A black phosphorus nanosheet analysis platform, a preparation method thereof and application thereof in inducing in-situ neural stem cell differentiation and proliferation

Abstract

This invention discloses a black phosphorus nanosheet analysis platform, its preparation method, and its application in inducing in situ neural stem cell differentiation and proliferation. It provides a method for inducing in situ neural stem cell differentiation and proliferation in adult mice and repairing nerve damage in gliomas. This invention utilizes black phosphorus nanomaterials that generate different reactive oxygen species (ROS) under different photoexcitations to construct a black phosphorus nanosheet analysis platform with controllable ROS release and quantitative analysis of ROS concentration. The platform is prepared as follows: black phosphorus nanosheets with a size of 100-150 nm are prepared by liquid-phase exfoliation; the released ROS are quantitatively analyzed using electrostatic adsorption and a ratiometric fluorescent probe; and polyethylene glycol derivatives that can cross the blood-brain barrier and target nerve trunks are loaded via electrostatic adsorption. The black phosphorus nanoplatform prepared by this invention crosses the blood-brain barrier after intravenous injection, generates different ROS under different photostimulations, and the concentration is precisely controlled by quantitative analysis using the loaded probe, achieving optimal conditions for nerve trunk differentiation into neurons. This provides a new method for nerve injury repair by continuously and controllably promoting nerve trunk differentiation.

Description

Technical Field

[0001] This invention belongs to the field of bioanalytical detection, and relates to a functionalized black phosphorus nanosheet analysis platform capable of separately and quantitatively analyzing singlet oxygen and hydroxyl radicals, which generate these two reactive oxygen species under light control, as well as its preparation method and its application in in vivo and in vitro neural stem cell differentiation, and as an effective method for nerve injury repair. Background Technology

[0002] Neural stem cells (NSCs) are a type of cell found in the subventricular zone (SVZ) and subgranular zone (SGZ) of the dentate gyrus in adult mammals. They possess strong proliferative capacity and the potential to differentiate into neurons, astrocytes, and oligodendrocytes, thus generating a large population of brain tissue cells. The differentiation of NSCs can replenish lost neurons and promote neurogenesis, representing a potential strategy for treating neurological injuries. Currently, NSCs are increasingly being used in the treatment of clinical diseases such as cerebral infarction, spinal cord injury, Parkinson's disease, and multiple sclerosis. Gliomas are common primary malignant tumors of the nervous system. During glioma development, physical compression and infiltration cause varying degrees of damage to adjacent normal brain tissue. This damage can further lead to long-term complications such as cerebral edema, resulting in impairment of functional areas of the brain. Common glioma treatments, including surgical resection, radiotherapy, and chemotherapy, often fail to restore the functional areas surrounding the damaged brain while simultaneously treating the tumor. The problem to be solved by this invention is to find a material that can release different ROS under different controlled conditions, and to find and test the optimal differentiation conditions for application in in situ differentiation of neural stem cells in vivo to treat nerve damage. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention proposes a black phosphorus nanoscale analytical platform capable of crossing the blood-brain barrier, releasing singlet oxygen and hydroxyl radicals upon photostimulation, and quantitatively analyzing these two reactive oxygen species, along with its preparation method and application in inducing in situ neural stem cell differentiation and proliferation. Ultrathin black phosphorus nanosheets are prepared via liquid-phase exfoliation and possess the characteristic of specifically generating hydroxyl radicals under ultraviolet light excitation and highly specifically generating singlet oxygen under visible light excitation. Singlet oxygen or hydroxyl radical probes and peptides that penetrate the blood-brain barrier and target neural stem cells are then electrostatically loaded onto the aforementioned ultrathin black phosphorus nanosheets. This enables the specific identification and quantitative analysis of singlet oxygen and hydroxyl radicals, while simultaneously generating these two reactive oxygen species under photocontrolled conditions. This technology can be applied to regulate and stimulate neural stem cell differentiation into neurons.

[0004] Ultrathin black phosphorus nanosheets exhibit a unique photocatalytic ability that allows for photoswitching in the generation of reactive oxygen species (ROS). Specifically, they can generate hydroxyl radicals under ultraviolet light excitation and singlet oxygen with high specificity under visible light excitation. This property determines that black phosphorus can be used as a research material to determine the effects of the types and amounts of ROS generated by different wavelengths of light excitation on neural stem cell differentiation.

[0005] This invention develops a high-performance probe capable of analyzing various reactive oxygen species (ROS) and combines it with black phosphorus nanosheets, a nanomaterial that generates multiple ROS under different light wavelength modulations. This ingeniously constructs a black phosphorus nanosheet analysis platform that combines the ability to stimulate neural stem cell differentiation with the ability to analyze ROS. This enables dynamic detection of ROS in the brain microenvironment, obtaining the redox state threshold that induces neural differentiation. From a dose-response perspective, it deeply analyzes the changes in ROS levels during the induction of endogenous neural stem cell differentiation and the positive role of inducing differentiation and forming new neural connections in the treatment of neural damage following glioma.

[0006] This invention is achieved through the following technical solution:

[0007] This invention provides a polyethylene glycol (PEG) derivative targeting neural stem cells, wherein the PEG derivative targeting neural stem cells is NH2-PEG. 2000 -NFL or NH2-PEG 2000 -ANG, where NFL is a polypeptide with the sequence YSSYSAPVSSSLSVRRSYSSSSGSC (SEQ ID NO.1); ANG is a polypeptide with the sequence TFFYGGSRGKRNNFKTEEYC (SEQ ID NO.2).

[0008] This invention also provides a method for preparing a polyethylene glycol polypeptide derivative targeting neural stem cells, the method comprising the following steps:

[0009] Step (1): The polypeptide NFL with the amino acid sequence YSSYSAPVSSSLSVRRSYSSSSGSC (SEQ ID NO.1) or the polypeptide ANG with the amino acid sequence TFFYGGSRGKRNNFKTEEYC (SEQ ID NO.2) was obtained by standard solid-phase polypeptide synthesis method.

[0010] Step (2): Dissolve the polypeptide NFL or polypeptide ANG obtained in step (1) in PBS buffer, and then react it with amino-polyethylene glycol maleimide (NH2-PEG) dissolved in PBS buffer. 2000-Maleimide), with TCEP (tris-(2-carboxyethyl)phosphine hydrochloride) as a reducing agent and catalyst, the reaction was stirred at 20-30℃ for 4-8 h to obtain an amino-modified polyethylene glycol polypeptide polyethylene glycol derivative (NH2-PEG). 2000 -NFL) or (NH2-PEG) 2000 -ANG).

[0011] In step (2), the polypeptide NFL (amino acid sequence as shown in SEQ ID NO.1) and aminopolyethylene glycol maleimide (NH2-PEG) are used. 2000 The molar ratio of -Maleimide is 1:(4-6), preferably 1:5.

[0012] In step (2), the polypeptide ANG (amino acid sequence as shown in SEQ ID NO.2) and aminopolyethylene glycol maleimide (NH2-PEG) are used. 2000 The molar ratio of -Maleimide is 1:(4-6), preferably 1:5.

[0013] In step (2), the amount of PBS buffer added is 5-15 mL, preferably 10 mL.

[0014] In step (2), the concentration of TCEP is 40-60 mM, preferably 50 mM.

[0015] In step (2), the reaction temperature is 20-30℃; preferably, it is 25℃.

[0016] In step (2), the reaction time is 4-8 hours; preferably, it is 5 hours.

[0017] In step (2), after the reaction is completed, the product is purified by semi-preparative high performance liquid chromatography and characterized by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS).

[0018] The present invention also provides a polyethylene glycol derivative of a polypeptide targeting neural stem cells prepared by the method, wherein the polyethylene glycol derivative of the polypeptide targeting neural stem cells includes an amino-polyethylene glycol modified polypeptide (NH2-PEG2000-NFL) or (NH2-PEG2000-ANG).

[0019] The present invention also provides a singlet oxygen / hydroxyl radical ratio fluorescent probe, wherein the ratio fluorescent probe is a singlet oxygen ratio fluorescent probe or a hydroxyl radical ratio fluorescent probe.

[0020] The present invention also provides a method for preparing the singlet oxygen / hydroxyl radical ratio fluorescent probe as described above, the preparation method comprising the following steps:

[0021] Step (1) 7-Hydroxy-2-oxo-2H-chromene-3-carboxylicacid is combined with a polyethylene glycol derivative (Fmoc-NH-PEG). 2000 -NH2), 2-succinimide-1,1,3,3-tetramethylurea tetrafluoroborate (TSTU), and N,N-diisopropylethylamine (DIPEA) were mixed and stirred in DMSO (dimethyl sulfoxide) to give a coumarin-polyethylene glycol derivative (coumarin-PEG). 2000 -NH-Fmoc);

[0022] Step (2) involves applying the coumarin-polyethylene glycol derivative (coumarin-PEG) obtained in step (1). 2000 -NH-Fmoc and N-(3-bromopropyl)carbamate (tert-Butyl-3-bromopropylcarbamate) were dissolved in DMF (N,N-dimethylformamide), and K2CO3 was added and stirred to react, yielding a Boc-protected coumarin-polyethylene glycol derivative (Boc-coumarin-PEG). 2000 -NH-Fmoc);

[0023] Step (3) involves applying the Boc-protected coumarin-polyethylene glycol derivative (Boc-coumarin-PEG) obtained in step (2). 2000 -NH-Fmoc) is dissolved in TFA / DCM (trifluoroacetic acid / dichloromethane) solution, and the reaction yields a propylamino-coumarin-polyethylene glycol derivative (NH2-coumarin-PEG). 2000 -NH-Fmoc);

[0024] Step (4) involves constructing a singlet oxygen ratio-based fluorescent probe by using the propylamino-coumarin-polyethylene glycol derivative (NH2-coumarin-PEG) obtained in step (3). 2000 -NH-Fmoc) was covalently coupled to the commercial singlet oxygen probe SOSG in DMSO via a TSTU and DIPEA-catalyzed amide reaction, thus preparing a FRET-based singlet oxygen ratiometric fluorescent probe precursor, SOSG-coumarin-PEG derivative (SOSG-coumarin-PEG). 2000 -NH-Fmoc); or,

[0025] To construct a hydroxyl radical ratio-based fluorescent probe, the propylamino-coumarin-polyethylene glycol derivative (NH2-coumarin-PEG) obtained in step (3) was used. 2000 -NH-Fmoc) was covalently coupled to the commercial hydroxyl radical probe HPF in DMSO via a TSTU and DIPEA-catalyzed amide reaction, thus preparing a hydroxyl radical ratiometric fluorescent probe precursor based on the FRET mechanism. This led to the preparation of the HPF-coumarin-PEG derivative HPF-coumarin-PEG. 2000 -NH-Fmoc;

[0026] Step (5), the SOSG-coumarin-PEG derivative obtained in step (4) 2000 -NH-Fmoc, the HPF-coumarin-polyethylene glycol derivative HPF-coumarin-PEG 2000 -NH-Fmoc, in a solution of Piperidine / DMF, is deprotected by Fmoc to yield SOSG-coumarin-PEG, a SOSG-coumarin-PEG derivative with terminal amino groups. 2000 -NH2, HPF-coumarin-PEG derivative HPF-coumarin-PEG 2000 -NH2.

[0027] In step (1), the 7-hydroxy-3-carboxycoumarin (7-Hydroxy-2-oxo-2H-chromene-3-carboxylic acid) and the polyethylene glycol derivative (Fmoc-NH-PEG) 2000 The molar ratio of -NH2), TSTU, and DIPEA is (1-1.5):(1-1.5):(1-1.5):(2-3); preferably, it is 1:1:1:2.

[0028] In step (1), the amount of DMSO added is 10-20 mL, preferably 15 mL; the volume / molar ratio of the amount of DMSO added to the raw material 7-hydroxy-2-oxo-2H-chromene-3-carboxylic acid is (10-20 mL): (1-2 mmol); preferably, it is 15 mL: 1 mmol.

[0029] In step (1), the reaction temperature is 20-30℃; preferably, it is 25℃.

[0030] In step (1), the reaction time is 20-40 min; preferably, it is 30 min.

[0031] In step (1), after the reaction is completed, the product is purified by semi-preparative high performance liquid chromatography and characterized by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS).

[0032] In step (2), the coumarin-polyethylene glycol derivative (coumarin-PEG) 2000 The molar ratio of -NH-Fmoc), N-(3-bromopropyl)carbamate tert-Butyl-3-bromopropylcarbamate, and K2CO3 is (1-1.5):(1-1.5):(1.3-2); preferably, it is 1:1:1.3.

[0033] In step (2), the amount of DMF added is 10-20 mL, preferably 15 mL; the volume / molar ratio of the amount of DMF added to the raw material N-(3-bromopropyl)carbamate is (5-10 mL): (1-2 mmol); preferably, it is 5 mL: 1 mmol.

[0034] In step (2), the reaction temperature is 50-60℃; preferably, it is 60℃.

[0035] In step (2), the reaction time is 24-48 hours; preferably, it is 24 hours.

[0036] In step (2), after the reaction is completed, the product is purified by semi-preparative high performance liquid chromatography and characterized by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS).

[0037] In step (3), the Boc-protected coumarin-polyethylene glycol derivative (Boc-coumarin-PEG) 2000 The molar / volume ratio of -NH-Fmoc), DCM, and TFA is (1-1.5 mmol):(3-5 mL):(0.5-2 mL); preferably, it is 1 mmol:4 mL:1 mL.

[0038] In step (3), the reaction temperature is 20-30℃; preferably, it is 25℃.

[0039] In step (3), the reaction time is 1-2 hours; preferably, it is 1 hour.

[0040] In step (3), after the reaction is completed, the product is washed with 5% NaHCO3 and saturated saline, purified by rotary evaporation using semi-preparative high performance liquid chromatography, and characterized by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS).

[0041] In step (4), the propylamino-coumarin-polyethylene glycol derivative (NH2-coumarin-PEG) 2000 The molar ratio of -NH-Fmoc), SOSG or HPF, TSTU, and DIPEA is (1-1.5):(1-1.5):(1-1.5):(2-3); preferably, it is 1:1:1:2.

[0042] In step (4), the amount of DMSO added is 10-20 mL, preferably 15 mL; the amount of DMSO added is related to the amount of propylaminocoumarin-polyethylene glycol derivative (NH2-coumarin-PEG). 2000 The volume / molar ratio of -NH-Fmoc is (10-20 mL): (1-2 mmol); preferably, it is 15 mL: 1 mmol.

[0043] In step (4), the reaction temperature is 20-30℃; preferably, it is 25℃.

[0044] In step (4), the reaction time is 20-40 min; preferably, it is 30 min.

[0045] In step (4), after the reaction is completed, the product is purified by semi-preparative high performance liquid chromatography and characterized by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS).

[0046] In step (5), the SOSG-coumarin-polyethylene glycol derivative (SOSG-coumarin-PEG) 2000 -NH-Fmoc) or HPF-coumarin-polyethylene glycol derivative (HPF-coumarin-PEG) 2000 The molar / volume / volume ratio of -NH-Fmoc to added Piperidine:DMF is 0.002-0.004 mmol:1 mL:(3-5) mL, preferably 0.003 mmol:1 mL:4 mL.

[0047] In step (5), the reaction temperature is 20-30℃; preferably, it is 25℃.

[0048] In step (5), the reaction time is 20-40 min; preferably, it is 30 min.

[0049] In step (5), after the reaction is completed, DMF is removed by vacuum, purified by semi-preparative high performance liquid chromatography, and the product is characterized by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS).

[0050] The present invention also provides a singlet oxygen / hydroxyl radical ratio fluorescent probe obtained by the above method, the probe comprising a SOSG-coumarin-PEG derivative with terminal amino groups. 2000 -NH2, HPF-coumarin-PEG derivative HPF-coumarin-PEG 2000 -NH2.

[0051] This invention also provides a method for preparing a black phosphorus nanosheet analysis platform, wherein the black phosphorus nanosheet analysis platform is capable of crossing the blood-brain barrier and targeting neural stem cells and quantitatively analyzing hydroxyl radicals / singlet oxygen. The preparation method includes the following steps:

[0052] Step (1) Disperse black phosphorus blocks in ultrapure water, purge with argon gas to eliminate dissolved oxygen in the water, sonicate the mixed solution in ice water, centrifuge the resulting brown suspension and take the supernatant to obtain ultrathin black phosphorus nanosheets dispersed in ultrapure water.

[0053] Step (2) By using a mixed ultrasound method, the singlet oxygen / hydroxyl radical ratio fluorescent probe described above and the polypeptide polyethylene glycol derivative described above that penetrates the blood-brain barrier and targets neural stem cells are electrostatically adsorbed onto the surface of black phosphorus nanosheets, thereby obtaining the black phosphorus nanosheet analysis platform that can quantitatively detect singlet oxygen / hydroxyl radicals and target neural stem cells in the brain to achieve in situ neural stem cell induction differentiation and proliferation.

[0054] In step (1), the dispersion system of the black phosphorus nanosheet dispersion is sterile ultrapure water, and the concentration of the black phosphorus nanosheet dispersion is 200-250 μg / mL.

[0055] The specific steps of step (1) are as follows: 25 mg of purchased black phosphorus blocks are added to 50 mL of ultrapure water (the concentration of black phosphorus blocks here is 0.5 mg / mL, which is the preparation concentration, different from the concentration of the dispersion system in the final step). The frequency of the ultrasound is set to 30-50 kHz. During the ultrasound process, the sample is kept in ice water. After ultrasound for 10-14 h, the obtained brown dispersion is centrifuged at 1000-1200 rpm for 10-15 minutes to obtain a black phosphorus nanosheet dispersion. Preferably, the ultrasound frequency is set to 50 kHz. After ultrasound in ice water for 12 h, the obtained brown dispersion is centrifuged at 1000 rpm for 10 minutes.

[0056] In step (2), 10 mg of the amino-polyethylene glycol-modified singlet oxygen / hydroxyl radical ratio fluorescent probe and the polypeptide-polyethylene glycol derivative are dispersed in 10-12.5 mL of black phosphorus nanosheets / H2O solution with a concentration of 200-250 μg / mL. The probe is sonicated for 30-40 minutes and stirred for 4-6 hours. The resulting mixture is then centrifuged in an ultrafiltration concentrator (MWCO 100 kDa; Millipore) at 4-8 °C at a speed of 3500-4000 rpm to remove excess polyethylene glycol. The washing is repeated 2-3 times. The resulting sample is then resuspended in PBS for later use and finally stored at 4-8 °C in the dark. Preferably, 10 mg of amino-polyethylene glycol-modified singlet oxygen / hydroxyl radical ratio fluorescent probe and polypeptide-polyethylene glycol derivative are dispersed in 10 mL of a 200 μg / mL black phosphorus nanosheet / H2O solution. The probe is sonicated for 30 minutes and stirred for 4 hours. The resulting mixture is then centrifuged in an ultrafiltration concentrator (MWCO 100 kDa; Millipore) at 4 °C and 3750 rpm to remove excess polyethylene glycol. The washing is repeated 3 times. The resulting sample is then resuspended in PBS for later use and finally stored at 4 °C in the dark.

[0057] In a specific embodiment, in step (1), the ultrathin black phosphorus nanosheets are prepared by liquid exfoliation of a modified solid black phosphorus sample. 25 mg of purchased black phosphorus blocks are added to 50 mL of ultrapure water, purged with argon gas to eliminate dissolved oxygen molecules, and then sonicated in ice water for 12 hours, using ice water to maintain a relatively low system temperature. The resulting brown dispersion is centrifuged at 1000 rpm for 10 minutes to remove any large, unremoved pieces of black phosphorus, and the supernatant containing the black phosphorus nanosheets is carefully collected.

[0058] In a specific embodiment, in step (2), the polyethylene glycol derivative and the singlet oxygen / hydroxyl radical ratio fluorescent probe are electrostatically adsorbed onto the surface of black phosphorus nanosheets. 10 mg of an amino-modified polyethylene glycol peptide (NH2-PEG) with the ability to penetrate the blood-brain barrier is added. 2000 -ANG), 10 mg of amino-modified polyethylene glycol peptide (NH2-PEG) derivative targeting neural stem cells. 2000 -NFL) and 10 mg singlet oxygen ratio-modified fluorescent probe (SOSG-coumarin-PEG) 2000 -NH2) or 10 mg of hydroxyl radical ratiometric fluorescent probe (HPF-coumarin-PEG) 2000The fluorescent probe (-NH2) was dispersed in 10 mL of black phosphorus nanosheets / H2O solution, with a black phosphorus nanosheet concentration of 200 μg / mL. After sonicating the probe for 30 minutes and stirring for 4 hours, the resulting mixture was washed for 30 minutes at 3750 rpm (4°C) in an Amicon tube (MWCO 100 kDa; Millipore) to remove excess polyethylene glycol molecules, and this washing was repeated twice. The resulting sample was resuspended in PBS for later use and finally stored at 4°C. The fluorescent probe was protected from light.

[0059] This invention also provides a black phosphorus nanosheet analysis platform prepared by the method described above. The black phosphorus nanosheets in the platform have a size of 100-150 nm and a thickness of 1.5-2.1 nm. They possess the ability to specifically generate hydroxyl radicals under ultraviolet light excitation and highly specifically generate singlet oxygen under visible light excitation. The loaded polypeptide polyethylene glycol derivative helps cross the blood-brain barrier and target neural stem cells in in vivo applications. The loaded hydroxyl radical or singlet oxygen fluorescent probes help monitor the concentration changes of these two reactive oxygen species during stem cell differentiation induction; and / or,

[0060] The black phosphorus nanosheet analysis platform quantitatively analyzes multiple reactive oxygen species generated simultaneously under light control; and / or

[0061] The black phosphorus nanosheet analysis platform can quantitatively detect changes in the concentration levels of local hydroxyl radicals and singlet oxygen, and can control the release of these two reactive oxygen species by controlling the light wavelength; and / or,

[0062] The black phosphorus nanosheet analysis platform generates singlet oxygen / hydroxyl radicals under stimulation with different wavelengths of light (405nm / 560nm).

[0063] This invention also provides a method for inducing neural stem cells to differentiate into neurons using the black phosphorus nanosheet analysis platform described above. The method includes isolating neural stem cells from the subgranular region of the dentate gyrus of an adult rat hippocampus, centrifuging them, dispersing them in a neural stem cell culture medium to obtain a cell suspension, then seeding them in a glass-bottomed culture dish, adding the prepared black phosphorus nanosheet analysis platform, and culturing in vitro with or without applying wavelengths of 405 nm or 560 nm and a power of 20 mW / cm². -2 Light stimulation induces neural stem cells to differentiate into neurons.

[0064] Specifically, the dispersion system of the black phosphorus nanosheet analysis platform is a fresh culture medium with a concentration of 5-50 μg / mL; preferably, it is 50 μg / mL.

[0065] Specifically, the inoculation volume of the cell suspension is 1-2 mL, containing (1-5) × 10⁻⁶ cells. 6The cells are cultured in vitro at 36.5-37.5℃ under saturated humidity conditions containing 4-6% CO2 for 6-10 days; preferably, the inoculation volume of the cell suspension is 1 mL, containing 5 × 10⁻⁶ cells. 6 Individual samples were cultured in vitro for 6 days at 37°C in a saturated humidity environment containing 5% CO2.

[0066] This invention also provides an application of the black phosphorus nanosheet analysis platform described above in stimulating the proliferation and differentiation of neural stem cells in vitro. The application includes the quantitative analysis of multiple reactive oxygen species by the black phosphorus nanosheet analysis platform, the simultaneous generation of reactive oxygen species under light control, and the generation of singlet oxygen / hydroxyl radicals under different wavelength light stimulation at 405nm / 560nm to stimulate the proliferation and differentiation of neural stem cells in vitro.

[0067] The present invention also provides an application of the black phosphorus nanosheet analysis platform described above in the in vitro quantitative detection of singlet oxygen / hydroxyl radicals, the application including the quantitative analysis of multiple reactive oxygen species generated simultaneously under light control by the black phosphorus nanosheet analysis platform.

[0068] This invention also provides an application of the black phosphorus nanoanalysis platform described above in stimulating the proliferation and differentiation of neural stem cells in a live mouse brain. The black phosphorus nanoanalysis platform generates singlet oxygen / hydroxyl radicals under stimulation with different wavelengths of light (405nm / 560nm) to stimulate the proliferation and differentiation of neural stem cells in a live mouse brain.

[0069] Specifically, the application utilizes glioma-induced model mice for stimulation therapy. The treatment includes intravenous injection via the tail vein of a black phosphorus nanosheet analysis platform carrying a blood-brain barrier-penetrating polypeptide polyethylene glycol derivative and a singlet oxygen / hydroxyl radical ratio fluorescent probe. All dose equivalents are 40-60 μg BP equivalents / kg. 10-14 hours post-injection, a head-implanted fiber optic array is activated to induce neural stem cell differentiation through light exposure. The induction parameters are 405 nm / 560 nm and 20 mW cm⁻¹. -2 30 min; preferably, all dose equivalents are 50 μg BP equivalent / kg, 12 h after injection.

[0070] In this invention, the "black phosphorus nanoanalysis platform" specifically refers to a platform modified with NH2-PEG. 2000 -ANG, NH2-PEG 2000 -NFL, and SOSG-coumarin-PEG 2000 -NH2 or HPF-coumarin-PEG 2000 -NH2 can be used to quantitatively detect singlet oxygen or hydroxyl radicals and generate two corresponding ROS, targeting neural stem cells in the brain.

[0071] Specifically, a method for in vitro induction of differentiation of neural stem cells isolated from the SGZ region of mice into neurons using a black phosphorus nanoplatform that releases singlet oxygen / hydroxyl radicals through light stimulation and quantitatively analyzes these two reactive oxygen species includes the following steps:

[0072] (1) Primary neural stem cells were isolated from the SGZ region of adult rat brain and passaged for subsequent experiments.

[0073] (2) After centrifuging the above-isolated and cultured neural stem cells, disperse them in culture medium to obtain a cell suspension, then inoculate them in a cell culture dish, add the black phosphorus nano-analysis platform prepared above, and incubate for 2 hours to allow the black phosphorus nanosheet analysis platform to be internalized and enter the cells.

[0074] (3) The cells after the above incubation were subjected to 405nm / 560nm (20mW cm⁻¹) treatment. -2 The neural stem cells were stimulated with light for 10 minutes and cultured in vitro for 6 days to induce differentiation into neurons.

[0075] (4) Differentiation and identification of neural stem cells. To investigate the effect on neuronal differentiation, neural stem cells were co-cultured with the black phosphorus nanosheet dispersion prepared above in a culture medium under different light conditions. Cell proliferation was detected by BrdU method, the types of differentiated cells were identified by immunofluorescence staining, and the number and proportion of different cell types after differentiation were analyzed by flow cytometry.

[0076] (5) The dendritic length and branch number of differentiated neurons were identified to assess the neuronal morphology. Data are presented as mean dendritic number / MAP2 positive cells and mean branch length / MAP2 positive cells. Dendritic counting and measurement were performed using ImageJ.

[0077] (6) Assess the electrical signal transmission function of neurons in different treatment groups and their connections with other synapses. Evaluate the degree of differentiation of neurons obtained from induced stem cells from the perspective of electrophysiological function and synaptic connections with other neurons.

[0078] (7) The black phosphorus nanoanalysis platform with ratiometric fluorescent probes loaded by the hybrid ultrasonic method was used to quantitatively detect the changes in the concentration levels of local hydroxyl radicals and singlet oxygen.

[0079] In step (1), the isolation method for neural stem cells includes obtaining the brain and periventricular region from adult mice via microscopic dissection, tissue preparation, and culture in a serum-free culture system. First, the harvested tissue was chemically digested using trypsin-EDTA, followed by mechanical dissociation to obtain a single-cell suspension. After antibody labeling and flow cytometry sorting, primary neural stem cells were obtained and cultured in 12-well plates. The resulting primary neural cells were passaged to reach the cell volume required for future experiments.

[0080] In step (2), the inoculation volume of the cell suspension is 1 mL, containing 5 × 10⁶ cells. 6 Cell culture dishes were pre-coated with poly-L-lysine and cultured in vitro for 12 hours at 37°C in a saturated, humid environment containing 5% CO2. After the cells adhered to the matrix, a 50 μg / mL black phosphorus nanosheet analysis platform dispersion prepared according to the above method was added to the cell culture medium. The dispersion system was fresh culture medium.

[0081] In step (3), after incubating the prepared black phosphorus nanosheets with the cells for 12 hours, the cells were washed three times to remove excess particles. Fresh culture medium was added, and the cells were treated with or without a wavelength of 405 nm or 560 nm and a power of 20 mW / cm². -2 The light stimulation, with an irradiation time of 10 minutes, specifically generated hydroxyl radicals or singlet oxygen, and this process lasted for 6 days, inducing neural stem cells to differentiate into neurons.

[0082] In step (4), cell proliferation was first detected using the BrdU method. Cells stimulated with light were added to a final concentration of 30 μM BrdU and incubated for 2 h. Cells were then fixed and permeabilized using 4% paraformaldehyde for 20 min and 0.2% Triton X-100 for 5 min. Cells were washed three times with PBS and the supernatant was removed. Cells were then treated with 0.5 ml of 0.1 M sodium tetraborate for 2 min. Cells were washed once with PBS / 1% BSA and the supernatant was removed. Cells were then incubated with a 1:500 diluted mouse anti-BrdU monoclonal antibody at room temperature in the dark for 2 h. Cells were washed once with PBS / 1% BSA and the supernatant was removed. Cells were then incubated with a 1:200 diluted goat anti-mouse secondary antibody-FITC at room temperature in the dark for 30 min. The supernatant was removed, and cells were resuspended in 0.5 ml PBS containing 20 μg / ml propidium iodide (PI stock solution). Cells were incubated at room temperature in the dark for half an hour and observed under a confocal fluorescence microscope.

[0083] Next, immunofluorescence staining was used to identify the differentiated cell types. Differentiated cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.2% Triton X-100 for 5 min, and then blocked with 10% (v / v) goat serum (PBS) at room temperature for 1 h or overnight at 4°C. Cells were then washed three times with PBS and incubated with primary antibody overnight at 4°C. After incubation with primary antibody, cells were washed three times with PBS (3 min each time), then incubated with the corresponding secondary antibody diluted 1:200 for 2 hours, washed with PBS for 5 min, and nuclei were analyzed using DAPI (0.5 μg / mL). Cells were observed under a confocal fluorescence microscope. Primary antibodies: mouse anti-Tuj 1 monoclonal antibody, rabbit anti-GFAP polyclonal antibody; Secondary antibodies: Alexa Fluor 488-labeled goat anti-rabbit IgG, Cy3-labeled goat anti-mouse IgG, Cy5-labeled goat anti-mouse IgG.

[0084] Finally, flow cytometry was used to analyze the number and proportion of different cell types after differentiation. To determine the proportion of differentiated neurons and astrocytes under different conditions, 1×10⁶ cells were harvested from each experimental group. 6 Cells were fixed, permeabilized, and blocked. They were then incubated with a 1:500 primary antibody for 2 hours, followed by incubation with a 1:200 secondary antibody for 2 hours. Cells were analyzed using a BDFACSAria flow cytometer, and data were analyzed using FlowJo. Primary antibodies: mouse anti-Tuj 1 monoclonal antibody; rabbit anti-GFAP polyclonal antibody; Secondary antibodies: Alexa Fluor 488-labeled goat anti-rabbit IgG and Cy3-labeled goat anti-mouse IgG. Alexa Fluor 488-labeled IgG and Cy3-labeled IgG were used as isotype controls to exclude false positives.

[0085] In step (5), as previously described, neural stem cells treated under different differentiation conditions were immunostained with MAP2 and DAPI. The number and length of dendrites for each MAP2-positive cell were obtained using ImageJ. At least 5 images were analyzed for each condition in each independent experiment, for a total of 245 images.

[0086] In step (6), to assess the autoconnectivity of individual cells, action potentials were recorded using current clamp. The pH was adjusted to 7.2. The membrane potential was maintained at approximately -65 to -70 mV, and a step current was injected to induce action potentials. Whole-cell currents were recorded at a hold potential of -70 mV. To demonstrate signal transduction between two cells, synaptic connectivity was studied using paired recording in voltage clamp mode. One neuron was stimulated with low-frequency (0.05–0.1 Hz) polarization via 1 ms steps of -70 mV to 0 mV, and the response of the other neuron was recorded.

[0087] In step (7), to obtain optimal differentiation conditions, the black phosphorus nanoparticle analysis platform loaded with the ratiometric fluorescent probe was used to quantitatively detect singlet oxygen / hydroxyl radicals under different differentiation conditions of neural stem cells. The specific detection steps were as follows: First, the spectral properties of the ratiometric singlet oxygen / hydroxyl radical fluorescent probe and its response to singlet oxygen / hydroxyl radicals were determined. Under simulated physiological conditions, the black phosphorus nanoparticle analysis platform was investigated for different concentrations of singlet oxygen / hydroxyl radicals. 1 The response of O2 and ·OH. A fluorescence spectrophotometer recorded the intensity ratio of fluorescence at 550 nm to 445 nm (I0.05). 550nm / I 445nm )Draw pairs 1 The linear response of O2 and the intensity ratio of fluorescence at 525 nm to 456 nm (I 525nm / I 456nm A linear response to ·OH was plotted. Then, to determine the relationship between the probe monitoring different levels of reactive oxygen species production and neural stem cell differentiation at the cellular level, neural stem cells were co-incubated with a black phosphorus nanoparticle analysis platform. This invention uses a 20mW cm⁻¹ power source. -2 The cells were irradiated with light of wavelengths of 405 nm and 560 nm for different durations. After completing the flow cytometry identification of the differentiated cell types and proportions in step (4) above, the generated ·OH or ·OH was recorded using the constructed ratiometric fluorescent probes. 1 The amount of O2 produced.

[0088] Specifically, a method for stimulating neural stem cell proliferation and differentiation to compensate for glioma damage in a live mouse brain using singlet oxygen / hydroxyl radicals generated by a black phosphorus nanoplatform under different wavelengths of light (405nm / 560nm) through photostimulation to release singlet oxygen and hydroxyl radicals and quantitatively analyze these two reactive oxygen species includes the following steps:

[0089] (1) Modeling glioma-bearing mice: The above-mentioned black phosphorus nanosheet platform was injected into the tail vein, and differentiation was induced by light using a fiber optic microarray (405nm / 560nm, 20mW cm⁻¹). -2 ).

[0090] (2) Immunofluorescence staining analysis of mouse brain slices after light stimulation under different wavelength light stimulation conditions, including cell proliferation marker BrdU, neurogenesis marker NeuN, and new neuron marker Dcx, to demonstrate the proliferation and differentiation of neural stem cells under light stimulation.

[0091] (3) The virus transfection method was used to label endogenous neural stem cells and demonstrate that the progeny neurons formed by proliferation and differentiation migrate to the tumor damage area.

[0092] (4) The black phosphorus nano-analysis platform with load ratio fluorescent probes was combined with fiber optic microarray to analyze the changes in brain regions and detect the local reactive oxygen species generated in the area where neural stem cells are located, which reached the threshold for neural repair.

[0093] (5) Verify the recovery of damaged neural connections in mice after treatment from behavioral and electrophysiological perspectives.

[0094] In step (1), the tumor size grows to 6-9 mm. 3 Following this, neural stem cell stimulation therapy was initiated. The treatment included a tail vein injection of a black phosphorus nanosheet analysis platform loaded with a functionalized polyethylene glycol derivative and a singlet oxygen / hydroxyl radical ratio fluorescent probe. All dose equivalents were 50 μg BP equivalents / kg. Twelve hours post-injection, a fiber optic microarray implanted in the head was activated to induce neural stem cell differentiation using light (405nm / 560nm / mixed wavelengths, 20mW cm⁻¹). -2 The photostimulation therapy is performed for 10 minutes at 10-minute intervals, for a total of 3 hours per day, with a total treatment cycle of 20 days. The fiber array consists of 12 fibers per group, each fiber being a 100μm core / 25μm cladding multimode fiber.

[0095] In step (2), mice in different treatment groups (n=5) were anesthetized with isoflurane gas at a concentration of 5 mg / mL. -1 The continuous anesthetic concentration is 2 mg / mL. -1 Simultaneously, the heart was perfused with PBS containing 4% (w / v) paraformaldehyde for fixation. The mouse whole brain was removed and stored in 4% paraformaldehyde fixative overnight at 4°C. The brain tissue was then incubated with 30% sucrose solution at 4°C for approximately 72 hours. Coronal sections (30 μm) were prepared using a cryostat microtome. After washing three times with PBS, the sections were blocked for 1 hour with PBST containing 2% goat serum (PBS + 0.3% Triton X-100), incubated overnight with primary antibody (1:1000) at 4°C, washed three times with PBST (5 minutes each time), and incubated with secondary antibody (1:500). The sections were incubated at room temperature for 2 hours. The sections were then rinsed with PBST. Confocal fluorescence microscopy images were obtained under a confocal fluorescence microscope (LEICA TCS SP8). Primary antibodies: mouse anti-BrdU monoclonal antibody, rabbit anti-Dcx polyclonal antibody, rabbit anti-NeuN polyclonal antibody, mouse anti-GFAP monoclonal antibody; Secondary antibodies: Alexa Fluor 488-labeled goat anti-rabbit IgG, Cy3-labeled goat anti-mouse IgG.

[0096] In step (3), a fluorescent viral tool, rAAV4-CMV-EGFP-pA, was used to track neural stem cells in the SGZ brain region of the treated mice, so that the progeny neurons obtained from proliferation and differentiation were labeled with EGFP green fluorescence. Virus injection method: Mice were anesthetized with isoflurane gas (ABS small animal gas anesthesia machine). The isoflurane induction concentration was 5 mg mL⁻¹, and the sustained anesthesia concentration was 2 mg mL⁻¹. During the operation, the mice were wrapped in a heating pad to maintain their body temperature at 37°C. Adult mice were anesthetized and mounted on a stereotactic device (RWD Life Sciences). Erythromycin ointment was used to protect the eyes. The head hair was shaved, the incision site was disinfected with povidone-iodine, and the skull above the dentate gyrus in the target area (stereotactic coordinates: AP-2.0 mm, ML+ / -1.45 mm) was drilled open. Inject 400-500 nL of virus, delivering it to the brain at a rate of 30 nL / min using stereotactic coordinates: the subgranular zone of the dentate gyrus in the hippocampus (SGZ, AP-2.0 mm, ML+ / -1.45 mm, DV2.0 mm). After injection, leave the pipette in place for a few minutes, then slowly remove it and use chemical sutures (3M). TM Vetbond TM The incision was closed with tissue adhesive, the scalp was sutured back, and the animals were monitored postoperatively until fully awake. Three weeks after viral injection, the mice were sacrificed for tracking imaging.

[0097] In step (4), probes were used to monitor the relationship between different reactive oxygen species (ROS) production levels and stem cell differentiation at the in vivo level. A fiber optic microarray combined with the probes was used to monitor the relationship at the in vivo level. 1 The generation of O2 or ·OH was investigated. To record fluorescence signals in the brains of tumor-bearing mice, a single 100μm core / 25μm cladding multimode optical fiber was implanted into the glioma region using a stereotaxic instrument, establishing a fluorescence fiber optic photometry system. Excitation light was generated by a fiber laser and transmitted through the fiber to the subgranular region of the hippocampal dentate gyrus, where neural stem cells reside. Different reactive oxygen species were generated by adjusting the wavelength of the therapeutic probe. Simultaneously, the probe at the other end of the fiber in the mouse brain was excited. The fluorescence signal from the probe was then acquired using the same fiber, and detected by a fluorescence spectrometer. To improve the radiation range, the fiber end was tapered using a laser drawing machine to optimize signal acquisition efficiency; the optimized fiber taper angle was 3.5°.

[0098] In step (5), after initiating photostimulation therapy for stem cell differentiation, glioma-bearing mice began the Morris water maze experiment. The maze diameter was 1.3 m, and the water in the maze was changed daily, maintaining a water temperature of 21 ± 2 °C. During training, a platform (approximately 6 cm in diameter) was placed at a fixed location 0.5 cm below the water surface. The trained mice were randomly placed at different locations on the edge of the maze. This training was conducted once a day for 5 days. If a mouse found the platform within 60 seconds, it could rest on the platform for 15 seconds. During the testing period, 24 hours after the last training session, the mice were tested without the platform to assess their memory retention. The trained mice were allowed to swim freely for 120 seconds from a starting point away from the platform, and the number of times each mouse traversed the original platform area and the time spent searching for the platform in each quadrant of the maze were recorded.

[0099] After the water maze test, mice in different treatment groups were fixed in a stereotaxic apparatus, and electrophysiological electrodes (30 μm) were implanted in the striatum region where the glioma was located (AP+1.0 mm, ML 2.0 mm, DV 3.0 mm) to record changes in local field potential. The data were processed using NEX 5.0 software.

[0100] The beneficial effects of this invention are as follows: This invention relates to a method for inducing the differentiation and proliferation of orthotopic neural stem cells in adult mice and repairing neural damage in gliomas, which can be used in vitro and / or in vivo to induce neural stem cells to differentiate into neurons. This invention utilizes black phosphorus, a nanomaterial capable of generating different types of reactive oxygen species (ROS) under different wavelengths of excitation, to construct a platform capable of controllable release of ROS and quantitative analysis of ROS concentration. The black phosphorus nanoplatform prepared by this invention crosses the blood-brain barrier after intravenous injection, generating different types of ROS under different wavelengths of light stimulation. The concentration of these ROS is quantitatively analyzed using a loaded fluorescent probe, enabling precise control of the generation of hydroxyl radicals and singlet oxygen. This achieves optimal ROS treatment conditions that maximize the differentiation of neural stem cells into neurons, continuously and effectively promoting the differentiation of neural stem cells into neurons in both time and space dimensions. This provides a new method for the repair and treatment of neural damage and offers new therapeutic ideas for clinical applications. Attached Figure Description

[0101] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0102] Figure 1Characterization of the modified black phosphorus nanosheet analytical platform. (a) Schematic diagram of the modified elements on the black phosphorus nanosheets. (b) AFM characterization of the thickness and size of the black phosphorus nanosheet platform before and after modification. (c) Raman spectroscopy showing shifts in the characteristic peaks of the black phosphorus nanosheets before and after modification. (d) Zeta potential detection of the change in surface potential of the black phosphorus nanosheet analytical platform before and after modification. (e) Infrared spectroscopy confirming the successful attachment of a polyethylene glycol derivative targeting neural stem cells that penetrates the blood-brain barrier and two reactive oxygen species probes to the black phosphorus nanosheets.

[0103] Figure 2 The black phosphorus nanosheet analysis platform generates different concentrations of ·OH or ·OH under light irradiation. 1 The effects of O2 on the viability and proliferation of neural stem cells. (a) Different concentrations 1 (b) Changes in the viability of neural stem cells under O2 treatment. (c) Changes in the viability of neural stem cells under different concentrations of OH treatment. 1 The proliferation of neural stem cells under O2 treatment. (d) The proliferation of neural stem cells under different concentrations of ·OH treatment was determined by the EdU method.

[0104] Figure 3 This describes the relationship between the black phosphorus nanosheet analysis platform and neural stem cell differentiation under different concentrations of reactive oxygen species generated by photoexcitation. (a) Differentiation of neural stem cells into neurons or glial cells under different concentrations of ·OH treatment. (b) Different concentrations of ·OH treatment... 1 (c) The differentiation of neural stem cells into neurons or glial cells under O2 treatment was analyzed using ImageJ software to determine the proportion of neural stem cells differentiating into neurons or glial cells, with mean ± SD, n = 10. (d) Different concentrations of O2 treatment... 1 The proportion of neural stem cells differentiating into neurons or glial cells under O2 treatment, mean ± SD, n = 10.

[0105] Figure 4 Flow cytometry is used to analyze the number and proportion of different cell types after differentiation, and immunofluorescence staining is used to assess the number and length of dendrites in differentiated neurons. Flow cytometry is also used to analyze different concentrations of ·OH(a) or 1 O2(b) The proportion of neural stem cells differentiating into neurons or glial cells under O2 treatment. (c, d, e, f) The average number and average length of dendrites of different MAP2-positive cells were obtained using ImageJ software.

[0106] Figure 5This study describes the in vivo application of black phosphorus nanosheet analysis platform-induced neural stem cell differentiation for the treatment of glioma. (a) Schematic diagram of the treatment process. (b) Immunofluorescence staining of brain slices after light stimulation of mouse brains under optimal differentiation conditions, showing neural stem cell marker MAP2 (green) and glial cell GFAP (red). (c) Proliferation of new neurons in the hippocampal SGZ region under optimal differentiation conditions compared to the control group. Co-staining of cell proliferation marker BrdU (green) and new neuron marker Dcx (red). (d) Neuralization in the striatum region under optimal differentiation conditions compared to the control and tumor growth groups. Co-staining of cell proliferation marker BrdU (green) and neurogenesis marker NeuN.

[0107] Figure 6 Viral transfection was used to label endogenous neural stem cells and demonstrate the migration of progeny neurons. (a) Schematic diagram of the migration process of labeled neural stem cells in the SGZ region of the hippocampus. (b) After viral implantation, fluorescence imaging was performed on continuous brain slices (300 μm each) spanning the hippocampus to the striatum using confocal microscopy to track the proliferation and differentiation of neural stem cells and the migration of differentiated cells.

[0108] Figure 7 Behavioral and electrophysiological studies validated the recovery of damaged neural connections in mice after treatment. (a) Swimming trajectories of mice in different treatment groups in a water maze with a platform, and heatmaps showing the time spent in different quadrants of the water maze. (b) In vivo electrophysiological studies of neural activity in the striatum of mice under different treatments. Detailed Implementation

[0109] The present invention will be further described in detail below with reference to the specific embodiments and accompanying drawings. Except for the contents specifically mentioned below, the processes, conditions, and experimental methods for implementing the present invention are all common knowledge and general knowledge in the art, and the present invention does not have any particular limitations.

[0110] The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the invention or its application or use. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention.

[0111] In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limitations. Therefore, other examples of exemplary embodiments may have different values.

[0112] This invention discloses a method for inducing the differentiation and proliferation of orthotopic neural stem cells in adult mice and repairing neural damage in gliomas, which can be used in vitro and / or in vivo to induce neural stem cells to differentiate into neurons. This invention utilizes black phosphorus, a nanomaterial capable of generating different types of reactive oxygen species (ROS) under different wavelength excitations, to construct a black phosphorus nanosheet analysis platform capable of controlled release of ROS and quantitative analysis of ROS concentration. The preparation process is as follows: black phosphorus nanosheets with a size of 100-150 nm were prepared using an improved liquid-phase exfoliation technique; ratiometric fluorescent probes were electrostatically adsorbed onto the black phosphorus nanosheets to achieve quantitative analysis of the two released ROS (singlet oxygen and hydroxyl radicals); a polypeptide polyethylene glycol derivative, which helps the nanomaterial cross the blood-brain barrier and target neural stem cells, was synthesized and similarly modified onto the black phosphorus nanosheets via electrostatic adsorption. The black phosphorus nanosheet analysis platform prepared in this invention crosses the blood-brain barrier after intravenous injection and generates different types of reactive oxygen species (ROS) under different wavelength light stimulation. The concentration of these ROS is quantitatively analyzed by the loaded ROS fluorescent probe, enabling precise control of the generation of hydroxyl radicals and singlet oxygen. This achieves optimal ROS treatment conditions that maximize the differentiation of neural stem cells into neurons, continuously and effectively promoting the differentiation of neural stem cells into neurons in both time and space. This provides a new method for the repair and treatment of nerve damage and offers new therapeutic ideas for clinical applications.

[0113] Unless otherwise specified, the experimental materials used in the examples are all conventional biochemical reagents.

[0114] Example 1

[0115] Preparation of a ratiometric fluorescent probe for quantitative analysis of singlet oxygen and hydroxyl radicals in this invention:

[0116] 7-Hydroxy-2-oxo-2H-chromene-3-carboxylic acid (1 mmol) was reacted with a polyethylene glycol derivative (Fmoc-NH-PEG). 2000 -NH2 (1 mmol), TSTU (1 mmol), and DIPEA (2 mmol) were dissolved in 10 mL of DMSO and stirred at 25 °C for 30 min. The mixture was then purified by semi-preparative high-performance liquid chromatography to obtain a coumarin-polyethylene glycol derivative (coumarin-PEG). 2000 -NH-Fmoc), yield 83%. Coumarin-polyethylene glycol derivative (coumarin-PEG) was used. 2000Boc-protected coumarin-polyethylene glycol derivative (Boc-coumarin-PEG) was obtained by mixing 1 mmol of N-(3-bromopropyl)carbamate (1 mmol) and K2CO3 (1.3 mmol) in 15 mL of DMF and stirring at 60 °C for 24 h. The mixture was then purified by semi-preparative high-performance liquid chromatography. 2000 -NH-Fmoc), yield 72%. The resulting Boc-protected coumarin-polyethylene glycol derivative (Boc-coumarin-PEG) was obtained. 2000 -NH-Fmoc (1 mmol) was reacted with stirring in 25% TFA / DCM, washed with 5% NaHCO3 and saturated brine, and purified by semi-preparative high performance liquid chromatography to obtain the propylamino-coumarin-polyethylene glycol derivative (NH2-coumarin-PEG). 2000 -NH-Fmoc), with a yield of 65%. Propylamino-coumarin-polyethylene glycol derivative (NH2-coumarin-PEG) 2000 SOSG (1 mmol), SOSG (1 mmol) or HPF (1 mmol), TSTU (1 mmol), and DIPEA (2 mmol) were mixed in 8 mL of DMSO and stirred at 25 °C for 30 min. The mixture was then purified by semi-preparative high-performance liquid chromatography to obtain the SOSG-coumarin-PEG derivative. 2000 -NH-Fmoc), with a yield of 54%; HPF-coumarin-polyethylene glycol derivative (HPF-coumarin-PEG) 2000 -NH-Fmoc), with a yield of 62%. The above-obtained Fmoc-protected SOSG-coumarin-polyethylene glycol derivative (SOSG-coumarin-PEG) 2000 -NH-Fmoc) and Fmoc-protected HPF-coumarin-polyethylene glycol derivatives (HPF-coumarin-PEG) 2000 SOSG-coumarin-PEG derivatives (HPF-coumarin-PEG) were obtained by reacting 25% Piperidine / DMF solution with stirring at 25°C for 30 min, followed by vacuum removal of DMF and purification by semi-preparative high-performance liquid chromatography. 2000 -NH2), with a yield of 68%; HPF-coumarin-polyethylene glycol derivative (HPF-coumarin-PEG) 2000 -NH2), with a yield of 66%.

[0117] This invention involves dissolving 1 mmol of either the polypeptide NFL (SEQ ID NO. 1) or the polypeptide ANG (SEQ ID NO. 1) obtained by standard solid-phase polypeptide synthesis in 10 mL of PBS buffer containing 50 mM TCEP, and then reacting it with amino-polyethylene glycol maleimide (NH2-PEG) dissolved in PBS. 2000 -Maleimide (5 mmol) was added, and TCEP was added as a reducing agent and catalyst. The mixture was stirred at 25 °C for 5 h, and purified by semi-preparative high performance liquid chromatography to obtain amino-polyethylene glycol modified peptide NFL (NH2-PEG). 2000 -NFL) or ANG(NH2-PEG) 2000 -ANG), with a yield of 71%.

[0118] This invention prepares a dispersion of black phosphorus nanosheets with an analytical platform capable of penetrating the blood-brain barrier and quantitatively analyzing hydroxyl radicals / singlet oxygen. The ultrathin black phosphorus nanosheets are prepared by liquid exfoliation of a modified solid black phosphorus sample. Black phosphorus bulk material is dispersed in ultrapure water. 25 mg of purchased black phosphorus is added to 50 ml of ultrapure water, and argon gas is introduced to eliminate dissolved oxygen molecules. The mixture is then sonicated in ice water for 12 hours, maintaining a relatively low system temperature using ice water. The resulting brown dispersion is centrifuged at 1000 rpm for 10 minutes to remove any remaining large pieces of black phosphorus, and the supernatant containing the black phosphorus nanosheets is carefully collected. Then, a singlet oxygen / hydroxyl radical ratio fluorescent probe and a polyethylene glycol (PEG) peptide targeting neural stem cells are electrostatically adsorbed onto the surface of the black phosphorus nanosheets using a mixed sonication method. 10 mg of an amino-modified PEG peptide (NH2-PEG) with the ability to penetrate the blood-brain barrier is added. 2000 -ANG), 10 mg of amino-modified polyethylene glycol peptide (NH2-PEG) derivative targeting neural stem cells. 2000 -NFL) and 10 mg singlet oxygen ratio-modified fluorescent probe (SOSG-coumarin-PEG) 2000 -NH2) or 10 mg of hydroxyl radical ratiometric fluorescent probe (HPF-coumarin-PEG) 2000 The fluorescent probe (-NH2) was dispersed in 10 mL of black phosphorus nanosheets / H2O solution, with a black phosphorus nanosheet concentration of 200 μg / mL. After sonicating the probe for 30 minutes and stirring for 4 hours, the resulting mixture was washed for 30 minutes at 3750 rpm (4°C) in an Amicon tube (MWCO 100 kDa; Millipore) to remove excess polyethylene glycol molecules, and this washing was repeated twice. The resulting sample was resuspended in PBS for later use and finally stored at 4°C. The fluorescent probe was protected from light.

[0119] Figure 1 Characterization of the modified black phosphorus nanosheet analysis platform. (a) Schematic diagram of the modified elements on the black phosphorus nanosheet analysis platform. (b) AFM characterization of the thickness and size of the black phosphorus nanosheet analysis platform before and after modification. (c) Raman spectroscopy showing shifts in the characteristic peaks of the black phosphorus nanosheet analysis platform before and after modification. (d) Zeta potential detection of the change in surface potential of the black phosphorus nanosheet analysis platform before and after modification. (e) Infrared spectroscopy confirming the successful connection of a blood-brain barrier-penetrating, neural stem cell-targeting polyethylene glycol derivative and a singlet oxygen / hydroxyl radical ratio fluorescent probe to the black phosphorus nanosheet analysis platform.

[0120] Example 2

[0121] This invention utilizes the black phosphorus nanosheet analysis platform prepared in Example 1 of this invention to evaluate the proliferative capacity of neural stem cells isolated from the SGZ region of adult mice under different ROS and concentrations in vitro. Primary neural stem cells were isolated from the SGZ region of adult mouse brains: the brain and periventricular region were obtained from adult mice through microscopic dissection, tissue preparation, and passage in a serum-free culture system for subsequent experiments. The isolated and cultured neural stem cells were centrifuged, dispersed in culture medium to obtain a cell suspension, and then seeded in cell culture dishes. The prepared black phosphorus nanosheet analysis platform was added, and incubated for 2 hours to allow the black phosphorus nanosheet analysis platform to internalize into the cells. The incubated cells were then subjected to 405nm / 560nm (20mW cm⁻¹) assays. -2 Neural stem cells were stimulated with light for 10 minutes and cultured in vitro for 6 days to induce proliferation. The viability of neural stem cells was assessed using the CCK8 assay, and cell proliferation was detected using the BrdU assay.

[0122] Figure 2 The black phosphorus nano-analysis platform generates different concentrations of ·OH or ·OH under light irradiation. 1 The effects of O2 on the viability and proliferation of neural stem cells. (a) Different concentrations 1 (b) Changes in the viability of neural stem cells under O2 treatment. (c) Changes in the viability of neural stem cells under different concentrations of OH treatment. 1 The proliferation of neural stem cells under O2 treatment. (d) The proliferation of neural stem cells under different concentrations of ·OH treatment was determined by the EdU method.

[0123] Example 3

[0124] This invention utilizes immunofluorescence staining to identify the types of differentiated cells and to determine the optimal types and concentrations of reactive oxygen species (ROS) applied at the cellular level to achieve the best neural stem cell differentiation effect. Differentiated cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.2% Triton X-100 for 5 min, and then blocked with 10% (v / v) goat serum (PBS) at room temperature for 1 h or overnight at 4°C. Cells were then washed three times with PBS and incubated overnight with primary antibody at 4°C. After incubation with primary antibody, cells were washed three times with PBS (3 min each time), then incubated with the corresponding secondary antibody diluted 1:200 for 2 hours, washed with PBS for 5 min, and nuclei were analyzed using DAPI (0.5 μg / mL). Cells were observed under a confocal fluorescence microscope. Primary antibodies: mouse anti-Tuj 1 monoclonal antibody, rabbit anti-GFAP polyclonal antibody; Secondary antibodies: Alexa Fluor 488-labeled goat anti-rabbit IgG, Cy3-labeled goat anti-mouse IgG, Cy5-labeled goat anti-mouse IgG.

[0125] Figure 3 This invention relates the black phosphorus nanosheet analysis platform prepared in Example 1 to the differentiation of neural stem cells under different concentrations of reactive oxygen species generated by photoexcitation. (a) Differentiation of neural stem cells into neurons or glial cells under different concentrations of ·OH treatment. (b) Different concentrations of ·OH treatment... 1 (c) The differentiation of neural stem cells into neurons or glial cells under O2 treatment was analyzed using ImageJ software to determine the proportion of neural stem cells differentiating into neurons or glial cells, with mean ± SD, n = 10. (d) Different concentrations of O2 treatment... 1 The proportion of neural stem cells differentiating into neurons or glial cells under O2 treatment, mean ± SD, n = 10.

[0126] Example 4

[0127] This invention uses flow cytometry to analyze the number and proportion of different cell types after differentiation, and immunofluorescence staining to assess the number and length of dendrites in differentiated neurons. To determine the ratio of differentiated neurons and astrocytes under different conditions, 1×10⁶ cells were harvested from each experimental group. 6Cells were fixed, permeabilized, and blocked. Cells were incubated with a 1:500 primary antibody for 2 hours, followed by incubation with a 1:200 secondary antibody for 2 hours. Cells were analyzed using a BD FACSAria flow cytometer, and data were analyzed using FlowJo. Primary antibodies: mouse anti-Tuj 1 monoclonal antibody; rabbit anti-GFAP polyclonal antibody; secondary antibodies: Alexa Fluor 488-labeled goat anti-rabbit IgG and Cy3-labeled goat anti-mouse IgG. Alexa Fluor 488-labeled IgG and Cy3-labeled IgG were used as isotype controls to exclude false positives. Neural stem cells treated under different differentiation conditions were immunostained with MAP2 and DAPI. The number and length of dendrites for each MAP2-positive cell were obtained using ImageJ. At least 5 images were analyzed for each condition in each independent experiment, for a total of 245 images.

[0128] Figure 4 Flow cytometry is used to analyze the number and proportion of different cell types after differentiation, and immunofluorescence staining is used to assess the number and length of dendrites in differentiated neurons. Flow cytometry is also used to analyze different concentrations of ·OH(a) or 1 O2(b) The proportion of neural stem cells differentiating into neurons or glial cells under O2 treatment. (c, d, e, f) The average number and average length of dendrites of different MAP2-positive cells were obtained using ImageJ software.

[0129] Example 5

[0130] This invention utilizes a black phosphorus nanosheet analysis platform prepared in Example 1 to stimulate the proliferation and differentiation of neural stem cells in the brain of living mice under optimal differentiation conditions, thus replenishing the damage caused by gliomas. The steps include: establishing a glioma-bearing mouse model. The tumor size grows to 6-9 mm. 3 Neural stem cell stimulation therapy was then initiated. The treatment involved intravenous injection via the tail vein of a black phosphorus nanosheet analysis platform loaded with a functionalized polyethylene glycol derivative and a singlet oxygen / hydroxyl radical ratio fluorescent probe. All dose equivalents were 50 μg BP equivalent / kg. Twelve hours post-injection, a fiber optic microarray implanted in the head was activated to induce neural stem cell differentiation through light stimulation, with irradiation every 10 minutes for 3 hours daily. The entire photostimulation treatment cycle lasted 20 days. After completing the 20-day treatment cycle, immunofluorescence staining analysis was performed on brain slices from the light-stimulated mice, including staining for cell proliferation markers such as BrdU, neurogenesis markers such as NeuN, and neonatal neuron markers such as Dcx, to demonstrate the proliferation and differentiation of neural stem cells under photostimulation.

[0131] Figure 5This study describes the in vivo application of black phosphorus nanosheet analysis platform-induced neural stem cell differentiation for the treatment of glioma. (a) Schematic diagram of the treatment process. (b) Immunofluorescence staining of brain slices after light stimulation of mouse brains under optimal differentiation conditions, showing neural stem cell marker MAP2 (green) and glial cell GFAP (red). (c) Proliferation of new neurons in the hippocampal SGZ region under optimal differentiation conditions compared to the control group. Co-staining of cell proliferation marker BrdU (green) and new neuron marker Dcx (red). (d) Neuralization in the striatum region under optimal differentiation conditions compared to the control and tumor growth groups. Co-staining of cell proliferation marker BrdU (green) and neurogenesis marker NeuN.

[0132] Example 6

[0133] This invention uses viral transfection to label endogenous neural stem cells, demonstrating the migration of progeny neurons from proliferation and differentiation to the tumor-damaged area. A fluorescent viral tool, rAAV4-CMV-EGFP-pA, was used to track neural stem cells in the SGZ brain region of treated mice, labeling the progeny neurons with EGFP green fluorescence. Virus injection method: Mice were anesthetized with isoflurane gas (ABS small animal gas anesthesia machine). The isoflurane induction concentration was 5 mg mL⁻¹, and the sustained anesthesia concentration was 2 mg mL⁻¹. During the procedure, the mice were wrapped in a heating pad to maintain a body temperature of 37°C. Adult mice were anesthetized and mounted on a stereotactic device (RWD Life Sciences). Erythromycin ointment was used to protect the eyes. The head hair was shaved, the incision site was disinfected with povidone-iodine, and the skull above the dentate gyrus in the target area (stereotactic coordinates: AP-2.0 mm, ML+ / -1.45 mm) was drilled open. Inject 400-500 nL of virus, delivering it to the brain at a rate of 30 nL / min using stereotactic coordinates: the subgranular zone of the dentate gyrus in the hippocampus (SGZ, AP-2.0 mm, ML+ / -1.45 mm, DV 2.0 mm). After injection, leave the pipette in place for a few minutes, then slowly remove it and use chemical sutures (3M). TM Vetbond TM The incision was closed with tissue adhesive, the scalp was sutured back, and the animals were monitored postoperatively until fully awake. Three weeks after viral injection, the mice were sacrificed for tracking imaging.

[0134] Figure 6Viral transfection was used to label endogenous neural stem cells and demonstrate the migration of progeny neurons. (a) Schematic diagram of the migration process of labeled neural stem cells in the SGZ region of the hippocampus. (b) After viral implantation, fluorescence imaging was performed on continuous brain slices (300 μm each) spanning the hippocampus to the striatum using confocal microscopy to track the proliferation and differentiation of neural stem cells and the migration of differentiated cells.

[0135] Example 7

[0136] The recovery of damaged neural connections in mice after treatment was verified from behavioral and electrophysiological perspectives. After 20 days of photostimulated stem cell differentiation therapy, glioma-bearing mice began the Morris water maze experiment. The maze diameter was 1.3 m, and the water in the maze was changed daily, maintaining a temperature of 21 ± 2℃. During training, a platform (approximately 6 cm in diameter) was placed at a fixed location 0.5 cm below the water surface. The trained mice were randomly placed at different locations on the edge of the maze. This training was conducted once daily for 5 days. If a mouse found a platform within 60 seconds, it could rest on the platform for 15 seconds. During the testing period, 24 hours after the last training session, the mice were tested without the platform to assess their memory retention. The trained mice were allowed to swim freely for 120 seconds from a starting point away from the platform, and the number of times each mouse traversed the original platform area and the time spent searching for the platform in each quadrant of the maze were recorded.

[0137] After the water maze test, mice in different treatment groups were fixed in a stereotaxic apparatus, and electrophysiological electrodes (30 μm) were implanted in the striatum region where the glioma was located (AP+1.0 mm, ML 2.0 mm, DV 3.0 mm) to record changes in local field potential. The data were processed using NEX 5.0 software.

[0138] Figure 7 Behavioral and electrophysiological studies validated the recovery of damaged neural connections in mice after treatment. (a) Swimming trajectories of mice in different treatment groups in a water maze with a platform, and heatmaps showing the time spent in different quadrants of the water maze. (b) In vivo electrophysiological studies of neural activity in the striatum of mice under different treatments.

[0139] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0140] As used in this invention, the terms "comprising" and "including" are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0141] As used in this invention, the term "and / or" includes any one or more of the related listed items and all combinations thereof.

[0142] The scope of protection of this invention is not limited to the above embodiments. Any variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of this invention are included in this invention and are protected by the appended claims.

Claims

1. A method for preparing a black phosphorus nanosheet analysis platform, characterized in that, The preparation method includes the following steps: Step (1): Disperse black phosphorus blocks in ultrapure water, purge with argon gas to eliminate dissolved oxygen in the water, sonicate the mixed solution in ice water, centrifuge the resulting brown suspension and take the supernatant to obtain ultrathin black phosphorus nanosheets dispersed in ultrapure water. Step (2) involves electrostatically adsorbing a singlet oxygen / hydroxyl radical ratio fluorescent probe and a polypeptide polyethylene glycol derivative that penetrates the blood-brain barrier and targets neural stem cells onto the surface of black phosphorus nanosheets using a mixed ultrasound method; thus preparing the black phosphorus nanoanalysis platform.

2. The preparation method according to claim 1, characterized in that, In step (1), the dispersion system of the black phosphorus nanosheet dispersion is sterile ultrapure water, and the concentration of the black phosphorus nanosheet dispersion is 200-250 μg / mL. The specific steps of step (1) are as follows: 25 mg of purchased black phosphorus blocks are added to 50 mL of ultrapure water; the frequency of the sonication is set to 30-50 kHz; the sample is kept in ice water during the sonication process; after sonication for 10-14 hours, the resulting brown dispersion is centrifuged at 1000-1200 rpm for 10-15 minutes to obtain a dispersion of the black phosphorus nanosheets; and / or, In step (2), 10 mg of the amino-polyethylene glycol modified singlet oxygen / hydroxyl radical ratio fluorescent probe and the polypeptide polyethylene glycol derivative are dispersed in 10-12.5 mL of black phosphorus nanosheets / H2O solution with a concentration of 200-250 μg / mL. The probe is sonicated for 30-40 minutes and stirred for 4-6 hours. The resulting mixture is then placed in an ultrafiltration concentration tube and centrifuged at 3500-4000 rpm at 4-8℃ to remove excess polyethylene glycol. The washing is repeated 2-3 times. The obtained samples were resuspended in PBS for later use, and finally stored at 4-8℃ in the dark.

3. The preparation method according to claim 1, characterized in that, In step (2), the preparation of the singlet oxygen / hydroxyl radical ratio fluorescent probe includes the following steps: Step (1) involves reacting 7-hydroxy-3-carboxycoumarin with the polyethylene glycol derivative Fmoc-NH-PEG. 2000 -NH2, 2-succinimide-1,1,3,3-tetramethylurea tetrafluoroborate, and N,N-diisopropylethylamine were mixed and stirred in DMSO to give the coumarin-polyethylene glycol derivative coumarin-PEG. 2000 -NH-Fmoc; Step (2) involves applying the coumarin-polyethylene glycol derivative coumarin-PEG obtained in step (1). 2000 -NH-Fmoc and N-(3-bromopropyl)carbamate tert-butyl ester were dissolved in DMF, and K2CO3 was added and the mixture was stirred to react, yielding the Boc-protected coumarin-polyethylene glycol derivative Boc-coumarin-PEG. 2000 -NH-Fmoc; Step (3) involves taking the Boc-protected coumarin-polyethylene glycol derivative Boc-coumarin-PEG obtained in step (2). 2000 -NH-Fmoc dissolved in TFA / DCM solution reacts to yield the propylamino-coumarin-polyethylene glycol derivative NH2-coumarin-PEG. 2000 -NH-Fmoc; Step (4) involves constructing a singlet oxygen ratio-based fluorescent probe by using the propylamino-coumarin-polyethylene glycol derivative NH2-coumarin-PEG obtained in step (3). 2000 -NH-Fmoc was covalently coupled to the commercial singlet oxygen probe SOSG in DMSO via a catalytic amide reaction of 2-succinimide-1,1,3,3-tetramethylurea tetrafluoroborate and N,N-diisopropylethylamine, thus preparing a FRET-based singlet oxygen ratiometric fluorescent probe precursor: SOSG-coumarin-PEG. 2000 -NH-Fmoc; and / or, To construct a hydroxyl radical ratio-based fluorescent probe, the propylamino-coumarin-polyethylene glycol derivative NH2-coumarin-PEG obtained in step (3) was used. 2000 -NH-Fmoc was covalently coupled to the commercial hydroxyl radical probe HPF in DMSO via an amide reaction catalyzed by 2-succinimide-1,1,3,3-tetramethylurea tetrafluoroborate and N,N-diisopropylethylamine, thus preparing a hydroxyl radical ratiometric fluorescent probe precursor based on the FRET mechanism. This process led to the preparation of the HPF-coumarin-PEG derivative HPF-coumarin. 2000 -NH-Fmoc; Step (5), the SOSG-coumarin-PEG derivative obtained in step (4) 2000 -NH-Fmoc, the HPF-coumarin-polyethylene glycol derivative HPF-coumarin-PEG 2000 -NH-Fmoc, in a Piperidine / DMF solution, was deprotected by Fmoc to yield the SOSG-coumarin-PEG derivative with terminal amino groups. 2000 -NH2, HPF-coumarin-PEG derivative HPF-coumarin-PEG 2000 -NH2.

4. The preparation method according to claim 3, characterized in that, In step (1), the 7-hydroxy-3-carboxycoumarin (7-Hydroxy-2-oxo-2H-chromene-3-carboxylic acid) and the polyethylene glycol derivative (Fmoc-NH-PEG) 2000 The molar ratio of DMSO to 7-hydroxy-2-oxo-2H-chromene-3-carboxylic acid is (1-1.5):(1-1.5):(1-1.5):(2-3); the volume / molar ratio of DMSO to the raw material 7-hydroxy-2-oxo-2H-chromene-3-carboxylic acid is (10-20 mL):(1-2 mmol); the reaction temperature is 20-30℃; the reaction time is 20-40 min. And / or, in step (2), the coumarin-polyethylene glycol derivative (coumarin-PEG) 2000 The molar ratio of DMF to tert-Butyl-3-bromopropylcarbamate (N-(3-bromopropyl)carbamate) and K2CO3 is (1-1.5):(1-1.5):(1.3-2); the volume / molar ratio of the added DMF to the raw material tert-Butyl-3-bromopropylcarbamate is (5-10 mL):(1-2 mmol); the reaction temperature is 50-60℃; the reaction time is 24-48 h. And / or, in step (3), the Boc-protected coumarin-polyethylene glycol derivative (Boc-coumarin-PEG) 2000 The molar / volume ratio of -NH-Fmoc), DCM, and TFA is (1-1.5 mmol):(3-5 mL):(0.5-2 mL); the reaction temperature is 20-30 °C; and the reaction time is 1-2 h. And / or, in step (4), the propylamino-coumarin-polyethylene glycol derivative (NH2-coumarin-PEG) 2000 The molar ratio of DMSO, SOSG or HPF, TSTU, and DIPEA is (1-1.5):(1-1.5):(1-1.5):(2-3); the amount of DMSO added is related to the amount of propylaminocoumarin-polyethylene glycol derivative (NH2-coumarin-PEG). 2000 The volume / molar ratio of -NH-Fmoc (10-20 mL): (1-2 mmol); the reaction temperature is 20-30 °C; the reaction time is 20-40 min; And / or, in step (5), the SOSG-coumarin-polyethylene glycol derivative (SOSG-coumarin-PEG) 2000 -NH-Fmoc) or HPF-coumarin-polyethylene glycol derivative (HPF-coumarin-PEG) 2000 The molar / volume / volume ratio of -NH-Fmoc to added Piperidine:DMF is 0.002-0.004 mmol:1 mL:(3-5) mL; the reaction temperature is 20-30 °C; and the reaction time is 20-40 min.

5. The preparation method according to claim 1, characterized in that, In step (2), the polypeptide polyethylene glycol derivative is NH2-PEG. 2000 -NFL and NH2-PEG 2000 -ANG mixture.

6. A black phosphorus nanosheet analysis platform prepared by the preparation method according to any one of claims 1-5, characterized in that, The black phosphorus nanosheet analysis platform uses black phosphorus nanosheets with a size of 100-150 nm and a thickness of 1.5-2.1 nm. These nanosheets are capable of specifically generating hydroxyl radicals under ultraviolet light excitation and highly specifically generating singlet oxygen under visible light excitation. The loaded polypeptide polyethylene glycol derivatives facilitate crossing the blood-brain barrier and targeting neural stem cells in in vivo applications. The loaded hydroxyl radical or singlet oxygen fluorescent probes help monitor the concentration changes of these two reactive oxygen species during stem cell differentiation induction; and / or, The black phosphorus nanosheet analysis platform quantitatively analyzes multiple reactive oxygen species generated simultaneously under light control; and / or The black phosphorus nanosheet analysis platform can quantitatively detect changes in the concentration levels of local hydroxyl radicals and singlet oxygen, and can control the release of these two reactive oxygen species by controlling the light wavelength; and / or, The black phosphorus nanosheet analysis platform generates singlet oxygen / hydroxyl radicals under stimulation with different wavelengths of light (405nm / 560nm).

7. A method for inducing neural stem cells to differentiate into neurons using the black phosphorus nanosheet analysis platform as described in claim 6, characterized in that, The method includes isolating neural stem cells from the subgranular region of the dentate gyrus of the adult rat hippocampus, centrifuging them, dispersing them in a neural stem cell culture medium to obtain a cell suspension, then seeding them in a glass-bottomed culture dish, adding the prepared black phosphorus nanosheet analysis platform, and culturing them in vitro with or without an applied wavelength of 405 nm or 560 nm and a power of 20 mW cm⁻¹. -2 Light stimulation induces neural stem cells to differentiate into neurons; The dispersion system of the black phosphorus nanosheet analysis platform is a fresh culture medium with a concentration of 5-50 μg / mL; And / or, the inoculation volume of the cell suspension is 1-2 mL, containing (1-5) × 10⁻⁶ cells. 6 The in vitro culture was carried out at 36.5-37.5℃ in a saturated humidity environment containing 4-6% CO2 for 6-10 days.

8. The application of the black phosphorus nanosheet analysis platform as described in claim 6 in stimulating the proliferation and differentiation of neural stem cells in vitro or in the quantitative detection of singlet oxygen / hydroxyl radicals in vitro.

9. An application of the black phosphorus nanoanalysis platform as described in claim 6 in stimulating the proliferation and differentiation of neural stem cells in the live mouse brain, characterized in that... The black phosphorus nanoanalysis platform generates singlet oxygen / hydroxyl radicals under different wavelengths of light (405nm / 560nm), which stimulate the proliferation and differentiation of neural stem cells in the live mouse brain.

10. The application as described in claim 9, characterized in that, The application utilizes glioma-induced model mice for stimulation therapy. The treatment includes a tail vein injection of a black phosphorus nanosheet analysis platform carrying a blood-brain barrier-penetrating polypeptide polyethylene glycol derivative and a singlet oxygen / hydroxyl radical ratio fluorescent probe. All dose equivalents are 50 μg BP equivalents / kg. Twelve hours post-injection, a head-implanted fiber optic array is activated to induce neural stem cell differentiation using light. The induction parameters are 405 nm / 560 nm and 20 mW cm⁻¹. -2 30 minutes.

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