Ultrathin two-dimensional As2Se3 nanosheet, preparation method, and application

Abstract

The present invention belongs to the field of nanomaterial technology, and specifically relates to an ultrathin two-dimensional As2Se3 nanosheet, its preparation method, and its application. The preparation method comprises subjecting As2Se3 to hydrothermal crystallization to obtain As2Se3 layered crystals; adding the As2Se3 layered crystals to a solvent, performing ultrasonic liquid phase exfoliation, and obtaining ultrathin two-dimensional As2Se3 nanosheets by differential centrifugation. The ultrathin two-dimensional As2Se3 nanosheets are used in the preparation of anti-tumor products, including at least one of products for reprogramming the tumor microenvironment, products for improving the efficacy of immunotherapy for tumors, products for photothermal therapy for tumors, products for combined photothermal therapy for tumors, and products for inhibiting mouse breast cancer 4T1 cells. The present invention successfully synthesizes ultrathin two-dimensional As2Se3 nanosheets, which are highly practical and have a simple preparation method.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanomaterials, and in particular relates to an ultra-thin two-dimensional As2Se3 nanosheet, a preparation method and an application thereof. Background Art

[0002] Arsenic-containing drugs have become first-line clinical treatments due to their remarkable efficacy in treating acute promyelocytic leukemia (APL). Arsenic trioxide (ATO) has achieved a complete remission rate of 83-95% in APL patients, garnering widespread attention in the scientific community. However, the conflict between the high efficacy and high systemic toxicity of arsenic-containing drugs severely limits their clinical efficacy in treating solid tumors. This dilemma urgently requires the development of new strategies to effectively detoxify arsenic and enhance its therapeutic efficacy.

[0003] Selenium (Se) is an essential trace element for maintaining human health. In 1938, Moxon et al. discovered that arsenic and selenium can detoxify each other, a finding subsequently validated in numerous experiments. Mechanistic studies have shown that arsenic and selenium react in the liver to form a detoxifying complex that is then excreted into the bile, ultimately achieving detoxification. Importantly, nutritional intake of Se can modulate immune function through the diverse redox activities of selenoproteins, reversing immunosuppression in the tumor microenvironment and promoting anti-tumor immune activation, thereby reducing cancer incidence. Pioneering work by Chen Tianfeng et al. in the development of Se therapy has revealed the enormous potential of Se in reprogramming the immune microenvironment in lung adenocarcinoma, as a vaccine nanoadjuvant, alleviating atopic dermatitis, identifying metallo-drugs, and reinvigorating cancer radioimmunotherapy. Therefore, the combination of Se and arsenic is a crucial step in anti-tumor immunotherapy, promising reduced toxic side effects and enhanced immune activation.

[0004] Two-dimensional nanomaterials, due to their typically ultrathin layered structure, have been shown to possess excellent optical properties and promising biomedical applications. Their ultrathin structure not only enables rapid response to light but also provides excellent in-plane electron mobility, enabling high photothermal conversion efficiency. Furthermore, their high surface area and high surface energy enable them to simultaneously achieve multiple functions, such as drug delivery and triggering intracellular biochemical reactions.

[0005] Photothermal therapy is a new, noninvasive cancer treatment method that converts light energy into heat to kill tumor cells. Two-dimensional materials from Group V elements, such as black phosphorus and arsenene, have been shown to exhibit excellent anti-tumor photothermal therapeutic effects. Furthermore, theoretical calculations by Xiaoying Zhuang et al. indicate that the bulk lattice band gap energy of arsenic selenide is 1.958 eV, while the band gap energy of double-layer arsenic selenide is 2.202 eV, which is comparable to the band gap energy of single-layer black phosphorus (2.0 eV). Although theoretical calculations predict that arsenic selenide nanosheets may be a promising photothermal agent for anti-tumor treatment, the synthesis of such ultrathin arsenic selenide (As2Se3) nanosheets has yet to be reported.

[0006] Therefore, the inventors studied ultra-thin two-dimensional arsenic selenide (As2Se3) nanosheets from multiple angles and found that they may play a positive role in clinical treatments such as anti-tumor. Summary of the Invention

[0007] In view of the shortcomings of the prior art described above, the first object of the present invention is to provide ultrathin two-dimensional As2Se3 nanosheets. The second object of the present invention is to provide a method for preparing ultrathin two-dimensional As2Se3 nanosheets. The third object of the present invention is to provide an application of ultrathin two-dimensional As2Se3 nanosheets. The significance of the present invention lies in the successful synthesis of ultrathin two-dimensional As2Se3 nanosheets, their high practicality, and the simple preparation method.

[0008] To achieve the above-mentioned and other related purposes, the present invention provides the following technical solutions:

[0009] In a first aspect of the present invention, an ultrathin two-dimensional As2Se3 nanosheet is provided, wherein the ultrathin two-dimensional As2Se3 nanosheet has a thickness of 2.3-2.6 nm and a lateral dimension of 150-300 nm.

[0010] Furthermore, the ultrathin two-dimensional As2Se3 nanosheets have a double-layer structure.

[0011] In a second aspect of the present invention, the method for preparing the ultrathin two-dimensional As2Se3 nanosheets comprises the following steps:

[0012] As2Se3 is hydrothermally crystallized to obtain As2Se3 layered crystals;

[0013] Ultrathin two-dimensional As2Se3 nanosheets were obtained by adding As2Se3 layered crystals to a solvent, performing ultrasonic liquid phase exfoliation, and then using differential centrifugation. The solvent included at least one of NMP, DMSO, and an isopropanol-water mixture. The isopropanol-water mixture can also be expressed as an isopropanol / water mixture, with a volume ratio of isopropanol to water of 3:1. DMSO is dimethyl sulfoxide.

[0014] It should be noted that the present invention uses a hydrothermal reaction to treat amorphous arsenic selenide to produce regular layered crystals, which are conducive to ultrasonic exfoliation to produce As2Se3 nanosheets with uniform thickness and lateral size.

[0015] In one embodiment of the present invention, the hydrothermal crystallization step includes: adding As2Se3 powder to a sodium hydroxide aqueous solution for a hydrothermal reaction. The mass-to-volume ratio of the As2Se3 powder to the sodium hydroxide aqueous solution is 0.9-1.2 g:9 mL. That is, 0.9-1.2 g of As2Se3 powder is added to 9 mL of the sodium hydroxide aqueous solution. Specifically, the As2Se3 powder is amorphous As2Se3 powder, and the sodium hydroxide aqueous solution has a pH of 12.

[0016] After the hydrothermal reaction, the mixture was cooled to room temperature, washed and dried to obtain As2Se3 layered crystals.

[0017] Furthermore, the specific steps of washing and drying include: repeatedly blowing the product cooled to room temperature into 20~50mL of ddH2O, centrifuging at a speed of 2000rpm for 5~10min, repeating 3 times, and then taking the precipitate and drying it at 60℃ for 3h, and then drying it at 30℃ for 12-24h.

[0018] In one embodiment of the present invention, the hydrothermal crystallization is carried out in a stainless steel autoclave with a polytetrafluoroethylene liner, the reaction temperature is 190-200° C., and the reaction time is 12-24 hours.

[0019] By adopting the above technical solution, the stainless steel autoclave with a polytetrafluoroethylene liner provides a high-pressure environment, and the present invention seals the As2Se3 powder and the sodium hydroxide aqueous solution in the high-pressure environment for reaction.

[0020] In one embodiment of the present invention, the ultrasonic liquid phase stripping step includes: performing ultrasonic liquid phase stripping twice, both times in ice water bath conditions, the first ultrasonic power is 1200~1600W, the ultrasonic time is 2~4h, and the ultrasonic power is ≥11000W. The pellet was collected by centrifugation at 0.5000 rpm and subjected to a second ultrasound with an ultrasound power of 600-650 W and an ultrasound time of 12-16 h.

[0021] Furthermore, during the ultrasonic liquid phase exfoliation process, the mass volume ratio of the As2Se3 layered crystals to the solvent NMP was 200 mg:100 mL during the first ultrasonic treatment at 1200 W. During the second ultrasonic treatment at 600 W, the mass volume ratio of the As2Se3 layered crystals to the solvent NMP was 200 mg:25 mL.

[0022] In one embodiment of the present invention, the differential centrifugation method is performed at 4°C, at a speed of 2500 rpm, for 5-10 minutes, and the supernatant is collected to obtain a dispersion of ultrathin two-dimensional As2Se3 nanosheets. The ultrathin two-dimensional As2Se3 nanosheet dispersion refers to ultrathin two-dimensional As2Se3 nanosheets aggregated in a solution.

[0023] The third aspect of the present invention is the application of the ultrathin two-dimensional As2Se3 nanosheets, and the application of the ultrathin two-dimensional As2Se3 nanosheets in the preparation of anti-tumor products. The anti-tumor products include at least one of products for reprogramming the tumor microenvironment, products for improving the effect of immunotherapy for tumors, products for photothermal therapy of tumors, products for photothermal and immunotherapy combined mode therapy of tumors, and products for inhibiting mouse breast cancer cells 4T1.

[0024] On the other hand, the application of the ultrathin two-dimensional As2Se3 nanosheets and the application of the ultrathin two-dimensional As2Se3 nanosheets in the preparation of drugs and / or systems, the drugs / systems have at least one of the following functions:

[0025] 1) Anti-tumor;

[0026] 2) Improve the efficacy of immunotherapy;

[0027] 3) Immunotherapy;

[0028] 4) Photothermal therapy;

[0029] 5) Photothermal and immunotherapy combined anti-tumor therapy;

[0030] 6) Activate immune response and inhibit tumor growth;

[0031] 7) Activate the immune response of tumor-bearing mice and inhibit the growth of tumors in tumor-bearing mice;

[0032] 8) Inhibit mouse breast cancer cell line 4T1;

[0033] 9) Promote the maturation and differentiation of dendritic cells in the tumor-draining lymph nodes of tumor-bearing mice;

[0034] 10) Increase spleen effector CD8 + T cell ratio;

[0035] 11) Infiltrating the tumor microenvironment;

[0036] 12) Reprogramming the tumor microenvironment.

[0037] The fourth aspect of the present invention is a product comprising the ultra-thin two-dimensional As2Se3 nanosheets,

[0038] The product has at least one of the following functions:

[0039] 1) Anti-tumor;

[0040] 2) Improve the efficacy of immunotherapy;

[0041] 3) Immunotherapy;

[0042] 4) Photothermal therapy;

[0043] 5) Photothermal and immunotherapy combined anti-tumor therapy;

[0044] 6) Activate immune response and inhibit tumor growth;

[0045] 7) Activate the immune response of tumor-bearing mice and inhibit the growth of tumors in tumor-bearing mice;

[0046] 8) Inhibit mouse breast cancer cell line 4T1;

[0047] 9) Promote the maturation and differentiation of dendritic cells in the tumor-draining lymph nodes of tumor-bearing mice;

[0048] 10) Increase spleen effector CD8 + T cell ratio;

[0049] 11) Infiltrating the tumor microenvironment;

[0050] 12) Reprogramming the tumor microenvironment.

[0051] It should be noted that in one embodiment of the present invention, the present inventors discovered a mechanism by which ultrathin two-dimensional As2Se3 nanosheets trigger a combination of light and heat, activating the immune response in tumor-bearing mice and inhibiting tumor growth in the mice. The tumors in the mice were 4T1 mouse breast cancer cells.

[0052] Furthermore, the present invention found that activating the immune response of tumor-bearing mice is to promote the maturation and differentiation of dendritic cells in the tumor-draining lymph nodes of tumor-bearing mice, increase the spleen effector CD8 + T cell proportion and infiltration into the tumor microenvironment.

[0053] Specifically, the present invention converts As2Se3 into exfoliable layered crystals. Ultrathin two-dimensional As2Se3 nanosheets are obtained by two consecutive liquid-phase exfoliations in the highly polar solvent NMP at varying powers. High-power ultrasonication (the first sonication) ensures the initial exfoliation. Low-power, prolonged ultrasonic exfoliation (the second sonication) further produces double-layered two-dimensional As2Se3 nanosheets with an average thickness of approximately 2.5 nm, while maintaining the nanosheets against oxidation. First, the thickness of the two-dimensional material is related to its band gap energy. Double-layer As2Se3 nanosheets are predicted in theoretical calculations to have a band gap energy similar to that of single-layer black phosphorus, which is conducive to the introduction of optical-assisted therapy. Second, the surface reactivity of two-dimensional materials increases with decreasing thickness, and they are more easily degraded into bioavailable components in the physiological environment, which helps to kill tumors. More importantly, selenium (Se) can detoxify arsenic (As) in the liver, which means that two-dimensional As2Se3 nanosheets can have higher biosafety in the body and can effectively resolve the contradiction between the high efficiency and high systemic toxicity of arsenic-containing drugs. In addition, while arsenic-based two-dimensional materials have been shown to activate the body's anti-tumor immune function, selenium (Se) is also an element that can effectively regulate the body's immune function. The combination of the two can also help to more effectively reprogram the tumor immune microenvironment and achieve more efficient tumor killing.

[0054] The beneficial effects of the present invention are:

[0055] First, the present invention successfully synthesized ultrathin two-dimensional As2Se3 nanosheets. Specifically: There has been no report on the synthesis of two-dimensional material As2Se3 nanosheets with a thickness of less than 10nm. The present invention synthesized ultrathin two-dimensional As2Se3 nanosheets with an average thickness of about 2.5nm for the first time. The present invention explored and concretized the preparation conditions of hydrothermal crystallization, ultrasonic liquid phase exfoliation and differential centrifugation, and confirmed that layered As2Se3 crystals can generate ultrathin nanosheets through liquid phase exfoliation.

[0056] Second, the present invention is simple to prepare. Specifically, the raw materials used are readily available and inexpensive, no inert gas protection is required, the production equipment, process conditions and steps are simple, and it is suitable for industrial production.

[0057] Third, the ultrathin two-dimensional As2Se3 nanosheets prepared by the present invention are highly practical, specifically:

[0058] 1) Ultrathin two-dimensional As2Se3 nanosheets have high surface reactivity and can be converted into bioavailable components in water and physiological environments.

[0059] 2) Ultra-thin two-dimensional As2Se3 nanosheets have higher biosafety than NaAsO2, an arsenic-containing drug ingredient currently widely used in clinical practice. This is because selenium can react with arsenic in the liver to form a detoxifying complex, which is then excreted into the bile, ultimately achieving a detoxifying effect.

[0060] 3) Compared with NaAsO2, an arsenic-containing drug ingredient currently widely used in clinical practice, ultrathin two-dimensional As2Se3 nanosheets have a more comprehensive tumor immune microenvironment reprogramming effect.

[0061] 4) Ultrathin two-dimensional As2Se3 nanosheets can be applied to anti-tumor immunotherapy systems, products, and drugs. The ultrathin two-dimensional As2Se3 nanosheets of the present invention have excellent photothermal conversion effects, not only achieving significant tumor suppression effects but also triggering photothermal combined anti-tumor immune activation in vivo. In addition, based on the mutual detoxification effect of selenium and arsenic in the liver, the ultrathin two-dimensional As2Se3 nanosheets demonstrated significantly better biosafety than other experimental groups, more effectively reducing toxic side effects. Therefore, ultrathin two-dimensional As2Se3 nanosheets are expected to serve as a safe and reliable nanoplatform for photothermal combined immunotherapy to achieve multimodal anti-tumor therapeutic effects.

[0062] Fourth, the present invention has been verified layer by layer and a large number of tests have been carried out, such as:

[0063] The present invention further studies the properties of the nanosheet in Examples 7 to 13, as follows:

[0064] 1) The composition and morphology of As2Se3 layered crystals and ultrathin two-dimensional As2Se3 nanosheets were verified. As2Se3 layered crystals have obvious layered stacking, which is helpful for the subsequent peeling step and can obtain few-layer nanosheets.

[0065] Through comparative tests, we found that:

[0066] 2) Ultrathin two-dimensional As2Se3 nanosheets have excellent photothermal conversion performance and are stable in vitro.

[0067] 3) In vitro cytotoxicity test: Under 635nm laser irradiation, ultrathin two-dimensional As2Se3 nanosheets can significantly induce cell death.

[0068] 4) Ultrathin two-dimensional As2Se3 nanosheets can achieve effective photothermal conversion at the tumor site in tumor-bearing mice.

[0069] 5) In vivo tumor inhibition test: Both the ultrathin two-dimensional As2Se3 nanosheets and the ultrathin two-dimensional As2Se3 nanosheets + 635nm laser irradiation test groups showed very significant inhibitory effects.

[0070] 6) In vivo biosafety test: No mice died in the ultrathin two-dimensional As2Se3 nanosheets and ultrathin two-dimensional As2Se3 nanosheets + 635nm laser irradiation test groups, confirming that ultrathin two-dimensional As2Se3 nanosheets can indeed achieve higher biosafety in vivo than the effective concentration of NaAsO2.

[0071] 7) Organ sections from the ultrathin two-dimensional As2Se3 nanosheets and ultrathin two-dimensional As2Se3 nanosheets + 635nm laser irradiation test groups showed normal tissue, further confirming that ultrathin two-dimensional As2Se3 nanosheets have stronger biosafety in the body than effective concentrations of NaAsO2.

[0072] 8) In vivo anti-tumor immune index test: ① CD80 of mice in the ultrathin two-dimensional As2Se3 nanosheet + 635nm laser irradiation experimental group + and CD86 + The number of dendritic cells increased significantly. ② The number of effector CD8+ T cells in the spleen of mice in the ultrathin two-dimensional As2Se3 nanosheet + 635nm laser irradiation experimental group increased. ③ The number of specific effector CD8+ T cells in the tumors of mice in the ultrathin two-dimensional As2Se3 nanosheet + 635nm laser irradiation experimental group was also significantly higher than that in the effective concentration NaAsO2 experimental group, confirming that under the synergistic effect of light and heat, ultrathin two-dimensional As2Se3 nanosheets exhibited a superior anti-tumor immune response than NaAsO2.

[0073] In summary, the present invention successfully synthesized ultrathin two-dimensional As2Se3 nanosheets, which are highly practical and have a simple preparation method. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention.

[0075] In the attached figure:

[0076] Figure 1 is a scanning electron microscope (SEM) image of the As2Se3 layered crystal of the present invention; wherein, Figure 1 Includes left and right pictures. The right picture is a partial enlarged picture of the left picture. The enlarged part has been marked in the left picture.

[0077] Figure 2 The X-ray powder diffraction patterns of the As2Se3 layered crystals, ultrathin two-dimensional As2Se3 nanosheets and As2Se3 cards of the present invention are as follows;

[0078] Figure 3 This is a transmission electron microscope (TEM) image of the ultrathin two-dimensional As2Se3 nanosheet of the present invention.

[0079] Figure 4 This is an atomic force microscope (AFM) image and data analysis diagram of the ultra-thin two-dimensional As2Se3 nanosheet of the present invention; wherein, Figure 4 It includes the left and right pictures. The left picture is the AFM picture, and the right picture is the data analysis picture. The right picture corresponds to a, b, and c in the left picture respectively.

[0080] Figure 5 This is the X-ray photoelectron spectroscopy (XPS) spectrum of ultrathin two-dimensional As2Se3 nanosheets;

[0081] Figure 6 This is the ultraviolet-visible (UV) spectrum of the ultrathin two-dimensional As2Se3 nanosheet of the present invention;

[0082] Figure 7 This is a comparison of the temperature-time curves of the ultrathin two-dimensional As2Se3 nanosheet dispersions and aqueous solutions of different concentrations under 635nm light;

[0083] Figure 8 The temperature-time curve of the 100 μg / mL ultrathin two-dimensional As2Se3 nanosheet dispersion of the present invention under 635 nm light irradiation is shown; wherein, a cycle means that the light source is removed and the dispersion is left to stand for 10 minutes after each 10 minutes of irradiation.

[0084] Figure 9 It is a cck8 detection comparison histogram of the ultrathin two-dimensional As2Se3 nanosheet of the present invention under light / dark conditions;

[0085] Figure 10 This is a cell comparison diagram of the ultrathin two-dimensional As2Se3 nanosheet of the present invention under different staining conditions;

[0086] Figure 11 This is a thermal image of the ultrathin two-dimensional As2Se3 nanosheet of the present invention and a control group;

[0087] Figure 12 It is a line comparison chart of the in vivo tumor inhibition effect test of the seven experimental groups of the present invention;

[0088] Figure 13 is a survival function graph of the in vivo biosafety test of seven experimental groups of the present invention;

[0089] Figure 14 This is a comparison of H&E staining of organ sections from the seven experimental groups of the present invention;

[0090] Figure 15 The CD80 in the 7 experimental groups of the present invention + and CD86 + Histogram comparison of dendritic cell content;

[0091] Figure 16The spleen CD8 + Histogram comparison of T cell ratios;

[0092] Figure 17 This is a bar graph comparing the proportion of CD8+ T cells in the tumor microenvironment in the seven experimental groups of the present invention;

[0093] Among them, NSs means nanosheets and Bulk means bulk. DETAILED DESCRIPTION

[0094] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0095] Unless otherwise specified, the reagents used in the examples can be easily obtained from commercial companies or prepared in-house.

[0096] NMP is N-methylpyrrolidone, purchased from Shanghai Bailingwei Chemical Technology Co., Ltd.;

[0097] As2Se3 amorphous powder was purchased from Shanghai Myrrel Biochemical Technology Co., Ltd., product number M54368-5G;

[0098] Sodium hydroxide was purchased from Sinopharm Shanghai Test;

[0099] Mice, BALB / c mice, were purchased from the Animal Model Research Center of Nanjing University.

[0100] Because there are powders and solutions, the present invention uses "mass-to-volume ratio" to express the added amount and indicates the units of mass and volume respectively.

[0101] Example 1

[0102] A method for preparing ultrathin two-dimensional As2Se3 nanosheets comprises the following steps:

[0103] (1) As2Se3 amorphous powder was added to a sodium hydroxide aqueous solution with a pH of 12 for hydrothermal reaction at a temperature of 190-200°C and a reaction time of 12-24 h. After the hydrothermal reaction, the mixture was cooled to room temperature, washed, and dried to obtain As2Se3 layered crystals. The mass-to-volume ratio of the As2Se3 amorphous powder to the sodium hydroxide aqueous solution was 0.9-1.2 g:9 mL.

[0104] (2) The As2Se3 layered crystals are added to a solvent, subjected to ultrasonic liquid phase exfoliation, and subjected to differential centrifugation to obtain ultrathin two-dimensional As2Se3 nanosheets, wherein the solvent comprises at least one of NMP, DMSO, and an isopropanol-water mixed solution. The volume ratio of isopropanol to water is 3:1. NMP is preferably the solvent.

[0105] Example 2

[0106] A method for preparing ultrathin two-dimensional As2Se3 nanosheets comprises the following steps:

[0107] The difference from Example 1 is that

[0108] In step (1), the mass volume ratio of As2Se3 amorphous powder and sodium hydroxide aqueous solution is 1g:9mL.

[0109] The remaining steps are the same as those in Example 1.

[0110] Example 3

[0111] A method for preparing ultrathin two-dimensional As2Se3 nanosheets comprises the following steps:

[0112] The difference from Example 1 / Example 2 is that,

[0113] In step (1), the hydrothermal reaction is carried out in a stainless steel autoclave with a polytetrafluoroethylene liner, the temperature of the hydrothermal reaction is 200° C., and the reaction time is 12 to 24 hours, preferably 24 hours.

[0114] The remaining steps are the same as Example 1 / Example 2.

[0115] Example 4

[0116] A method for preparing ultrathin two-dimensional As2Se3 nanosheets comprises the following steps:

[0117] The difference from Examples 1 to 3 is that

[0118] In step (1), the specific steps of cooling to room temperature, washing and drying are as follows: after cooling to room temperature, the reaction products are repeatedly blown into 20-50 mL of ddH2O, centrifuged at a speed of 2000 rpm for 5-10 minutes, preferably 10 minutes, and repeated 3 times. The precipitate is then dried at 60°C for 3 hours and then dried at 30°C for 12 hours.

[0119] The remaining steps are the same as any one of Examples 1 to 3.

[0120] Example 5

[0121] A method for preparing ultrathin two-dimensional As2Se3 nanosheets comprises the following steps:

[0122] The difference from Examples 1 to 4 is that

[0123] In step (2), the specific steps of ultrasonic liquid phase stripping are as follows: perform ultrasonic liquid phase stripping twice, both times in ice water bath conditions, the first ultrasonic power is 1200~1600W, preferably 1200W, the ultrasonic time is 2~4h, preferably 4h, the speed is ≥11000 Centrifugation, preferably 11,000 g g, collect the precipitate, and perform a second ultrasonication with an ultrasonic power of 600-650 W, preferably 600 W, and an ultrasonication time of 12-16 h, preferably 16 h.

[0124] During the first ultrasound, the mass volume ratio of As2Se3 layered crystals and solvent NMP was 200 mg:100 mL.

[0125] During the second ultrasound, the mass volume ratio of As2Se3 layered crystals and solvent NMP was 200 mg:25 mL.

[0126] The remaining steps are the same as any one of Examples 1 to 4.

[0127] Example 6

[0128] A method for preparing ultrathin two-dimensional As2Se3 nanosheets comprises the following steps:

[0129] The difference from Examples 1 to 5 is that

[0130] In step (2), the specific steps of the differential centrifugation method are as follows: performing the centrifugation at 4°C, a centrifugal speed of 2500 rpm, and a centrifugal time of 10 minutes, collecting the supernatant to obtain an ultrathin two-dimensional As2Se3 nanosheet dispersion, that is, obtaining ultrathin two-dimensional As2Se3 nanosheets. The ultrathin two-dimensional As2Se3 nanosheet dispersion means that the ultrathin two-dimensional As2Se3 nanosheets are aggregated in the solution.

[0131] The remaining steps are the same as any one of Examples 1 to 5.

[0132] Through the preparation method of the above-mentioned Examples 1 to 6, the present invention prepares an ultra-thin two-dimensional As2Se3 nanosheet, and the thickness of the ultra-thin two-dimensional As2Se3 nanosheet is 2.3-2.6nm and the lateral size is 150-300nm.

[0133] Furthermore, the present invention studies the properties of the ultrathin two-dimensional As2Se3 nanosheets prepared by the present invention in the following Examples 7 to 13.

[0134] Example 7

[0135] In this example, the composition and morphology of ultrathin two-dimensional As2Se3 nanosheets were detected and analyzed.

[0136] 1. Scanning electron microscope (SEM) images of As2Se3 layered crystals Figure 1 shown.

[0137] Depend on Figure 1 Under high temperature, high pressure, and alkaline conditions, amorphous As2Se3 powder crystallizes into As2Se3 layered crystals. Scanning electron microscopy images show distinct layered stacking, which facilitates subsequent exfoliation to yield few-layer nanosheets. The high temperature and high pressure are provided by the Teflon-lined stainless steel autoclave, along with the reaction time and temperature. The alkaline condition is provided by a sodium hydroxide solution at a pH of 12.

[0138] 2. X-ray powder diffraction patterns of As2Se3 layered crystals and ultrathin two-dimensional As2Se3 nanosheets Figure 2 shown.

[0139] Depend on Figure 2 It can be seen that both the As2Se3 layered crystals and the ultrathin two-dimensional As2Se3 nanosheets have the characteristic peaks shown by As2Se with the card number PDF#75-0739, which confirms that the composition of the layered crystals and ultrathin two-dimensional nanosheets synthesized by Examples 1 to 6 is As2Se3.

[0140] 3. Transmission electron microscopy (TEM) images of ultra-thin two-dimensional As2Se3 nanosheets Figure 3 shown.

[0141] Depend on Figure 3 It can be seen that after two ultrasonic treatments, the As2Se3 layered crystals were peeled off to form a thin sheet structure in the strong polar solvent NMP.

[0142] 4. Atomic force microscopy (AFM) images of ultra-thin two-dimensional As2Se3 nanosheets Figure 4 shown.

[0143] Three locations on the ultrathin two-dimensional As2Se3 nanosheet were selected for analysis, and the results were Figure 4 .

[0144] Depend on Figure 4 It can be seen that the ultrathin two-dimensional As2Se3 nanosheets synthesized in Examples 1 to 6 have a thickness of 2.3 to 2.6 nm and a lateral size of 150 to 300 nm.

[0145] 5. X-ray photoelectron spectroscopy (XPS) analysis results of ultrathin two-dimensional As2Se3 nanosheets Figure 5 shown.

[0146] Depend on Figure 5It can be seen that the valence state of the As element in the ultrathin two-dimensional As2Se3 nanosheets synthesized in Examples 1 to 6 is +3, and the valence state of the Se element is -2.

[0147] Example 8

[0148] In this example, the in vitro photothermal conversion performance of ultrathin two-dimensional As2Se3 nanosheets was tested and analyzed.

[0149] 1. Ultraviolet-visible (UV) spectrum of ultrathin two-dimensional As2Se3 nanosheets Figure 6 shown.

[0150] Depend on Figure 6 It can be seen that the ultrathin As2Se3 nanosheets of different concentrations and all synthesized from Examples 1 to 6 exhibit broad peak absorption near 600 nm, which proves that they are expected to undergo photothermal conversion under the excitation of red light / infrared light with strong tissue penetration.

[0151] 2. Temperature-time curve of ultrathin two-dimensional As2Se3 nanosheet dispersion under 635nm light Figure 7-8 shown.

[0152] Depend on Figure 7 It can be seen that, considering the spectrum and tissue penetration of laser, the inventors chose a 635nm laser and tested the photothermal conversion of ultrathin two-dimensional As2Se3 nanosheets. It was found that within 10 minutes, the temperature of the ultrathin two-dimensional As2Se3 nanosheet dispersion at 100μg / mL could reach 60.7℃; 200μg / mL could reach even higher.

[0153] Depend on Figure 8 It can be seen that the photothermal conversion of 100 μg / mL ultrathin two-dimensional As2Se3 nanosheets can remain stable for 6 cycles.

[0154] Example 9

[0155] In this example, the in vitro cytotoxicity of ultrathin two-dimensional As2Se3 nanosheets was tested and analyzed.

[0156] 1. The cck8 test results of ultra-thin two-dimensional As2Se3 nanosheets are as follows Figure 9 shown.

[0157] Depend on Figure 9 It can be seen that under 635nm laser irradiation conditions, the IC50 of ultrathin two-dimensional As2Se3 nanosheets is 0.76±0.32μg / mL; under dark conditions, the IC50 of ultrathin two-dimensional As2Se3 nanosheets is 16.38±1.85 μg / mL, and the phototoxicity index PI value reaches 21.55, confirming that there is a significant difference between phototoxicity and dark toxicity.

[0158] 2. Live / dead cell staining experimental results of ultrathin two-dimensional As2Se3 nanosheets Figure 10 shown.

[0159] The cytotoxicity was verified by Calcein AM and PI staining, and four experimental conditions were set: a control group (control), only 635nm laser irradiation without the addition of ultrathin two-dimensional As2Se3 nanosheets (635nm), only the addition of ultrathin two-dimensional As2Se3 nanosheets (As2Se3NSs), and 635nm laser irradiation with the addition of ultrathin two-dimensional As2Se3 nanosheets (As2Se3NSs+635nm).

[0160] Depend on Figure 10 It can be seen that under 635nm laser irradiation conditions, ultrathin two-dimensional As2Se3 nanosheets can significantly induce cell death, while the experimental groups under other conditions cannot.

[0161] Example 10

[0162] In this example, the in vivo photothermal conversion performance of ultrathin two-dimensional As2Se3 nanosheets was tested and analyzed.

[0163] There are two experimental conditions: control experimental group: only 635nm laser irradiation (635nm); 635nm laser irradiation and addition of ultrathin two-dimensional As2Se3 nanosheets (As2Se3NSs+635nm).

[0164] Depend on Figure 11 It can be seen that under the conditions of 635nm laser irradiation, the temperature of the control experimental group only rose by 1.5℃ (from 38.6℃ to 40.1℃), and the temperature of the experimental group injected with ultrathin two-dimensional As2Se3 nanosheets rose by 13.6℃ (from 37.8℃ to 51.4℃), confirming that ultrathin two-dimensional As2Se3 nanosheets can achieve effective photothermal conversion at the tumor site of tumor-bearing mice.

[0165] Example 11

[0166] In this example, the in vivo tumor-suppressing effect of ultrathin two-dimensional As2Se3 nanosheets was tested and analyzed.

[0167] Depend on Figure 12 Based on the cell-based experiments, mice were implanted with tumors in their left axilla to create a 4T1 tumor-bearing mouse model. Tumor implantation methods are well-known in the art and will not be detailed here. The same method was used for all mice to avoid influencing the results. After dosing on days 0, 3, 6, and 10 and monitoring tumor volume in the mice, it was found that both the ultrathin two-dimensional As2Se3 nanosheets and the ultrathin two-dimensional As2Se3 nanosheets combined with 635nm laser irradiation groups exhibited highly significant inhibitory effects.

[0168] Example 12

[0169] In this example, the in vivo biosafety of ultrathin two-dimensional As2Se3 nanosheets was tested and analyzed.

[0170] The present invention selected seven experimental conditions: control (blank control group), NaAsO2, Na2Se, bulk As2Se3, 635nm laser irradiation alone, ultrathin two-dimensional As2Se3 nanosheets, and ultrathin two-dimensional As2Se3 nanosheets combined with 635nm laser irradiation. These seven experimental conditions were chosen because NaAsO2 served as a cation control, Na2Se served as an anion control, bulk As2Se3 served as a morphology control for non-two-dimensional materials of the same substance, 635nm laser irradiation alone served as a laser-only control, ultrathin two-dimensional As2Se3 nanosheets served as a material-only control, and ultrathin two-dimensional As2Se3 nanosheets combined with 635nm laser irradiation served as both material and light irradiation. NaAsO2 is a widely used arsenic-containing pharmaceutical ingredient in clinical practice.

[0171] 1. Survival rate of ultrathin two-dimensional As2Se3 nanosheets Figure 13 shown.

[0172] Depend on Figure 13 It can be seen that after monitoring the survival of mice during the treatment process, it was found that Na2Se drugs have strong physiological toxicity. After the end of the treatment cycle, the survival rate was only 37.5%. The survival rate of NaAsO2 and bulk As2Se3 was 75%. However, there was no mouse death in the ultra-thin two-dimensional As2Se3 nanosheets and ultra-thin two-dimensional As2Se3 nanosheets + 635nm laser irradiation experimental groups, confirming that ultra-thin two-dimensional As2Se3 nanosheets can indeed achieve higher biosafety in the body than the effective concentration of NaAsO2.

[0173] 2. H&E staining of organ sections of ultrathin two-dimensional As2Se3 nanosheets Figure 14 shown.

[0174] Depend on Figure 14 It can be seen that after the treatment, the mice were dissected and sliced, and the slices were stained with H&E. The organs were divided into spleen, liver, heart, lung, and kidney.

[0175] The results showed that Na2Se could cause damage to the spleen, severe damage to liver tissue, and inflammatory response in the lungs. NaAsO2 could also cause damage to the liver and inflammatory response in the lungs of mice. However, the organ slices of the ultra-thin two-dimensional As2Se3 nanosheets and ultra-thin two-dimensional As2Se3 nanosheets + 635nm laser irradiation experimental groups showed normal tissues, further confirming that ultra-thin two-dimensional As2Se3 nanosheets have stronger biosafety in the body than effective concentration NaAsO2.

[0176] Example 13

[0177] In this example, the in vivo anti-tumor immune indicators of ultrathin two-dimensional As2Se3 nanosheets were tested and analyzed.

[0178] The present invention selects 7 experimental conditions: Control (blank control group), NaAsO2, Na2Se, BulkAs2Se3, 635nm laser irradiation only, ultrathin two-dimensional As2Se3 nanosheets, and ultrathin two-dimensional As2Se3 nanosheets + 635nm laser irradiation.

[0179] 1. The maturation and differentiation ratio of dendritic cells in tumor-bearing mice treated with ultrathin two-dimensional As2Se3 nanosheets and 635nm laser irradiation is as follows: Figure 15 shown.

[0180] Depend on Figure 15 It can be seen that 24 hours after administration, the tumor-bearing mice were dissected and the lymph nodes in the tumor drainage area were taken. It was found that the CD80 + and CD86 + The content of dendritic cells increased significantly.

[0181] 2. The effector CD8+T cell ratio of mouse spleen treated with ultrathin two-dimensional As2Se3 nanosheets + 635nm laser irradiation is as follows: Figure 16 shown.

[0182] Depend on Figure 16 It can be seen that after the administration was completed, the mice were dissected and the spleen was ground for testing, and it was found that the content of effector CD8+T cells in the spleen of mice in the ultra-thin two-dimensional As2Se3 nanosheets + 635nm laser irradiation experimental group increased.

[0183] 3. The specific effector CD8+T cell ratio in the tumor microenvironment of mice treated with ultrathin two-dimensional As2Se3 nanosheets + 635nm laser irradiation is shown in Figure 3. Figure 17 shown.

[0184] Depend on Figure 17After the administration, the mice were dissected and the tumor tissue was ground for testing. The results showed that the levels of specific effector CD8+ T cells in the tumors of mice in the ultrathin two-dimensional As2Se3 nanosheets and ultrathin two-dimensional As2Se3 nanosheets + 635nm laser irradiation groups were significantly increased. In particular, the levels of specific effector CD8+ T cells in the tumors of mice in the ultrathin two-dimensional As2Se3 nanosheets + 635nm laser irradiation group were significantly higher than those in the effective concentration NaAsO2 group, confirming that the ultrathin two-dimensional As2Se3 nanosheets exhibited a superior anti-tumor immune response than NaAsO2 under the synergistic effect of light and heat.

[0185] In summary, Examples 1 to 6 of the present invention successfully prepared ultrathin two-dimensional As2Se3 nanosheets. The present invention explored and concretized the preparation conditions of hydrothermal crystallization, ultrasonic liquid phase exfoliation, and differential centrifugation, and confirmed that layered As2Se3 crystals can be generated into ultrathin nanosheets through liquid phase exfoliation.

[0186] The present invention further studies the properties of the nanosheet in Examples 7 to 13, as follows:

[0187] 1) The composition and morphology of As2Se3 layered crystals and ultrathin two-dimensional As2Se3 nanosheets were verified. As2Se3 layered crystals have obvious layered stacking, which is helpful for the subsequent peeling step and can obtain few-layer nanosheets.

[0188] Through comparative tests, we found that:

[0189] 2) Ultrathin two-dimensional As2Se3 nanosheets have excellent photothermal conversion performance and are stable in vitro.

[0190] 3) In vitro cytotoxicity test: Under 635nm laser irradiation, ultrathin two-dimensional As2Se3 nanosheets can significantly induce cell death.

[0191] 4) Ultrathin two-dimensional As2Se3 nanosheets can achieve effective photothermal conversion at the tumor site in tumor-bearing mice.

[0192] 5) In vivo tumor inhibition test: Both the ultrathin two-dimensional As2Se3 nanosheets and the ultrathin two-dimensional As2Se3 nanosheets + 635nm laser irradiation test groups showed very significant inhibitory effects.

[0193] 6) In vivo biosafety test: No mice died in the ultrathin two-dimensional As2Se3 nanosheets and ultrathin two-dimensional As2Se3 nanosheets + 635nm laser irradiation test groups, confirming that ultrathin two-dimensional As2Se3 nanosheets can indeed achieve higher biosafety in vivo than the effective concentration of NaAsO2.

[0194] 7) Organ sections from the ultrathin two-dimensional As2Se3 nanosheets and ultrathin two-dimensional As2Se3 nanosheets + 635nm laser irradiation test groups showed normal tissue, further confirming that ultrathin two-dimensional As2Se3 nanosheets have stronger biosafety in the body than effective concentrations of NaAsO2.

[0195] 8) In vivo anti-tumor immune index test: ① CD80 of mice in the ultrathin two-dimensional As2Se3 nanosheet + 635nm laser irradiation experimental group + and CD86 + The content of dendritic cells increased significantly. ② The effector CD8 in the spleen of mice in the ultra-thin two-dimensional As2Se3 nanosheet + 635nm laser irradiation experimental group + ③ The specific effector CD8 in the tumor of mice in the ultrathin two-dimensional As2Se3 nanosheet + 635nm laser irradiation experimental group + The T cell content was also significantly increased compared to the effective concentration NaAsO2 experimental group, confirming that under the synergistic effect of light and heat, ultra-thin two-dimensional As2Se3 nanosheets exhibited a better anti-tumor immune response than NaAsO2.

[0196] As can be seen from the above, the ultrathin two-dimensional As2Se3 nanosheets prepared by this invention can be used in anti-tumor immunotherapy systems, products, and drugs. Further, anti-tumor products include products that reprogram the tumor microenvironment, products that improve the efficacy of immunotherapy for tumors, products that use photothermal therapy for tumors, products that use a combined photothermal and immunotherapy approach for tumor treatment, and products that inhibit the growth of mouse breast cancer 4T1 cells.

[0197] The mechanism involved is: ultra-thin two-dimensional As2Se3 nanosheets trigger the combination of light and heat, activate the immune response of tumor-bearing mice, and inhibit the growth of tumors in tumor-bearing mice. Among them, the tumors in tumor-bearing mice are mouse breast cancer cells 4T1; the activation of the immune response of tumor-bearing mice is to promote the maturation and differentiation of dendritic cells in the lymph nodes of the tumor-draining area of tumor-bearing mice, and increase the spleen effector CD8 + T cell proportion and infiltration into the tumor microenvironment.

[0198] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein with equivalents. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A method for preparing ultrathin two-dimensional As2Se3 nanosheets, characterized in that: The steps include: As2Se3 is hydrothermally crystallized to obtain As2Se3 layered crystals; As2Se3 layered crystals were added to a solvent, subjected to ultrasonic liquid phase exfoliation and differential centrifugation to obtain ultrathin two-dimensional As2Se3 nanosheets. The hydrothermal crystallization step includes: adding As2Se3 powder to a sodium hydroxide aqueous solution for hydrothermal reaction, wherein the mass volume ratio of the As2Se3 powder to the sodium hydroxide aqueous solution is 0.9-1.2 g:9 mL; the reaction temperature is 190-200° C., and the reaction time is 12-24 hours.

2. The method for preparing ultrathin two-dimensional As2Se3 nanosheets according to claim 1, characterized in that: The hydrothermal crystallization is carried out in a stainless steel autoclave with a polytetrafluoroethylene liner; The solvent includes at least one of NMP, DMSO, and an isopropyl alcohol-water mixture.

3. The method for preparing ultrathin two-dimensional As2Se3 nanosheets according to claim 1, characterized in that: The ultrasonic liquid phase stripping step includes: performing ultrasonic liquid phase stripping twice, both times in ice water bath conditions, the first ultrasonic power is 1200-1600W, the ultrasonic time is 2-4h, the speed is ≥11000 The pellet was collected by centrifugation at 0.5000 g and subjected to a second ultrasonic treatment at a power of 600-650 W and a time of 12-16 h.

4. The method for preparing ultrathin two-dimensional As2Se3 nanosheets according to claim 1, characterized in that: The conditions of the differential centrifugation method include: performing at 4°C, a centrifugal speed of 2500 rpm, a centrifugal time of 5 to 10 minutes, collecting the supernatant, and obtaining an ultrathin two-dimensional As2Se3 nanosheet dispersion, that is, obtaining ultrathin two-dimensional As2Se3 nanosheets.

5. An ultrathin two-dimensional As2Se3 nanosheet, characterized in that: The ultrathin two-dimensional As2Se3 nanosheet is prepared according to the preparation method of the ultrathin two-dimensional As2Se3 nanosheet according to any one of claims 1 to 4. The ultrathin two-dimensional As2Se3 nanosheet has a thickness of 2.3 to 2.6 nm and a lateral size of 150 to 300 nm.

6. The ultrathin two-dimensional As2Se3 nanosheet according to claim 5, characterized in that The ultra-thin two-dimensional As2Se3 nanosheet has a double-layer structure.

7. An application of the ultrathin two-dimensional As2Se3 nanosheet according to claim 6, characterized in that: The ultra-thin two-dimensional As2Se3 nanosheets are used in the preparation of anti-tumor products, which include at least one of products for reprogramming the tumor microenvironment, products for improving the effect of immunotherapy for tumors, products for photothermal therapy for tumors, products for photothermal and immunotherapy combined mode therapy for tumors, and products for inhibiting mouse breast cancer cell 4T1.

8. An application of the ultrathin two-dimensional As2Se3 nanosheet according to claim 6, characterized in that: The use of the ultrathin two-dimensional As2Se3 nanosheets in the preparation of drugs and / or systems, wherein the drugs / systems have at least one of the following functions: 1) Anti-tumor; 2) Improve the efficacy of immunotherapy; 3) Immunotherapy; 4) Photothermal therapy; 5) Photothermal and immunotherapy combined anti-tumor therapy; 6) Activate immune response and inhibit tumor growth; 7) Activate the immune response of tumor-bearing mice and inhibit the growth of tumors in tumor-bearing mice; 8) Inhibit mouse breast cancer cell line 4T1; 9) Promote the maturation and differentiation of dendritic cells in the tumor-draining lymph nodes of tumor-bearing mice; 10) Increase spleen effector CD8 + T cell ratio; 11) Infiltrating the tumor microenvironment; 12) Reprogramming the tumor microenvironment.

9. A product, characterized in that It comprises the ultra-thin two-dimensional As2Se3 nanosheet according to claim 6, and the product has at least one of the following functions: 1) Anti-tumor; 2) Improve the efficacy of immunotherapy; 3) Immunotherapy; 4) Photothermal therapy; 5) Photothermal and immunotherapy combined anti-tumor therapy; 6) Activate immune response and inhibit tumor growth; 7) Activate the immune response of tumor-bearing mice and inhibit the growth of tumors in tumor-bearing mice; 8) Inhibit mouse breast cancer cell line 4T1; 9) Promote the maturation and differentiation of dendritic cells in the tumor-draining lymph nodes of tumor-bearing mice; 10) Increase spleen effector CD8 + T cell ratio; 11) Infiltrating the tumor microenvironment; 12) Reprogramming the tumor microenvironment.

Images