Two-dimensional titanium hydroxide nanosheet as well as preparation method and application thereof

Abstract

The invention relates to a two-dimensional titanium hydroxide nanosheet as well as a preparation method and application thereof. The two-dimensional titanium hydroxide nanosheet is prepared by combining an alkali-assisted hydrolysis method with an ultrasonic-assisted liquid phase crushing method. The transverse size of the two-dimensional titanium hydroxide nanosheet is 10-200nm, and the thickness of the two-dimensional titanium hydroxide nanosheet is less than 10nm. The two-dimensional titanium hydroxide nanosheet prepared by the invention has better catalytic oxidation resistance, biocompatibility and stability, and can be widely applied to the fields of catalysis and biomedicine.

Description

Technical Field

[0001] The present invention belongs to the field of nanomaterial technology and biomedicine, and relates to a two-dimensional titanium hydroxide nanosheet and a preparation method and application thereof, and specifically relates to a two-dimensional titanium hydroxide nanosheet with catalytic antioxidant properties and a preparation method thereof and application thereof in the preparation of a drug for treating diabetic cardiomyopathy. Background Art

[0002] The pathogenesis of diabetic cardiomyopathy (DCM) is closely related to the overexpression of reactive oxygen species (ROS), especially hydrogen peroxide (H2O2). Continuous high glucose can cause ROS overexpression and increased oxidative stress levels through various pathways such as mitochondrial dysfunction, induce cardiomyocyte apoptosis, and further induce inflammation, causing macrophages to transform into pro-inflammatory M1 type, secrete pro-inflammatory factors such as IL-6, IL-1β and TNF-α, and then lead to myocardial structural and functional lesions. Clearing the overexpressed ROS in the lesion area to relieve oxidative stress is of great significance for the treatment of DCM. However, there are very few international studies on DCM and it is still in the preliminary stage. Commonly used antioxidants such as N-acetylcysteine ​​and coenzyme Q10 have limited therapeutic effects on DCM. Summary of the invention

[0003] In view of the above problems, the present invention provides a two-dimensional titanium hydroxide nanosheet with catalytic antioxidant properties, a preparation method thereof, and an application of the two-dimensional titanium hydroxide nanosheet in the preparation of a drug for treating diabetic cardiomyopathy.

[0004] The inventors found in the research process that titanium hydroxide nanosheets have good catalytic antioxidant properties and can be used as nanocatalysts to trigger catalytic reactions in situ in the lesion area to remove ROS. In this process, the structure and composition of titanium hydroxide nanosheets do not change before and after the reaction, so they have sustainable antioxidant effects. In addition, cardiovascular diseases usually cause vascular endothelial damage, so titanium hydroxide nanosheets can be enriched in diseased myocardial tissues through enhanced penetration and retention effects, remove ROS, and alleviate oxidative stress damage.

[0005] In the present invention, the titanium hydroxide nanosheet is a two-dimensional nanomaterial with a huge specific surface area, which can expose as many active sites as possible, which is conducive to the reaction; for catalytic antioxidant therapy, the two-dimensional titanium hydroxide nanosheet has a unique advantage, which can improve the catalytic antioxidant efficiency, thereby improving the treatment efficiency. In addition, the two-dimensional titanium hydroxide nanosheet has good biocompatibility and stability, and can be widely used in the fields of catalysis and biomedicine.

[0006] In a first aspect, the present invention provides a two-dimensional titanium hydroxide nanosheet, which is prepared by an alkali-assisted hydrolysis method combined with an ultrasound-assisted liquid phase crushing method; the lateral size of the two-dimensional titanium hydroxide nanosheet is 10 to 200 nm and the thickness is less than 10 nm.

[0007] In a second aspect, the present invention provides a method for preparing the above-mentioned two-dimensional titanium hydroxide nanosheets, comprising: adding an alkaline solution dropwise into a titanium salt solution, and after the reaction is completed, performing a first centrifugal treatment to obtain a titanium hydroxide precipitate; The obtained titanium hydroxide precipitate is redispersed in water and ultrasonicated in an ice bath for 20 to 40 minutes using an ultrasonic cell pulverizer. The obtained mixed solution is subjected to a second centrifugal treatment for 2 to 10 minutes to obtain the two-dimensional titanium hydroxide nanosheets.

[0008] Preferably, the molar ratio of the titanium salt solution to the alkaline solution is 1:(0.3-2).

[0009] Preferably, the titanium salt in the titanium salt solution is selected from at least one of titanium sulfate, titanyl sulfate, titanium nitrate and titanium chloride; preferably titanium sulfate; the solvent of the titanium salt solution is deionized water; the concentration of the titanium salt solution is 0.01-1 mol / L.

[0010] Preferably, the solute in the alkaline solution is at least one of sodium hydroxide, potassium hydroxide, urea and ammonia water; the solvent of the alkaline solution is deionized water; and the concentration of the alkaline solution is 0.01-2 mol / L.

[0011] Preferably, the titanium salt solution is heated to 40-100° C. before adding the alkali solution. The purpose is to promote the formation of a two-dimensional sheet structure of titanium hydroxide.

[0012] Preferably, the reaction time is 5 to 120 minutes.

[0013] Preferably, the rotation speed of the first centrifugal treatment is 6000-15000 rpm.

[0014] The power of the ultrasonic cell pulverization is 100-1200W; The rotation speed of the second centrifugal treatment is 8000 to 23000 rpm.

[0015] In a third aspect, the present invention provides a use of the above-mentioned two-dimensional titanium hydroxide nanosheets in the preparation of a drug for treating diabetic cardiomyopathy.

[0016] Beneficial effects: The two-dimensional titanium hydroxide nanosheets prepared by the present invention have good catalytic antioxidant properties, can be enriched in the diabetic cardiomyopathy lesion area, remove excess hydrogen peroxide, protect myocardial cells, inhibit myocardial cell apoptosis, regulate the abnormal inflammatory microenvironment in the lesion area, and achieve catalytic antioxidant treatment for diabetic cardiomyopathy. Antioxidants on the market are often used as reactants to neutralize active oxygen, while the present invention catalyzes the spontaneous removal of active oxygen as a catalyst, so the dosage can be greatly reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a transmission electron microscopy image of the two-dimensional titanium hydroxide nanosheet prepared in Example 1; Figure 2 Element mapping of the two-dimensional titanium hydroxide nanosheets prepared in Example 1; Figure 3 A specific catalytic pathway diagram of the two-dimensional titanium hydroxide nanosheets prepared in Example 1; Figure 4 The antioxidant protective effect of two-dimensional titanium hydroxide nanosheets of different concentrations on myocardial cells in Example 2; Figure 5 The effect of the two-dimensional titanium hydroxide nanosheets in Example 3 on the expression of M1 macrophage markers (IL-6, IL-1β and TNF-α) and M2 macrophage markers (Arg-1 and IL-10) of M1 macrophages; wherein the control group represents: no reagent or material is added; the treatment group represents: only 1ppm LPS is added; the material group represents: 1ppm LPS and 50ppm titanium hydroxide solution are added; Figure 6 The effect of the anti-inflammatory effect of two-dimensional titanium hydroxide nanosheets on the viability of cardiomyocytes was evaluated by the indirect co-culture method in Example 4; wherein, the control group represented: the control group in Example 3 was cultured for 12 hours, and then the culture medium was replaced with fresh culture medium, and then the supernatant was centrifuged after 24 hours for culturing cardiomyocytes; the treatment group represented: the treatment group in Example 3 was cultured for 12 hours, and then the culture medium was replaced with fresh culture medium, and then the supernatant was centrifuged after 24 hours for culturing cardiomyocytes; the material group represented: the material group in Example 3 was cultured for 12 hours, and then the culture medium was replaced with fresh culture medium, and then the supernatant was centrifuged after 24 hours for culturing cardiomyocytes; Figure 7 It is a quantitative index measured by echocardiography of the diabetic cardiomyopathy model mice in Example 5 before and after tail vein injection of phosphate buffered saline (PBS) containing two-dimensional titanium hydroxide nanosheets for 14 days; wherein, the control group represents: mice fed with normal feed; the treatment group represents: type 2 diabetic cardiomyopathy mice fed with high fat and injected with streptozotocin; the material group represents: mice in the treatment group injected with phosphate buffered saline (10 mg / kg) containing two-dimensional titanium hydroxide nanosheets; Figure 8 The hematoxylin-eosin staining pictures of the heart tissue of the diabetic cardiomyopathy model mice in Example 5 before and after the tail vein injection of PBS containing two-dimensional titanium hydroxide nanosheets for 14 days; wherein, the control group represents: mice fed with normal feed; the treatment group represents: type 2 diabetic cardiomyopathy mice fed with high fat and injected with streptozotocin; the material group represents: mice in the treatment group injected with phosphate buffer (10 mg / kg) containing two-dimensional titanium hydroxide nanosheets; Fig. 9 This is a transmission electron microscopy image of the titanium hydroxide nanosheet prepared in Comparative Example 1; Fig.10 This is a transmission electron microscopy image of the titanium hydroxide nanosheet prepared in Comparative Example 2; Fig.11 This is a transmission electron microscopy image of the titanium hydroxide nanosheets prepared in Comparative Example 3. DETAILED DESCRIPTION

[0018] To further explain the content, features and practical effects of the present invention, the present invention is described in detail below in conjunction with the embodiments. It should be pointed out that the modification method of the design of the present invention is not limited to these specific implementation methods. Without departing from the spirit and connotation of the design of the present invention, the equivalent replacement and modification made by those skilled in the art on the basis of reading the content of the present invention are also within the scope of the present invention.

[0019] The present invention aims at the technical bottleneck in the existing treatment of diabetic cardiomyopathy, and uses alkali-assisted hydrolysis combined with ultrasound-assisted liquid phase crushing to prepare a two-dimensional titanium hydroxide nanosheet with catalytic antioxidant properties. The two-dimensional titanium hydroxide nanosheet has good catalytic antioxidant properties, and the amorphous structure exposes more catalytic active sites, thereby improving the catalytic ability. At the same time, the two-dimensional titanium hydroxide nanosheet also has good myocardial cell protection and anti-inflammatory properties.

[0020] In the present invention, the two-dimensional titanium hydroxide nanosheet can catalyze the disproportionation reaction of H2O2 and remove active oxygen. The titanium in the two-dimensional titanium hydroxide nanosheet is tetravalent, and the entire catalytic reaction is based on Ti IV / Ti III The specific catalytic pathway is that a molecule of hydrogen peroxide is first adsorbed on the material, one of the hydrogen atoms combines with the hydroxyl group coordinated with the titanium ion to form a weak hydrogen bond, and then breaks down to generate a molecule of water. At the same time, the charge is transferred, and Ti IV Become Ti IIIThe active species then combines with the hydroxyl group on another titanium ion to form a hydrogen bond, which is further broken down to generate another molecule of water and oxygen. Then another molecule of hydrogen peroxide is adsorbed on the material, and the two hydroxyl groups in it coordinate with the two titanium ions respectively, the OO bond is broken, and it returns to the initial structure (such as Figure 3 shown).

[0021] The two-dimensional titanium hydroxide nanosheet of the present invention only acts as a catalyst rather than a reactant in the process of catalytic antioxidant removal of ROS, and the structure and composition do not change before and after the reaction, so it has a sustainable antioxidant effect. At the same time, the two-dimensional titanium hydroxide nanosheet shows good blood, tissue compatibility and biosafety.

[0022] In the present invention, large titanium hydroxide nanosheets are formed by reacting titanium salt with alkaline solution, and then ultrasonic cell disruption is used to obtain the two-dimensional titanium hydroxide nanosheets. The following is an exemplary description of the preparation method of the two-dimensional titanium hydroxide nanosheets provided by the present invention.

[0023] The titanium salt is added into deionized water, and a uniformly dispersed titanium salt solution is obtained by magnetic stirring and ultrasonic treatment.

[0024] In an optional embodiment, the magnetic stirring time is 5 to 60 minutes.

[0025] In an optional embodiment, the titanium salt in the titanium salt solution is selected from at least one of titanium sulfate, titanyl sulfate, titanium nitrate, and titanium chloride; preferably titanium sulfate; the solvent of the titanium salt solution is deionized water; and the concentration of the titanium salt solution is 0.01 to 1 mol / L.

[0026] The titanium salt solution is heated, and an alkali solution is added dropwise thereto to carry out a reaction.

[0027] In an optional embodiment, the heating temperature is 40-100° C. In the present invention, the purpose of heating the titanium salt solution before the reaction is to promote the formation of a sheet structure of titanium hydroxide nanosheets. If not heated, a two-dimensional sheet structure cannot be formed, but large agglomerates are formed.

[0028] In an optional embodiment, the molar ratio of the titanium salt solution to the alkaline solution is 1:(0.3-2). If the amount of alkaline solution added is excessive, a two-dimensional sheet structure cannot be formed, but particle agglomerates are formed. The reaction time is 5-120 minutes.

[0029] After the reaction is completed, the mixed solution is centrifuged for the first time for 5 to 20 minutes to obtain a white product, and the product is washed twice with deionized water and anhydrous ethanol respectively. The obtained product is redispersed in deionized water and ultrasonicated in an ice bath for 20 to 40 minutes using an ultrasonic cell crusher. The obtained mixed solution is centrifuged for a second time for 2 to 10 minutes, and the obtained precipitate is a nano-scale two-dimensional titanium hydroxide nanosheet.

[0030] In an optional embodiment, the rotation speed of the first centrifugation is 6000-15000 rpm.

[0031] In an optional embodiment, the power of the ice bath ultrasound is 100-1200 W. The rotation speed of the second centrifugation is 8000-23000 rpm.

[0032] In the present invention, the lateral size of the two-dimensional titanium hydroxide nanosheet is 10 to 200 nm, and the thickness is less than 10 nm, preferably, the thickness is 1 to 20 nm. The two-dimensional titanium hydroxide nanosheet can be uniformly dispersed in an aqueous solution. The two-dimensional titanium hydroxide nanosheet is an amorphous structure, which can expose more catalytic active sites, which is conducive to the catalytic reaction.

[0033] The preparation method of the two-dimensional titanium hydroxide nanosheet provided by the invention is simple, has high yield and is easy for batch production; the prepared two-dimensional titanium hydroxide nanosheet has good catalytic antioxidant properties.

[0034] The following further examples are given to illustrate the present invention in detail. It should also be understood that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention belong to the scope of protection of the present invention. The specific process parameters and the like in the following examples are also only examples within a suitable range, that is, those skilled in the art can make a selection within a suitable range through the description herein, and are not limited to the specific values ​​​​exemplified below.

[0035] Example 1 0.5 g of titanium sulfate was added to 40 mL of deionized water, and a uniformly dispersed titanium sulfate solution was obtained by magnetic stirring and ultrasound. Then, the titanium sulfate solution was heated to 80 ° C, and 15 drops of 2M NaOH (the molar ratio of titanium salt solution to alkaline solution was 1: 0.72) were added dropwise thereto, and the reaction was carried out for 15 minutes. After the reaction was completed, the mixed solution was centrifuged for 5 minutes, and the obtained white product was washed twice with deionized water and anhydrous ethanol. Subsequently, the obtained product was redispersed in deionized water, and ultrasonicated in an ice bath with an ultrasonic cell crusher for 30 minutes. The obtained mixed solution was centrifuged for 5 minutes, and the obtained precipitate was a nano-scale two-dimensional titanium hydroxide nanosheet.

[0036] Figure 1 This is a transmission electron microscope image of the two-dimensional titanium hydroxide nanosheet prepared in Example 1. As can be seen from the figure, the obtained two-dimensional titanium hydroxide nanosheet has good dispersibility and a size of about 100 nm.

[0037] Figure 2 This is the element mapping of the two-dimensional titanium hydroxide nanosheet prepared in Example 1. As can be seen from the figure, the two elements Ti and O are evenly distributed on the nanosheet.

[0038] Example 2 Aqueous solutions of the two-dimensional titanium hydroxide nanosheets prepared in Example 1 with different concentrations (1, 2, 4 and 8 ppm) were prepared, 2 mM hydrogen peroxide was added thereto to induce myocardial apoptosis, and the catalytic antioxidant ability to protect myocardial cells was detected.

[0039] Figure 4 The figure shows the antioxidant protective effect of different concentrations of two-dimensional titanium hydroxide nanosheets on cardiomyocytes in Example 2. As can be seen from the figure, the addition of 2mM hydrogen peroxide leads to a significant decrease in cardiomyocyte activity, and the addition of the two-dimensional titanium hydroxide nanosheets prepared by the present invention restores cardiomyocyte activity and shows concentration dependence. As the concentration of the two-dimensional titanium hydroxide nanosheets increases, the cardiomyocyte activity is restored higher, indicating that the two-dimensional titanium hydroxide nanosheets gradually enhance their ability to scavenge reactive oxygen species.

[0040] Example 3 Raw264.7 cells were induced into M1 macrophages by lipopolysaccharide, and then two-dimensional titanium hydroxide nanosheets (50 ppm) were added to the culture medium for co-culture for 24 hours. The relative expression levels of M1 macrophage markers (IL-6, IL-1β and TNF-α) and M2 macrophage markers (Arg-1 and IL-10) in the cells were detected by RT-PCR.

[0041] Figure 5 The effect of the two-dimensional titanium hydroxide nanosheets in Example 3 on the expression of M1 macrophage markers (IL-6, IL-1β and TNF-α) and M2 macrophage markers (Arg-1 and IL-10) of M1 macrophages. As can be seen from the figure, after 24 hours of co-culture with the prepared two-dimensional titanium hydroxide nanosheets, the expression of M1 macrophage markers (IL-6, IL-1β and TNF-α) of macrophages decreased, and the expression of M2 macrophage markers (IL-10 and Arg-1) increased. That is, the two-dimensional titanium hydroxide nanosheets prepared by the present invention can reduce the expression of M1 macrophage markers and increase the expression of M2 macrophage markers, that is, the macrophages are transformed from the pro-inflammatory M1 phenotype to the M2 anti-inflammatory M2 phenotype.

[0042] Example 4 The culture medium of M1 macrophages was added to cardiomyocytes by indirect co-culture method, and then two-dimensional titanium hydroxide nanosheets (50ppm) were added to the culture medium for co-culture for 24 hours. The CCK-8 method was used to detect the anti-inflammatory effect of two-dimensional titanium hydroxide nanosheets on the viability of cardiomyocytes.

[0043] Figure 6 The anti-inflammatory effect of the two-dimensional titanium hydroxide nanosheets on the viability of myocardial cells was evaluated by the indirect co-culture method in Example 4. As can be seen from the figure, the two-dimensional titanium hydroxide nanosheets prepared by the present invention can restore the viability of myocardial cells to a large extent.

[0044] Example 5 Phosphate buffer (PBS) containing two-dimensional titanium hydroxide nanosheets (the concentration of two-dimensional titanium hydroxide nanosheets in phosphate buffer was 1000ppm) was injected into the diabetic cardiomyopathy model mice via the tail vein at an injection dose of 10mg / kg. After 14 days, Doppler ultrasound, cardiac tissue section staining and other analytical methods were used to explore the improvement of myocardial function by two-dimensional titanium hydroxide nanosheets, and the therapeutic effect of the two-dimensional titanium hydroxide nanosheets on diabetic cardiomyopathy in vivo was systematically evaluated.

[0045] Figure 7 It is a quantitative index measured by echocardiography of the diabetic cardiomyopathy model mice in Example 5 before and after the tail vein injection of phosphate buffered saline (PBS) containing two-dimensional titanium hydroxide nanosheets for 14 days. As can be seen from the figure, by directly injecting PBS containing two-dimensional titanium hydroxide nanosheets into the diabetic cardiomyopathy model mice through the tail vein, the left ventricular ejection fraction and the left ventricular short axis shortening rate of the mice were improved after 14 days, while the left ventricular end-diastolic diameter and the left ventricular end-systolic diameter were reduced, indicating that the heart contraction and diastolic function were restored. That is, the heart contraction and diastolic function of the mice were improved after the injection of PBS containing two-dimensional titanium hydroxide nanosheets.

[0046] Figure 8 The following are hematoxylin-eosin staining images of the heart tissue of the diabetic cardiomyopathy model mice in Example 5 before and after the tail vein injection of PBS containing two-dimensional titanium hydroxide nanosheets for 14 days. As can be seen from the figure, the cardiomyocytes of the mice without injection were hypertrophic and disordered, while the cardiomyocytes of the mice were restored after the injection of PBS containing two-dimensional titanium hydroxide nanosheets.

[0047] Comparative Example 1

[0048] The preparation method of titanium hydroxide nanosheets in this comparative example 1 is the same as that in Example 1, except that ultrasonic-assisted liquid phase crushing is not performed after the white product is obtained.

[0049] Fig. 9This is a transmission electron microscope image of the titanium hydroxide nanosheets prepared in Comparative Example 1. As can be seen from the image, without ultrasonic-assisted liquid phase fragmentation, it is found that some titanium hydroxide nanosheets are agglomerated.

[0050] Comparative Example 2

[0051] The preparation method of titanium hydroxide nanosheets in this comparative example 2 is the same as that in Example 1, except that the titanium sulfate solution is not heated before adding NaOH.

[0052] Fig.10 This is a transmission electron microscope image of titanium hydroxide nanosheets prepared in Comparative Example 2. As can be seen from the figure, when NaOH is added to the titanium sulfate solution at room temperature, titanium hydroxide nanosheets with a two-dimensional sheet structure cannot be prepared after the reaction, but large agglomerates with a size of several microns are formed.

[0053] Comparative Example 3

[0054] The preparation method of titanium hydroxide nanosheets in this comparative example 3 is the same as that in Example 1, except that 20 mL of 2M NaOH is added to the titanium sulfate solution.

[0055] Fig.11 The transmission electron microscope image of the titanium hydroxide nanosheet prepared in comparative example 3. As can be seen from the figure, after adding an excess of alkali (the molar ratio of the titanium salt solution to the alkali solution is 1:19), two-dimensional titanium hydroxide nanosheets are not formed, but rather larger particles are formed and agglomerated together.

Claims

1. A two-dimensional titanium hydroxide nanosheet, characterized in that: The two-dimensional titanium hydroxide nanosheet is prepared by an alkali-assisted hydrolysis method combined with an ultrasound-assisted liquid phase crushing method; the lateral size of the two-dimensional titanium hydroxide nanosheet is 10 to 200 nm, and the thickness is less than 10 nm.

2. A method for preparing the two-dimensional titanium hydroxide nanosheet according to claim 1, comprising: The alkaline solution is added dropwise into the titanium salt solution, and after the reaction is completed, a titanium hydroxide precipitate is obtained by a first centrifugation treatment; The obtained titanium hydroxide precipitate is redispersed in water and ultrasonicated in an ice bath for 20 to 40 minutes using an ultrasonic cell pulverizer. The obtained mixed solution is subjected to a second centrifugal treatment for 2 to 10 minutes to obtain the two-dimensional titanium hydroxide nanosheets.

3. The preparation method according to claim 2, characterized in that: The molar ratio of the titanium salt solution to the alkaline solution is 1:(0.3-2).

4. The preparation method according to claim 2 or 3, characterized in that: The titanium salt in the titanium salt solution is selected from at least one of titanium sulfate, titanyl sulfate, titanium nitrate and titanium chloride; preferably titanium sulfate; the solvent of the titanium salt solution is deionized water; the concentration of the titanium salt solution is 0.01-1 mol / L.

5. The preparation method according to any one of claims 2 to 4, characterized in that: The solute in the alkaline solution is at least one of sodium hydroxide, potassium hydroxide, urea and ammonia water; the solvent of the alkaline solution is deionized water; and the concentration of the alkaline solution is 0.01-2 mol / L.

6. The preparation method according to any one of claims 2 to 5, characterized in that: Before adding the alkali solution, heat the titanium salt solution to 40-100°C.

7. The preparation method according to any one of claims 2 to 6, characterized in that: The reaction time is 5 to 120 minutes.

8. The preparation method according to any one of claims 2 to 7, characterized in that: The rotation speed of the first centrifugal treatment is 6000 to 15000 rpm; The power of the ultrasonic cell pulverization is 100-1200W; The rotation speed of the second centrifugal treatment is 8000 to 23000 rpm.

9. Use of the two-dimensional titanium hydroxide nanosheet according to claim 1 in preparing a drug for treating diabetic cardiomyopathy.

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