A two-dimensional TiO2 nanomaterial with peroxidase-like activity and its application in sonodynamic therapy

Abstract

The present invention belongs to the technical field of sonodynamic therapy, and discloses a two-dimensional TiO2 nanomaterial with peroxidase activity and its application in sonodynamic therapy. The present invention firstly proposes to design ultrathin 2D TiO2 nanosheets with peroxidase activity as efficient sonosensitizers for sonodynamic cancer therapy. The ultrathin Ti3C2 nanosheets obtained by acid etching treatment are oxidized to 2D TiO2 through simple heat treatment. Surprisingly, 2D TiO2 has peroxidase activity, and when used as a sonosensitizer, the 2D TiO2 nanosheets exhibit excellent ROS generation activity under US irradiation. In vitro experiments show that the 2D TiO2 nanosheets can effectively induce apoptosis of cells under US irradiation.

Description

Technical Field

[0001] The present invention belongs to the technical field of sonodynamic therapy, and more specifically, relates to a two-dimensional TiO2 nanomaterial with peroxidase activity and its application in sonodynamic therapy. Background Art

[0002] Sonodynamic Therapy (SDT) is a non-invasive new non-invasive tumor treatment method. SDT uses ultrasound (US) to activate sonosensitizers to generate reactive oxygen species (ROS) to induce tumor cell death, thereby achieving the treatment purpose. Based on the deep tissue penetration ability of ultrasound, SDT can achieve the treatment of deeper tumors and has broad prospects for clinical translation. Although sonodynamic therapy can specifically form ROS at the tumor site and induce oxidative stress in tumors, there are still challenges in developing new and efficient sonosensitizers. Organic sonosensitizers have disadvantages such as poor water solubility, fast in vivo clearance, low enrichment degree at the tumor site, and unsatisfactory efficacy. Inorganic sonosensitizers have good chemical stability and low phototoxicity. TiO2 is a representative of inorganic nano-sonosensitizers, but the rapid recombination of its electrons (e-) and holes (h+) in the energy band structure limits its efficacy. Therefore, how to prevent the rapid recombination of electrons (e-) and holes (h+) of TiO2 nano-sonosensitizers induced by ultrasonic irradiation and further improve the efficiency of sonocatalysis still faces great challenges.

[0003] Many nanocatalysts have peroxidase activity and can react with excessive hydrogen peroxide in tumors to produce ROS. The in-situ catalytic reaction caused by introducing nanocatalysts into the tumor microenvironment or cancer cells will cause cancer cells to undergo severe oxidative damage and apoptosis. Based on nanocatalytic tumor therapy with peroxidase activity and further combined with other treatment modes, a synergistic treatment mode based on catalytic tumor therapy can be achieved. Two-dimensional (2D) layered nanomaterials have been proven to be a promising nanoplatform for various biomedical applications due to their excellent biocompatibility, acid-sensitive biodegradability, different chemical compositions and structures. However, the use of 2D TiO2 with peroxidase activity as a sonosensitizer for SDT has not been reported. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the above problems existing in the prior art, and first provide a preparation method of a two-dimensional TiO2 nanomaterial with peroxidase activity.

[0005] The second object of the present invention is to provide the two-dimensional TiO2 nanomaterial obtained by the above method.

[0006] The third object of the present invention is to provide the application of the above two-dimensional structure TiO2 nanomaterial.

[0007] The object of the present invention is achieved by the following technical solutions:

[0008] A preparation method of a two-dimensional TiO2 nanomaterial, comprising the following steps:

[0009] S1. Pour lithium fluoride into a 3-12 mol / L hydrochloric acid solution, and stir magnetically in an ice-water bath until dissolved. Then add Ti3AlC2 powder in batches, keep the temperature at 30-50 °C, and stir for 24-72 hours to obtain a Ti3C2 stock solution;

[0010] S2. Add the Ti3C2 stock solution to the solution in multiple times, stir evenly, then perform ultrasonic oscillation, centrifuge, and collect the upper layer liquid to obtain Ti3C2 with a concentration of 100-10,000 ppm;

[0011] S3. Heat the obtained Ti3C2 liquid to 60-80 °C, keep it warm for 12-24 h, then heat it to 80-100 °C, keep it warm for 12-24 h, and then cool it to room temperature, and centrifuge and freeze-dry to obtain a two-dimensional TiO2 nanomaterial.

[0012] Preferably, in step S3, the cooling rate is 1-5 °C / min.

[0013] Preferably, in step S1, the molar ratio of lithium fluoride to the hydrochloric acid solution is 1-3:3-12.

[0014] Preferably, in step S2, the solution is selected from PBS, pure water, deionized water, alcohol, and lithium fluoride; the volume ratio of the Ti3C2 stock solution to the solution is 3-5:1-5.

[0015] The present invention also provides a two-dimensional structure TiO2 nanomaterial obtained by the above method. The two-dimensional TiO2 nanomaterial is fusiform, with a length of 100-400 nm and a width of 20-100 nm.

[0016] The present invention also provides the application of the two-dimensional structure TiO2 nanomaterial as a photosensitizer in sonodynamic therapy.

[0017] The present invention also provides the application of the two-dimensional structure TiO2 nanomaterial in the preparation of a functional product for treating tumors, and the functional product can induce apoptosis of tumor cells under the excitation of ultrasound; preferably, the conditions of the ultrasound are 0.5-2 W / cm 2 .

[0018] Compared with the prior art, the present invention also has the following beneficial effects:

[0019] The present invention first proposes to design 2D TiO2 nanosheets as efficient sonosensitizers for sonodynamic cancer therapy. The ultrathin Ti3C2 nanosheets synthesized by hydrothermal method are oxidized to 2D TiO2 through simple acid etching treatment. Surprisingly, when used as a sonosensitizer, the 2D TiO2 nanosheets have peroxidase activity and exhibit excellent ROS generation activity under US irradiation. In vitro experiments show that under US irradiation, the 2D TiO2 nanosheets can effectively induce apoptosis of cells. Brief Description of the Drawings

[0020] Figure 1 Characterization of the two-dimensional structure TiO2 nanosheets of the present invention; Figure 1 A: TEM image of TiO2; 1B: HRTEM image of TiO2; 1C: AFM image of TiO2; Figure 1 D: XRD pattern of TiO2; Figure 1 E: Band gap diagram of TiO2; Figure 1 F: ROS formation ability of TiO2 after ultrasonic treatment at different times;

[0021] Figure 2 A is for detecting ·OH using MB probe in the presence of TiO2 and H2O2; Figure 2 B is the ESR spectrum of TiO2 (the capturer is TMP); Figure 2 C is the ESR spectrum of TiO2 (the capturer is DMPO); Figure 2 D is the dynamic recording of DCFH-DA fluorescence signal by Incucyte live cell analysis system after tumor cells are incubated with different concentrations of TiO2 and ultrasonic treatment; Figure 2 E is the cell survival rate after tumor cells are incubated with different concentrations of TiO2 and ultrasonic treatment;

[0022] Figure 3 A is the ability of TiO2 to form ROS at different concentrations; Figure 3 B is the sonodynamic therapy of TiO2 to kill liver cancer cells; Figure 3 C is the apoptosis analysis induced in tumor cells under different conditions. Detailed Embodiments

[0023] The following further describes the detailed embodiments of the present invention. It should be noted here that the description of these embodiments is for helping to understand the present invention, but does not constitute a limitation to the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0024] Example 1

[0025] The preparation method of two-dimensional TiO2 nanosheets includes the following steps:

[0026] a. Pour lithium fluoride into a 5 mol / L hydrochloric acid solution, and stir magnetically in an ice bath for about 10 minutes until it dissolves. Then, slowly add the Ti3AlC2 powder in batches. After that, place the solution in a heating magnetic stirrer and keep it at a constant temperature (30 °C) and stir magnetically for 24 hours to obtain the Ti3C2 stock solution.

[0027] b. Add 50 ml of the Ti3C2 stock solution to 40 ml of deionized water in 5 portions (10 mL each time). After stirring evenly, ultrasonically vibrate for 20 s, centrifuge at 5000 revolutions for 5 minutes, collect the upper layer liquid, and finally collect the solution to 3000 mL. Divide the solution into tubes of 30 mL each, and the concentration of each tube is 4000 ppm.

[0028] c. Heat the Ti3C2 liquid from room temperature to 60 °C, keep it warm for 24 h, then heat it to 80 °C, keep it warm for 12 h, and then cool it to room temperature at a cooling rate of 1 °C / min. Centrifuge and freeze-dry to obtain two-dimensional nanostructured TiO2.

[0029] Example 2

[0030] A method for preparing two-dimensional TiO2 nanosheets, comprising the following steps:

[0031] a. Pour lithium fluoride into a 7 mol / L hydrochloric acid solution, and stir magnetically in an ice bath for about 20 minutes until it dissolves. Then, slowly add the Ti3AlC2 powder in batches. After that, place the solution in a heating magnetic stirrer and keep it at a constant temperature (40 °C) and stir magnetically for 36 hours to obtain the Ti3C2 stock solution.

[0032] b. Add 40 ml of the Ti3C2 stock solution to 40 ml of deionized water in 4 portions (10 mL each time). After stirring evenly, ultrasonically vibrate for 30 s, centrifuge at 5000 revolutions for 10 minutes, collect the upper layer liquid, and finally collect the solution to 3000 mL. Divide the solution into tubes of 30 mL each, and the concentration of each tube is 6000 ppm.

[0033] c. Heat the Ti3C2 liquid from room temperature to 70 °C, keep it warm for 18 h, then heat it to 90 °C, keep it warm for 18 h, and then cool it to room temperature at a cooling rate of 3 °C / min. Centrifuge and freeze-dry to obtain two-dimensional nanostructured TiO2.

[0034] I. Structure characterization

[0035] It can be seen from the TEM image that the size of the TiO2 nanosheets is 100 - 200 nm. In addition, we used an X-ray powder diffractometer to analyze the crystal structure of the TiO2 nanosheets, and the diffraction peaks ( Figure 1 D) are basically the same as those of the TiO2 nanosheets reported in previous literature.

[0036] Compared with common TiO2, the two-dimensional structured TiO2 synthesized by this method has a band gap energy of 1.9 eV( Figure 1 E). Compared with normal TiO2 (3.2 eV), the band gap energy is significantly reduced, making the sonocatalytic activity of monolayer TiO2 better. To detect singlet oxygen during the sonodynamic therapy process, we used 1,3-diphenylisobenzofuran (DPBF) for the detection of ROS. The results confirmed that through ultrasonic excitation, ROS increased with the prolongation of ultrasonic time.

[0037] II. Mechanism of action

[0038] The in-situ catalytic reaction triggered by introducing nano-catalysts into the tumor microenvironment or cancer cells will cause severe oxidative damage to cancer cells and lead to apoptosis. Further combining nano-catalytic tumor therapy with other treatment modalities can achieve a synergistic treatment mode based on catalytic tumor therapy. Therefore, we studied the peroxidase-like activity of two-dimensional structured TiO2 through a series of experiments. First, the property of two-dimensional structured TiO2 reacting with H2O2 to generate ·OH was studied using MB. The results showed that two-dimensional structured TiO2 had the catalytic activity of reducing H2O2( Figure 2 A), and ESR measurement( Figure 2 B, 2C) also consistently showed that two-dimensional structured TiO2 could convert H2O2 into ·OH and 1 O2, which was manifested as characteristic peaks in ESR, and the production of ·OH and 1 O2 from H2O2 was higher under ultrasonic excitation, indicating that the peroxidase activity of two-dimensional structured TiO2 and sonodynamic therapy could promote each other.

[0039] To explore how two-dimensional structured TiO2 generates oxygen free radicals through ultrasonic excitation and thus plays a role in sonodynamic therapy, we co-incubated 20 μg / mL TiO2 with tumor cells, and then performed ultrasonic treatment at 1 W / cm 2 for 3 min according to the grouping. After the treatment, DCFH-DA staining was used and the cells were cultured in an Incucyte live cell analysis system to dynamically record the fluorescence signals of each treatment group. As Figure 2 shown in D, compared with the blank control group, the fluorescence intensity of tumor cells increased with time, and the intensity of ROS generated after ultrasonic excitation was significantly improved. As the concentration increased, the fluorescence intensity increased with time. By MTS analysis, TiO2 was co-incubated with cells at different Ti concentrations for 48 h. As Figure 2 shown in E, US irradiation treatment of cells showed high toxicity, and the combination of TiO2 and US irradiation showed the highest cytotoxicity. Flow cytometry apoptosis assay also confirmed the synergistic treatment effect( Figure 3 C). The results also showed that the treatment effect of the TiO2 + US irradiation group was the best.

[0040] III. Inducing apoptosis of tumor cells

[0041] After TiO2 was co-incubated with cells at different Ti concentrations (12.5 - 100 μg / mL), it was stained with DCFH-DA (2 μg / mL) and observed and photographed under a laser confocal microscope. It was found that the fluorescence intensity increased with the increase in concentration. Under the synergistic action with ultrasound, the highest amount of ROS was formed ( Figure 3 A). We also observed and photographed images through a laser confocal microscope, demonstrating that ROS could be generated inside the cells after 3 minutes of ultrasound treatment at 1.0 W / cm 2 . Figure 3 B is the fluorescence image of tumor cells incubated with TiO2 (12.5 - 100 μg / mL) and after different treatments, stained with calcein (green, live cells) and propidium iodide (red, dead cells). Under the laser confocal microscope, the green fluorescence represents live cells and the red fluorescence represents dead cells. The death of tumor cells increased significantly after the combined treatment of laser and ultrasound, indicating that the ultrasound-excited treatment could achieve an ideal tumor cell killing effect. And it was confirmed that TiO2 could induce apoptosis of tumor cells under ultrasound excitation. At the same time, obvious apoptosis of tumor cells could be induced after ultrasound excitation. It shows that the peroxidase-like activity of two-dimensional TiO2 combined with sonodynamic therapy can achieve a tumor synergistic treatment effect.

[0042] Example 3

[0043] Preparation method of two-dimensional TiO2 nanosheets, including the following steps:

[0044] a. Pour lithium fluoride into 12 mol / L hydrochloric acid solution and stir magnetically in an ice bath for about 30 minutes until dissolved. Then, slowly add Ti3AlC2 powder in batches. Then, place the solution in a thermostatic magnetic stirrer (50 °C) and stir magnetically for 48 hours to obtain Ti3C2 stock solution.

[0045] b. Add 30 mL of Ti3C2 stock solution to 40 mL of deionized water in 3 portions (10 mL each time). After stirring evenly, shake it ultrasonically for 50 s, centrifuge at 5000 rpm for 15 minutes, collect the upper layer liquid, and finally collect the solution to 3000 mL. Divide the solution into 30 mL / tube, and the concentration of each tube is 1000 ppm.

[0046] c. Heat the Ti3C2 liquid from room temperature to 80 °C, keep it warm for 24 h, then heat it to 100 °C and keep it warm for 24 h, and then cool it to room temperature at a cooling rate of 5 °C / min. Centrifuge and freeze-dry to obtain two-dimensional nanostructured TiO2.

[0047] The above has described the embodiments of the present invention in detail, but the present invention is not limited to the described embodiments. For those skilled in the art, without departing from the principles and spirit of the present invention, various changes, modifications, substitutions, and variations made to these embodiments still fall within the protection scope of the present invention.

Claims

1. A method for preparing a two-dimensional TiO2 nanomaterial, characterized in that, It includes the following steps: S1. Pour lithium fluoride into a 3 - 12 mol / L hydrochloric acid solution. After magnetic stirring in an ice - water bath until dissolved, add Ti3AlC2 powder in batches, keep the temperature at 30 - 50 °C, and stir for 24 - 72 hours to obtain the Ti3C2 stock solution; S2. Add the Ti3C2 stock solution into the solution in multiple times. After stirring evenly, perform ultrasonic oscillation, centrifuge, and collect the upper - layer liquid to obtain Ti3C2 with a concentration of 100 - 10000 ppm. In step S2, the solution is selected from PBS, pure water, deionized water, and alcohol, and the volume ratio of the Ti3C2 stock solution to the solution is 3 - 5:1 - 5; S3. Heat the obtained Ti3C2 liquid to 60 - 80 °C, keep it warm for 12 - 24 h, then heat it to 80 - 100 °C, keep it warm for 12 - 24 h, and then cool it to room temperature. After centrifugal freeze - drying, a two - dimensional TiO2 nanomaterial is obtained.

2. The preparation method of the two-dimensional TiO2 nanomaterial according to claim 1, characterized in that, In step S3, the cooling rate is 1 - 5 °C / min.

3. The preparation method of the two-dimensional TiO2 nanomaterial according to claim 1, characterized in that, In step S1, the molar ratio of lithium fluoride to the hydrochloric acid solution is 1 - 3:3 - 12.

4. The two-dimensional TiO2 nanomaterial obtained by the method according to any one of claims 1 to 3, characterized in that The two - dimensional TiO2 nanomaterial is spindle - shaped, with a length of 100 - 400 nm and a width of 20 - 100 nm.

5. Application of the two - dimensional TiO2 nanomaterial according to claim 4 in the preparation of a photosensitizer for sonodynamic therapy.

6. Use of the two-dimensional TiO2 nanomaterial according to claim 4 in the preparation of a functional product for treating tumors, characterized in that, The functional product can induce apoptosis of tumor cells under the excitation of ultrasound.

7. The application according to claim 6, characterized in that The conditions of the ultrasound are 0.5 - 2 W / cm 2 .

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