Example 10

[0093] This embodiment proposes a ThT fluorescence method for testing Aβ, which involves taking equal portions of the sample. 1-42 One tube was equilibrated at room temperature for 10 minutes, then 10 μl of DMSO was added and sonicated in an ice-water bath for 10 minutes. 50 μl of PBS (16.7 μM) was added and mixed well. Cur, PEG-Tar@Cur, and BP-PEG-Tar@Cur solutions (all at 0.5 μg / ml) were added to EP tubes and incubated at 37°C with a shaker at 100 rpm for 72 h. After the reaction, the sample was mixed with 400 μl of ThT (25 μM). Fluorescence intensity was detected using a fluorescence spectrometer (model: HITACHI F-4500, Japan) at 25°C, with a slit width of 5 nm and excitation and emission wavelengths of 440 and 480 nm, respectively. The results are shown in Figure 5D, using Aβ-free... 1-42 Using a blank solution as background, containing only Aβ 1-42 The solution was at 100% fluorescence intensity. Higher fluorescence intensity indicates a higher content of protofibrils. The results showed that the fluorescence intensity of the BP-PEG-Tar@Cur treatment group decreased to about 35%, indicating that the material can effectively inhibit the aggregation of Aβ protofibrils.

[0094] Example 11

[0095] This embodiment proposes a method for cell-targeted uptake experiments, in which N2a cells are placed at a density of 2 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells per confocal dish. After cell adhesion, Aβ was added.1-42 After incubation at (25 μM) for 12 hours, 10 μg / ml of Cur, PEG-Tar@Cur, and BP-PEG-Tar@Cur solutions were added, and incubation continued for another 12 hours. After incubation, the culture medium was changed and the cells were washed three times with PBS (pH 7.4, 1X). Cells were fixed with 4% paraformaldehyde (wt) for 10 minutes, followed by DAPI staining for 5 minutes. Cells were observed using a laser scanning confocal microscope at an excitation wavelength of 488 nm. The results are shown below. Figure 6 As shown in the confocal microscope images, the material group with added targeting substance Tar showed a significant increase in cellular uptake and was able to target Aβ.

[0096] Example 12

[0097] This embodiment proposes a method for eliminating mitochondrial ROS. N2a cells in logarithmic growth phase were selected, and single-cell suspensions were prepared using 500 μl of culture medium supplemented with 100 U / ml double antibiotics, 23 ml of DMEM (10% FBS), and 23 ml of Opti-MEM. Cells were then cultured at a rate of 2 × 10⁻⁶ cells / year. 5 Cells were seeded in confocal dishes, with 1 ml of culture medium added to each dish. The dishes were placed in a cell culture incubator and incubated for 12 hours. Then, 25 μM Aβ fibrils were added and incubated for another 12 hours. Next, 5 μg / ml of Cur, PEG-Tar@Cur, and BP-PEG-Tar@Cur solutions were added and incubated for 12 hours respectively. Cells were washed twice with PBS (pH 7.4, 1X), and incubated in the dark at 37°C with 1 ml of working solution containing 5 μM MitoSOX superoxide indicator. The cells were then stained and incubated in a cell culture incubator at 37°C for 10 minutes. Cells were fixed with 4% paraformaldehyde (wt) for 15 minutes, washed twice with PBS (pH 7.4, 1X), and counterstained with 1 μM DAPI for 5 minutes. Fluorescent images of ROS scavenging were obtained using a laser scanning confocal microscope. Results are as follows: Figure 7 As shown, the ROS content of cells treated with Aβ fibrils increased significantly, while the ROS content decreased significantly after drug treatment. This indicates that the material can effectively reduce ROS generated by Aβ fibril aggregation.

[0098] Example 13

[0099] This embodiment proposes a Transwell in vitro invasion assay method. At the cellular level, immortalized mouse brain microvascular endothelial cells (bEnd.3 cells) are used to simulate the in vitro environment of the blood-brain barrier (BBB) ​​to study the BBB penetration ability of BP-PEG-Tar@Cur under photothermal effects. bEnd.3 (1×10⁻⁶ cells) were used to simulate the BBB environment. 4Cells were seeded on the front side of gelatin-coated 6-well Transwell chambers and cultured in DMEM containing 10% FBS and 1% penicillin / streptomycin. Transendothelial resistance (TEER) was measured every 3-4 days, and the integrity of the BBB monolayer was assessed using a World Precision Instruments (Sarasota, FL, USA) voltmeter (EVOM). TEER values ​​were measured before and after the experiment, and experiments could begin once the TEER values ​​stabilized. To ensure accuracy, the entire process was performed at a constant temperature of 37°C. Three points were taken from each cell insert in different directions, and measurements were repeated three times. After several days, the cells fused to form a reliable BBB monolayer, resulting in a tight monolayer (TEER exceeding 200 Ωcm). The same concentrations of Cur, BP-PEG-Tar@Cur, and NIR-irradiated BP-PEG-Tar@Cur materials were incubated in the top donor chamber at 37℃ and 5% CO2 for 2 hours. 200 μl samples were collected from the outside of the substrate, and the Cur content was determined by UV spectrophotometry. The initial Cur content of all three treated materials was 43.5 μg. After 2 hours, the Cur transmittance of the BP-PEG-Tar@Cur material group was 11.30 μg, with a transmittance of 25.9%; the Cur transmittance of the NIR-irradiated BP-PEG-Tar@Cur material group was 14.08 μg, with a transmittance of 32.4%. The results are as follows: Figure 8 As shown, under NIR irradiation, BP-PEG-Tar@Cur can effectively open the blood-brain barrier and enhance the permeability of Cur. Simultaneously, cells treated with Cur, BP-PEG-Tar@Cur, and BP-PEG-Tar@Cur under NIR irradiation were distinguished as live cells from dead cells using Calcein-AM / PI staining. Figure 8 As shown, the microscopic findings of the three treatment groups were not significantly different, with no excessive dead cells, indicating that NIR irradiation does not increase the toxicity of BP-PEG-Tar@Cur.

[0100] Definitions of abbreviations and key terms

[0101] BP: Black phosphorus, a black semiconductor crystal with a metallic luster, has a density of 2.70 g / cm³. 3 It has a hardness of 2. Its crystal lattice consists of two atomic layers, each composed of tortuous chains 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 of the phosphorus allotropes and does not spontaneously combust in air.

[0102] PEG: C18-PEG-NH2, amino polyethylene glycol stearic acid.

[0103] Curcumin is a polyphenolic compound derived from the plant turmeric, which is naturally found in turmeric, a food and medicine widely used in India and China.

[0104] Tar: 4-(dimethylamino)cinnamic acid, used as a targeting material for targeting Aβ.

[0105] PEG-Tar: A combination of polyethylene glycol stearic acid and cinnamic acid.

[0106] PEG-Tar@Cur: A combination of polyethylene glycol stearate, cinnamic acid, and curcumin.

[0107] BP-PEG-Tar@Cur: A cinnamic acid black phosphorus nanocomposite targeting Aβ.

[0108] NHS: N-hydroxysuccinimide, white to off-white crystals, used in the synthesis of amino acid protectants, semi-synthetic kanamycin, and pharmaceutical intermediates.

[0109] EDC: 1-Ethyl-(3-dimethylaminopropyl)carbodiimide is an organic compound with the chemical formula C8H17N3.

[0110] AD: Alzheimer's disease is a progressive neurodegenerative disease with an insidious onset. Clinically, it is characterized by comprehensive dementia manifestations such as memory impairment, aphasia, apraxia, agnosia, visuospatial skill impairment, executive dysfunction, and personality and behavioral changes. The cause remains unknown.

[0111] Aβ (amyloid β-protein): β-amyloid protein, with a molecular weight of about 4kDa, is derived from the hydrolysis of β-amyloid precursor protein (APP). It is secreted by cells and has a strong neurotoxic effect after precipitating and accumulating in the cytosol.

[0112] FTIR: Fourier Transform Infrared Spectrometer.

[0113] DLS: Dynamic Light Scattering Instrument.

[0114] TEM: Transmission electron microscope.

[0115] HFIP: Hexafluoroisopropanol.

[0116] SpeedVac: Vacuum concentrator.

[0117] DMSO: Dimethyl sulfoxide.

[0118] CLSM: Laser confocal scanning microscope.

[0119] PBS: Phosphate Buffer Solution, is one of the most widely used buffer solutions in biochemical research. Its main components are Na2HPO4, KH2PO4, NaCl, and KCl. It is generally used as a solvent to dissolve and protect reagents.

[0120] N2a cells: N2a cells, short for mouse neuroblastoma N2a cells, are mouse-derived neuroblastoma blasts.

[0121] DAPI (4,6-diamidinyl-2-phenylindole) is a fluorescent dye that binds strongly to DNA and is commonly used in fluorescence microscopy.

[0122] NIR: Near-infrared spectrometer.

[0123] CCK-8: Cell Counting Kit-8.

[0124] ROS: Reactive oxygen species are oxygen-containing chemically reactive substances, including peroxides, superoxides, hydroxyl radicals, singlet oxygen, and alpha oxygen.

[0125] FBS: Fetal bovine serum.

[0126] DMEM: is a culture medium containing various amino acids and glucose.

[0127] Opti-MEM: Used for culturing hematopoietic cells.

[0128] MitoSOX: Superoxide indicator is a novel fluorescent dye that specifically targets mitochondria in living cells.

[0129] BBB: Blood-brain barrier, refers to the barrier between blood plasma and brain cells formed by the walls of brain capillaries and glial cells, and the barrier between blood plasma and cerebrospinal fluid formed by the choroid plexus. These barriers prevent certain substances (mostly harmful) from entering brain tissue from the blood.

[0130] 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 cleaved by intracellular esterases into membrane-impermeable polar molecules called Calcein, which emit green fluorescence. Dead cells lack esterases and cannot be cleaved, thus they do not emit light. PI cannot pass through the cell membrane of living cells, but can only pass through the disordered region of the dead cell membrane to reach the cell nucleus and embed itself into the cell's DNA double helix, thus producing red fluorescence. Staining with both dyes simultaneously can distinguish between living and dead cells.

[0131] Obviously, the embodiments described above are only some embodiments of this application, not all embodiments. The accompanying drawings show preferred embodiments of this application, but do not limit the patent scope of this application. This application can be implemented in many different forms; rather, the purpose of providing these embodiments is to provide a more thorough and comprehensive understanding of the disclosure of this application. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing specific embodiments, or make equivalent substitutions for some of the technical features. Any equivalent structures made using the content of this application's specification and drawings, directly or indirectly applied to other related technical fields, are similarly within the scope of patent protection of this application.

Claims

1. A cinnamic acid-black phosphorus nanocomposition targeting Aβ, characterized in that, It is composed of drug-loaded black phosphorus nanosheets, modified material aminopolyethylene glycol stearic acid, targeting material 4-(dimethylamino)cinnamic acid, and therapeutic material curcumin; wherein, the 4-(dimethylamino)cinnamic acid reacts with the aminopolyethylene glycol stearic acid to form a targeting complex, and the targeting complex is then connected to the black phosphorus nanosheets through phosphorus-oxygen bonds. The 4-(dimethylamino)cinnamic acid serves as a group that targets Aβ; Furthermore, the drug loading rate of the nanocomposition is 60%~70%, and the cumulative release rate reaches 50% in phosphate buffer at pH 7.4 after 24 hours. The cinnamic acid black phosphorus nanocomposition targeting Aβ is obtained by a preparation method comprising the following steps: (1) Mix 4-(dimethylamino)cinnamic acid with N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide in a certain proportion, add solvent to dissolve and react, and then add amino polyethylene glycol stearic acid to continue the reaction. (2) After adding curcumin to the solution obtained in step (1) and reacting, add it dropwise to water that is being stirred rapidly, and heat until the odor is completely volatilized. After centrifugation, take the upper layer solution. (3) The upper solution obtained in step (2) is reacted with black phosphorus nanosheets to generate the final product, a cinnamic acid black phosphorus nanocomposite targeting Aβ. The molar ratio of the four materials in step (1), namely 4-(dimethylamino)cinnamic acid, amino polyethylene glycol stearic acid, N-hydroxysuccinimide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide, is 2:1:2:

2.

2. The cinnamic acid black phosphorus nanocomposition targeting Aβ according to claim 1, characterized in that, The solvent used in step (1) is tetrahydrofuran, and the reaction takes 4 to 6 hours after the solvent is added.

3. The cinnamic acid black phosphorus nanocomposition targeting Aβ according to claim 2, characterized in that, After adding amino polyethylene glycol stearic acid as described in step (1), a hydrophobic reaction is carried out for 20 to 25 hours.

4. The cinnamic acid black phosphorus nanocomposition targeting Aβ according to claim 2, characterized in that, Step (2) specifically includes adding curcumin to the solution obtained in step (1) and reacting for 10 to 15 hours. The resulting product is then dropped into rapidly stirred water, heated in a water bath until the odor is completely volatilized, and the upper layer solution is taken after centrifugation.

5. The cinnamic acid black phosphorus nanocomposition targeting Aβ according to claim 2, characterized in that, Step (3) specifically involves reacting the solution obtained in step (2) with black phosphorus nanosheets through phosphorus-oxygen bonds for 10 to 15 hours, followed by repeated centrifugation twice to obtain the final product, cinnamic acid black phosphorus nanocomposite particles targeting Aβ.

6. The use of the cinnamic acid black phosphorus nanocomposition targeting Aβ according to claim 1 in the preparation of a drug for treating Alzheimer's disease.

Images