A fluorescent nanomaterial, its preparation method and application

Abstract

The present invention discloses a fluorescent nanomaterial, a preparation method thereof and an application thereof. The method includes: obtaining calcium sulfide nanoparticles doped with rare earth ions; dispersing the calcium sulfide nanoparticles doped with rare earth ions in cyclohexane, adding polyoxyethylene nonyl phenyl ether, concentrated ammonia water and tetraethoxysilane, centrifuging after the reaction ends to obtain calcium sulfide nanoparticles doped with rare earth ions coated with silica; mixing the calcium sulfide nanoparticles doped with rare earth ions coated with silica and an amino-modified silicon source in ethanol to obtain calcium sulfide nanoparticles doped with rare earth ions coated with amino-modified silica; activating the carboxyl group of glucose oxidase, and then adding the calcium sulfide nanoparticles doped with rare earth ions coated with amino-modified silica to react to obtain the fluorescent nanomaterial. The fluorescent nanomaterial has near-infrared second-region (NIR-II) fluorescence performance and can decompose in a glucose solution to release calcium ions and hydrogen sulfide.

Description

Technical Field

[0001] The present invention relates to the technical field of preparation of fluorescent nanomaterials, and particularly relates to a fluorescent nanomaterial, a preparation method thereof, and an application thereof. Background Art

[0002] Cancer, also known as malignant tumor, can cause tumor cells to proliferate infinitely and metastasize throughout the body, thereby triggering various diseases and seriously endangering the health of patients. Therefore, the early detection, diagnosis, and treatment of cancer are the focus of current clinical physicians. Early cancer diagnosis mainly relies on doctor palpation and imaging examinations. Currently widely used imaging methods, such as X-ray, computed tomography (CT), magnetic resonance imaging (MRI), ultrasound, etc., all have their own advantages. However, their sensitivity, spatial resolution, and signal specificity are limited, making it difficult to achieve high-sensitivity detection of small tumors in the early stage of tumor development. In this context, in vivo fluorescence imaging has received wide attention in the biomedical field.

[0003] Currently, the clinically approved indocyanine green (ICG) as a near-infrared region I (NIR-I, 700 - 1000 nm) fluorescent probe has been widely used in tumor fluorescence imaging and surgical guidance. However, there are still some problems and challenges. Due to the liver metabolism of ICG, it cannot accumulate at the tumor site. At the same time, the disadvantage of low signal-to-noise ratio seriously affects the accuracy of surgery. Near-infrared region II (NIR-II, 1000 - 1700 nm) fluorescent probes have deeper tissue penetration ability compared to traditional near-infrared fluorescent probes, and are more likely to obtain imaging information with high signal-to-noise ratio and high spatial resolution. In recent years, they have been widely used as cancer diagnosis tools. With the exploration of NIR-II imaging technology, more and more imaging probes with excellent performance have emerged in this field. Among them, rare earth ion-doped inorganic nanoparticles, as novel inorganic functional materials, have superior optical properties, including strong photostability, narrow emission peaks, and long luminescence lifetimes. Inorganic nanoparticles doped with rare earth element ions (such as Nd 3+ 、Er 3+ 、Tm 3+ ) aggregate at the tumor site through the enhanced permeability and retention (EPR) effect of solid tumors and can obtain clear imaging of the tumor site under laser irradiation.

[0004] Although NIR-II fluorescent probes have good application prospects, they still face some difficulties and challenges. For example, the probes can accumulate in the body and have long-term toxicity. Therefore, how to enable the probes to be rapidly metabolized in the body without affecting the imaging effect has become a key problem to be solved urgently.

[0005] In summary, it is of great significance to develop a NIR-II fluorescent nanomaterial with in vivo metabolism and good biocompatibility for early diagnosis and treatment of cancer. Summary of the Invention

[0006] The object of the present invention is to provide a fluorescent nanomaterial, a preparation method and an application thereof. The fluorescent nanomaterial has near-infrared second region (NIR-II) fluorescence performance, can decompose in a glucose solution and release calcium ions and hydrogen sulfide, can be metabolized in vivo, and has good biocompatibility.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] In the first aspect of the present invention, a preparation method of a fluorescent nanomaterial is provided, and the method includes:

[0009] Obtaining calcium sulfide nanoparticles doped with rare earth ions;

[0010] Dispersing the calcium sulfide nanoparticles doped with rare earth ions in a reaction solvent, adding polyoxyethylene nonylphenyl ether, concentrated ammonia water and tetraethoxysilane, centrifuging after the stirring reaction ends, and obtaining calcium sulfide nanoparticles doped with rare earth ions coated with silica;

[0011] Mixing the calcium sulfide nanoparticles doped with rare earth ions coated with silica and an amino-modified silicon source in ethanol or water, and stirring until the reaction ends to obtain calcium sulfide nanoparticles doped with rare earth ions coated with amino-modified silica;

[0012] Activating the carboxyl group of glucose oxidase and then adding the calcium sulfide nanoparticles doped with rare earth ions coated with amino-modified silica for reaction to obtain the fluorescent nanomaterial.

[0013] Further, the calcium sulfide nanoparticles doped with rare earth ions are selected from neodymium-doped calcium sulfide nanoparticles, ytterbium / erbium co-doped calcium sulfide nanoparticles, thulium / ytterbium co-doped calcium sulfide nanoparticles, cerium / erbium co-doped calcium sulfide nanoparticles.

[0014] Further, the reaction solvent includes at least one of cyclohexane, ethanol, isopropanol and water.

[0015] Further, the mass ratio range of the calcium sulfide nanoparticles doped with rare earth ions to the tetraethoxysilane is 1:9 to 1:315.

[0016] Further, the amino-modified silicon source is selected from (3-aminopropyl)triethoxysilane, 3-(2-aminoethylamino)propyltrimethoxysilane.

[0017] Further, the mass ratio of the calcium sulfide nanoparticles doped with rare earth ions coated with silica to the amino-modified silicon source is 1:2 to 1:10.

[0018] Further, the mass ratio of the glucose oxidase to the calcium sulfide nanoparticles doped with rare earth ions and coated with amino-modified silica is 1:20 to 1:100.

[0019] In a second aspect of the present invention, there is provided a fluorescent nanomaterial prepared by the method described above.

[0020] In a third aspect of the present invention, there is provided the use of the fluorescent nanomaterial described above in the preparation of a NIR-II fluorescence imaging reagent.

[0021] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0022] 1. A method for preparing a fluorescent nanomaterial provided by the present invention can first solve the problem of water quenching of calcium sulfide nanoparticles doped with rare earth ions by silica coating, and at the same time improve its dispersibility in aqueous solution; then amino modification provides a binding site for subsequent glucose oxidase; then activated glucose oxidase is added to obtain a fluorescent nanomaterial, abbreviated as CaS:Ln@SiO2-GO x NPs. The inventors of the present application found through experiments that glucose oxidase can consume glucose in tumor cells to produce gluconic acid, acidifying the cell environment, promoting the decomposition of the nanomaterial and releasing calcium ions and hydrogen sulfide; at the same time, glucose oxidase consumes glucose in cells, which can achieve the effect of starvation therapy. Therefore, the nanomaterial of the present invention can be used as a fluorescent nanomaterial that can decompose in glucose solution to realize early diagnosis and treatment of tumors.

[0023] 2. A fluorescent nanomaterial provided by the present invention has NIR-II fluorescence properties and can be used in the application of a nanomaterial that decomposes in glucose solution and releases calcium ions and hydrogen sulfide in organisms. Irradiated with an 808 nm laser, the nanomaterial exhibits strong fluorescence in NIR-II and can be used for bioimaging; the fluorescent nanomaterial has uniform size and good dispersibility in water, decomposes and releases calcium ions and hydrogen sulfide in cells, can accumulate at the tumor site for NIR-II fluorescence imaging, improves the imaging signal-to-noise ratio and sensitivity at the tumor site, and solves the problem of long-term toxicity of the nanomaterial in organisms, making the fluorescent nanomaterial described in the present invention have good application prospects in the early diagnosis and treatment of cancer. Description of the Drawings

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0025] Figure 1 Transmission electron micrograph of CaS:2Nd@SiO2-GOx NPs;

[0026] Figure 2 pH change of CaS:2Nd@SiO2-GOx NPs in glucose solution;

[0027] Figure 3 Calcium ion release of CaS:2Nd@SiO2-GOx NPs in glucose solution;

[0028] Figure 4 Hydrogen sulfide release of CaS:2Nd@SiO2-GOx NPs in glucose solution;

[0029] Figure 5 Emission spectrum of CaS:2Nd@SiO2-GOx NPs;

[0030] Figure 6 Emission spectrum of CaS:Yb / Er@SiO2-GOx NPs;

[0031] Figure 7 Emission spectrum of CaS:Yb / Tm@SiO2-GOx NPs;

[0032] Figure 8 Emission spectrum of CaS:Ce / Er@SiO2-GOx NPs;

[0033] Figure 9 Emission spectrum of CaS:Eu@SiO2-GOx NPs;

[0034] Figure 10 Calcium ion release of CaS:2Nd@SiO2-GOx NPs in tumor cells;

[0035] Figure 11 Hydrogen sulfide release of CaS:2Nd@SiO2-GOx NPs in tumor cells. Detailed implementation manners

[0036] The present invention will be specifically described below in conjunction with the detailed implementation manners and examples, and the advantages and various effects of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these detailed implementation manners and examples are for illustrating the present invention, rather than limiting the present invention.

[0037] Throughout the specification, unless otherwise specifically stated, the terms used herein shall be construed to have the meanings as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as the general understanding of those skilled in the art to which the present invention pertains. In case of any contradiction, the present specification shall prevail.

[0038] Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment, etc. used in the present invention can be obtained through market purchase or by existing methods.

[0039] According to a typical implementation manner of an embodiment of the present invention, a preparation method of a fluorescent nanomaterial is provided, and the synthesis route is as follows:

[0040]

[0041] The method includes:

[0042] Step S1: Obtain calcium sulfide nanoparticles doped with rare earth ions, abbreviated as CaS:Ln NPs;

[0043] In the step S1,

[0044] The calcium sulfide nanoparticles doped with rare earth ions are selected from neodymium-doped calcium sulfide nanoparticles, ytterbium / erbium co-doped calcium sulfide nanoparticles, thulium / ytterbium co-doped calcium sulfide nanoparticles, and cerium / erbium co-doped calcium sulfide nanoparticles.

[0045] As a specific implementation manner, the obtaining of the calcium sulfide nanoparticles doped with rare earth ions is neodymium-doped calcium sulfide nanoparticles, and its preparation method includes:

[0046] Mix calcium acetate, neodymium acetate, oleic acid, oleylamine, and trioctylamine, heat to 100 °C - 160 °C under N2 protection for reaction, and then cool to room temperature to obtain a reaction product;

[0047] Dissolve diphenylthiourea in ethanol, add the reaction product, stir and react under N2 protection, and then obtain calcium sulfide nanoparticles doped with rare earth ions through sedimentation and centrifugal separation.

[0048] As an optional implementation manner, the molar ratio of calcium acetate, neodymium acetate, and diphenylthiourea is 0.98:0.02:3.

[0049] As a specific implementation manner, the obtaining of the calcium sulfide nanoparticles doped with rare earth ions is ytterbium / erbium co-doped calcium sulfide nanoparticles, and the preparation steps are as follows:

[0050] Take calcium acetate monohydrate, erbium acetate tetrahydrate, and ytterbium acetate hydrate in a three-necked flask; add oleic acid, oleylamine, and trioctylamine, heat and stir to 90 - 110 °C, and evacuate for 10 - 30 min; raise the temperature of the solution to 110 - 130 °C, evacuate to remove oxygen and water, and under N₂ protection, raise the temperature of the solution to 150 - 170 °C and stir for 20 - 40 min; cool to room temperature, dissolve diphenylthiourea in ethanol, and add it all to the three-necked flask, evacuate at 70 - 90 °C for 20 - 40 min;

[0051] Under N₂ protection, raise the temperature of the solution to 310 - 330 °C and stir rapidly for 0.5 - 2 h; after the reaction is completed, add ethanol for sedimentation and centrifuge at 9000 - 12000 rpm for 8 - 20 min to separate CaS:Yb / Er NPs.

[0052] As a specific embodiment, the obtained calcium sulfide nanoparticles doped with rare earth ions are thulium / ytterbium co-doped calcium sulfide nanoparticles, and the preparation steps are as follows:

[0053] Take calcium acetate monohydrate, thulium acetate hexahydrate, and ytterbium acetate hydrate in a three-necked flask; add oleic acid, oleylamine, and trioctylamine to the three-necked flask, heat and stir to 90 - 110 °C, and evacuate for 10 - 30 min; raise the temperature of the solution to 110 - 130 °C, evacuate to remove oxygen and water, and under N₂ protection, raise the temperature of the solution to 150 - 170 °C and stir for 20 - 40 min; cool to room temperature, dissolve diphenylthiourea in ethanol, and add it all to the three-necked flask, evacuate at 70 - 90 °C for 20 - 40 min; under N₂ protection, raise the temperature of the solution to 310 - 330 °C and stir rapidly for 0.5 - 2 h; after the reaction is completed, add ethanol for sedimentation and centrifuge at 9000 - 12000 rpm for 8 - 20 min to separate CaS:Tm / Yb NPs.

[0054] As a specific embodiment, the obtained calcium sulfide nanoparticles doped with rare earth ions are cerium / erbium co-doped calcium sulfide nanoparticles, and the preparation steps are as follows:

[0055] Take calcium acetate monohydrate, erbium acetate tetrahydrate and cerium acetate hydrate in a three-necked flask; add oleic acid, oleylamine and trioctylamine to the three-necked flask, heat and stir to 90-110 °C, evacuate for 10-30 min; heat the solution to 110-130 °C, evacuate to remove oxygen and water, under N2 protection, heat the solution to 150-170 °C, stir for 20-40 min; cool to room temperature, dissolve diphenylthiourea in ethanol, and add all of it to the three-necked flask, evacuate at 70-90 °C for 20-40 min; under N2 protection, heat the solution to 310-330 °C, stir rapidly for 0.5-2 h; after the reaction is completed, add ethanol for sedimentation and centrifuge at 9000-12000 rpm for 8-20 min to separate CaS:Ce / Er NPs.

[0056] As a specific embodiment, the obtained calcium sulfide nanoparticles doped with rare earth ions are europium-doped calcium sulfide nanoparticles, and the preparation steps are as follows:

[0057] Take calcium acetate monohydrate, erbium acetate tetrahydrate and europium acetate hydrate in a three-necked flask; add oleic acid, oleylamine and trioctylamine to the three-necked flask, heat and stir to 90-110 °C, evacuate for 10-30 min; heat the solution to 110-130 °C, evacuate to remove oxygen and water, under N2 protection, heat the solution to 150-170 °C, stir for 20-40 min; cool to room temperature, dissolve diphenylthiourea in ethanol, and add all of it to the three-necked flask, evacuate at 70-90 °C for 20-40 min; under N2 protection, heat the solution to 310-330 °C, stir rapidly for 0.5-2 h; after the reaction is completed, add ethanol for sedimentation and centrifuge at 9000-12000 rpm for 8-20 min to separate CaS:Eu NPs.

[0058] Step S2: Disperse the calcium sulfide nanoparticles doped with rare earth ions in a reaction solvent, add polyoxyethylene nonylphenyl ether, concentrated ammonia water and tetraethoxysilane, and after stirring the reaction is completed, centrifuge to obtain calcium sulfide nanoparticles doped with rare earth ions coated with silica, abbreviated as CaS:Ln@SiO2 NPs;

[0059] In the step S2,

[0060] The reaction solvent includes at least one of cyclohexane, ethanol, isopropanol and water.

[0061] The mass ratio range of the calcium sulfide nanoparticles doped with rare earth ions to the tetraethoxysilane is 1:9-1:315.

[0062] The concentration range of the concentrated ammonia water is 22-25%.

[0063] The polyoxyethylene nonyl phenyl ether, as a non-ionic surfactant, can activate anions, cations, and non-ionic surfactants, reduce the surface tension between the calcium sulfide nanoparticles doped with rare earth ions and tetraethoxysilane, and enable tetraethoxysilane to better undergo hydrolysis and polycondensation reactions on the surface of the nanoparticles, resulting in nanoparticles coated with a dense silica layer.

[0064] The reaction principle of step S2 is as follows: The polyoxyethylene nonyl phenyl ether reduces the surface tension between the calcium sulfide nanoparticles doped with rare earth ions and tetraethoxysilane, enabling tetraethoxysilane to more readily undergo hydrolysis and polycondensation reactions on the surface of the nanoparticles.

[0065] The reaction equation of step S2 is as follows:

[0066] Hydrolysis reaction:

[0067] Condensation reaction:

[0068] The calcium sulfide nanoparticles doped with rare earth ions have the problem of quenching when exposed to water. The inventors of this application can solve the above-mentioned problem of quenching when exposed to water through silica coating, and at the same time improve its dispersibility in aqueous solution.

[0069] Step S3: Mix the silica-coated calcium sulfide nanoparticles doped with rare earth ions and the amino-modified silicon source in ethanol, stir until the reaction is completed, and obtain amino-modified silica-coated calcium sulfide nanoparticles doped with rare earth ions, abbreviated as CaS:Ln@SiO2-NH2 NPs;

[0070] In step S3,

[0071] The amino-modified silicon source is selected from (3-aminopropyl)triethoxysilane and 3-(2-aminoethylamino)propyltrimethoxysilane.

[0072] The mass ratio of the silica-coated calcium sulfide nanoparticles doped with rare earth ions to the amino-modified silicon source is 1:2 to 1:10.

[0073] The reason for amino modification is to provide a binding site for glucose oxidase in the subsequent steps;

[0074] Step S4: Activate the carboxyl group of glucose oxidase, and then add the amino-modified silica-coated calcium sulfide nanoparticles doped with rare earth ions to react, obtaining a fluorescent nanomaterial, abbreviated as CaS:Ln@SiO2-GO x NPs.

[0075] In step S4,

[0076] The specific steps for activating the carboxyl group of glucose oxidase are as follows: Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and glucose oxidase to ultrapure water, and stir at room temperature for 10 - 60 min. The mass ratio of the EDC, NHS, and glucose oxidase is (4 - 5):(6 - 8):(0.3 - 1).

[0077] The mass ratio of the carboxyl group of the glucose oxidase to the amino-modified calcium sulfide nanoparticles coated with silica and doped with rare earth ions is 1:20 - 1:100.

[0078] The inventors of the present application found through experiments that glucose oxidase can consume glucose in tumor cells to produce gluconic acid, acidify the cellular environment, promote the decomposition of the nanomaterial and release calcium ions and hydrogen sulfide; at the same time, glucose oxidase consumes glucose in cells, which can achieve the effect of starvation therapy.

[0079] According to another typical implementation manner of the embodiments of the present invention, a fluorescent nanomaterial obtained by using the method is provided.

[0080] According to another typical implementation manner of the embodiments of the present invention, the application of the fluorescent nanomaterial in the preparation of NIR-II fluorescence imaging reagents is provided.

[0081] The nanomaterial has uniform size and good dispersibility in water, decomposes in cells and releases calcium ions and hydrogen sulfide, can accumulate at the tumor site for NIR-II fluorescence imaging, improve the imaging signal-to-noise ratio and sensitivity at the tumor site, and solve the problem of long-term toxicity of the nanomaterial in vivo. The fluorescent nanomaterial described in the present invention has good application prospects in the early diagnosis and treatment of cancer.

[0082] The preparation method of a fluorescent nanomaterial of the present application will be described in detail below with reference to examples, comparative examples and experimental data.

[0083] Example 1, Fluorescent Nanomaterial CaS:2Nd@SiO2-GOx NPs and Its Preparation Method

[0084] Step 1, Obtain calcium sulfide nanoparticles doped with rare earth ions CaS:2Nd NPs, and the preparation steps are as follows:

[0085] (1) Weigh 0.1727 g of calcium acetate monohydrate (Ca(CH3COO)2·H2O) and 8.2 mg of neodymium acetate pentahydrate (Nd(CH3COO)3·5H2O) into a three-necked flask;

[0086] (2) Add 2 mL of oleic acid, 12 mL of oleylamine, and 6 mL of trioctylamine into a three-necked flask, heat and stir to 100 °C, and evacuate for 20 min;

[0087] (3) Heat the solution to 120 °C, evacuate to remove oxygen and water, and under N2 protection, heat the solution to 160 °C and stir for 30 min;

[0088] (4) Cool to room temperature. Dissolve 0.6849 g of diphenylthiourea (DPTU) in 10 mL of ethanol, then add all of it into the three-necked flask, and evacuate at 80 °C for 30 min;

[0089] (5) Under N2 protection, heat the solution to 320 °C and stir rapidly for 1 h; after the reaction is completed, add ethanol for sedimentation and then centrifuge at 10000 rpm for 10 min to separate CaS:2Nd NPs, that is, calcium sulfide nanoparticles doped with rare earth ions.

[0090] Step 2: Obtain calcium sulfide nanoparticles doped with rare earth ions coated with silica, CaS:2Nd@SiO2 NPs: Disperse 12 mg of CaS:2Nd NPs in 1 mL of cyclohexane, add it together with 2 mL of CO-520 into 50 mL of cyclohexane, and ultrasonicate for 10 min; add 0.5 mL of concentrated ammonia water and stir for 30 min; slowly dropwise add 0.8 mL of TEOS, stir for 24 h and then centrifuge at 10000 rpm for 10 min to separate CaS:2Nd@SiO2 NPs.

[0091] Step 3: Obtain amino-modified calcium sulfide nanoparticles doped with rare earth ions coated with silica, CaS:2Nd@SiO2-NH2 NPs: Add 12 mg of CaS:2Nd@SiO2 NPs and 50 μL of APTES into 20 mL of ethanol, stir at room temperature for 24 h and then centrifuge at 10000 rpm for 10 min to separate CaS:2Nd@SiO2-NH2 NPs.

[0092] Step 4: Obtain fluorescent nanomaterials CaS:2Nd@SiO2-GOx NPs: Add 4.7 mg of EDC, 6.9 mg of NHS, and 0.5 mg of GOx (glucose oxidase) into 2 mL of ultrapure water, stir at room temperature for 40 min. After dissolving 20 mg of CaS:2Nd@SiO2-H2NPs in 2 mL of ultrapure water, add it into the above mixed solution, stir at room temperature for 8 h and then centrifuge at 10000 rpm for 10 min. After centrifugation, take 2 mL of ultrapure water for dispersion to obtain the target CaS:2Nd@SiO2-GOx NPs.

[0093] Example 2: Fluorescent nanomaterial CaS:Yb / Er@SiO2-GOx NPs and its preparation method

[0094] Step 1: Obtain calcium sulfide nanoparticles CaS:Yb / Er NPs doped with rare earth ions. The preparation steps are as follows:

[0095] (1) Weigh 0.1727 g of calcium acetate monohydrate (Ca(CH3COO)2·H2O), 4.2 mg of erbium acetate tetrahydrate (Er(CH3COO)3·4H2O), and 3.5 mg of ytterbium acetate hydrate (Yb(CH3COO)3·xH2O) into a three-necked flask;

[0096] (2) Add 2 mL of oleic acid, 12 mL of oleylamine, and 6 mL of trioctylamine to the three-necked flask, heat and stir to 100 °C, and evacuate for 20 min;

[0097] (3) Raise the temperature of the solution to 120 °C, evacuate to remove oxygen and water, and under N2 protection, raise the temperature of the solution to 160 °C and stir for 30 min;

[0098] (4) Cool to room temperature, dissolve 0.6849 g of DPTU in 10 mL of ethanol, then add it all to the three-necked flask, and evacuate at 80 °C for 30 min;

[0099] (5) Under N2 protection, raise the temperature of the solution to 320 °C and stir rapidly for 1 h; after the reaction is completed, add ethanol for sedimentation and centrifuge at 10000 rpm for 10 min to separate CaS:Yb / Er NPs.

[0100] Steps 2 - 4 are the same as in Example 1.

[0101] Example 3: Fluorescent nanomaterial CaS:Tm / Yb@SiO2-GOx NPs and its preparation method

[0102] Step 1: Obtain calcium sulfide nanoparticles CaS:Tm / Yb NPs doped with rare earth ions. The preparation steps are as follows:

[0103] (1) Weigh 0.1224 g of calcium acetate monohydrate (Ca(CH3COO)2·H2O), 0.1362 g of thulium acetate hexahydrate (Tm(CH3COO)3·6H2O), and 1.8 mg of ytterbium acetate hydrate (Yb(CH3COO)3·xH2O) into a three-necked flask;

[0104] (2) Add 2 mL of oleic acid, 12 mL of oleylamine, and 6 mL of trioctylamine to the three-necked flask, heat and stir to 100 °C, and evacuate for 20 min;

[0105] (3) Heat the solution to 120 °C, evacuate to remove oxygen and water, and under N2 protection, heat the solution to 160 °C and stir for 30 min;

[0106] (4) Cool to room temperature, dissolve 0.6849 g of DPTU in 10 mL of ethanol, add it all to the three-necked flask, and evacuate at 80 °C for 30 min;

[0107] (5) Under N2 protection, heat the solution to 320 °C and stir rapidly for 1 h; after the reaction is completed, add ethanol for sedimentation and centrifuge at 10000 rpm for 10 min to separate CaS:Tm / Yb NPs.

[0108] Steps 2 - 4 are the same as in Example 1.

[0109] Example 4, Fluorescent Nanomaterial CaS:Ce / Er@SiO2-GOx NPs and Its Preparation Method

[0110] Step 1. Obtain calcium sulfide nanoparticles CaS:Ce / Er NPs doped with rare earth ions, and the preparation steps are as follows:

[0111] (1) Weigh 0.1752 g of calcium acetate monohydrate (Ca(CH3COO)2·H2O), 2.1 mg of erbium acetate tetrahydrate (Er(CH3COO)3·4H2O), and 0.2 mg of cerium acetate hydrate (Ce(CH3COO)3·1.5H2O) into a three-necked flask;

[0112] (2) Add 2 mL of oleic acid, 12 mL of oleylamine, and 6 mL of trioctylamine to the three-necked flask, heat and stir to 100 °C, and evacuate for 20 min;

[0113] (3) Heat the solution to 120 °C, evacuate to remove oxygen and water, and under N2 protection, heat the solution to 160 °C and stir for 30 min;

[0114] (4) Cool to room temperature, dissolve 0.6849 g of DPTU in 10 mL of ethanol, add it all to the three-necked flask, and evacuate at 80 °C for 30 min;

[0115] (5) Under N2 protection, heat the solution to 320 °C and stir rapidly for 1 h; after the reaction is completed, add ethanol for sedimentation and centrifuge at 10000 rpm for 10 min to separate CaS:Ce / Er NPs.

[0116] Steps 2 - 4 are the same as in Example 1.

[0117] Example 5, Fluorescent Nanomaterial CaS:Eu@SiO2-GOx NPs and Its Preparation Method

[0118] Step 1. Obtain calcium sulfide nanoparticles doped with rare earth ions CaS:Eu NPs, and the preparation steps are as follows:

[0119] (1) Weigh 0.1760 g of calcium acetate monohydrate (Ca(CH3COO)2·H2O) and 60 μL of europium acetate aqueous solution (0.01 M) into a three-necked flask.

[0120] (2) Add 2 mL of oleic acid, 12 mL of oleylamine, and 6 mL of trioctylamine into the three-necked flask, heat and stir to 100 °C, and evacuate for 20 min.

[0121] (3) Heat the solution to 120 °C, evacuate to remove oxygen and water, and under N2 protection, heat the solution to 160 °C and stir for 30 min.

[0122] (4) Cool to room temperature, dissolve 0.6849 g of DPTU in 10 mL of ethanol, then add all of it to the three-necked flask, and evacuate at 80 °C for 30 min.

[0123] (5) Under N2 protection, heat the solution to 320 °C and stir rapidly for 1 h. After the reaction is completed, add ethanol for sedimentation and then centrifuge at 10000 rpm for 10 min to separate CaS:Eu NPs.

[0124] Steps 2 - 4 are the same as in Example 1.

[0125] Experimental Example 1. Transmission electron microscope image of the fluorescent nanomaterial CaS:2Nd@SiO2-GOx NPs in Example 1

[0126] We took transmission electron microscope pictures of the prepared nanomaterials. The specific test steps are as follows: Drop 10 μL of the sample solution with a concentration of 0.5 mg / mL on a copper grid, and perform morphology testing after the sample is dried. The image is as Figure 1 shown. It can be seen from the figure that the nanomaterial has an obvious core-shell structure. The tetragonal CaS:2Nd NPs are wrapped by silica spheres on the outside, and it has good dispersibility in aqueous solution. Its particle size is about 90 nm, which is beneficial to enter cells, further decompose rapidly in the organism, and release calcium ions and hydrogen sulfide.

[0127] Experimental Example 2. pH value change of the fluorescent nanomaterial CaS:2Nd@SiO2-GOx NPs in Example 1 in glucose solution

[0128] Disperse 2.5 mg of CaS:2Nd@SiO2-GOx NPs in 5 mL of ultrapure water and glucose solution (2 mM), and measure the pH value at different time intervals. As Figure 2As shown, in a glucose solution, the pH of CaS:2Nd@SiO2-GOx NPs can drop to 4.24 within 1 h, and the glucose concentration in tumor cells ranges from 0.86 to 7.11 mM. Therefore, glucose oxidase can promote the decomposition and release of CaS:2Nd@SiO2-GOx NPs in tumor cells.

[0129] Experimental Example 3: Release of calcium ions of the fluorescent nanomaterial CaS:2Nd@SiO2-GOx NPs of Example 1 in a glucose solution

[0130] Disperse 10 mg of CaS:2Nd@SiO2-GOx NPs in 1 mL of ultrapure water (10 mg / mL) and place it in a dialysis bag; immerse it in 25 mL of a glucose solution (2 mM) in a 50 mL beaker and shake it using a shaker; take 5 mL of the dialysis solution after 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h, and supplement 5 mL of the glucose solution; measure the calcium ion concentration in the dialysis solution by inductively coupled plasma optical emission spectrometer (ICP-OES). The image of the calcium ion release amount in the glucose solution is as Figure 3 shown. It can be seen from the figure that the calcium ion release amount of CaS:2Nd@SiO2-GOx NPs reaches 90% within 24 h. Therefore, CaS:2Nd@SiO2-GOx NPs can release calcium ions in tumor cells.

[0131] Experimental Example 4: Release of hydrogen sulfide of the fluorescent nanomaterial CaS:2Nd@SiO2-GOx NPs of Example 1 in a glucose solution

[0132] In this experiment, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) was used as an indicator. Dissolve 20 mg of DTNB in 5 mL of PBS to prepare an Ellamn solution.

[0133] Add 50 μg of CaS:2Nd@SiO2-GOx NPs (25 μg / mL) to a cuvette containing 2 mL of a glucose solution (2 mM), add 100 μL of the Ellman solution and mix well, seal it and measure the absorbance change value at 412 nm at different times. The experimental results are as Figure 4 shown. In the glucose solution, CaS:2Nd@SiO2-GOx NPs rapidly decompose and release hydrogen sulfide. Therefore, CaS:2Nd@SiO2-GOx NPs can release hydrogen sulfide in tumor cells.

[0134] Experimental Example 5: Luminescence spectrum of the fluorescent nanomaterial CaS:2Nd@SiO2-GOx NPs of Example 1

[0135] Dilute CaS:2Nd@SiO2-GOx NPs to 2 mg / mL with ultrapure water and irradiate with an 808 nm laser (1 W / cm 2 ), receive the signal and plot the spectrogram. As Figure 5 shown, CaS:2Nd@SiO2-GOx NPs have excellent luminescence ability in NIR-II.

[0136] Experimental Example 6, Luminescence Spectrum of Fluorescent Nanomaterial CaS:Yb / Er@SiO2-GOx NPs in Example 2

[0137] Dilute CaS:Yb / Er@SiO2-GOx NPs to 2 mg / mL with ultrapure water and irradiate with a 980 nm laser (1 W / cm 2 ), receive the signal and plot the spectrogram. As Figure 6 shown, CaS:Yb / Er@SiO2-GOx NPs have excellent luminescence ability in NIR-II.

[0138] Experimental Example 7, Luminescence Spectrum of Fluorescent Nanomaterial CaS:Tm / Yb@SiO2-GOx NPs in Example 3

[0139] Dilute CaS:Yb / Tm@SiO2-GOx NPs to 2 mg / mL with ultrapure water and irradiate with a 980 nm laser (1 W / cm 2 ), receive the signal and plot the spectrogram. As Figure 7 shown, CaS:Yb / Tm@SiO2-GOx NPs have excellent luminescence ability in NIR-II.

[0140] Experimental Example 8, Luminescence Spectrum of Fluorescent Nanomaterial CaS:Ce / Er@SiO2-GOx NPs in Example 4

[0141] Dilute CaS:Ce / Er@SiO2-GOx NPs to 2 mg / mL with ultrapure water and irradiate with a 980 nm laser (1 W / cm 2 ), receive the signal and plot the spectrogram. As Figure 8 shown, CaS:Ce / Er@SiO2-GOx NPs have excellent luminescence ability in NIR-II.

[0142] Experimental Example 9, Luminescence Spectrum of Fluorescent Nanomaterial CaS:Eu@SiO2-GOx NPs in Example 5

[0143] Dilute CaS:Eu@SiO2-GOx NPs to 2 mg / mL with ultrapure water and irradiate with a 280 nm xenon lamp, receive the signal and plot the spectrogram. As Figure 9As shown, CaS:Eu@SiO2-GOx NPs have excellent luminescence ability.

[0144] Experimental Example 10, Release of hydrogen sulfide by the fluorescent nanomaterial CaS:2Nd@SiO2-GOx NPs of Example 1 in tumor cells

[0145] Mouse breast cancer 4T1 cells were cultured in DMEM culture medium containing CaS:2Nd@SiO2-GOx NPs (20 μg / mL). After incubation in a 5% CO2, 37 °C incubator for 24 h, the hydrogen sulfide probe (WSP-5) (50 μM) was added, and the cells were cultured for another 1 h. After rinsing three times with PBS buffer solution, fluorescence imaging was performed. The experiment was divided into an experimental group and a blank control group. From Figure 10 the cell imaging pictures, it can be seen that CaS:2Nd@SiO2-GOx NPs can release hydrogen sulfide in tumor cells. The fluorescent nanomaterials of Examples 2 - 5 can also release hydrogen sulfide in tumor cells.

[0146] Experimental Example 11, Release of calcium ions by the fluorescent nanomaterial CaS:2Nd@SiO2-GOx NPs of Example 1 in tumor cells

[0147] Mouse breast 4T1 cancer cells were cultured in DMEM culture medium containing CaS:2Nd@SiO2-GOx NPs (20 μg / mL). After incubation in a 5% CO2, 37 °C incubator for 24 h, the calcium ion fluorescent probe (Fluo-4 AM) (5 μM) was added, and the cells were cultured for another 1 h. After rinsing three times with PBS buffer solution, fluorescence imaging was performed. The experiment was divided into an experimental group and a blank control group. From Figure 11 the cell imaging pictures, it can be seen that CaS:2Nd@SiO2-GOx NPs can release calcium ions in tumor cells.

[0148] The fluorescent nanomaterials of Examples 2 - 5 can also release calcium ions in tumor cells.

[0149] Finally, it should also be noted that the term "comprising", "including" or any other variation thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements, but also includes other elements not expressly listed, or elements inherent to such process, method, article or device.

[0150] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they learn the basic creative concept. Therefore, the appended claims are intended to be construed as including the preferred embodiments and all changes and modifications falling within the scope of the present invention.

[0151] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. A method for preparing a fluorescent nanomaterial, characterized in that, The method includes: Obtaining calcium sulfide nanoparticles doped with rare earth ions; Dispersing the calcium sulfide nanoparticles doped with rare earth ions in a reaction solvent, adding polyoxyethylene nonyl phenyl ether, concentrated ammonia water and tetraethoxysilane, centrifuging after the stirring reaction ends, to obtain calcium sulfide nanoparticles doped with rare earth ions coated with silica; the calcium sulfide nanoparticles doped with rare earth ions are selected from neodymium-doped calcium sulfide nanoparticles, ytterbium / erbium co-doped calcium sulfide nanoparticles, thulium / ytterbium co-doped calcium sulfide nanoparticles, cerium / erbium co-doped calcium sulfide nanoparticles; Mixing the calcium sulfide nanoparticles doped with rare earth ions coated with silica and an amino-modified silicon source in ethanol or water, stirring until the reaction ends, to obtain calcium sulfide nanoparticles doped with rare earth ions coated with silica and modified with amino groups; Activating the carboxyl group of glucose oxidase and then adding the calcium sulfide nanoparticles doped with rare earth ions coated with silica and modified with amino groups for reaction, to obtain a fluorescent nanomaterial.

2. The method according to claim 1, wherein The reaction solvent includes at least one of cyclohexane, ethanol, isopropanol and water.

3. The method according to claim 1, wherein The mass ratio of the calcium sulfide nanoparticles doped with rare earth ions to the tetraethoxysilane is 1:9 to 1:

315.

4. The method according to claim 1, wherein The amino-modified silicon source is selected from (3-aminopropyl)triethoxysilane, 3-(2-aminoethylamino)propyltrimethoxysilane.

5. The method according to claim 1, wherein The mass ratio of the calcium sulfide nanoparticles doped with rare earth ions coated with silica to the amino-modified silicon source is 1:2 to 1:

10.

6. The method according to claim 1, characterized in that, The mass ratio of the glucose oxidase to the calcium sulfide nanoparticles doped with rare earth ions coated with silica and modified with amino groups is 1:20 to 1:

100.

7. A fluorescent nanomaterial prepared by the method according to any one of claims 1-6.

8. The fluorescent nanomaterial according to claim 7, wherein the particle size of the fluorescent nanomaterial is 80-100 nm.

9. Use of the fluorescent nanomaterial according to any one of claims 7-8 in the preparation of a NIR-II fluorescence imaging reagent.

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