CN117224503A - Abeta-targeting cinnamic acid black phosphorus nano composition and preparation method and application thereof - Google Patents
Abeta-targeting cinnamic acid black phosphorus nano composition and preparation method and application thereof Download PDFInfo
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- CN117224503A CN117224503A CN202310985793.6A CN202310985793A CN117224503A CN 117224503 A CN117224503 A CN 117224503A CN 202310985793 A CN202310985793 A CN 202310985793A CN 117224503 A CN117224503 A CN 117224503A
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Abstract
The application discloses an Abeta-targeted cinnamic acid black phosphorus nano composition and a preparation method and application thereof. The composition takes 4- (dimethylamino) cinnamic acid as a group for targeting Abeta, and consists of a drug-carrying main body BP (black phosphorus nano-sheet), 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 comprises the following steps: (1) preparation of PEG-Tar, (2) preparation of PEG-Tar@Cur, and (3) preparation of BP-PEG-Tar@Cur composition. According to the application, the Cur (curcumin) is loaded by utilizing the black phosphorus nanosheets, the drug loading capacity of the Cur is obviously improved by utilizing the good specific surface area, 4- (dimethylamino) cinnamic acid is used as a group for targeting Abeta, the diffuse distribution of the drug in the brain is avoided, the drug utilization rate of the Cur is greatly improved, the synthesis process is simple, the repeatability is high, the industrial large-scale application is easy, and the active effect is realized in the field of treating Alzheimer's disease.
Description
Technical Field
The application relates to the technical field of biomedical materials, in particular to an Abeta-targeted cinnamic acid black phosphorus nano composition and a preparation method and application thereof.
Background
The onset of Alzheimer's Disease (AD) is thought to be associated with abnormal deposition of beta amyloid (Abeta) in the brain. Abeta is produced by hydrolyzing A Precursor Protein (APP) of the Abeta through secretase, and is transported to the brain, abnormal aggregation produces Abeta oligomers, and the Abeta oligomers cause neurotoxic reactions such as inflammation, ROS oxidative stress, calcium overload and the like, damage synaptic membranes and finally cause nerve cell death. Thus reducing Aβ production, promoting Aβ clearance, regulating Aβ transport, inhibiting Aβ aggregation, and combating its neurotoxicity, and is clinically effective in treating AD.
Black Phosphorus (BP) is a novel nanomaterial consisting of a single Phosphorus element, and has the following advantages: the singlet oxygen is generated under the irradiation of near infrared light and can be used as a photosensitizer for photodynamic therapy; the light absorption can be widely carried out in a longer wavelength region, and the thermal property of the near infrared light can be applied to photothermal treatment; the black phosphorus nano-sheet has higher specific surface area and unique fold structure, so that the black phosphorus nano-sheet has extremely high drug loading capacity and higher biological activity and biological degradability.
Curcumin is a hydrophobic polyphenol component extracted from plants of Curcuma, araceae, etc., and has been proved to have anti-inflammatory, antioxidant, antiviral, etc. The research shows that curcumin can play a role in neuroprotection in Parkinson's disease through various ways of protecting dopaminergic neurons, resisting inflammation, resisting oxidation, resisting apoptosis, resisting mitochondrial injury, regulating autophagy and the like. Compared with chemical medicines, the traditional Chinese medicine has good tolerance and safety, small adverse reaction and wide applicable population, but has low in vivo absorptivity caused by low water solubility of curcumin and low bioavailability, thus greatly limiting the application of curcumin in the medicine field.
At present, curcumin and peptide are formed into a composition for improving the biocompatibility of curcumin, such as curcumin-peptide complex proposed by Chinese patent CN105358179B (grant publication date: 2018.12.07). Although the curcumin and peptide complex increases the solubility of curcumin, the main limitation is that the curcumin is easily cut and degraded by protease and lack of specificity, so that a part of the curcumin is degraded before reaching the brain, thereby greatly reducing the utilization rate of the medicine. Another problem is that the use of peptides to entrap drugs has low loading and cannot achieve large volume drug delivery. Moreover, pure drug delivery without targeting the target substance can lead to diffuse distribution of the drug in the brain, nonspecifically bind other sites, and thus lead to reduced drug availability.
Disclosure of Invention
In order to overcome the defects of the prior art, the application aims to provide an Abeta-targeted cinnamic acid black phosphorus nano composition, a preparation method and application thereof.
The first aspect of the application provides a cinnamic acid black phosphorus nanometer composition for targeting Abeta, which consists of a medicine carrying main body black phosphorus nanometer sheet, a modifying material amino polyethylene glycol stearic acid, a targeting material 4- (dimethylamino) cinnamic acid and a therapeutic material Cur (curcumin).
The application relates to a beta-targeting cinnamic acid black phosphorus nano composition, which takes 4- (dimethylamino) cinnamic acid as a beta-targeting group.
The second aspect of the application provides a preparation method of an Abeta-targeted cinnamic acid black phosphorus nano composition, which comprises the following steps:
mixing 4- (dimethylamino) cinnamic acid with N-hydroxysuccinimide and 1-ethyl- (3-dimethylaminopropyl) carbodiimide in a certain proportion, adding a solvent for dissolution reaction, and then adding amino polyethylene glycol stearic acid for continuous reaction.
And (3) adding curcumin into the solution obtained in the step (1) for reaction, dripping the solution into water which is rapidly stirred, heating until the smell is completely volatilized, and centrifuging to obtain an upper layer solution.
And (3) reacting the upper layer solution obtained in the step (2) with the black phosphorus nanosheets to generate the final product of the cinnamic acid black phosphorus nanosubstance targeting Abeta.
In the application, in the step (1), the molar ratio of the four materials of 4- (dimethylamino) cinnamic acid, aminopolyglycol stearic acid, N-hydroxysuccinimide and 1-ethyl- (3-dimethylaminopropyl) carbodiimide is (1-2): 1: (1-2): (1-2).
More preferably, the four materials in the step (1) are in a molar ratio of 2:1:2:2.
in the application, the solvent in the step (1) is tetrahydrofuran, and the reaction is carried out for 4-6 hours after the solvent is added.
In the application, the amino polyethylene glycol stearic acid is added in the step (1) and then subjected to a hydrophobic reaction for 20-25 hours.
In the application, the step (2) specifically comprises the steps of adding curcumin into the solution obtained in the step (1) for reaction for 10-15 hours, dripping the obtained product into water which is rapidly stirred, heating in a water bath until the smell is completely volatilized, and centrifuging to obtain an upper layer solution.
In the application, the step (3) specifically comprises the steps of reacting the solution obtained in the step (2) with the black phosphorus nanosheets through phosphorus-oxygen bonds for 10-15 hours, and repeating centrifugation twice to obtain the final product of the target Abeta cinnamic acid black phosphorus nanosomposition particles.
The third aspect of the application provides an application of an Abeta-targeted cinnamic acid black phosphorus nano composition in preparation of a medicament for treating Alzheimer's disease, wherein the composition is prepared by any one of the methods.
Compared with the prior art, the application has the following beneficial effects:
1. according to the application, cinnamic acid is added as a targeting substance, so that the accurate targeting of Abeta can be realized, the diffuse distribution of the medicine in the brain is avoided, the medicine utilization rate is greatly improved, the advantages of the novel nano material black phosphorus are combined, the nano material black phosphorus is nontoxic, the nano material black phosphorus has good biodegradability and biocompatibility, the high surface volume ratio enables the medicine carrying capacity of the black phosphorus nano sheet to curcumin to be obviously improved, and the problems that the medicine is easy to degrade in the in-vivo transportation process and the loading rate is low when the peptide is used for carrying the medicine are overcome. The cinnamic acid black phosphorus composite nano composition has the dual functions of opening a blood brain barrier by photo-heat under NIR irradiation and targeting Abeta, and has wide prospect in the field of medicaments for treating Alzheimer's disease.
2. According to the application, a complex synthesis procedure is not needed, only BP is added as a carrier, cinnamic acid is used as a target substance, the drug loading rate and targeting capability of Cur can be greatly improved, the synthesis process is simple, the repeatability is high, and the industrial large-scale application is easy.
Drawings
In order to more clearly illustrate the application or the solutions of the prior art, a brief description will be given below of the drawings used in the description of the embodiments or the prior art, it being obvious that the drawings in the description below are some embodiments of the application and that other drawings can be obtained from them without the inventive effort of a person skilled in the art.
FIG. 1 is a graph of the ultraviolet spectrum of example 1 showing that Cur and BP are not bound;
FIG. 2 is the ultraviolet spectrum of example 2;
FIG. 3 is a graph of the ultraviolet spectrum of example 3;
FIG. 4A is a BP microscopy topography;
FIG. 4B is a BP-PEG-Tar@Cur microscopic topography;
FIG. 4C is a diagram of various material potentials;
FIG. 4D is a graph of particle size distribution density of various materials;
FIG. 4E is a Fourier infrared spectrum of each material;
FIG. 4F is spectrophotometry values for each material solution;
FIG. 4G shows the Raman spectrum values of the materials;
FIG. 4H is a graph of dimensional stability of various materials in PBS;
FIG. 4I is a graph of dimensional stability of various materials in DMEM;
FIG. 4J is an in vitro drug release model of curcumin;
FIG. 5A is a schematic diagram of the results of a cell viability test;
FIG. 5B is a graph of A.beta.depolymerization fluorescence experiments;
FIG. 5C is a schematic diagram showing the quantitative assay results of BCA protein;
FIG. 5D is a schematic illustration of the results of the ThT fluorescence test;
FIG. 6 is a schematic diagram of the results of a cell targeted uptake assay;
FIG. 7 is a graphical representation of mitochondrial ROS scavenging assay results;
FIG. 8 is a schematic representation of the results of a Transwell experiment.
Description of the embodiments
In order that those skilled in the art will better understand the present application, a technical solution in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings in which it is apparent that the described embodiments are only some embodiments of the present application, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present application without making any inventive effort, shall fall within the scope of the present application.
In order to make the person skilled in the art better understand the solution of the present application, the technical solution of the embodiment of the present application will be clearly and completely described below with reference to the accompanying drawings.
Example 1
The embodiment shows a preparation method of a black phosphorus nanocomposite, which specifically comprises the following steps:
step 1: 2mg of Cur is weighed and dissolved in 1ml of absolute ethyl alcohol, auxiliary dissolution is carried out by using ultrasonic, and then 8ml of absolute ethyl alcohol is slowly added for dissolution.
Step 2: the above solutions were placed on average in two separate tubes in 5ml EP tubes, 0.5ml BP at a concentration of 1mg/ml was added to one of the tubes, mixed well and placed on a shaker overnight.
Step 3: the EP tube was removed and centrifuged in a high-speed centrifuge at 7830r/min for 20 minutes and then filtered.
Step 4: the material synthesis result is judged by detecting the ultraviolet spectrum, as shown in figure 1, the ultraviolet spectrum of the solution added with BP and the ultraviolet spectrum of the solution not added with BP are not greatly different, and it is proved that Cur and BP are not combined.
Example 2
The embodiment shows a preparation method of a cinnamic acid black phosphorus nanometer composition, which specifically comprises the following steps:
step 1, adding 4- (dimethylamino) cinnamic acid, N-hydroxysuccinimide and 1-ethyl- (3-dimethylaminopropyl) carbodiimide into a flask, adding 2ml of tetrahydrofuran for dissolution, and activating hydroxyl groups in the 4- (dimethylamino) cinnamic acid.
Step 2: after 5 hours of reaction, aminopolyethylene glycol stearic acid was added to form a hydrophobic chain-wrapped Cur, which was reacted for 24 hours. The molar ratio of the four materials of 4- (dimethylamino) cinnamic acid, aminopolyethylene glycol stearic acid, N-hydroxysuccinimide and 1-ethyl- (3-dimethylaminopropyl) carbodiimide is 1:1:1:1.
step 3: 10mg of Cur was added to the flask for reaction, and after 12 hours of reaction, the reaction solution was slowly dropped into 12ml of water which was rapidly stirred, and heated in a water bath at 40℃until the odor was completely volatilized.
Step 4: after the odor volatilizes completely, the solution is placed in a high-speed centrifuge for centrifugation, the rotation speed is 6000r/min, the centrifugation is carried out for 5 minutes, and after the completion, the supernatant is pumped out and the volume is fixed to 14ml.
Step 5: half of the PEG-Tar@Cur supernatant was withdrawn to determine the UV spectrophotometry, the other half was placed in a flask, 4ml of BP at a concentration of 1mg/ml was centrifuged, resuspended in 1ml of water and added to the flask for reaction.
Step 6: after reacting for 12 hours, pumping out the reaction liquid in the flask, centrifuging in a high-speed centrifuge at a rotating speed of 7000r/min for 5 minutes, pumping out the supernatant after centrifugation is finished to measure ultraviolet spectrophotometry value, then adding 5ml of water for resuspension, repeating the centrifugation steps, washing off unbound PEG-Tar@Cur to obtain 5.3ml of suspension of a final product, measuring absorbance value by an ultraviolet spectrophotometer, measuring the Cur content of the final suspension by ultraviolet spectrometry, measuring the drug loading amount of curcumin according to the step of example 4, and finally drying to obtain 1mg of BP-PEG-Tar@Cur nanoparticle.
Step 7: the synthesis of materials was judged by detecting the uv spectra of different products and the encapsulation and loading rates were calculated by detecting the Cur content of the supernatant as shown in table 1, with encapsulation and loading rates of 11.72% and 45.84%, respectively, both of which were relatively low.
Table 1 encapsulation and drug loading results for example 2
Example 3
The embodiment shows a preparation method of a cinnamic acid black phosphorus nanometer composition, which specifically comprises the following steps:
step 1: first, 4- (dimethylamino) cinnamic acid, N-hydroxysuccinimide, 1-ethyl- (3-dimethylaminopropyl) carbodiimide and 2ml of tetrahydrofuran were added to the flask to dissolve the N-hydroxysuccinimide and activate the hydroxyl groups in 4- (dimethylamino) cinnamic acid.
Step 2: after 5 hours of reaction, aminopolyethylene glycol stearic acid was added to form a hydrophobic chain-wrapped Cur, which was reacted for 24 hours. The molar ratio of the four materials of 4- (dimethylamino) cinnamic acid, aminopolyethylene glycol stearic acid, N-hydroxysuccinimide and 1-ethyl- (3-dimethylaminopropyl) carbodiimide is 2:1:2:2.
step 3: 10mgCur was added to the flask for reaction, and after 12 hours of reaction, the reaction solution was slowly dropped into 12ml of water which was rapidly stirred, and heated in a water bath at 40℃until the odor was completely volatilized.
Step 4: after the odor volatilizes completely, the solution is placed in a high-speed centrifuge for centrifugation, the rotation speed is 6000r/min, the centrifugation is carried out for 5 minutes, and after the completion, the supernatant is pumped out and the volume is fixed to 14ml.
Step 5: half of the PEG-Tar@Cur reaction supernatant was withdrawn into the flask, and 5ml of BP at a concentration of 1mg/ml was centrifuged, resuspended in 1ml of water and added to the flask for reaction.
Step 6: after reacting for 12 hours, pumping out the reaction liquid in the flask, centrifuging in a high-speed centrifuge at a rotating speed of 7000r/min for 5 minutes, pumping out the supernatant after centrifugation is finished to measure ultraviolet spectrophotometry value, adding 5ml of water for resuspension, repeating the centrifugation steps, washing off unbound PEG-Tar@Cur to obtain 5.3ml of suspension of a final product, measuring the Cur content of the final suspension by using ultraviolet spectroscopy, measuring the drug loading amount of curcumin according to the step of example 4, and finally drying to obtain 1.36mg of BP-PEG-Tar@Cur nanoparticle.
Step 7: the synthesis of the materials was judged by detecting the uv spectra of the different products and the encapsulation and loading rates were calculated by detecting the Cur content of the supernatant as shown in table 3, and after adjusting the ratio between the materials, both the encapsulation and loading rates were improved by 22.75% and 68.51%, respectively.
Table 2 results of encapsulation and drug loading calculations for example 3
Characterization analysis of BP, PEG-Tar, PEG-Tar@Cur and BP-PEG-Tar@Cur
Taking the morphology shooting of BP and BP-PEG-Tar@Cur microscope, dispersing part of BP and BP-PEG-Tar@Cur sample into ethanol solution for ultrasound, then taking a plurality of dispersed liquid drops by drops on a copper mesh, airing, using a TEM transmission electron microscope with the model of American FEI Talos F200X G2, accelerating the voltage by 200kV, taking the morphology (high resolution) of EDS super-X, and the result is shown in figures 4A and 4B.
2. The particle diameters and potentials of the respective materials were measured by a laser particle size analyzer (model number Vern Nanosizer Nano ZS), and the results are shown in FIGS. 4C, 4D, 4H, and 4I.
3. The Fourier IR spectrum of each material was measured by a Fourier IR spectrometer (model number Thermo Science Nicolet iS). In a dry environment, the ATR accessory is placed in the light path of a spectrometer, the air background is scanned, 1 drop of liquid is dripped on the crystal surface of the ATR accessory by using a dropper, then the infrared spectrum of the sample is collected, the resolution is 4cm < -1 >, the scanning times are 32 times, the testing wave number range is 400-4000cm < -1 >, and the result is shown in figure 4E.
4. Spectrophotometry values of the respective materials were measured by an ultraviolet spectrophotometer (model: UV-2450), and the results are shown in FIG. 4F.
5. The Raman spectral values of the materials were determined by using a high resolution confocal microscopic laser Raman instrument (model: WITec alpha300R, germany, laser: 532 nm). Single spectrum test conditions: the laser energy was 36mW, 1800G/mm grating, olympus20 times objective (Olympus 20 x/0.25), integration time was 30s, and the number of times was 10, and the result was shown in FIG. 4G.
Example 4
The present example proposes a method of measuring the drug loading of curcumin in the BP-PEG-tar@cur nanoparticles of examples 2 and 3 with an ultraviolet spectrophotometer, first drawing a standard curve of curcumin, dissolving 1mg of curcumin in absolute ethanol and diluting to different concentrations (0.625, 1.25, 2.5, 5, 10, 20 μg/ml), measuring the absorbance value of each concentration solution obtained at 425nm according to the ultraviolet spectrophotometer, and drawing a concentration-absorbance standard curve of curcumin. To calculate the curcumin loading of the synthetic material, the BP-PEG-tar@cur solvent was centrifuged and resuspended in 1ml absolute ethanol and the absorbance value measured at 425nm, the amount of curcumin in the nanoparticles divided by the amount of total nanoparticles multiplied by the percentage to give the drug loading percentage. The resulting product of example 2, BP-PEG-Tar@Cur, was tested for curcumin concentration of 458.45 μg/ml in the absolute ethanol solvent and the product of example 3, BP-PEG-Tar@Cur, was tested for curcumin concentration of 931.74 μg/ml in the absolute ethanol solvent.
Example 5
This example presents a method for in vitro cumulative release of curcumin from BP-PEG-Tar@Cur nanoparticles by adding 0.5ml of a 435mg/ml curcumin loaded material solution to a dialysis bag having a molecular weight cut-off of 500 and sealing with a clip. The dialysis bag was placed in 50ml of absolute ethanol (0.01 m, ph=7.4) release medium, placed on a shaking table with a constant temperature of 37 ℃, and 3ml of release liquid was removed at predetermined time intervals (0, 2, 4,6, 8, 10, 12, 24 hours) while supplementing the same volume of absolute ethanol. The absorbance value of the release solution from each extraction was measured at 425nm using an ultraviolet spectrophotometer, and from each result, the cumulative release profile of curcumin was calculated, as shown in fig. 4J, by initially releasing about 20% cur from BP-PEG-tar@cur nanoparticles in 2 hours, and then continuing to release to about 50% in 24 hours.
Example 6
This example provides a method for preparing A beta monomers and fibrils1mg frozen Abeta 1-42 Equilibration was carried out at room temperature for 30 min to prevent peptide condensation. 222 μl of HFIP was added to Abeta 1-42 1mM solution was obtained. After sonicating the resulting solution in a water bath for 10 minutes, after incubation with shaking for 2 hours at 4 ℃, the peptide solution was vortexed to take 10 μl aliquots, all aliquots were placed in a SpeedVac at 45 ℃ for 30 minutes to remove any residual HFIP. Finally, pretreated solid Abeta 1-42 The monomers were stored in small EP tubes and stored at-20℃until use. Before use, these dried aliquots were resuspended in anhydrous DMSO to a concentration of 1mM (amount of material 10 nmol), sonicated for about 10 minutes, and then diluted to the desired concentration using PBS (pH 7.4, 10 mM), and the prepared solutions were immediately used in the experiment. Aβ 1-42 The monomers were incubated at 37℃for 72 hours to form fibrils.
Example 7
This example proposes a method for determining the effect of Cur, PEG-Tar@Cur, BP-PEG-Tar@Cur on inhibition of Abeta fibril aggregation by BCA protein quantification, taking Abeta with a volume of 100. Mu.l and a concentration of 25. Mu.M 1-42 One of the aliquots was used as a blank, the other three aliquots were incubated with Cur, PEG-Tar@Cur, BP-PEG-Tar@Cur, respectively, except for the blank, the Cur concentration in each solution was ensured to be 5. Mu.g/ml, the four aliquots were centrifuged at 20000rpm/min for 20 minutes after shaking at 100rpm for 72 hours in PBS (PH= 7.4,1X) buffer at 37℃and 75% of the supernatant was extracted for determination of the concentration of soluble Abeta by BCA protein quantification. After resuspension of aliquots with 10 μl dmso, PBS (ph= 7.4,1X) was added to dilute the total volume to 100 μl. Mu.l each was added to the EP tube and 75. Mu.l PBS (pH= 7.4,1X) was added to the tube in a total volume of 100. Mu.l. As shown in FIG. 5C, the monomer content of the untreated group is about 30%, and the monomer content of the treated group with the material is sequentially increased, and the monomer content of the BP-PEG-Tar@Cur group is even 80%, so that the BP-PEG-Tar@Cur Cur can effectively inhibit the aggregation of the Abeta fibrils.
Example 8
This example provides a method for testing ThT fluorescence, taking out aliquotsSample Aβ 1-42 One tube was equilibrated for 10 minutes at room temperature, then sonicated in a 10. Mu.l DMSO ice-water bath for 10 minutes, 50. Mu.l PBS (16.7. Mu.M) was added and mixed well. The Cur, PEG-Tar@Cur and BP-PEG-Tar@Cur solutions with Cur concentrations of 0.5 mug/ml were added to the EP tube, and the tubes were placed in a constant temperature shaker at 37℃and incubated at 100rpm for 72 hours. After the reaction was completed, the sample was mixed with 400. Mu.l of ThT (25. Mu.M). Fluorescence intensity was measured using a fluorometer (model: HITACHI F-4500, japan) at 25℃with a slit of 5nm and excitation and emission wavelengths of 440.480nm, respectively. The results are shown in FIG. 5D to contain no Abeta 1-42 Is used as background and contains Abeta only 1-42 The solution of (2) is 100% in fluorescence intensity, the higher the fluorescence intensity is, the more fibrils are formed, and the result shows that the fluorescence intensity of BP-PEG-Tar@Cur treatment group is reduced to about 35%, which indicates that the material can effectively inhibit Abeta fibril aggregation.
Example 9
The present example provides a method for cell targeted uptake experiments, where N2a cells were grown at 2X 10 cells per well 5 The density of individual cells was seeded into confocal dishes. After the cells adhere to the wall, Aβ is added 1-42 After incubation for 12 hours (25. Mu.M), 10. Mu.g/ml of Cur, PEG-Tar@Cur, BP-PEG-Tar@Cur solution were added, respectively, and incubation was continued for a further 12 hours. After the incubation, the medium was changed and washed 3 times with PBS (ph= 7.4,1X). Fixation with wt4% paraformaldehyde for 10 min followed by staining with DAPI for 5 min. When the cells are observed by using a laser scanning confocal microscope at the excitation wavelength of 488nm, the result is shown in fig. 6, and the image under the confocal microscope shows that the cell uptake of the material group added with the targeting substance Tar is obviously increased and the Abeta can be targeted.
Example 10
This example provides a cytotoxicity test method by inoculating a 96-well plate with a cell suspension (100. Mu.l), about 5000 cells/well, culturing the cells for 12 hours, and adding Abeta 1-42 The fibrils were incubated for 6 hours. After adding 5 types of Cur, PEG-Tar@Cur and BP-PEG-Tar@Cur solutions with concentrations of 0.1, 0.5, 1, 2 and 4 mug/ml respectively, incubating for 30 minutes, performing NIR irradiation for 3 minutes (0.75W cm-2) to achieve the maximum targeting effect, and then continuously incubating for 2 minutes4 hours. 10. Mu.l of CCK8 solution was added to each well, and the blank wells were set with wells to which equal amounts of cell culture, drug and CCK8 solution were added but no cells were added. As a result, as shown in fig. 5A, the material showed lower toxicity.
Example 11
This example shows a method for removing mitochondrial ROS by selecting N2a cells in logarithmic growth phase, preparing single cell suspension from culture solution containing 100U/ml double antibody cell culture medium (500 μl), 10% FBS DMEM (23 ml), opti-MEM (23 ml) per 50ml, and mixing with the culture solution according to 2×10 5 Cells/dish were inoculated into copolymer Jiao Min, 1ml of medium was added to each dish, placed in a cell incubator, incubated for 12 hours, 25 μ M A β fibrils were added for 12 hours, 5 μg/ml of Cur, PEG-tar@cur, BP-PEG-tar@cur after 12 hours incubation, washed twice with PBS (ph= 7.4,1X), and 1ml working solution containing superoxide indicator MitoSOX 5 μm was added in the dark at 37 ℃, stained in a 37 ℃ cell incubator for 10 minutes, 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 fluorescent images of ROS removal were obtained with a laser scanning confocal microscope. As a result, as shown in FIG. 7, the ROS content of the cells treated with Abeta fibrils was greatly increased, while the ROS content was significantly reduced after the drug treatment. The material is shown to be effective in reducing ROS produced by aggregation of Abeta fibrils.
Example 12
The embodiment provides a Transwell in-vitro invasion experimental method, which simulates the in-vitro environment of a BBB by using immortalized mouse brain microvascular endothelial cells (bEnd.3 cells) on the cellular level, and researches the BBB penetration capacity of BP-PEG-Tar@Cur under the photo-thermal effect. bEnd.3 (1X 10) 4 Individual cells) were inoculated on the front side of a gelatin-coated 6-well plate Transwell chamber and cultured with DMEM containing 10% fbs, 1% penicillin/streptomycin. Transendothelial resistance (TEER) was measured every 3-4 days and the integrity of the BBB monolayers was assessed using a voltameter (EVOM) of World Precision Instruments (Sarasota, FL, USA). The transendothelial resistance (TEER) values were measured before and after the experiment and the experiment was started after the TEER values stabilized. To ensure the accuracy of the numerical valueThe whole process is carried out at a constant temperature of 37 ℃. Three spots were taken for each cell insert in different directions and the measurements were repeated 3 times. After several days, the cells coalesce to form a reliable BBB monolayer, forming a tight monolayer (transendothelial resistance (TEER) exceeding 200 Ω cm). The same concentration of Cur, BP-PEG-Tar@Cur and BP-PEG-Tar@Cur material under NIR irradiation are treated at 37 ℃ and 5% CO 2 Under the condition that the samples are respectively added into a top donor chamber for incubation for 2 hours, 200 mu l of samples are collected from the outer side of the substrate, and the content of Cur is detected by an ultraviolet spectrophotometer. The initial Cur content of the three treatment materials is 43.5 mug, the transmittance content of BP-PEG-Tar@Cur material group Cur after 2 hours is 11.30 mug, and the transmittance is 25.9%; the BP-PEG-Tar@Cur material group Cur under NIR irradiation has a transmittance of 14.08 mug and a transmittance of 32.4%, and the result is shown in figure 8, and the BP-PEG-Tar@Cur material group Cur under NIR irradiation can effectively open the blood brain barrier and enhance the transmittance of Cur. Meanwhile, living cells and dead cells of the cells treated by the BP-PEG-Tar@Cur under the irradiation of Cur, BP-PEG-Tar@Cur and NIR are differentiated by the Calcein-AM/PI staining, as shown in figure 8, the conditions under the three treatment groups are not much different, and no excessive dead cells exist, which indicates that the toxicity of the BP-PEG-Tar@Cur Cur is not increased by the irradiation of NIR.
Abbreviation and key term definitions
BP: black phosphorus, a black metal-glossy semiconductor crystal having a density of 2.70 g/cm 3 The hardness was 2. Its lattice is composed of diatomic layers, each of which is composed of a tortuous chain of phosphorus atoms. In these chains, the P-P-P bond angle is 90℃and the phosphorus-phosphorus bond distance is 2.17 angstroms. Black phosphorus is the least reactive among phosphorus allotropes and does not spontaneously ignite in air.
PEG：C18-PEG-NH 2 Amino polyethylene glycol stearic acid.
Cur: curcumin, a polyphenol compound derived from plants, naturally occurs in turmeric, which is a food and drug widely used in india and china.
Tar:4- (dimethylamino) cinnamic acid as a targeting material for targeting aβ.
PEG-Tar: polyethylene glycol stearic acid cinnamic acid composition.
PEG-Tar@Cur: polyethylene glycol stearic acid cinnamic acid curcumin composition.
BP-PEG-Tar@Cur: abeta-targeted cinnamic acid black phosphorus nanocomposites.
NHS: n-hydroxysuccinimide, white to off-white crystals, are used for the synthesis of amino acid protectants, semisynthetic kanamycin and pharmaceutical intermediates.
EDC: 1-ethyl- (3-dimethylaminopropyl) carbodiimide is an organic compound having the formula C8H17N3.
AD: alzheimer's disease is a progressive degenerative disease of the nervous system with hidden disease development. Clinically, global dementia characterized by memory impairment, aphasia, disuse, disrecognition, impairment of visual space skills, executive dysfunction, personality and behavioral changes, etc., has heretofore been unknown in etiology.
Aβ (amylase β -protein): beta-amyloid, having a molecular weight of about 4kDa, is hydrolyzed from beta-amyloid precursor protein (beta-amyloid precursor protein, APP), secreted by cells, and has a potent neurotoxic effect after accumulation of cellular matrix precipitations.
FTIR: fourier transform infrared instrument.
DLS: dynamic light scattering instrument.
TEM: transmission electron microscope.
HFIP: hexafluoroisopropanol.
SpeedVac: vacuum concentrator.
DMSO: dimethyl sulfoxide.
CLSM: confocal laser scanning microscope.
PBS: phosphate buffer solution, which is the most widely used buffer solution in biochemical research, contains Na as the main component 2 HPO 4 、KH 2 PO 4 NaCl and KCl generally act as solvents to solubilize the protective agent.
N2a cells: n2a cells, collectively mouse neuroblastoma N a cells, are mouse-derived neuroma cells.
DAPI:4, 6-diamidino-2-phenylindole is a fluorescent dye capable of binding with DNA strongly and is commonly used for fluorescent microscopy.
NIR: near infrared spectrometer.
CCK-8: cell Counting Kit-8 cell counting reagent.
ROS: reactive oxygen species are oxygen-containing chemically reactive chemicals including peroxides, superoxides, hydroxyl radicals, singlet oxygen, and alpha-oxygen.
FBS: fetal bovine serum.
DMEM: is a culture medium containing various amino acids and glucose.
Opti-MEM: for culturing hematopoietic cells.
Mitosox: the superoxide indicator is a novel fluorescent dye specifically targeted to mitochondria in living cells.
BBB: the blood brain barrier, which refers to the barrier between the brain capillary wall and the plasma formed by glial cells and brain cells and the barrier between the plasma formed by the choroid plexus and cerebrospinal fluid, can prevent certain substances (most likely harmful) from entering the brain tissue from the blood.
Calcein-AM/PI staining: calcetin AM has hydrophobicity, can enter the living cell membrane easily, calcetin AM (self does not fluoresce) is cut into non-permeable polar molecule Calcetin by intracellular esterase and emits green fluorescence, and dead cells lack esterase and cannot be sheared so as not to emit light; PI cannot cross the cell membrane of living cells, can only cross disordered regions of dead cell membranes to reach the nucleus, and intercalates into DNA duplex of cells to generate red fluorescence, and two dyes are simultaneously stained to distinguish between living and dead cells.
It is apparent that the above-described embodiments are only some embodiments of the present application, but not all embodiments, and the preferred embodiments of the present application are shown in the drawings, which do not limit the scope of the patent claims. This application may be embodied in many different forms, but rather, embodiments are provided in order to provide a thorough and complete understanding of the present disclosure. Although the application has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described in the foregoing description, or equivalents may be substituted for elements thereof. All equivalent structures made by the content of the specification and the drawings of the application are directly or indirectly applied to other related technical fields, and are also within the scope of the application.
Claims (10)
1. A beta-targeting cinnamic acid black phosphorus nanometer composition is characterized by comprising a medicine carrying main body black phosphorus nanometer sheet, a modifying material amino polyethylene glycol stearic acid, a targeting material 4- (dimethylamino) cinnamic acid and a therapeutic material curcumin.
2. The aβ -targeting black phosphorus cinnamate nanocomposite of claim 1 wherein 4- (dimethylamino) cinnamic acid is used as the aβ -targeting group.
3. A preparation method of a beta-targeting cinnamic acid black phosphorus nanometer composition is characterized by comprising the following preparation steps:
mixing 4- (dimethylamino) cinnamic acid with N-hydroxysuccinimide and 1-ethyl- (3-dimethylaminopropyl) carbodiimide in a certain proportion, adding a solvent for dissolution reaction, and then adding amino polyethylene glycol stearic acid for continuous reaction;
adding curcumin into the solution obtained in the step (1) for reaction, dripping the solution into water which is rapidly stirred, heating the solution until the smell is completely volatilized, and centrifuging the solution to obtain an upper layer solution;
and (3) reacting the upper layer solution obtained in the step (2) with the black phosphorus nanosheets to generate the final product of the cinnamic acid black phosphorus nanosubstance targeting Abeta.
4. The method for preparing the beta-targeting cinnamic acid black phosphorus nano composition according to claim 3, wherein in the step (1), the four materials including 4- (dimethylamino) cinnamic acid, aminopolyglycol stearic acid, N-hydroxysuccinimide and 1-ethyl- (3-dimethylaminopropyl) carbodiimide have a molar ratio of (1-2): 1: (1-2): (1-2).
5. The method for preparing the aβ -targeting cinnamic acid black phosphorus nano composition according to claim 3, wherein in the step (1), the molar ratio of the four materials of 4- (dimethylamino) cinnamic acid, aminopolyglycol stearic acid, N-hydroxysuccinimide and 1-ethyl- (3-dimethylaminopropyl) carbodiimide is 2:1:2:2.
6. the method for preparing the Abeta-targeted cinnamic acid black phosphorus nano composition according to claim 3, wherein the solvent in the step (1) is tetrahydrofuran, and the reaction is carried out for 4-6 hours after the solvent is added.
7. The method for preparing the Abeta-targeted cinnamic acid black phosphorus nano composition according to claim 3, wherein the step (1) is performed with a hydrophobic reaction for 20-25 hours after adding amino polyethylene glycol stearic acid.
8. The method for preparing the Abeta-targeted cinnamic acid black phosphorus nano composition according to claim 3 is characterized in that the step (2) specifically comprises the steps of adding curcumin into the solution obtained in the step (1) for reaction for 10-15 hours, dripping the obtained product into water which is rapidly stirred, heating in a water bath until the smell is completely volatilized, and centrifuging to obtain an upper solution.
9. The method for preparing the beta-targeting cinnamic acid black phosphorus nano composition according to claim 3, wherein the step (3) specifically comprises the steps of reacting the solution obtained in the step (2) with black phosphorus nano sheets for 10-15 hours through phosphorus-oxygen bonds, and repeating centrifugation for two times to obtain the beta-targeting cinnamic acid black phosphorus nano composition particles.
10. Application of Abeta-targeted cinnamic acid black phosphorus nanometer composition in preparation of medicine for treating Alzheimer's disease.
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| CN115607523A (en) * | 2022-09-30 | 2023-01-17 | 广东省第二人民医院（广东省卫生应急医院） | Composition for relieving and/or treating Alzheimer's disease and preparation method and application thereof |
| CN115887694A (en) * | 2022-08-20 | 2023-04-04 | 河南省龙星生物科技有限公司 | Preparation method and application of targeted nano-drug delivery system |
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| WO2018095017A1 (en) * | 2016-11-25 | 2018-05-31 | 深圳大学 | Targeted photothermal black phosphorus nano preparation and preparation method and application thereof |
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