Use of barium titanate-gold composite nanoparticles for promoting proliferation and / or differentiation of neural stem cells

Abstract

The application provides application of barium titanate-gold composite nanoparticles in promoting proliferation and / or differentiation of neural stem cells, and belongs to the technical field of medicines. The barium titanate-gold composite nanoparticles have a core-shell structure, and comprise a barium titanate nano core and a gold shell layer wrapped on the surface of the barium titanate nano core. The barium titanate-gold composite nanoparticles can generate weak current stimulation under near-infrared light stimulation to promote proliferation and differentiation of neural stem cells, and can especially promote differentiation of neural stem cells into mature neurons.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the application of barium titanate-gold composite nanoparticles in promoting the proliferation and / or differentiation of neural stem cells. Background Technology

[0002] Alzheimer's disease (AD) is a common neurodegenerative disease in the elderly. The cognitive function of AD patients gradually declines as the disease progresses. Patients in the late stage often lack the ability to take care of themselves, which brings huge care and economic pressure to patients' families and society. The main pathological feature of AD is the death of a large number of neurons.

[0003] Neural stem cells (NSCs) are cells in the nervous system with self-renewal capacity and multi-lineage differentiation potential (differentiating into neurons, astrocytes, and oligodendrocytes). During embryonic development, the process of NSCs generating neurons is fundamental to human brain development; however, substantial evidence suggests that NSCs and neurons persist throughout the lifespan of mammals, including humans. In the adult brain, two recognized neurogenic regions exist: the subventricular region of the lateral ventricle and the subgranular region of the dentate gyrus of the hippocampus. These regions contain NSCs. Utilizing endogenous neural stem cells to promote the efficient regeneration of new neurons to replace lost neurons in the brain offers new hope for the treatment of neurodegenerative diseases, including Alzheimer's disease (AD), and nerve injuries. Therefore, achieving efficient, targeted, and controllable differentiation of NSCs into target neurons is particularly important for the treatment of neurodegenerative diseases and nerve injuries.

[0004] Currently, inducing NSC differentiation mainly relies on two methods: chemical induction (such as neurotrophic factors and small molecule compounds) and physical stimulation (such as electrical, magnetic, and mechanical stimulation). While chemical induction is widely used, it suffers from poor in vivo stability, insufficient targeting, high cost, and the potential to elicit unintended cellular responses. NSCs, due to their unique electrophysiological characteristics (excitation generated by rapid depolarization of the resting membrane potential, with cell membrane potential regulation occurring during cell migration, proliferation, differentiation, and excitation), are significantly regulated by external electrical stimulation. Therefore, using nanomaterials to mediate electrical stimulation to promote NSC differentiation, migration, and synaptic growth has attracted widespread attention from researchers. Designing and preparing nanomaterials that can effectively mediate radio stimulation to promote NSC differentiation into mature neurons is a pressing technical problem in this field. Summary of the Invention

[0005] The purpose of this invention is to provide the application of barium titanate-gold composite nanoparticles in promoting the proliferation and / or differentiation of neural stem cells. The barium titanate-gold composite nanoparticles of this invention can generate weak current stimulation under near-infrared light stimulation to promote the proliferation and differentiation of neural stem cells, especially to promote the differentiation of neural stem cells into mature neurons.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of barium titanate-gold composite nanoparticles in the preparation of formulations that promote the proliferation and / or differentiation of neural stem cells. The barium titanate-gold composite nanoparticles have a core-shell structure, comprising a barium titanate nanocore and a gold shell layer encapsulating the surface of the barium titanate nanocore.

[0007] Preferably, the differentiation includes differentiating into neurons and promoting neuronal maturation.

[0008] Preferably, the proliferation includes promoting neurosphere growth.

[0009] This invention provides the application of barium titanate-gold composite nanoparticles in the preparation of formulations that promote the generation of reactive oxygen species in neural stem cells. The barium titanate-gold composite nanoparticles have a core-shell structure, including a barium titanate nanocore and a gold shell layer wrapped around the surface of the barium titanate nanocore.

[0010] This invention provides the application of barium titanate-gold composite nanoparticles in the preparation of formulations that promote the increase of calcium ions in neural stem cells. The barium titanate-gold composite nanoparticles have a core-shell structure, including a barium titanate nanocore and a gold shell layer wrapped around the surface of the barium titanate nanocore.

[0011] Preferably, the neural stem cells include neural stem cells cultured in vitro.

[0012] Preferably, the barium titanate-gold composite nanoparticles function under light irradiation at wavelengths of 600-808 nm.

[0013] Preferably, the barium titanate-gold composite nanoparticles have a zeta potential of 5-10 mV, the particle size of the barium titanate-gold composite nanoparticles is 80-100 nm, and the particle size of the barium titanate nanocore is 55-85 nm.

[0014] Preferably, the mass ratio of barium titanate to gold in the barium titanate-gold composite nanoparticles is 0.85~0.95:0.05~0.15.

[0015] Preferably, the surface of the gold shell in the barium titanate-gold composite nanoparticles is further modified with polyethylene glycol, and the content of polyethylene glycol in the barium titanate-gold composite nanoparticles is 7.5~9.5wt%.

[0016] Beneficial Effects: The barium titanate-gold composite nanoparticles of this invention effectively unify photothermal metal gold and pyroelectric barium titanate into a single nanomaterial. Under near-infrared light stimulation, these barium titanate-gold composite nanoparticles can generate a weak current to promote the proliferation and differentiation of neural stem cells, particularly promoting their differentiation into mature neurons. Specifically, the barium titanate-gold composite nanoparticles are a composite material formed by combining pyroelectric nanomaterials and photothermal metals. Under near-infrared light irradiation, the gold shell absorbs near-infrared light to generate heat, and the pyroelectric barium titanate nanomaterials generate active charges with temperature changes. Then, under near-infrared light irradiation (specifically 808nm near-infrared light in this embodiment), a large number of pyroelectric-induced reactive oxygen species are generated, thereby inducing neural stem cell differentiation. The combination of barium titanate and gold can also effectively inhibit electron-hole recombination, thus effectively improving the ability of the barium titanate-gold composite nanoparticles to generate reactive oxygen charges, i.e., to generate a weak current. These currents can stimulate neural stem cells, triggering the activation of calcium ion channels and a cascade of signaling pathways related to intracellular calcium ion influx, thereby affecting the life activities of neural stem cells. In this invention, barium titanate-gold composite nanoparticles can significantly enhance the proliferation and differentiation capacity of neural stem cells and promote their differentiation into neurons. Therefore, this invention utilizes near-infrared irradiation of barium titanate-gold composite nanoparticles to generate active charges, thereby promoting the proliferation and differentiation of neural stem cells. Attached Figure Description

[0017] Figure 1 Transmission electron microscope image of barium titanate-gold composite nanoparticles; Figure 2 The image shows the elemental mapping of barium titanate-gold composite nanoparticles using scanning transmission electron microscopy (STEM) and energy-dispersive X-ray spectroscopy (EDS). Figure 3 The fluorescence image shows the generation of reactive oxygen species in cells by barium titanate-gold composite nanoparticles under near-infrared light irradiation. Figure 4 Fluorescence and quantitative average fluorescence intensity of barium titanate-gold composite nanoparticles promoting neural stem cell proliferation; Figure 5 Photographs of the neurospheres used to promote neural stem cell proliferation by barium titanate-gold composite nanoparticles and a quantitative image of the neurosphere diameter on day 5. Figure 6 Nestin fluorescence staining image and quantitative average fluorescence intensity image of neural stem cell markers that promote neural stem cell differentiation by barium titanate-gold composite nanoparticles. Figure 7Fluorescence staining and quantitative average fluorescence intensity of Tuj1, an early neuronal marker for promoting neural stem cell differentiation by barium titanate-gold composite nanoparticles. Figure 8 Map2 fluorescence staining image and average fluorescence intensity quantification image of mature neurons that are markers of neural stem cell differentiation promoted by barium titanate-gold composite nanoparticles; Figure 9 Fluorescence staining image and quantitative average fluorescence intensity image of GFAP, a marker of astrocyte differentiation promoted by barium titanate-gold composite nanoparticles; Figure 10 Fluorescence staining and quantitative average fluorescence intensity diagrams show how barium titanate-gold composite nanoparticles promote the increase of intracellular calcium ion concentration in neural stem cells. Detailed Implementation

[0018] This invention provides the application of barium titanate-gold composite nanoparticles in the preparation of formulations that promote the proliferation and / or differentiation of neural stem cells. The barium titanate-gold composite nanoparticles have a core-shell structure, comprising a barium titanate nanocore and a gold shell layer encapsulating the surface of the barium titanate nanocore.

[0019] Barium titanate (BaTiO3, BTO) nanomaterials are thermoelectric and piezoelectric materials with good biocompatibility, excellent thermoelectric conversion performance and high piezoelectric coefficient. As a nanomedicine material, it has been widely used in the biomedical field, such as sterilization and tissue regeneration. As a thermoelectric material, the self-polarization of barium titanate nanomaterials decreases when heated. Therefore, the asymmetrically distributed electrons and holes on the surface of the material are released and react with the surrounding medium to generate reactive oxygen free radicals. As a piezoelectric material, barium titanate nanomaterials can generate a weak current instantaneously when subjected to external mechanical stress, thereby inducing cell differentiation and proliferation. However, this material still has some disadvantages, such as: (1) As a thermoelectric material, BaTiO3 itself does not have the ability to generate heat. Therefore, the heat required to generate free radicals can only rely on external physical heating; (2) As a thermoelectric material, BaTiO3 has a weak response to external temperature changes and a relatively long response time; (3) Electrons and holes generated under thermal excitation are prone to recombination, thereby reducing the pyroelectric performance of BaTiO3 nanomaterials.

[0020] Gold nanoparticles (AuNPs) are widely used in the biomedical field as drug carriers, imaging probes, and photothermal therapeutic agents due to their excellent biocompatibility, ease of surface functionalization, and unique optical properties. Particularly in photothermal conversion, AuNPs can efficiently generate heat under irradiation with specific wavelengths of light (especially near-infrared light, which has good tissue penetration), a property that has been used in tumor ablation. However, most related technologies limit the photothermal conversion capabilities of AuNPs to thermotherapy or heat-based controlled drug release, greatly neglecting their enormous potential in neuromodulation, particularly as photoelectric cell stimulators.

[0021] This invention combines pyroelectric nanomaterials with photothermal metals, effectively addressing some shortcomings of barium titanate nanomaterials by directly driving electrothermal activity. - and h + The movement to inhibit e - and h + The rapid combination of these components enables efficient light-heat-electricity conversion, thereby effectively regulating the life activities of neural stem cells and promoting their proliferation and differentiation.

[0022] As one embodiment of the present invention, the differentiation may include differentiation into neurons and promotion of neuronal maturation. That is, the barium titanate-gold composite nanoparticles of the present invention can promote the differentiation of neural stem cells into neurons, especially promote the differentiation of neural stem cells into mature neurons.

[0023] In one embodiment of the present invention, the proliferation includes promoting neurosphere growth, that is, the barium titanate-gold composite nanoparticles of the present invention can promote the increase of neurosphere diameter.

[0024] This invention provides the application of barium titanate-gold composite nanoparticles in the preparation of formulations that promote the generation of reactive oxygen species in neural stem cells. The barium titanate-gold composite nanoparticles have a core-shell structure, including a barium titanate nanocore and a gold shell layer wrapped around the surface of the barium titanate nanocore.

[0025] This invention provides the application of barium titanate-gold composite nanoparticles in the preparation of formulations that promote the increase of calcium ions in neural stem cells. The barium titanate-gold composite nanoparticles have a core-shell structure, including a barium titanate nanocore and a gold shell layer wrapped around the surface of the barium titanate nanocore.

[0026] In one embodiment of the present invention, the neural stem cells include neural stem cells cultured in vitro.

[0027] As one embodiment of the present invention, the barium titanate-gold composite nanoparticles can function under light irradiation at wavelengths of 600-808nm, and further can function under light irradiation at wavelengths of 800-808nm. In this embodiment, irradiation with 808nm near-infrared light is used as an example for illustration.

[0028] In one embodiment of the present invention, the zeta potential of the barium titanate-gold composite nanoparticles (BTO@Au) can be 5~10mV, specifically 7.9±0.9mV; the particle size of the barium titanate-gold composite nanoparticles can be 80~100nm, specifically 90±6.7nm; and the particle size of the barium titanate nanocore can be 55~85nm, specifically 69.2±9.1nm. In another embodiment of the present invention, the mass ratio of barium titanate to gold in the barium titanate-gold composite nanoparticles can be 0.85~0.95:0.05~0.15, specifically 0.89:0.11.

[0029] In one embodiment of the present invention, the surface of the gold shell of the barium titanate-gold composite nanoparticles can be modified with polyethylene glycol, which is beneficial to improving the biocompatibility of the barium titanate-gold composite nanoparticles and enhancing their dispersibility in physiological solutions. In another embodiment, the polyethylene glycol can specifically be thiolated polyethylene glycol (PEG-SH), and the weight-average molecular weight (Mw) of the PEG-SH can be 5000; the gold shell and the polyethylene glycol can be connected via Au-S bonds. In another embodiment, the polyethylene glycol content in the polyethylene glycol-modified barium titanate-gold composite nanoparticles (pBTO@Au) can be 7.5~9.5wt%, specifically 8.7wt%; the zeta potential of the polyethylene glycol-modified barium titanate-gold composite nanoparticles can be -3~1mV, specifically -0.94±1.6mV, and the hydrodynamic particle size can be 100~120nm, specifically 113.2±10.3nm.

[0030] The preparation methods of barium titanate-gold composite nanoparticles and barium titanate-gold composite nanoparticles modified with polyethylene glycol described in this invention will be described in detail below.

[0031] In this invention, the preparation method of the barium titanate-gold composite nanoparticles includes the following steps: Barium titanate was mixed with an aqueous H2O2 solution and subjected to hydroxylation modification to obtain hydroxylated barium titanate. The hydroxylated barium titanate, 3-aminopropyltrimethoxysilane, and ethanol were mixed and subjected to amination modification to obtain amination-modified barium titanate. The barium titanate aminated, colloidal gold nanoparticles, NaCl and water were mixed to carry out the first stage reaction to obtain a colloidal gold nanoparticle / barium titanate aminated composite suspension. K2CO3 and HAuCl4 are mixed with water to carry out a second-stage reaction, resulting in a K2CO3 / HAuCl4 plating solution. The colloidal gold nanoparticle / aminated barium titanate composite suspension, the K2CO3 / HAuCl4 plating solution, and formaldehyde aqueous solution are mixed to carry out a third-stage reaction, resulting in the barium titanate-gold composite nanoparticles.

[0032] This invention involves mixing barium titanate with an aqueous H₂O₂ solution and performing a hydroxylation modification treatment to obtain hydroxylated barium titanate. In one embodiment, the concentration of the H₂O₂ aqueous solution can be 9-10 M; the ratio of barium titanate to the H₂O₂ aqueous solution can be 380-400 mg: 20-25 mL; the mixing is preferably carried out under ultrasonic treatment conditions, and the ultrasonic treatment time can be 15-20 min. In another embodiment, the hydroxylation modification treatment is preferably carried out under reflux conditions, specifically at a temperature of 110°C in this example; the hydroxylation modification treatment time can be 3-4 h; after the hydroxylation modification treatment, the resulting liquid is preferably centrifuged, the solid material is collected, and washed with water and ethanol respectively to obtain the hydroxylated barium titanate (BTO-OH).

[0033] After obtaining hydroxylated barium titanate, the present invention mixes the hydroxylated barium titanate, 3-aminopropyltrimethoxysilane, and ethanol, and performs an amination modification treatment to obtain aminated barium titanate. Preferably, the hydroxylated barium titanate is dispersed in ethanol, and then 3-aminopropyltrimethoxysilane is added to the resulting dispersion. Based on the mass of barium titanate, the ratio of barium titanate to ethanol can be 380-400 mg: 20-25 mL, and the volume ratio of barium titanate to 3-aminopropyltrimethoxysilane can be 380-400 mg: 380-400 μL. In one embodiment of the present invention, the amination modification treatment temperature can be 75-80°C, and the time can be 22-24 h. After the amination modification treatment, the resulting liquid is preferably centrifuged, the solid material is collected, and washed with water and ethanol respectively to obtain the aminated barium titanate (BTO-OH).

[0034] After obtaining barium titanate aminated, the present invention mixes the barium titanate aminated, colloidal gold nanoparticles, NaCl, and water to carry out a first-stage reaction, thereby obtaining a colloidal gold nanoparticle / barium titanate aminated composite suspension. Preferably, the present invention mixes an aqueous NaCl solution with the aqueous suspension of colloidal gold nanoparticles, and then adds an aqueous dispersion of barium titanate aminated under ultrasonic conditions to the resulting mixture to carry out the first-stage reaction; the volume ratio of the aqueous NaCl solution, the aqueous suspension of colloidal gold nanoparticles, and the aqueous dispersion of barium titanate aminated can be 0.8~1 mL: 8~10 mL: 0.8~1 mL; the concentration of the aqueous NaCl solution can be 0.8~1 M; based on the mass of barium titanate, the volume ratio of barium titanate to water in the aqueous dispersion of barium titanate aminated can be 380~400 mg: 8~10 mL. In one embodiment of the present invention, the preparation method of the colloidal gold nanoparticle aqueous suspension includes the following steps: mixing NaOH aqueous solution, tetramethylphosphoric chloride aqueous solution, and water; then adding HAuCl4 aqueous solution to the resulting mixture; and undergoing a reduction reaction to obtain the colloidal gold nanoparticle aqueous suspension. The volume ratio of NaOH aqueous solution, tetramethylphosphoric chloride aqueous solution, water, and HAuCl4 aqueous solution can be 1~1.2:3.5~4:170~180:6.5~6.75; the concentration of NaOH aqueous solution can be 0.8~1M; the concentration of tetramethylphosphoric chloride aqueous solution can be 1~1.2mM; and the concentration of HAuCl4 aqueous solution can be 23~25mM. The reduction reaction can be carried out rapidly at room temperature, and the color of the solution changes from colorless to brown during the reduction reaction. In another embodiment of the present invention, the first stage reaction can be carried out at room temperature, and the reaction time can be 10~12h. No post-processing is required after the first stage reaction, and the colloidal gold nanoparticle / barium titanate aminocarboxylate complex (BTO-NH2-Au) suspension is obtained directly.

[0035] This invention involves mixing K₂CO₃, HAuCl₄, and water to conduct a second-stage reaction, yielding a K₂CO₃ / HAuCl₄ plating solution. Preferably, the second-stage reaction is carried out by mixing an aqueous K₂CO₃ solution and an aqueous HAuCl₄ solution; the volume ratio of the K₂CO₃ solution to the HAuCl₄ solution can be 180-200:2.5-3; the concentration of the K₂CO₃ solution can be 1.6-1.8 mM; and the concentration of the HAuCl₄ solution can be 23-25 ​​mM. In one embodiment, the second-stage reaction can be carried out at room temperature for 10-12 hours; preferably, it is carried out in darkness; no post-processing is required after the second-stage reaction, and the K₂CO₃ / HAuCl₄ plating solution is obtained directly.

[0036] After obtaining the colloidal gold nanoparticle / barium titanate composite suspension and the K2CO3 / HAuCl4 plating solution, this invention mixes the colloidal gold nanoparticle / barium titanate composite suspension, the K2CO3 / HAuCl4 plating solution, and a formaldehyde aqueous solution to carry out a third-stage reaction, thereby obtaining the barium titanate-gold composite nanoparticles. Preferably, this invention adds the colloidal gold nanoparticle / barium titanate composite suspension to the K2CO3 / HAuCl4 plating solution under ultrasonic conditions, and then adds a formaldehyde aqueous solution to the resulting mixture under stirring conditions; the volume ratio of the K2CO3 / HAuCl4 plating solution, the colloidal gold nanoparticle / barium titanate composite suspension, and the formaldehyde aqueous solution can be 140~150:15~16:0.4~0.5; the concentration of the formaldehyde aqueous solution can be 35~37wt%. In one embodiment of the present invention, the third-stage reaction can be carried out at room temperature, and the reaction time can be 8-10 min. After the third-stage reaction, the resulting liquid is preferably centrifuged, the solid material is collected, washed with water, and freeze-dried to obtain the barium titanate-gold composite nanoparticles (BTO@Au).

[0037] In this invention, the preparation method of the barium titanate-gold composite nanoparticles modified with polyethylene glycol includes the following steps: A mercaptolated polyethylene glycol aqueous solution was mixed with an aqueous suspension of barium titanate-gold composite nanoparticles for modification treatment to obtain the barium titanate-gold composite nanoparticles modified with polyethylene glycol.

[0038] In one embodiment of the present invention, the volume ratio of the thiolated polyethylene glycol aqueous solution to the barium titanate-gold composite nanoparticle aqueous suspension can be 0.8~1:8~10; the concentration of the thiolated polyethylene glycol aqueous solution can be 0.8~1 mg / mL, and the weight-average molecular weight (Mw) of the thiolated polyethylene glycol in the thiolated polyethylene glycol aqueous solution can be 5000; the concentration of the barium titanate-gold composite nanoparticle aqueous suspension can be 450~500 μg / mL. In another embodiment of the present invention, the modification treatment can be carried out at room temperature for 10~12 h; the modification treatment is preferably carried out in the dark; after the modification treatment, the resulting liquid is preferably centrifuged, the solid material is collected, washed with water, and freeze-dried to obtain the polyethylene glycol-modified barium titanate-gold composite nanoparticles (pBTO@Au).

[0039] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0040] Preparation Example 1 The preparation of barium titanate-gold composite nanoparticles includes the following steps: (1) Preparation of 3-aminopropyltrimethoxysilane (APTMS) functionalized barium titanate (BTO-NH2): 400 mg BTO was weighed and dispersed in 25 mL of 10 M H2O2 aqueous solution, sonicated for 20 min, and then heated to 110 °C under stirring and continuously refluxed for 4 h; after the reaction, the resulting liquid was centrifuged at 10000 rpm for 5 min, the solid material was collected, and washed twice with water and ethanol respectively. The washed solid material (i.e. BTO-OH) was dispersed in 25 mL of ethanol to obtain BTO-OH ethanol dispersion; 400 μL APTMS was added to the BTO-OH ethanol dispersion, and the reaction was stirred at 80 °C for 24 h; after the reaction, the resulting liquid was centrifuged at 10000 rpm for 5 min, the solid material was collected, and washed twice with ethanol and water respectively. The washed solid material (i.e. BTO-NH2) was dispersed in 10 mL of water to obtain BTO-NH2 aqueous dispersion.

[0041] (2) Preparation of colloidal gold nanoparticles (GNPs): 1.2 mL of 1 M NaOH aqueous solution and 4 mL of 1.2 mM tetrahydroxymethyl phosphorus chloride (THPC) aqueous solution were added to 180 mL of water. Then, 6.75 mL of 25 mM HAuCl4 aqueous solution was added to the mixture. The color of the solution changed from colorless to brown, and a GNPs suspension was obtained.

[0042] (3) Preparation of BTO-NH2-Au: Mix 1 mL of 1 M NaCl aqueous solution with 10 mL of the GNP suspension, and then add 1 mL of the BTO-NH2 aqueous dispersion to the mixture under ultrasonic conditions. React at room temperature (25 °C) for 12 h to obtain BTO-NH2-Au suspension.

[0043] (4) Preparation of barium titanate-gold composite nanoparticles (BTO@Au): 200 mL of 1.8 mM K2CO3 aqueous solution and 3 mL of 25 mM HAuCl4 aqueous solution were stirred and reacted at room temperature in the dark for 12 h. The color of the solution changed from light yellow to colorless, and K2CO3 / HAuCl4 plating solution was obtained. 16 mL of the BTO-NH2-Au suspension was added to 150 mL of the K2CO3 / HAuCl4 plating solution under ultrasonic conditions. Then, 500 μL of 37 wt% formaldehyde (HCHO, Mw=30.03) aqueous solution was added under stirring conditions, and the reaction was carried out at room temperature for 10 min. After the reaction was completed, the obtained solution was centrifuged at 10000 rpm for 10 min, washed twice with water, and freeze-dried to obtain BTO@Au.

[0044] In this preparation example, the barium titanate-gold composite nanoparticles include a barium titanate nanocore and a gold shell coating the surface of the barium titanate nanocore; the zeta potential of the barium titanate-gold composite nanoparticles is 7.9±0.9mV; the particle size of the barium titanate-gold composite nanoparticles is 90±6.7nm; the particle size of the barium titanate nanocore is 69.2±9.1nm; and the mass ratio of barium titanate to gold in the barium titanate-gold composite nanoparticles is 0.89:0.11.

[0045] Figure 1 The image shows a transmission electron microscope image of barium titanate-gold composite nanoparticles, revealing that these nanoparticles possess a core-shell nanostructure.

[0046] Figure 2 The scanning transmission electron microscopy (STEM) and energy-dispersive X-ray spectroscopy (EDS) elemental mappings of barium titanate-gold composite nanoparticles show that Ba, Ti, and O elements are confined in the center, while Au elements surround the outer layer of BTO.

[0047] Preparation Example 2 The preparation of polyethylene glycol (PEG) modified BTO@Au (abbreviated as pBTO@Au) includes the following steps: 1 mL of 1 mg / mL thiolated polyethylene glycol (PEG-SH, Mw=5000) aqueous solution was mixed with 10 mL of 500 μg / mL BTO@Au aqueous suspension, and the mixture was stirred and reacted at room temperature in the dark for 12 h. After the reaction was completed, the resulting solution was centrifuged at 8000 rpm for 8 min, washed twice with water, and freeze-dried to obtain pBTO@Au.

[0048] In this preparation example, the polyethylene glycol content in the polyethylene glycol-modified barium titanate-gold composite nanoparticles is 8.7 wt%; the zeta potential of the polyethylene glycol-modified barium titanate-gold composite nanoparticles is -0.94 ± 1.6 mV, and the hydrodynamic particle size is 113.2 ± 10.3 nm.

[0049] Preparation Example 3 The preparation of Rhodamine B (RhB) labeled pBTO@Au (abbreviated as pBTO@Au-RhB) includes the following steps: 20 mL of a 500 μg / mL pBTO@Au aqueous suspension was sonicated for 1 h, and then 20 mL of a 500 μg / mL SH-PEG-NH2 aqueous solution was added dropwise. The mixture was stirred and reacted in the dark for 24 h. The resulting solution was then centrifuged at 7000 rpm, and the solid material (pBTO@Au-NH2) was collected and washed with water. The solid material was then redispersed in 20 mL of pure water to obtain a pBTO@Au-NH2 aqueous suspension. 1 mL of 2 mg / mL RhB aqueous solution, 1 mL of 2.4 mg / mL 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) aqueous solution, and 1 mL of 2 mg / mL N-hydroxysuccinimide (NHS) aqueous solution were stirred at room temperature for 24 h. The resulting solution was then centrifuged, and the solid material was collected and washed three times with water. The washed solid material was mixed with the pBTO@Au-NH2 aqueous suspension and stirred continuously for 24 h. The resulting solution was then centrifuged, washed three more times with water, and freeze-dried to obtain pBTO@Au-RhB.

[0050] Preparation Example 4 The preparation of pBTO@Au (abbreviated as pBTO@Au-Cy7) labeled with cyanogen dye (Cy7) was carried out by following the preparation method of pBTO@Au-RhB, except that the RhB aqueous solution was replaced with the Cy7 aqueous solution, and pBTO@Au-Cy7 was finally obtained.

[0051] Example 1 This embodiment investigates the ability of barium titanate-gold composite nanoparticles to generate reactive oxygen species (ROS) within cells under near-infrared light irradiation. As a thermoelectric material, barium titanate nanomaterials exhibit reduced polarizability upon heating. Consequently, electrons and holes asymmetrically distributed on the surface of the barium titanate nanomaterials are released and react with the surrounding medium to generate ROS. The specific steps are as follows: (1) Cell culture Digested neural stem cells were divided into groups of 5 × 10⁻⁶. 4Cells were seeded at a density of 1 cells / well in 12-well plates pretreated with poly-L-lysine and cultured overnight. After the cells adhered, barium titanate-gold composite nanoparticles were diluted with culture medium to a concentration of 25 μg / mL and added to the cells. The cells were cultured for another 6 hours and then irradiated with 808 nm infrared light for 10 minutes. After the infrared light irradiation was completed, the cells were returned to the incubator and cultured for another 24 hours.

[0052] (2) Detection of intracellular reactive oxygen species production Intracellular total reactive oxygen species (ROS) production was detected using an ROS detection kit. The ROS fluorescent probe (DCFH-DA, APF) was diluted to a concentration of 1 μM with complete culture medium. The old culture medium in the cells was removed, and the diluted DCFH-DA solution was added. The cells were incubated for 30 min, washed three times with PBS, and then incubated at 37°C for 15 min with Hoechst 33342 staining solution. The cell nuclei were stained, and fluorescence images were captured using an inverted fluorescence microscope.

[0053] Figure 3 The image shows the fluorescence of reactive oxygen species (ROS) generated in cells by barium titanate-gold composite nanoparticles under near-infrared light irradiation. The results show that the ROS fluorescence intensity in the barium titanate-gold composite nanoparticle group treated with near-infrared light irradiation was significantly higher than that in the other three groups, indicating that barium titanate-gold composite nanoparticles can increase the production of ROS in neural stem cells under near-infrared light irradiation.

[0054] Example 2 This embodiment examines the ability of barium titanate-gold composite nanoparticles to promote neural stem cell proliferation under near-infrared light irradiation. Barium titanate-gold composite nanoparticles have a certain effect on promoting neural stem cell proliferation. As a thermoelectric material, barium titanate generates a microcurrent upon heating, promoting cell proliferation. Therefore, this embodiment uses Ki67 staining experiments and measurements of neurosphere diameter to detect the effect of barium titanate-gold composite nanoparticles on promoting neural stem cell proliferation. The specific steps are as follows: (1) Ki67 detection The cell culture method was the same as that in Example 1. The cells were then fixed with 4% paraformaldehyde, washed three times with PBS, permeabilized and blocked with 5% BSA and 0.4% Triton-100, washed three times with PBST, incubated overnight at 4°C with Ki67 antibody according to the instructions, washed three times with PBST, incubated with secondary antibody at room temperature for 1 hour, and finally stained the cell nuclei with DAPI and captured fluorescence images using a fluorescence microscope.

[0055] Figure 4The images show fluorescence and quantitative average fluorescence intensity of barium titanate-gold composite nanoparticles promoting neural stem cell proliferation. The left side shows the fluorescence image, and the right side shows the quantitative average fluorescence intensity. The results indicate that the number of Ki67-positive cells in the barium titanate-gold composite nanoparticle treatment group was significantly higher than in the other three groups, suggesting that barium titanate-gold composite nanoparticles can promote neural stem cell proliferation under near-infrared light irradiation.

[0056] (2) Measurement of the diameter of the nerve ball Digested neural stem cells were divided into groups of 5 × 10⁻⁶. 4 The cells were seeded at a density of cells / well in 12-well plates and cultured. On the second day, the barium titanate-gold composite nanoparticles were diluted with culture medium to a concentration of 25 μg / mL and added to the cells. After the cells were cultured for another 6 hours, they were irradiated with 808 nm infrared light for 10 min. The irradiation was repeated every other day. The cells were photographed under a microscope after 2, 3, 4 and 5 days of growth.

[0057] Figure 5 Images show the neurospheres observed after barium titanate-gold composite nanoparticles promoted neural stem cell proliferation, along with a quantitative image of the neurosphere diameter on day 5. The left image shows the neurospheres observed, and the right image shows the quantitative image of the neurosphere diameter on day 5. The results indicate that the neurosphere diameter in the barium titanate-gold composite nanoparticle-treated group was significantly higher than that in the other three groups, demonstrating that barium titanate-gold composite nanoparticles can promote neurosphere growth under near-infrared light irradiation.

[0058] Example 3 This embodiment examines the ability of barium titanate-gold composite nanoparticles to promote neural stem cell differentiation under near-infrared light irradiation. Barium titanate-gold composite nanoparticles have a certain effect on promoting neural stem cell differentiation. As a thermoelectric material, barium titanate generates a weak current upon heating, promoting cell differentiation. Therefore, the effect of barium titanate-gold composite nanoparticles on promoting neural stem cell differentiation can be detected by immunofluorescence staining of differentiated cells for relevant markers. The specific steps are as follows: (1) Place cell crawling smears in 4-well plates, treat with pre-cooled poly-L-lysine, and digest the neurospheres into single cells at 1×10⁻⁶. 5 The cells were seeded at a density of 1 cell / well in a four-well plate and differentiation medium was added for differentiation. (2) On the second day, the barium titanate-gold composite nanoparticles were diluted with culture medium to a concentration of 25 μg / mL and added to the cells. After the cells were cultured for 6 hours, they were irradiated with 808 nm infrared light for 10 minutes. The irradiation was repeated every other day. (3) After differentiation for 7 days, fixation, permeation and blocking were performed according to the steps of immunofluorescence staining. Antibodies of Nestin, Tuj1, Map2 and GFAP were diluted according to the instructions and added to the cells. The cells were incubated overnight at 4°C. (4) Wash the cells three times with PBST, add diluted fluorescent secondary antibody and incubate for 1 hour, wash the cells three times with PBST, place the cell smear upside down on a glass slide containing DAPI anti-fluorescence quencher, seal the slide, and then take a fluorescence image under a fluorescence microscope.

[0059] Figure 6 The images show the Nestin fluorescence staining and average fluorescence intensity quantification of neural stem cell markers promoted by barium titanate-gold composite nanoparticles. The left side shows the Nestin fluorescence staining, and the right side shows the average fluorescence intensity quantification. The results indicate that the average fluorescence intensity of Nestin, a neural stem cell marker, in the barium titanate-gold composite nanoparticle-treated group was significantly lower than that in the other three groups, suggesting that barium titanate-gold composite nanoparticles can promote the differentiation of neural stem cells into different types of neural cells under near-infrared light irradiation.

[0060] Figure 7 The images show the fluorescence staining and quantitative mean fluorescence intensity of Tuj1, an early neuronal marker promoting neural stem cell differentiation, induced by barium titanate-gold composite nanoparticles. The left side shows the Tuj1 fluorescence staining image, and the right side shows the quantitative mean fluorescence intensity. The results indicate that the mean fluorescence intensity of Tuj1, an early neuronal marker, in the barium titanate-gold composite nanoparticle-treated group was significantly higher than that in the other three groups, suggesting that near-infrared light irradiation can promote the differentiation of neural stem cells into early neurons.

[0061] Figure 8 The images show the Map2 fluorescence staining and average fluorescence intensity quantification maps of the markers for mature neurons influencing neural stem cell differentiation by barium titanate-gold composite nanoparticles. The left side shows the Map2 fluorescence staining map, and the right side shows the average fluorescence intensity quantification map. The results indicate that the average fluorescence intensity of Map2, a marker for mature neurons, in the barium titanate-gold composite nanoparticle-treated group was significantly higher than that in the other three groups, suggesting that barium titanate-gold composite nanoparticles can promote the differentiation of neural stem cells into mature neurons under near-infrared light irradiation.

[0062] Figure 9The images show fluorescence staining and quantitative mean fluorescence intensity of GFAP, a marker of astrocyte differentiation, promoted by barium titanate-gold composite nanoparticles in promoting neural stem cell differentiation. The left side shows the GFAP fluorescence staining image, and the right side shows the quantitative mean fluorescence intensity. The results indicate that the mean fluorescence intensity of GFAP in the barium titanate-gold composite nanoparticle-treated group was significantly lower than that in the other three groups, suggesting that near-infrared light irradiation of barium titanate-gold composite nanoparticles can reduce the differentiation of neural stem cells into astrocytes.

[0063] Example 4 This embodiment investigates the ability of barium titanate-gold composite nanoparticles to promote intracellular calcium ion increase under near-infrared light irradiation. As a photo-thermal-electric composite material, barium titanate-gold composite nanoparticles generate a weak current under near-infrared light irradiation, thereby promoting an increase in intracellular calcium ion concentration. Therefore, by detecting the intracellular calcium ion concentration in differentiated cells, the effect of barium titanate-gold composite nanoparticles on promoting intracellular calcium ion increase can be evaluated. The specific steps are as follows: (1) After digestion, the neural stem cells were divided into groups of 8×10 4 The cells / well were seeded at a density of 1 cell / well and incubated overnight in 4-well plates that had been pretreated with poly-L-lysine. (2) After the cells adhered to the cell wall, the barium titanate-gold composite nanoparticles were diluted with culture medium to a concentration of 25 μg / mL and added to the cells. The cells were cultured for 6 hours and then irradiated with 808 nm infrared light for 10 min. The cells were cultured for 5 consecutive days and irradiated once every 2 days.

[0064] (3) After the culture is completed, use Fluo-4 AM Ca 2+ The fluorescent probe was diluted according to the instructions and incubated with cells at 37°C for 30 min. Cells were then washed three times with PBS, and Ca-free PBS was added. 2+ Incubate with HBSS solution for another 20 minutes, and then take fluorescence images under a fluorescence microscope.

[0065] Figure 10 The images show fluorescence staining and quantitative average fluorescence intensity maps of barium titanate-gold composite nanoparticles promoting increased intracellular calcium ion concentration in neural stem cells. The left side shows the fluorescence staining map, and the right side shows the quantitative average fluorescence intensity map. The results indicate that barium titanate-gold composite nanoparticles significantly increased intracellular calcium ion concentration in neural stem cells treated with near-infrared light. 2+ The average fluorescence intensity was significantly higher than that of the other three groups, indicating that barium titanate-gold composite nanoparticles can promote intracellular Ca2+ under near-infrared light irradiation. 2+ Increase in concentration.

[0066] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of barium titanate-gold composite nanoparticles in the preparation of formulations that promote the proliferation and / or differentiation of neural stem cells, wherein the barium titanate-gold composite nanoparticles have a core-shell structure, comprising a barium titanate nanocore and a gold shell layer encapsulating the surface of the barium titanate nanocore.

2. The application according to claim 1, characterized in that, The differentiation includes differentiating into neurons and promoting neuronal maturation.

3. The application according to claim 1, characterized in that, The proliferation includes promoting neurosphere growth.

4. Application of barium titanate-gold composite nanoparticles in the preparation of formulations that promote the generation of reactive oxygen species in neural stem cells, wherein the barium titanate-gold composite nanoparticles have a core-shell structure, including a barium titanate nanocore and a gold shell layer wrapped around the surface of the barium titanate nanocore.

5. Application of barium titanate-gold composite nanoparticles in the preparation of formulations that promote the increase of calcium ions in neural stem cells, wherein the barium titanate-gold composite nanoparticles have a core-shell structure, comprising a barium titanate nanocore and a gold shell layer encapsulating the surface of the barium titanate nanocore.

6. The application according to any one of claims 1 to 5, characterized in that, The neural stem cells include neural stem cells cultured in vitro.

7. The application according to claim 6, characterized in that, The barium titanate-gold composite nanoparticles function under light irradiation at wavelengths of 600–808 nm.

8. The application according to any one of claims 1 to 5, characterized in that, The barium titanate-gold composite nanoparticles have a zeta potential of 5-10 mV, a particle size of 80-100 nm, and a particle size of 55-85 nm for the barium titanate nanocore.

9. The application according to claim 8, characterized in that, The mass ratio of barium titanate to gold in the barium titanate-gold composite nanoparticles is 0.85~0.95:0.05~0.

15.

10. The application according to claim 8, characterized in that, The gold shell surface of the barium titanate-gold composite nanoparticles is further modified with polyethylene glycol, and the content of polyethylene glycol in the barium titanate-gold composite material is 7.5~9.5wt%.

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