Polypeptide modified black phosphorus nanosheet composite material for brain targeting and preparation method thereof

Abstract

The invention discloses a polypeptide-modified black phosphorus nanosheet composite material for brain targeting and a preparation method thereof, the invention provides the polypeptide-modified black phosphorus nanosheet composite material for brain targeting and the preparation method thereof, and the black phosphorus nanosheet composite material has good drug loading capacity and excellent photo-thermal capacity, and can be applied to brain targeting. The problem of solubility of the resveratrol is solved, and the bioavailability of the resveratrol is improved. The black phosphorus nanosheet composite material disclosed by the invention is modified by ANG polypeptide, penetrates through a blood brain barrier through LRP-1 receptor mediated endocytosis and enters the brain, and has an obvious brain targeting characteristic. Meanwhile, under near-infrared illumination, the blood-brain barrier permeability of the black phosphorus nanosheet composite material can be further improved. The black phosphorus nanosheet composite material disclosed by the invention has a remarkable treatment effect on ischemic brain injury under the conditions of polypeptide modification and near-infrared illumination.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and in particular relates to a brain-targeted polypeptide-modified black phosphorus nanosheet composite material and a preparation method thereof. Background Art

[0002] The blood-brain barrier (BBB) ​​is a structure that limits the free exchange of substances between the blood and brain tissue. It is based on the imperforate endothelium of brain capillaries, the complete and continuous basement membrane, and the perivascular feet of astrocytes. It has different permeabilities for different substances. For example, fat-soluble substances, O2, CO2, and certain anesthetics are easy to pass through, while the permeability for water-soluble substances varies greatly. For example, the permeability for glucose and amino acids is relatively high, while the permeability for mannitol, sucrose, and certain ions is very low or even cannot pass through. However, there is no blood-brain barrier in some parts of the brain, such as the pineal gland and the posterior pituitary gland, so the permeability of the capillary walls there is relatively high. The existence of the barrier is of great significance in maintaining the stability of the internal environment around the brain tissue and preventing harmful substances in the blood from entering the brain tissue.

[0003] With the aging of society and the increasing pressure of competition, central nervous system (CNS) diseases have become the second largest disease after cardiovascular diseases. However, the success rate of CNS drug research and development is very low. Compared with the 20% success rate of cardiovascular disease drugs, the success rate of CNS drugs is only 8%. A major factor affecting the success of CNS drug research and development is the blood-brain barrier, which blocks almost 100% of large molecule drugs and more than 98% of small molecule drugs. Therefore, in addition to having good activity and metabolic properties and low toxicity, CNS drugs also need to overcome the blood-brain barrier and achieve sufficient exposure in the central nervous system, which is the key prerequisite for the successful development of CNS drugs.

[0004] Nanomaterials have special properties in terms of specific surface area, surface energy, and surface atoms, which give them nano effects. Nanotechnology can be applied to the study of drug delivery to the brain by using the nano effect. At the same time, nanomaterials can be modified with surface functionalization to actively transport through transcytosis, thereby improving BBB transport and improving drug permeability.

[0005] Black phosphorus (BP), also known as phosphorene, has a unique structure and properties and is becoming a rising star since its first successful exfoliation in 2014. BP has a broadband tunable direct band gap, ensuring that it has a broad spectrum of strong light absorption. Due to its strong interaction with light, BP has a high extinction coefficient in the near-infrared region, high photothermal conversion efficiency, and high fluorescence quantum yield, with potential performance in the fields of photothermal therapy, photoacoustic imaging, and fluorescence imaging. Further experiments showed that BP has significant fluorescence properties as well as negligible cytotoxicity. More importantly, due to its ultra-thin thickness and unique wrinkled lattice structure, BP nanosheets are very suitable as a drug platform.

[0006] The most important feature of BP is its complete biodegradation under physiological conditions. BP can be degraded into phosphate, which is a ubiquitous metabolite in the body. Illumination and modification can effectively control the degradation rate from 1 hour to 1 month, indicating the great potential of BP as a next-generation smart drug delivery platform. With so many excellent properties, BP is an excellent carrier for brain disease applications. In addition, because of their photothermal effect, BP nanosheets show good BBB permeability under near-infrared irradiation. At the same time, BP nanosheets reduce intracellular ROS content and increase mitochondrial membrane potential (MMP). BP shows excellent biocompatibility and stability in vitro and in vivo. Therefore, BP can be regarded as a promising neuroprotective drug delivery system.

[0007] Angiopep-2 (TFFYGGSRGKRNNFKTEEY, ANG) is a new type of polypeptide composed of 19 amino acid molecules. It is a ligand for low-density lipoprotein receptor related protein-1 (LRP-1) on the blood-cerebrospinal fluid barrier. Angiopep-2 penetrates the BBB into the brain through LRP-1 receptor-mediated endocytosis and has obvious brain targeting characteristics. Experiments have shown that the blood-brain barrier permeability of drugs is increased by liposomes, polymer nanoparticles and other carriers modified with Angiopep-2. Angiopep-2 has the advantages of easy synthesis, low immunogenicity and little interference with the internal environment of the receptor, so it is widely used in the modification of targeted BBB materials.

[0008] Resveratrol (RES) is a polyphenolic compound and a natural antitoxin widely found in plants such as Polygonum cuspidatum, Veratrum, and grapes. Studies have shown that resveratrol has antioxidant and free radical scavenging drug activity and high biocompatibility, making it an ideal neuroprotective drug. However, due to the poor solubility of resveratrol in water and its rapid metabolism in the body, most of the drugs entering the systemic circulation are blocked by the BBB, resulting in resveratrol under normal administration routes not having a long-term therapeutic effect. An efficient dosage form is urgently needed to improve the ability of resveratrol to resist ischemic brain damage.

[0009] In summary, the present invention is designed to prepare low-layer and uniformly sized black phosphorus nanosheets by liquid phase exfoliation, and at the same time, to improve the blood-brain barrier permeability after polypeptide ANG modification, and to connect resveratrol to construct a complete black phosphorus nanosheet drug delivery system. Using small animal in vivo imaging, the accumulation of drugs in the brain after administration of black phosphorus nanosheet composites under different dosage forms and conditions is explored, and its therapeutic effect against ischemic brain damage is evaluated, providing experimental and theoretical basis for the application development and clinical use of black phosphorus nanosheet composites that can improve the permeability of the blood-brain barrier in brain diseases. Summary of the invention

[0010] The purpose of the present invention is to overcome the shortcomings of the above-mentioned prior art and provide a brain-targeted polypeptide-modified black phosphorus nanosheet composite material and a preparation method thereof. The black phosphorus nanosheet composite material of the present invention has good drug loading capacity and excellent photothermal capacity, solves the problem of drug solubility, improves bioavailability, and allows drugs to effectively pass through the blood-brain barrier, and has good application prospects in the treatment of brain diseases.

[0011] To achieve the above-mentioned purpose, the technical solution adopted by the present invention is: a polypeptide-modified black phosphorus nanosheet composite material for brain targeting, wherein the black phosphorus nanosheet composite material is a sheet-shaped layered structure, the black phosphorus nanosheet is modified with ANG to form BP-ANG, and the model drug resveratrol (RES) is loaded in the sheet layer of the black phosphorus nanosheet by electrostatic adsorption, that is, the black phosphorus nanosheet composite material (ANG-BP-RES).

[0012] In some embodiments of the present invention, the mass ratio of the black phosphorus nanosheets to the ANG is 1:5.

[0013] In some preferred embodiments of the present invention, the mass ratio of the BP-ANG to the RES is 1:10.

[0014] In some more preferred embodiments of the present invention, the average size of the black phosphorus nanosheets is 100-150 nm.

[0015] In some more preferred embodiments of the present invention, the particle size of the black phosphorus nanosheets modified with PEG is 200 to 230 nm.

[0016] In some more preferred embodiments of the present invention, the particle size of the black phosphorus nanosheet composite material is 240-250 nm.

[0017] The present invention provides a method for preparing a brain-targeted polypeptide-modified black phosphorus nanosheet composite material, comprising the following steps:

[0018] Take an appropriate amount of resveratrol and dissolve it with an equal volume of solvent. Centrifuge the ANG-modified black phosphorus nanosheet suspension at high speed and low temperature for 15 minutes, remove the supernatant, add resveratrol solution, shake in the dark at low temperature, centrifuge at high speed and low temperature, collect the supernatant, add ultrapure water and resuspend to obtain a black phosphorus nanosheet composite material (ANG-BP-RES) solution.

[0019] In some embodiments of the present invention, the solvent is selected from at least one of PEG400, ethanol, and DMSO.

[0020] In some preferred embodiments of the present invention, the high-speed low-temperature centrifugation conditions are 10,000 rpm, 4° C., and the light-proof shaking time is 24 h.

[0021] In some preferred embodiments of the present invention, the method for preparing the black phosphorus nanosheets comprises: weighing black phosphorus powder and dispersing it in an organic solvent, placing it in a mortar, grinding it at low temperature in the dark, transferring all the ground suspension to an EP tube, and adding an ice organic solvent. The obtained black phosphorus suspension is ultrasonicated in an ice water bath in an ultrasonic machine in the dark, and the water is continuously changed during the period to ensure the low temperature state, and then the ultrasound is continued in a cell disruptor. After completion, the suspension is taken out and allowed to stand at 4°C overnight. Take the upper layer of the suspension after standing, centrifuge it at low speed and low temperature for 5 minutes, take the upper liquid, and then replace NMP with an equal volume of ultrapure water after two high-speed low-temperature centrifugations to obtain a black phosphorus nanosheet suspension solution.

[0022] In some preferred embodiments of the present invention, the organic solvent is selected from any one of N-methylpyrrolidone (NMP), dimethylformamide (DMF) and dimethyl sulfoxide (DMSO).

[0023] In some preferred embodiments of the present invention, the grinding time is 1 h, the ultrasonic power is 500 W, the ice water bath ultrasonic time is 6 h, and the cell disruptor ultrasonic time is 1 h.

[0024] In some preferred embodiments of the present invention, the low-speed low-temperature centrifugation conditions are 2000 rpm and 4°C, and the high-speed low-temperature centrifugation conditions are 15000 rpm and 4°C.

[0025] In some more preferred embodiments of the present invention, the method for preparing ANG-modified black phosphorus nanosheets includes: taking a black phosphorus nanosheet (BP) suspension, adding ANG (ANG-NH2), ultrasonicating, vortex mixing, shaking in the dark under low temperature conditions, centrifuging at high speed under low temperature conditions for 15 minutes, and removing the supernatant to obtain the ANG-modified black phosphorus nanosheets (BP-ANG).

[0026] In some more preferred embodiments of the present invention, the ultrasonic power is 500 W and the ultrasonic time is 30 min.

[0027] In some more preferred embodiments of the present invention, the light-proof shaking time is 8 hours, and the high-speed low-temperature centrifugation conditions are 12000 rpm and 4°C.

[0028] The invention provides a preparation for improving the permeability of the blood-brain barrier, comprising the black phosphorus nanosheet composite material.

[0029] In the present invention, the black phosphorus nanosheet composite material can improve the blood-brain barrier permeability of poorly soluble drugs under near-infrared light conditions.

[0030] In the present invention, the black phosphorus nanosheet composite material has a significant therapeutic effect on ischemic brain injury under the conditions of polypeptide modification and near-infrared light irradiation.

[0031] The present invention provides a black phosphorus nanosheet composite material for brain targeting and enhancing the therapeutic effect of ischemic brain injury. The black phosphorus nanosheet composite material is the above-mentioned black phosphorus nanosheet composite material.

[0032] The beneficial effects of the present invention are:

[0033] The present invention utilizes the principle of electrostatic adsorption to functionalize and modify RES and ANG molecules onto the surface of negatively charged black phosphorus nanosheets, and can control drug release through the photothermal effect of black phosphorus nanosheets, improve the blood-brain barrier permeability of drugs through near-infrared assistance, and enhance the therapeutic level of drugs in the brain.

[0034] The black phosphorus nanosheet composite material of the invention can be degraded into phosphate in vivo and has good biological safety and low toxicity.

[0035] The black phosphorus nanosheet composite material of the present invention may be used as a universal carrier of drugs to load drugs for treating other brain diseases for the treatment of target diseases, becoming a new strategy for treating brain diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0037] Figure 1The figures are a scanning electron microscopy image of black phosphorus nanosheets in an embodiment of the present invention (A), a scanning electron microscopy image of black phosphorus nanosheets modified with ANG (B), a scanning electron microscopy image of a black phosphorus nanosheet composite material (C), a transmission electron microscopy image of a black phosphorus nanosheet (D), a transmission electron microscopy image of a black phosphorus nanosheet modified with ANG (E), and a transmission electron microscopy image of a black phosphorus nanosheet composite material (F).

[0038] Figure 2 The particle size distribution diagram (A) of the black phosphorus nanosheets, the black phosphorus nanosheets modified by ANG, and the black phosphorus nanosheet composite material in the embodiments of the present invention, and the Zeta potential distribution diagram (B) of the black phosphorus nanosheets, the black phosphorus nanosheets modified by ANG, and the black phosphorus nanosheet composite material.

[0039] Figure 3 The ultraviolet full-wavelength scanning image (A) of the black phosphorus nanosheets, the black phosphorus nanosheets modified by ANG, and the black phosphorus nanosheet composite material in the embodiments of the present invention, and the infrared spectrum image (B) of the black phosphorus nanosheets, the black phosphorus nanosheets modified by ANG, and the black phosphorus nanosheet composite material.

[0040] Figure 4 Graphs showing in vitro drug release of black phosphorus nanosheet composite materials under different conditions in the embodiments of the present invention, wherein Graph A is a group not irradiated with near-infrared radiation, and Graph B is a group irradiated with near-infrared radiation.

[0041] Figure 5 The graphs are the UV full-wavelength scanning results of the black phosphorus nanosheets (A), the black phosphorus nanosheets modified by ANG (B), and the black phosphorus nanosheet composite material (C) in the embodiment of the present invention before and after being stored in PBS solution for one week; the graphs are the UV full-wavelength scanning results of the black phosphorus nanosheets (D), the black phosphorus nanosheets modified by ANG (E), and the black phosphorus nanosheet composite material (F) before and after five near-infrared irradiation cycles.

[0042] Figure 6 These are in vivo imaging images of small animals at different time points after injection of the black phosphorus nanosheet composite material in the embodiment of the present invention, where a is ANG-BP-Cy5.5+NIP; b is ANG-BP-Cy5.5; c is BP-Cy5.5+NIR; d is BP-Cy5.5; and e is Cy5.5+NIR.

[0043] Figure 7 This is a fluorescence intensity signal diagram of the main organs of mice 6 hours after injection of the black phosphorus nanosheet composite material in an embodiment of the present invention, where a is ANG-BP-Cy5.5+NIP; b is ANG-BP-Cy5.5; c is BP-Cy5.5+NIR; d is BP-Cy5.5; and e is Cy5.5+NIR.

[0044] Figure 8Figure 1 is a graph showing behavioral scores of the blank group, model group, resveratrol-administered group, and black phosphorus nanosheet composite material-administered group in the embodiment of the present invention (A); a graph showing brain water content of the blank group, model group, resveratrol-administered group, and black phosphorus nanosheet composite material-administered group (B); a graph showing TTC staining results of the blank group, model group, resveratrol-administered group, and black phosphorus nanosheet composite material-administered group (C); a graph showing cerebral infarction area of ​​the blank group, model group, resveratrol-administered group, and black phosphorus nanosheet composite material-administered group (D).

[0045] Fig. 9 Graph showing the cytotoxicity results of resveratrol (A), black phosphorus nanosheets (B), black phosphorus nanosheets modified with ANG (C), and black phosphorus nanosheet composite materials (D) in the embodiments of the present invention.

[0046] Fig.10 This is a diagram showing the H&E staining results of various tissues one week after injection of various components in the examples of the present invention. DETAILED DESCRIPTION

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

[0048] The experimental methods used in the examples of the present invention are all conventional methods unless otherwise specified; the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0049] In the embodiment of the present invention, the preparation method of the black phosphorus nanosheet composite material is as follows:

[0050] S1: Preparation of black phosphorus nanosheets (BP):

[0051] Black phosphorus nanosheets were prepared by liquid phase exfoliation. 0.1 g of black phosphorus powder was thoroughly ground in a mortar, and then ultrasonicated in an ultrasonic cleaner (power: 500 W) for 12 h in the dark, with water constantly changed to ensure low temperature. Then, ultrasonicated for 1 h in a cell disruptor at 500 W. The upper layer of the suspension was taken and stirred at 2000 r / min. -1 , centrifuged at 4 °C for 5 min, the upper layer was taken and centrifuged twice at 15 000 r / min -1 , and then centrifuge at 4°C, and replace NMP with an equal volume of PBS to obtain BP suspension.

[0052] S2: Preparation of peptide ANG modified black phosphorus nanosheets (BP-ANG)

[0053] 10 mg ANG-PEG-NH2 was added to 5 ml BP / PBS solution (200 μg / ml BP), stirred for 8 h, and then spun at 10,000 rpm for 15 min at 4°C to remove excess ANG-PEG-NH2. The sample was washed twice with PBS as above. The precipitate was taken, resuspended in PBS, and stored at 4°C.

[0054] S3: Preparation of black phosphorus nanosheet composites (ANG-BP-RES)

[0055] The above ANG-BP solution and RES (2 mg / ml ethanol, 5 ml) were stirred continuously at room temperature for 24 h. The sample was centrifuged at 10,000 rpm for 15 min at 4°C, and the supernatant was discarded to remove free RES. The sample was washed twice with PBS using the same method. The precipitate, i.e., ANG-BP-RES, was then resuspended in PBS and stored at 4°C.

[0056] Test Example 1

[0057] This example analyzes the structural characteristics and properties of the intermediates and products in the preparation process of the example.

[0058] The black phosphorus nanosheets, ANG-modified black phosphorus nanosheets (BP-ANG), and black phosphorus nanosheet composites (ANG-BP-RES) were observed by electron microscopy. Figure 1 shown.

[0059] from Figure 1 It can be seen that the average size of the black phosphorus nanosheets is about 200nm and has a layered structure; the size increases slightly after modification and drug loading, and it can be seen that the drug is loaded between the layers of the black phosphorus nanosheets. Before and after modification and drug loading, the sheet structure does not change.

[0060] The particle size and potential change of the obtained black phosphorus nanosheets were investigated by Malvern particle size analyzer. The results are as follows Figure 2 shown.

[0061] from Figure 2It can be seen that the size of the black phosphorus nanosheets is 125.2±9.2nm, the size of BP-ANG is 200.6±8.5nm, and the size of ANG-BP-RES is 238.4±5.8nm. There is no obvious volume change of the black phosphorus nanosheets after ANG modification, and stacking polymerization occurs after drug loading, resulting in a larger particle size measured by Malvern. The potential results show that the Zeta potential of the black phosphorus nanosheets is -13.83±0.96eV, the Zeta potential of BP-ANG is -17.80±0.54eV, and the Zeta potential of ANG-BP-RES is -21.23±0.37eV. Under the same conditions, the absolute value of the Zeta potential of ANG-BP-RES is significantly higher than that of BP and BP-ANG. This shows that after BP is modified with peptides and loaded with drugs, the electrostatic repulsion between its electrons increases, which enhances the electronic stability of the drug delivery system.

[0062] The black phosphorus nanosheets (BP), ANG-modified black phosphorus nanosheets (BP-ANG), and black phosphorus nanosheet composites (ANG-BP-RES) were scanned at full UV wavelength, and Fourier transform infrared spectrometer was used to observe the changes in the characteristic peaks of the drug delivery system. The results are as follows Figure 3 shown.

[0063] from Figure 3 The results of mid-ultraviolet full-wavelength scanning showed that BP-ANG had a characteristic peak of ANG at 201nm, and ANG-BP-RES had a characteristic peak of RES at 306nm. The results of Fourier transform infrared spectroscopy showed that the drug delivery system ANG-BP-RES had a characteristic peak at 1650cm -1 The characteristic peak of C=O stretching vibration is enhanced, 1538cm -1 The NH bending vibration peak at 1045 cm -1 The CO stretching vibration peak at was enhanced. The above results confirmed that BP was successfully modified by ANG and loaded with RES.

[0064] In order to explore the differences in drug release ability of black phosphorus nanosheet drug delivery system under NIR irradiation and different pH conditions, three groups of pH 5, pH 6, and pH 7 were divided and in vitro drug release experiments were carried out under two conditions: NIR irradiation and non-NIR irradiation. The results are shown in Figure 2. Figure 4 shown.

[0065] The 24h drug release rate of the pH5 group without NIR irradiation was 38.83±1.89%, and the 24h drug release rate of the pH5 group under NIR irradiation was 65.82±2.08%; the 24h drug release rate of the pH6 group without NIR irradiation was 52.80±1.24%, and the 24h drug release rate of the pH6 group under NIR irradiation was 85.64±3.08%; the 24h drug release rate of the pH7 group without NIR irradiation was 41.82±6.36%, and the 24h drug release rate of the pH7 group under NIR irradiation was 71.02±5.72%. The above experimental results confirm that the black phosphorus nanosheet drug delivery system has the best drug release ability at pH 6 and under NIR irradiation.

[0066] In order to investigate the stability of black phosphorus nanosheets (BP), ANG-modified black phosphorus nanosheets (BP-ANG), and black phosphorus nanosheet composites (ANG-BP-RES) after being stored in PBS solution for one week and before and after near-infrared irradiation, UV full-wavelength scanning was performed on black phosphorus nanosheets (BP), ANG-modified black phosphorus nanosheets (BP-ANG), and black phosphorus nanosheet composites (ANG-BP-RES) after being stored in PBS solution for one week and subjected to five near-infrared irradiation cycles. The results are shown in Figure 5. Figure 5 shown.

[0067] The absorbance of BP decreased significantly over time, but after being modified with peptides and loaded with drugs, its absorbance did not change much. This is because the surface of BP is not covered with compounds, making it very easy to oxidize. After being modified with ANG, it forms a core-shell structure, which slows down oxidation and increases the stability of the black phosphorus nanosheet composite material.

[0068] Test Example 2

[0069] This test example evaluated the effect of fluorescently labeled black phosphorus nanosheets (BP), ANG-modified black phosphorus nanosheets (BP-ANG), and black phosphorus nanosheet composites (ANG-BP-RES) on improving the permeability of the blood-brain barrier.

[0070] According to the preparation method of ANG-modified black phosphorus nanosheets (BP-ANG) in Example S2, BP was linked to the fluorescent protein CY5.5-PEG-NH2 to obtain BP-PEG-CY5.5. Male mice weighing 25-30 g were randomly selected and marked, and the animals were randomly divided into five groups (n=3): (1) Cy5.5+NIR irradiation; (2) BP-Cy5.5; (3) BP-Cy5.5+NIR irradiation; (4) ANG-BP-Cy5.5 and (5) ANG-BP-Cy5.5+NIR irradiation. The animals were given the designated treatment (Cy5 1 mg / kg, BP 6 mg / kg). After 30 min, all animals except those in Group 2 and Group 4 were subjected to NIR irradiation (808 nm, 1 W·cm-2 , 5min). Animals were imaged at 2, 4, 6, 8, 12, and 24h after treatment. Every 6 hours, a small group of animals (n=3) were anesthetized and perfused with saline, and then major organs (brain, heart, liver, spleen, lungs, and kidneys) were isolated and fluorescently imaged. Figure 6 , Figure 7 shown.

[0071] according to Figure 6 The results of in vivo imaging of small animals showed that the fluorescence intensity of the brain of mice was the strongest 6 hours after the tail vein injection of the reagent. Therefore, the mice were perfused and dissected 6 hours after the administration. Figure 7 The results show that the fifth group, that is, the ANG-BP-Cy5.5+NIR irradiation group, has the strongest fluorescence intensity in the brain tissue of mice. Comparing the results of each group, it can be seen that the blood-brain barrier permeability of the drug in the peptide-modified nanosheet composite material is greater than that of the unmodified one, and the blood-brain barrier permeability of the drug in the near-infrared irradiated nanosheet composite material is greater than that of the unirradiated one.

[0072] Test Example 3

[0073] This test example simply evaluates the anti-ischemic brain damage effect of black phosphorus nanosheets (BP) and black phosphorus nanosheet composites (ANG-BP-RES).

[0074] In order to investigate the therapeutic effect of black phosphorus nanosheet composites on ischemic brain injury, male mice weighing 25-30g were randomly selected and marked and divided into 4 groups; sham operation blank group: the mice were anesthetized by intraperitoneal injection of 5% chloral hydrate, and after being fixed in the supine position, the local cerebral blood flow of the mice was monitored using a laser Doppler blood flow detector, and a sterile cotton swab dipped in iodine was smeared on the middle suture of the neck, the skin of the middle suture was cut open with ophthalmic scissors, and the muscle tissue was bluntly separated with ophthalmic forceps until the common carotid artery (CCA) was exposed. After the CCA and vagus nerve were carefully separated, the skin was sutured, and 0.3mL of normal saline was injected intraperitoneally. MCAO reperfusion modeling group: After separating the vagus nerve, ligate the proximal end of the CCA, the distal end of the external carotid artery (ECA), and the internal carotid artery (ICA), cut a small incision on the ECA, insert the nylon head of the thread plug into the ECA, open the ICA ligation, slowly advance the thread plug in the direction of the ICA until the black mark on the tail is immersed in the blood vessel, ligate the proximal end of the ECA and open the proximal end of the CCA ligation, start timing when the local blood flow decreases by 70% or more and stabilizes, suture the skin, leave the tail of the thread plug outside the suture, apply iodine to disinfect, and inject 0.3mL of normal saline into the abdomen. After 2 hours, pull out the thread plug to the ligature for reperfusion, and the local blood flow is considered a successful model when it recovers to 80% or more of normal. RES group: Immediately after reperfusion, inject 30mg / kg of RES suspension (PBS solution) into the tail vein and irradiate at 808nm, 2W / cm 2ANG-BP-RES group: ANG-BP-RES suspension (PBS) at a dose of 30 mg / kg RES was injected into the tail vein immediately after reperfusion. ANG-BP-RES+NIR group: ANG-BP-RES suspension (PBS) at a dose of 30 mg / kg RES was injected into the tail vein immediately after reperfusion and irradiated at 808 nm, 2 W / cm 2 The brain tissues of the four groups of mice were taken for TTC staining, the changes in cerebral infarction area were investigated, and the water content of the brain tissues of the mice in each group was evaluated. Figure 8 shown.

[0075] from Figure 8 (A) It can be seen that compared with the blank group, the mice in the model group showed obvious neurobehavioral disorders 24 hours after surgery; the mice in the RES group and ANG-BP-RES group still had symptoms of neurobehavioral disorders, but compared with the model group, the RES group could improve the symptoms of neurobehavioral disorders in mice, and the ANG-BP-RES+NIR group could significantly reduce the neurobehavioral disorders in mice.

[0076] from Figure 8 (B) It can be seen that compared with the blank group, the water content of the ischemic side of the brain tissue of the mice in the model group increased significantly 24 hours after surgery; compared with the model group, the water content of the brain tissue of the mice in the RES group and the ANG-BP-RES group was significantly reduced, and the therapeutic effect of the ANG-BP-RES+NIR group was significantly higher than that of the other drug-treated groups.

[0077] from Figure 8 (C) and (D) show that 24 hours after reperfusion of MCAO model in mice, the model group had obvious infarction in the ischemic side of the brain tissue. Compared with the model group, the infarction volume of the RES group and ANG-BP-RES group was significantly reduced, and the infarction area of ​​the ANG-BP-RES+NIR group was significantly smaller than that of the other drug-treated groups.

[0078] Test Example 4

[0079] This test example simply evaluates the biosafety of black phosphorus nanosheets (BP) and black phosphorus nanosheet composites (ANG-BP-RES).

[0080] In order to investigate the cytotoxicity of black phosphorus nanosheets (BP) and black phosphorus nanosheet composites (ANG-BP-RES), a cytotoxicity experiment was performed. SH-SY5Y cells were selected and inoculated in a 96-well plate and cultured at 37°C and 5% CO2 for 24 hours. The original culture medium was aspirated, and then 200 μL of culture medium containing materials of different concentrations (0-200 μg / mL) was added and cultured for another 24 hours. A blank group was also set up, and the cells in the blank control group were co-cultured with PBS solution. After the co-culture, the original culture medium was aspirated, and the PBS was rinsed twice. The culture medium in the 96-well plate was replaced with 200 μL of fresh culture medium containing 10% fetal bovine serum, and 20 μL of CCK-8 solution was added to each well. After culturing in the incubator for 2 hours, the liquid was aspirated into the 96-well plate, and the absorbance of each well at 450 nm was measured with an enzyme reader. The group containing no cells but only culture medium and CCK8 was set as the blank control group. The cell survival rate was calculated, and the results are shown in the figure. Fig. 9 shown.

[0081] from Fig. 9 It can be seen that even at a high drug concentration of 200 μg / mL, the cell activity of each group of cells is not less than 75%, proving that its cytotoxicity is low and relatively safe.

[0082] To investigate the tissue toxicity of black phosphorus nanosheets (BP) and black phosphorus nanosheet composites (ANG-BP-RES), H&E staining was performed on the animal tissues of the RES group, ANG-BP-RES, and ANG-BP-RES+NIR groups after continuous injection of the maximum dose for one week. The results are shown in Figure 2. Fig.10 shown.

[0083] The results showed that no obvious inflammatory response was observed in the major tissues of mice in the RES, ANG-BP-RES, and ANG-BP-RES+NIR groups after continuous daily injection of the maximum dose for one week, indicating that each group of drugs produced almost no obvious tissue toxicity.

Claims

1. A peptide-modified black phosphorus nanosheet composite material for brain targeting, characterized in that The black phosphorus nanosheet composite material is a sheet-shaped layered structure, the black phosphorus nanosheet is modified by the polypeptide Angiopep-2 (TFFYGGSRGKRNNFKTEEY, ANG), and the model drug resveratrol is loaded in the sheet of the black phosphorus nanosheet by electrostatic adsorption. The black phosphorus nanosheet composite material can improve its blood-brain barrier permeability and promote the release of the model drug resveratrol in the brain under the condition of polypeptide modification and near-infrared light irradiation, and has good stability.

2. The black phosphorus nanosheet composite material according to claim 1, characterized in that: The particle size of the black phosphorus nanosheets is 100-150 nm.

3. The black phosphorus nanosheet composite material according to claim 1, characterized in that: The particle size of the ANG-modified black phosphorus nanosheets is 200-230 nm.

4. The black phosphorus nanosheet composite material according to claim 1, characterized in that: The particle size of the black phosphorus nanosheet composite material is 240-250 nm.

5. The black phosphorus nanosheet composite material according to claim 1, characterized in that: The mass ratio of the black phosphorus nanosheets to the ANG is 1:

5.

6. The black phosphorus nanosheet composite material according to claim 1, characterized in that: The mass ratio of the black phosphorus nanosheets to the resveratrol is 1:

10.

7. A method for preparing the black phosphorus nanosheet composite material according to claims 1 to 6, characterized in that: The preparation method of the black phosphorus nanosheet composite material is as follows: taking an appropriate amount of resveratrol, dissolving it with an equal volume of solvent, centrifuging the ANG-modified black phosphorus nanosheet suspension at high speed and low temperature for 10 minutes, removing the supernatant, adding resveratrol solution, shaking in the dark at low temperature, centrifuging at high speed and low temperature, collecting the supernatant, adding ultrapure water and resuspending to obtain a black phosphorus nanosheet composite material (BP-PEG-RES) solution.

8. The method for preparing the black phosphorus nanosheet composite material according to claim 7, characterized in that: The solvent is selected from at least one of PEG400, ethanol, and dimethyl sulfoxide (DMSO).

9. The method for preparing the black phosphorus nanosheet composite material according to claim 7, characterized in that: The high-speed low-temperature centrifugation conditions are 12000 rpm, 4°C, and the light-proof shaking time is 48 hours.

10. A brain targeting preparation, characterized in that: The invention comprises the black phosphorus nanosheet composite material according to any one of claims 1 to 6.

11. Use of the black phosphorus nanosheet composite material according to any one of claims 1 to 6 in a drug for improving the permeability of the blood-brain barrier.

12. Use of the black phosphorus nanosheet composite material according to any one of claims 1 to 6 in resisting ischemic brain damage.

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