Preparation method and application of biotin-modified ultrathin two-dimensional As2Te3 nano material

Abstract

The invention discloses a preparation method and an application of a biotin modified ultrathin two-dimensional As2Te3 nano material. The preparation method comprises the following steps: performing hydrothermal crystallization on As2Te3 amorphous powder, performing ultrasonic liquid phase stripping to obtain the ultrathin two-dimensional As2Te3 nano material, and finally performing biotin modification to obtain the biotin-modified ultrathin two-dimensional As2Te3 nano material. The biotin-modified ultrathin two-dimensional As2Te3 nano material prepared by the invention has an excellent chemotherapy effect, and can effectively target tumor tissues to realize a tumor inhibition effect; meanwhile, anti-tumor immune response can be activated, tumor growth is inhibited, an excellent anti-tumor treatment effect is achieved, and a new scheme is provided for novel targeted chemotherapy and immunotherapy.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomaterials, and in particular to a preparation method and application of a biotin-modified ultrathin two-dimensional As2Te3 nanomaterial. Background Art

[0002] Arsenic-containing drugs, such as arsenic trioxide (ATO), have achieved remarkable efficacy in the treatment of acute promyelocytic leukemia (APL) and have become a first-line clinical treatment. Studies have shown that ATO can achieve a complete remission rate of 83%-95% in APL patients, a highly effective treatment that has attracted widespread attention in the scientific community. However, the high efficacy of arsenic-containing drugs is accompanied by high systemic toxicity, a paradox that severely limits their clinical application in the treatment of solid tumors. For example, arsenic agents may cause serious side effects such as cardiopulmonary dysfunction, liver and kidney damage, and gastrointestinal reactions during treatment. These side effects not only affect patients' quality of life but can also be life-threatening. Therefore, developing new strategies, such as enhancing drug targeting through nanotechnology or combining them with other drugs for combination therapy, to improve their efficacy in the treatment of solid tumors is a key focus of future research.

[0003] Two-dimensional nanomaterials, due to their unique ultrathin layered structures, show great potential in optical properties and biomedical applications. The high specific surface area and high surface energy of these materials give them significant advantages in multiple functions, such as drug delivery and triggering intracellular biochemical reactions. In recent years, arsenene, a single-element two-dimensional nanomaterial from Group V, has been reported to possess excellent optoelectronic properties and loading capacity. Arsenene and its modified nanoplatforms have been shown to be capable of acting as chemotherapy, photodynamic therapy, and immunotherapy drugs, exhibiting excellent anti-tumor effects, giving arsenic-containing two-dimensional nanomaterials broad application prospects in tumor treatment.

[0004] Tellurium (Te), as an important narrow-bandgap p-type semiconductor, has attracted much attention due to its practicality in many fields such as photoresponse, electrochemistry, and optoelectronics. In recent years, many studies have been conducted on the biomedical applications of tellurium nanomaterials. For example, tellurium nanorods, nanodots, and nanosheets have been used as effective photothermal or photodynamic therapeutic agents in tumor treatment. Existing studies have shown that tellurium nanowires can react with hydrogen peroxide (H2O2) in tumor tissue to generate highly toxic TeO6 6- , used for selective cancer chemotherapy. In addition, tellurium-containing nanomaterials have been reported to induce immunogenic cell death in tumor cells in vivo and activate downstream immune responses, effectively enhancing anti-tumor immune effects.

[0005] Arsenic telluride is widely used in semiconductor devices, nanomaterial preparation, electrocatalysis, and flexible two-dimensional electronics. However, little research has been devoted to exploring the biomedical behavior of AsT, particularly its role in cancer cells. Furthermore, the AsT crystals developed so far are primarily clustered in the three-dimensional domain. Despite theoretical calculations predicting a two-dimensional morphology, the synthesis of such ultrathin AsT nanomaterials has yet to be reported. Therefore, developing a material combining tellurium and arsenic for anti-tumor immunotherapy to enhance tumor treatment and anti-tumor immune activation is of great significance. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention provides a method for preparing and applying a biotin-modified ultrathin two-dimensional As2Te3 nanomaterial. The biotin-modified ultrathin two-dimensional As2Te3 nanomaterial prepared by the present invention exhibits excellent anti-tumor immunotherapy effects and can effectively kill 4T1 tumor cells.

[0007] The technical solutions of the present invention are as follows:

[0008] A method for preparing a biotin-modified ultrathin two-dimensional As2Te3 nanomaterial, the preparation method comprising the following steps:

[0009] (1) Preparation of layered As2Te3 crystals: As2Te3 amorphous powder is added to an alkaline solution for hydrothermal reaction. After the reaction is completed, the mixture is cooled to room temperature, washed and dried to obtain layered As2Te3 crystals;

[0010] (2) Preparation of ultrathin two-dimensional As2Te3 nanomaterial dispersion: adding layered As2Te3 crystals to a solvent, subjecting the mixture to ultrasonic liquid phase exfoliation, and collecting the supernatant by differential centrifugation to obtain an ultrathin two-dimensional As2Te3 nanomaterial dispersion;

[0011] (3) Biotin modification: The ultrathin two-dimensional As2Te3 nanomaterial dispersion was dispersed in methanol after solvent replacement, and then mixed with DSPE-PEG 2000 -Biotin methanol solution was mixed, ultrasonically dispersed, and then subjected to rotary evaporation to obtain biotin-modified ultrathin two-dimensional As2Te3 nanomaterials.

[0012] Furthermore, in step (1), the alkali solution is a sodium hydroxide aqueous solution with a pH of 11-13; and the concentration of the As2Te3 amorphous powder in the alkali solution is 0.10-0.12 g / mL.

[0013] Furthermore, in step (1), the temperature of the hydrothermal reaction is 180-200° C., and the reaction time is 12-24 h.

[0014] Furthermore, in step (2), the solvent is dimethyl sulfoxide; and the concentration of the layered As2Te3 crystals in the solvent is 0.4 to 0.6 mg / mL.

[0015] Furthermore, in step (2), the ultrasonic liquid phase stripping is specifically performed as follows: performing ultrasonic liquid phase stripping once at high power, collecting the precipitate by centrifugation, and then performing ultrasonic liquid phase stripping twice at low power.

[0016] Furthermore, the power of the first ultrasonic liquid phase stripping is 1450-1550W, and the time is 3.5-4.5h; the power of the second ultrasonic liquid phase stripping is 550-650W, and the time is 16-20h; the temperature of the ultrasonic liquid phase stripping is 0-4°C; the speed of the centrifugation is 11000rpm, the temperature is 20-25°C, and the time is 10-30min.

[0017] Furthermore, in step (2), the speed of the differential centrifugation is 5000 rpm, the temperature is 20-25° C., and the time is 5-10 min.

[0018] Furthermore, in step (3), the ultrathin two-dimensional As2Te3 nanomaterial and DSPE-PEG 2000 -The mass ratio of Biotin is 1:5-10.

[0019] Furthermore, in step (3), the power of the ultrasound is 500W, the temperature is 0-4°C, and the time is 15-20 minutes; the temperature of the rotary evaporation is 36-42°C, and the time is 0.2-0.5 hours.

[0020] An antitumor drug comprises the biotin-modified ultrathin two-dimensional As2Te3 nanomaterial prepared by the preparation method.

[0021] The beneficial technical effects of the present invention are:

[0022] The invention uses readily available and inexpensive raw materials, does not require inert gas protection, has simple production equipment, process conditions and steps, and is suitable for industrial production.

[0023] By optimizing the conditions of ultrasonic liquid phase exfoliation, the present invention synthesized ultrathin two-dimensional As2Te3 nanosheets with an average thickness of less than 10nm for the first time, confirming that layered arsenic telluride crystals can be generated into ultrathin nanosheets through liquid phase exfoliation.

[0024] DSPE-PEG prepared by the present invention 2000 -Biotin-modified ultra-thin two-dimensional As2Te3 nanomaterials have higher biosafety and more comprehensive tumor immune microenvironment reprogramming effects compared to sodium arsenite, an arsenic-containing drug ingredient currently widely used in clinical practice.

[0025] DSPE-PEG prepared by the present invention 2000 -Biotin-modified ultra-thin two-dimensional As2Te3 nanosheets, after being modified with biotin polymer, have better water solubility, improved blood circulation half-life, and further enhanced biocompatibility. At the same time, the surface-loaded biotin can specifically target tumor tissues in vivo, which is beneficial to the enrichment of drugs in tumors. While maintaining the anti-tumor immunotherapy effect, the drug concentration can be further reduced to reduce toxic side effects.

[0026] The present invention obtains As2Te3 layered crystals by hydrothermal crystallization of arsenic telluride As2Te3 powder. Then, the As2Te3 layered crystals are added to the solvent DMSO and subjected to secondary ultrasonic liquid phase exfoliation. Ultrathin two-dimensional As2Te3 nanomaterials are further obtained by differential centrifugation. Finally, the biotin polymer DSPE-PEG is dispersed by ultrasonic dispersion. 2000 -Biotin modified nanomaterials to obtain As2Te3@DPB. On the one hand, the ultra-thin two-dimensional As2Te3 nanomaterial combines the anti-tumor functions of As and Te, effectively inhibiting tumor growth in vivo with the morphology of ultra-thin two-dimensional materials, and effectively activating the immune response of mice; on the other hand, the biotin polymer DSPE-PEG 2000 -Biotin modification makes the originally poorly soluble nanomaterials water-soluble, improves the blood circulation half-life of the drug in the body, and takes advantage of the fact that tumor cells overexpress sodium complex vitamin transporters that can absorb biotin, thereby improving the drug's targeting in the body and reducing toxic side effects. During the treatment process, the ultra-thin two-dimensional As2Te3 nanomaterial group and the biotin polymer DSPE-PEG 2000 -Biotin modified nanomaterials to obtain As2Te3@DPB did not cause mouse death.

[0027] The ultrathin two-dimensional As2Te3 nanomaterial prepared by the present invention has an average thickness of 6.75nm, and the biotin polymer DSPE-PEG 2000 -Biotin modified nanomaterials obtained As2Te3@DPB dynamic light scattering particle size of 156.3nm, with excellent anti-tumor immunotherapy effect, can effectively kill tumor cells 4T1, the IC50 before and after biotin modification were 0.57μg / mL and 0.41μg / mL, respectively.

[0028] The ultrathin two-dimensional As2Te3 nanomaterials and modified As2Te3 nanomaterials prepared by the present invention are both suitable for anti-tumor immunotherapy, but the modified As2Te3 nanomaterials have better anti-tumor effects. The ultrathin two-dimensional As2Te3 nanomaterials and the modified As2Te3 nanomaterials achieve anti-tumor effects by inducing ferroptosis in 4T1 cancer cells. First, the materials disrupt the iron homeostasis in cancer cells by upregulating the Hmox1 protein. It also inhibits the uptake of cystine by cancer cells, resulting in insufficient synthesis of the reducing substance glutathione in the cells, making it impossible to prevent lipid peroxidation in the cells, and ultimately inducing ferroptosis. Because the surface of the modified As2Te3 nanomaterial is covered with biotin that can be specifically taken up by cancer cells, the modified As2Te3 nanomaterial is more effective in inducing ferroptosis in cancer cells and has stronger biocompatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The layered As2Te3 crystals and As2Te3NS prepared in Example 1 of the present invention S and X-ray powder diffraction patterns of As2Te3@DPB.

[0030] Figure 2 As2Te3NS prepared in Example 1 and Comparative Examples 1-2 of the present invention S Transmission electron micrograph of .

[0031] In the figure: A, As2Te3NS prepared in Example 1 S Transmission electron micrograph of As2Te3NS prepared in Comparative Example 1 S Transmission electron micrograph of As2Te3NS prepared in Comparative Example 2 S Transmission electron micrograph of .

[0032] Figure 3 As2Te3NS prepared in Example 1 of the present invention S Atomic force microscope photo.

[0033] Figure 4 As2Te3NS prepared in Example 1 of the present invention S and As2Te3@DPB and biotin polymer DSPE-PEG 2000 -Infrared absorption spectrum of Biotin.

[0034] Figure 5 As2Te3NS prepared in Example 1 of the present invention S X-ray photoelectron spectrum.

[0035] Figure 6This is the X-ray photoelectron spectrum of As2Te3@DPB prepared in Example 1 of the present invention.

[0036] Figure 7 As2Te3NS prepared in Example 1 of the present invention S and dynamic light scattering particle size analysis of As2Te3@DPB.

[0037] Figure 8 As2Te3NS prepared in Example 1 of the present invention S And the cytotoxicity test results of As2Te3@DPB on mouse breast cancer cells.

[0038] Figure 9 The NaAsO2, Na2TeO3, As2Te3 Bulk and As2Te3NS prepared in Example 1 are used in the present invention. S Comparative imaging of live / dead staining fluorescence of mouse breast cancer cells after treatment with As2Te3@DPB.

[0039] Figure 10 As2Te3NS prepared in Example 1 of the present invention S The cytotoxicity test results of As2Te3@DPB on mouse gastric cancer cells, mouse pancreatic cancer cells, human cervical squamous cell carcinoma cells, human breast cancer cells and human pancreatic cancer cells.

[0040] Figure 11 As2Te3NS prepared in Example 1 of the present invention S And As2Te3@DPB uptake by mouse breast cancer cells (4T1) in vitro.

[0041] Figure 12 As2Te3NS prepared in Example 1 of the present invention S and the enrichment of As2Te3@DPB in tumor tissues in vivo.

[0042] Figure 13 The present invention uses NaAsO2, Na2TeO3, As2Te3 Bulk and As2Te3NS prepared in Example 1 S Tumor volume of mice treated with As2Te3@DPB.

[0043] Figure 14 The present invention uses NaAsO2, Na2TeO3, As2Te3 Bulk and As2Te3NS prepared in Example 1 S Flow cytometric analysis of DC cell maturation and differentiation in the tumor-draining lymph nodes of mice after treatment with As2Te3@DPB.

[0044] Figure 15The present invention uses NaAsO2, Na2TeO3, As2Te3 Bulk and As2Te3NS prepared in Example 1 S CD4 in mouse tumor tissue after treatment with As2Te3@DPB + / CD8 + T cell flow cytometry plot.

[0045] Figure 16 The present invention uses NaAsO2, Na2TeO3, As2Te3 Bulk and As2Te3NS prepared in Example 1 S Spleen-specific effector CD8 in mice treated with As2Te3@DPB + T cell flow cytometry plot.

[0046] Figure 17 The present invention uses NaAsO2, Na2TeO3, As2Te3 Bulk and As2Te3NS prepared in Example 1 S Figure 3. Changes in mouse body weight after treatment with As2Te3@DPB.

[0047] Figure 18 The present invention uses NaAsO2, Na2TeO3, As2Te3 Bulk and As2Te3NS prepared in Example 1 S Blood biochemistry and blood routine indicators of mice after treatment with As2Te3@DPB. DETAILED DESCRIPTION

[0048] The present invention will be described in detail below with reference to the accompanying drawings and embodiments.

[0049] The raw material As2Te3 amorphous powder (As2Te3 Bulk) in the following examples of the present invention was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. with the product number A860760; DSPE-PEG 2000 -Biotin (DPB) was purchased from Shanghai Pengshuo Biotechnology Co., Ltd., where the molecular weight of PEG was 2000.

[0050] The method for constructing the experimental animal 4T1 tumor-bearing mouse model used in the following test examples of the present invention is as follows: 4T1 cell suspension PBS (1×10 6 cells / mouse) were injected subcutaneously into the axilla of female Balb / c mice. When the tumor grew to 150 mm 3 At this time, the 4T1 cancer cell model was established, and the 4T1 tumor-bearing mouse model was obtained for subsequent experiments.

[0051] Example 1

[0052] A method for preparing a biotin-modified ultrathin two-dimensional As2Te3 nanomaterial comprises the following steps:

[0053] (1) Preparation of layered As2Te3 crystals: 3 g of As2Te3 bulk was added to 27 mL of a sodium hydroxide aqueous solution with a pH of 12, and a hydrothermal reaction was carried out at 180°C for 24 h. After the reaction was completed, the mixture was cooled to room temperature, washed, and dried to obtain layered As2Te3 crystals.

[0054] (2) Preparation of ultrathin two-dimensional As2Te3 nanomaterials: 100 mg of layered As2Te3 crystals were added to 250 mL of ultra-dry dimethyl sulfoxide. In an ice-water bath, ultrasonic liquid phase stripping was performed at 1500 W for 4 h, and then the precipitate was collected by centrifugation at 11000 rpm for 10 min. Ultrathin liquid phase stripping was performed at 600 W for 16 h. Differential centrifugation was performed at 25 ° C and 5000 rpm for 5 min. The supernatant was collected and concentrated at room temperature to obtain an ultrathin two-dimensional As2Te3 nanomaterial (As2Te3NS) with a concentration of 0.2 mg / mL. S ) dispersion.

[0055] (3) Biotin modification: 1.0 mL of 0.2 mg / mL ultrathin two-dimensional As2Te3 nanomaterial dispersion was dispersed in methanol after solvent replacement, and then mixed with 10 mL of 0.1 mg / mL DSPE-PEG 2000 -Biotin methanol solution was mixed, ultrasonically dispersed at 0℃ and 500W for 15min, and then rotary evaporated at 42℃ for 0.2h to obtain biotin-modified ultrathin two-dimensional As2Te3 nanomaterial As2Te3@DPB.

[0056] Example 2

[0057] A method for preparing a biotin-modified ultrathin two-dimensional As2Te3 nanomaterial comprises the following steps:

[0058] (1) Preparation of layered As2Te3 crystals: 3 g of As2Te3 bulk was added to 27 mL of a sodium hydroxide aqueous solution with a pH of 11, and a hydrothermal reaction was carried out at 190°C for 20 h. After the reaction was completed, the mixture was cooled to room temperature, washed, and dried to obtain layered As2Te3 crystals.

[0059] (2) Preparation of ultrathin two-dimensional As2Te3 nanomaterials: 100 mg of layered As2Te3 crystals were added to 250 mL of ultra-dry dimethyl sulfoxide. In an ice-water bath, ultrasonic liquid phase stripping was performed at 1450 W for 4.5 h, and then the precipitate was collected by centrifugation at 11000 rpm for 20 min. Ultrathin liquid phase stripping was then performed at 550 W for 20 h. Differential centrifugation was performed at 25 ° C and 5000 rpm for 5 min. The supernatant was collected and concentrated at room temperature to obtain an ultrathin two-dimensional As2Te3 nanomaterial (As2Te3NS) with a concentration of 0.25 mg / mL. S ) dispersion.

[0060] (3) Biotin modification: 1.0 mL of ultrathin two-dimensional As2Te3 nanomaterial dispersion with a concentration of 0.25 mg / mL was dispersed in methanol after solvent replacement, and then mixed with 15 mL of DSPE-PEG with a concentration of 0.15 mg / mL. 2000 -Biotin methanol solution was mixed, ultrasonically dispersed at 2°C and 500W for 18 minutes, and then rotary evaporated at 40°C for 0.35h to obtain biotin-modified ultrathin two-dimensional As2Te3 nanomaterials As2Te3@DPB.

[0061] Example 3

[0062] A method for preparing a biotin-modified ultrathin two-dimensional As2Te3 nanomaterial comprises the following steps:

[0063] (1) Preparation of layered As2Te3 crystals: 3 g of As2Te3 bulk was added to 27 mL of a sodium hydroxide aqueous solution with a pH of 13, and a hydrothermal reaction was carried out at 200°C for 12 h. After the reaction was completed, the mixture was cooled to room temperature, washed, and dried to obtain layered As2Te3 crystals.

[0064] (2) Preparation of ultrathin two-dimensional As2Te3 nanomaterials: 100 mg of layered As2Te3 crystals were added to 250 mL of ultra-dry dimethyl sulfoxide. In an ice-water bath, ultrasonic liquid phase stripping was performed at 1550 W for 3.5 h, and then centrifuged at 11000 rpm for 10 min to collect the precipitate. Ultrathin liquid phase stripping was then performed at 650 W for 16 h. Differential centrifugation was performed at 20 ° C and 5000 rpm for 10 min. The supernatant was collected and concentrated at room temperature to obtain an ultrathin two-dimensional As2Te3 nanomaterial (As2Te3NS) with a concentration of 0.3 mg / mL. S ) dispersion.

[0065] (3) Biotin modification: 1.0 mL of 0.3 mg / mL ultrathin two-dimensional As2Te3 nanomaterial dispersion was dispersed in methanol after solvent replacement, and then mixed with 10 mL of 0.2 mg / mL DSPE-PEG2000 -Biotin methanol solution was mixed, ultrasonically dispersed at 4°C and 500W for 20 minutes, and then rotary evaporated at 36°C for 0.5h to obtain biotin-modified ultrathin two-dimensional As2Te3 nanomaterials As2Te3@DPB.

[0066] Comparative Example 1

[0067] A method for preparing As2Te3 nanomaterials comprises the following steps:

[0068] (1) Preparation of layered As2Te3 crystals: 3 g of As2Te3 bulk was added to 27 mL of a sodium hydroxide aqueous solution with a pH of 12, and a hydrothermal reaction was carried out at 180°C for 24 h. After the reaction was completed, the mixture was cooled to room temperature, washed, and dried to obtain layered As2Te3 crystals.

[0069] (2) Preparation of ultrathin two-dimensional As2Te3 nanomaterials: 100 mg of layered As2Te3 crystals were added to 250 mL of ultra-dry NMP. In an ice-water bath, ultrasonic liquid phase stripping was performed at 1500 W for 4 h, and then the precipitate was collected by centrifugation at 11000 rpm for 10 min. Ultrasonic liquid phase stripping was then performed at 600 W for 16 h. Differential centrifugation was performed at 25 ° C and 5000 rpm for 5 min. The supernatant was collected and concentrated at room temperature to obtain As2Te3 nanomaterials (As2Te3NS) with a concentration of 0.2 mg / mL. S ) dispersion.

[0070] Comparative Example 2

[0071] A method for preparing As2Te3 nanomaterials comprises the following steps:

[0072] (1) Preparation of layered As2Te3 crystals: 3 g of As2Te3 bulk was added to 27 mL of a sodium hydroxide aqueous solution with a pH of 12, and a hydrothermal reaction was carried out at 180°C for 24 h. After the reaction was completed, the mixture was cooled to room temperature, washed, and dried to obtain layered As2Te3 crystals.

[0073] (2) Preparation of As2Te3 nanomaterials: 100 mg of layered As2Te3 crystals were added to 250 mL of ultra-dry DMSO. In an ice-water bath, ultrasonic liquid phase stripping was performed at 1500 W for 4 h, and then centrifuged at 5000 rpm for 10 min to collect the supernatant. The supernatant was placed in an activated dialysis bag with a molecular weight cutoff of 7 kDa and placed in a 60 g / L PEG aqueous solution. The peripheral aqueous solution was changed every 8 h. After 24 h, it was placed in a 100 g / L PEG aqueous solution for concentration to obtain As2Te3 nanomaterials (As2Te3NS S ) dispersion.

[0074] Comparative Example 3

[0075] A method for preparing As2Te3 nanomaterials comprises the following steps:

[0076] (1) Preparation of layered As2Te3 crystals: Same as step (1) in Example 1.

[0077] (2) Preparation of ultrathin two-dimensional As2Te3 nanomaterials: 100 mg of layered As2Te3 crystals were added to 250 mL of ultra-dry dimethyl sulfoxide. In an ice-water bath, ultrasonic liquid phase stripping was performed at 1700 W for 4 h, and then the precipitate was collected by centrifugation at 11000 rpm for 10 min. Ultrasonic liquid phase stripping was then performed at 600 W for 16 h. Differential centrifugation was performed at 25 ° C and 5000 rpm for 5 min. The supernatant was collected and concentrated at room temperature to obtain As2Te3 nanomaterials (As2Te3NS) with a concentration of 0.2 mg / mL. S ) dispersion.

[0078] Results: The As2Te3 nanomaterials obtained in this comparative example were small in size, no obvious flake structure could be observed, and the tellurium-arsenic ratio was unbalanced, with some materials being oxidized.

[0079] Comparative Example 4

[0080] A method for preparing As2Te3 nanomaterials comprises the following steps:

[0081] (1) Preparation of layered As2Te3 crystals: Same as step (1) in Example 1.

[0082] (2) Preparation of ultrathin two-dimensional As2Te3 nanomaterials: 100 mg of layered As2Te3 crystals were added to 250 mL of ultra-dry dimethyl sulfoxide. In an ice-water bath, ultrasonic liquid phase stripping was performed at 1300 W for 4 h, and then the precipitate was collected by centrifugation at 11000 rpm for 10 min. Ultrasonic liquid phase stripping was then performed at 600 W for 16 h. Differential centrifugation was performed at 25 ° C and 5000 rpm for 5 min. The supernatant was collected and concentrated at room temperature to obtain As2Te3 nanomaterials (As2Te3NS) with a concentration of 0.2 mg / mL. S ) dispersion.

[0083] Results: The As2Te3 nanomaterials obtained in this comparative example have a relatively uniform size, but the yield is relatively low.

[0084] A method for preparing an ultrathin two-dimensional As2Te3 nanomaterial comprises the following steps:

[0085] (1) Preparation of layered As2Te3 crystals: Same as step (1) in Example 1.

[0086] (2) Preparation of ultrathin two-dimensional As2Te3 nanomaterials: 100 mg of layered As2Te3 crystals were added to 250 mL of ultra-dry dimethyl sulfoxide. In an ice-water bath, ultrasonic liquid phase stripping was performed at 1500 W for 4 h, and then the precipitate was collected by centrifugation at 11000 rpm for 10 min. Ultrasonic liquid phase stripping was then performed at 400 W for 16 h. Differential centrifugation was performed at 25 ° C and 5000 rpm for 5 min. The supernatant was collected and concentrated at room temperature to obtain As2Te3 nanomaterials (As2Te3NS) with a concentration of 0.2 mg / mL. S ) dispersion.

[0087] Results: The As2Te3 nanomaterials obtained in this comparative example had relatively uniform size, but the yield was low.

[0088] Comparative Example 6

[0089] A method for preparing an ultrathin two-dimensional As2Te3 nanomaterial comprises the following steps:

[0090] (1) Preparation of layered As2Te3 crystals: Same as step (1) in Example 1.

[0091] (2) Preparation of ultrathin two-dimensional As2Te3 nanomaterials: 100 mg of layered As2Te3 crystals were added to 250 mL of ultra-dry dimethyl sulfoxide. In an ice-water bath, ultrasonic liquid phase stripping was performed at 1500 W for 4 h, and then the precipitate was collected by centrifugation at 11000 rpm for 10 min. Ultrathin liquid phase stripping was performed at 800 W for 16 h. Differential centrifugation was performed at 25 ° C and 5000 rpm for 5 min. The supernatant was collected and concentrated at room temperature to obtain an ultrathin two-dimensional As2Te3 nanomaterial (As2Te3NS) with a concentration of 0.2 mg / mL. S ) dispersion.

[0092] Results: The As2Te3 nanomaterial obtained in this comparative example has an unbalanced tellurium-arsenic ratio, and part of the material is oxidized.

[0093] Test Case

[0094] (1) Composition and morphology characterization

[0095] The test objects for composition and morphology characterization include the layered As2Te3 crystals prepared in Example 1, As2Te3NS S Dispersion, As2Te3@DPB and As2Te3NS prepared in Comparative Examples 1-2 S Dispersion, in which layered As2Te3 crystals and As2Te3@DPB were dissolved and diluted to 0.5 mg / mL using PBS, and As2Te3NS S The dispersion was concentrated to 0.5 mg / mL at room temperature to obtain the corresponding test sample. The specific test was as follows:

[0096] Take the layered As2Te3 crystals and As2Te3NS prepared in Example 1 with a concentration of 0.5 mg / mL S and As2Te3@DPB, the composition and crystal structure of the particles were observed by X-ray diffractometer. Figure 1 As shown. Figure 1 The results show that the layered As2Te3 crystals and As2Te3NS S The material composition is As2Te3, and the As2Te3@DPB modified with biotin polymer does not destroy the crystal structure of the particles.

[0097] Take As2Te3NS prepared in Example 1 and Comparative Examples 1-2 at a concentration of 0.5 mg / mL S , its structure was observed by transmission electron microscopy, and the results were as follows Figure 2 As shown. Figure 2 The results show that after two ultrasonic liquid-phase exfoliation treatments, the layered As2Te3 crystals were exfoliated into thin flakes in the extremely strong solvent DMSO. When liquid-phase exfoliation was performed using NMP as the solvent, it was found that NMP, a less polar solvent than DMSO, was unable to effectively disperse the As2Te3 crystals under ultrasonic probes of equivalent power. Furthermore, when dialysis was used to replace the solution to obtain an As2Te3 NSs dispersion, it was found that the dialysis process caused the material to aggregate, resulting in a loss of its original morphology.

[0098] Take the As2Te3NS prepared in Example 1 with a concentration of 0.5 mg / mL S , its thickness was measured by atomic force microscopy, and the results were as follows Figure 3 As shown. Figure 3 The results show that As2Te3NS S The average thickness is about 6.75nm.

[0099] Take the As2Te3NS prepared in Example 1 with a concentration of 0.5 mg / mL S and As2Te3@DPB, and raw material DSPE-PEG 2000 -Biotin, detected by infrared spectrometer, the results are as follows Figure 4 As shown. Figure 4 The results show that As2Te3@DPB has both As2Te3NS S and biotin polymer DSPE-PEG 2000 -The characteristic infrared peak of Biotin, which confirms the As2Te3NS S DSPE-PEG with biotin polymer 2000 -Biotin successfully combined.

[0100] Take the As2Te3NS prepared in Example 1 with a concentration of 0.5 mg / mL S , detected by X-ray photoelectron spectroscopy, the results are as follows Figure 5 As shown. Figure 5 The results show that As2Te3NS S The As element has two binding energies of 40.65eV and 42.10eV, with a peak area ratio of 2:3, corresponding to As atoms in two different chemical environments; the two binding energies of Te are 573.08eV and 572.75eV, corresponding to Te 2- 3D 3 / 2 and 3D 5 / 2 track.

[0101] The As2Te3@DPB prepared in Example 1 was taken at a concentration of 0.5 mg / mL and detected by X-ray photoelectron spectrometer. The results are as follows: Figure 6 As shown. Figure 6 The results show that in addition to the two binding energies of 40.65eV and 42.10eV, As in As2Te3@DPB also has a higher binding energy of 44.53eV. Due to the modification of biotin polymer, some of the peripheral As and DSPE-PEG 2000 -Biotin forms an As-O bond. Te element, in addition to Te 2- 3D 3 / 2 and 3D 5 / 2 In addition to the two binding energies corresponding to the orbitals, there is another binding energy of 576 eV, which is close to the theoretical value of TeO2.

[0102] Take the As2Te3NS prepared in Example 1 with a concentration of 0.5 mg / mL S and As2Te3@DPB, were detected by dynamic light scattering nanoparticle size analyzer, and the results were as follows Figure 7 As shown. Figure 7 The results show that As2Te3NS S The average particle size of As2Te3@DPB is 122.0nm, and the average particle size of As2Te3@DPB is 156.3nm.

[0103] (2) In vitro cytotoxicity test

[0104] Mouse breast cancer cells (4T1) were used as experimental subjects and the cells were cultured to a density of 5×10 4 cells / mL, add As2Te3NS prepared in Example 1 of the present invention at a concentration of 0.1-4 μg / mL S and As2Te3@DPB (prepared or diluted with PBS) for 24 h, and the As2Te3NS prepared in Example 1 of the present invention was detected.S and As2Te3@DPB to 4T1 median lethal dose (IC50) toxicity, the results are as follows Figure 8 As shown. Figure 8 The results show that As2Te3NS S The IC50 of As2Te3@DPB to 4T1 is 0.57μg / mL, and the IC50 of As2Te3@DPB to 4T1 is 0.41μg / mL. The toxicity of As2Te3@DPB to 4T1 is higher than that of As2Te3NS S About 1.4 times of .

[0105] Mouse breast cancer cells (4T1) were used as experimental subjects and the cells were cultured to a density of 5×10 4 cells / mL, and then divided into 6 groups, including control group (Control), NaAsO2 group, Na2TeO3 group, As2Te3 Bulk group, As2Te3NSs group, and As2Te3@DPB group; the NaAsO2 group was treated with 0.488μg / mL NaAsO2 solution (solvent: PBS) for 24 h, the Na2TeO3 group was treated with 1.25μg / mL Na2TeO3 solution (solvent: PBS) for 24 h, and the As2Te3 Bulk group was treated with 1μg / mL As2Te3 4T1 cells were treated with bulk solution (solvent: PBS) for 24 h, As2Te3NSs group was treated with 1 μg / mL As2Te3NSs (solvent: PBS) prepared in Example 1 of the present invention for 24 h, As2Te3@DPB group was treated with 1 μg / mL As2Te3@DPB prepared in Example 1 of the present invention for 24 h, and the control group was treated with an equal amount of PBS for 24 h. The cells were then collected and stained with calcein AM and propidium iodide to detect cytotoxicity. The results are shown in Figure 2. Figure 9 As shown. Figure 9 The results show that Te itself is highly toxic. While treating cells with an equivalent amount of Te to that in the As2Te3@DPB group effectively kills tumor cells, this toxicity is indiscriminate. The introduction of Te significantly enhances the cytotoxicity of the As2T3 NSs group. Furthermore, modification with DSPE-PEG2000-Biotin does not reduce the cytotoxicity of As2T3 NSs, but rather further enhances their cytotoxicity by increasing their water solubility and effectively binding to biotin receptors on the cancer cell surface.

[0106] Other cancer cell lines, including mouse gastric cancer cells (MFC), mouse pancreatic cancer cells (Pan02), human cervical squamous cell carcinoma cells (SiHa), human breast cancer cells (MCF-7), and human pancreatic cancer cells (Panc-1), were used as experimental subjects. The cells were cultured to a density of 5×10 4 cells / mL, then the As2Te3NS prepared in Example 1 of the present invention was added to the SiHa cells at a concentration of 0.001-0.16 μg / mL. S and As2Te3@DPB (prepared or diluted with PBS) for 24 h, MFC cells, Pan02 cells, MCF-7 cells, and Panc-1 cells were added with As2Te3NS prepared in Example 1 of the present invention at a concentration of 0.1-16 μg / mL. S and As2Te3@DPB (prepared or diluted with PBS) for 24 h, and the As2Te3NS prepared in Example 1 of the present invention was detected. S and As2Te3@DPB to its median lethal dose (IC50) toxicity, the results are as follows Figure 10 and as shown in Table 1.

[0107] Table 1

[0108]

[0109]

[0110] from Figure 9 As can be seen from the results in Table 1, As2Te3NS S As2Te3@DPB and As2Te3@DPB have high killing activity against various cancer cells. Among them, As2Te3@DPB is more active against Pan02, SiHa and MCF-7 cancer cells.

[0111] (3) Targeted testing

[0112] Mouse breast cancer cells (4T1) were used as experimental subjects to observe the As2Te3NS prepared in Example 1 of the present invention. S The results of As2Te3@DPB uptake by mouse breast cancer cells (4T1) in vitro are as follows: Figure 10 As shown. Figure 11 The results show that at 12h, the uptake of As2Te3@DPB by mouse breast cancer cells (4T1) far exceeds that of As2Te3NS S .

[0113] The 4T1 tumor-bearing mouse model was used as the experimental object to observe the As2Te3NS prepared in Example 1 of the present invention. S The enrichment of As2Te3@DPB in tumor tissues in vivo is shown in the following figure. Figure 12 As shown. Figure 12 The results show that the enrichment of As2Te3@DPB in the tumor area is higher than that of As2Te3NS 24 hours and even 7 days after administration. S .

[0114] The 4T1 tumor-bearing mouse model was used as the experimental object and divided into control group (Control), NaAsO2 group, Na2TeO3 group, As2Te3 Bulk group, As2Te3NSs group, and As2Te3@DPB group. The NaAsO2 group, Na2TeO3 group, As2Te3 Bulk group, As2Te3NSs group, and As2Te3@DPB group were injected with NaAsO2, Na2TeO3, As2Te3 Bulk, As2Te3NSs prepared in Example 1 of the present invention at a dose of 1.5 mg / kg, respectively. S and As2Te3@DPB, and the 4T1 tumor-bearing mouse model injected with the same amount of normal saline was used as the control group. The drugs were administered on days 0, 3, 6, and 10, and the tumor volume was detected. The results are shown in Figure 5. Figure 13 As shown. Figure 13 The results show that As2Te3NS S Both As2Te3@DPB treatment showed a very significant inhibitory effect on the tumor volume of mice.

[0115] (4) Immune index detection

[0116] The 4T1 tumor-bearing mouse model was used as the experimental object and divided into control group (Control), NaAsO2 group, Na2TeO3 group, As2Te3 Bulk group, As2Te3NSs group, and As2Te3@DPB group. The NaAsO2 group, Na2TeO3 group, As2Te3 Bulk group, As2Te3NSs group, and As2Te3@DPB group were injected with NaAsO2, Na2TeO3, As2Te3 Bulk, As2Te3NSs prepared in Example 1 of the present invention at a dose of 1.5 mg / kg, respectively. S and As2Te3@DPB, and the 4T1 tumor-bearing mice injected with an equal amount of saline were used as the control group. 24 hours after administration, the mice were dissected and the lymph nodes in the tumor drainage area were taken to detect CD80 + Dendritic cells, CD86 + Dendritic cell content, take tumor area tissue to detect CD4 + T cells and CD8 + T cell content, take the mouse spleen to detect effector CD8 + T cell content, the results are as follows Figure 14-16 As shown. Figure 14The results showed that the CD80 + Dendritic cells and CD86 + The content of dendritic cells increased significantly. Figure 15 The results showed that the CD8 + T cell content increased. Figure 16 The results showed that the effector CD8 + The T cell content increased. This shows that the cytotoxic T cell content in mice treated with As2Te3@DPB is higher and the immune activity is stronger.

[0117] (5) Biosafety testing

[0118] The 4T1 tumor-bearing mouse model was used as the experimental object and divided into control group (Control), NaAsO2 group, Na2TeO3 group, As2Te3 Bulk group, As2Te3NSs group, and As2Te3@DPB group. The NaAsO2 group, Na2TeO3 group, As2Te3 Bulk group, As2Te3NSs group, and As2Te3@DPB group were injected with NaAsO2, Na2TeO3, As2Te3 Bulk, As2Te3NSs prepared in Example 1 of the present invention at a dose of 1.5 mg / kg, respectively. S and As2Te3@DPB, and the 4T1 tumor-bearing mice model injected with the same amount of normal saline was used as the control group. The weight changes, blood biochemistry and blood routine tests of the mice were detected. The results are as follows Figure 17-18 As shown. Figure 17 The results show that As2Te3NS S There was no significant difference in the body weight of mice treated with As2Te3@DPB compared with the control group. Figure 18 The results showed that the blood biochemistry and blood routine indicators of As2Te3@DPB treated mice were not significantly different from those of the control group mice; while As2Te3NS S Several indicators of blood biochemistry and routine blood tests (including WBC, RBC, HGB, HCT, MCV, MCH, MCHC, PL, ALT, AST, GOT, UREA, LDH) of treated mice showed slight differences compared with those of control mice. During the in vivo experiment, we found that when Na2TeO3 PBS solution with the same Te element concentration as As2Te3 NSs and As2Te3@DPB was intravenously injected into mice, it caused acute toxic death of mice. Subsequent experiments with lower Na2TeO3 concentrations showed that blood biochemical indicators were still abnormal ( Figure 17). Therefore, the introduction of Te improves the activity of arsenic drugs and avoids the indiscriminate toxicity of Te itself. The blood biochemical indicators of mice treated with As2Te3@DPB are closest to those of normal mice. S The killing effect of As2Te3@DPB on tumor cells is similar to that of As2Te3@DPB, but As2Te3@DPB has a stronger immune activation effect on 4T1 tumor-bearing mice and better biocompatibility.

[0119] The above description is merely a preferred embodiment of the present invention, and the present invention is not limited to the above embodiment. It is understood that other improvements and variations directly derived or imagined by those skilled in the art without departing from the spirit and concept of the present invention should be considered to be included in the scope of protection of the present invention.

Claims

1. A method for preparing a biotin-modified ultrathin two-dimensional As2Te3 nanomaterial, characterized in that: The preparation method comprises the following steps: (1) Preparation of layered As2Te3 crystals: As2Te3 amorphous powder is added to an alkaline solution for hydrothermal reaction. After the reaction is completed, the mixture is cooled to room temperature, washed and dried to obtain layered As2Te3 crystals; (2) Preparation of ultrathin two-dimensional As2Te3 nanomaterial dispersion: adding layered As2Te3 crystals to a solvent, subjecting the mixture to ultrasonic liquid phase exfoliation, and collecting the supernatant by differential centrifugation to obtain an ultrathin two-dimensional As2Te3 nanomaterial dispersion; (3) Biotin modification: The ultrathin two-dimensional As2Te3 nanomaterial dispersion was dispersed in methanol after solvent replacement, and then mixed with DSPE-PEG 2000 -Biotin methanol solution was mixed, ultrasonically dispersed, and then subjected to rotary evaporation to obtain biotin-modified ultrathin two-dimensional As2Te3 nanomaterials.

2. The preparation method according to claim 1, characterized in that In step (1), the alkali solution is a sodium hydroxide aqueous solution with a pH of 11-13; and the concentration of the As2Te3 amorphous powder in the alkali solution is 0.10-0.12 g / mL.

3. The preparation method according to claim 1, characterized in that In step (1), the temperature of the hydrothermal reaction is 180-200° C., and the reaction time is 12-24 hours.

4. The preparation method according to claim 1, characterized in that In step (2), the solvent is dimethyl sulfoxide; the concentration of the layered As2Te3 crystals in the solvent is 0.4 to 0.6 mg / mL.

5. The preparation method according to claim 1, characterized in that In step (2), the ultrasonic liquid phase stripping is specifically performed as follows: performing ultrasonic liquid phase stripping once at high power, collecting the precipitate by centrifugation, and then performing ultrasonic liquid phase stripping twice at low power.

6. The preparation method according to claim 5, characterized in that The power of the first ultrasonic liquid phase stripping is 1450-1550W, and the time is 3.5-4.5h; the power of the second ultrasonic liquid phase stripping is 550-650W, and the time is 16-20h; the temperature of the ultrasonic liquid phase stripping is 0-4°C; the speed of the centrifugation is 11000rpm, the temperature is 20-25°C, and the time is 10-30min.

7. The preparation method according to claim 1, characterized in that In step (2), the speed of the differential centrifugation is 5000 rpm, the temperature is 20-25° C., and the time is 5-10 min.

8. The preparation method according to claim 1, characterized in that In step (3), the ultrathin two-dimensional As2Te3 nanomaterial and DSPE-PEG 2000 -The mass ratio of Biotin is 1:5-10.

9. The preparation method according to claim 1, characterized in that In step (3), the power of the ultrasound is 500W, the temperature is 0-4°C, and the time is 15-20 minutes; the temperature of the rotary evaporation is 36-42°C, and the time is 0.2-0.5 hours.

10. An anti-tumor drug, characterized in that: The anti-tumor drug comprises the biotin-modified ultra-thin two-dimensional As2Te3 nanomaterial prepared by the preparation method according to any one of claims 1 to 9.

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