Hafnium diselenide nanosheet and application of hafnium diselenide nanosheet in treatment of colitis

Abstract

The invention discloses a hafnium diselenide nanosheet and application of the hafnium diselenide nanosheet in treatment of colitis. The hafnium diselenide nanosheet is synthesized by a liquid phase stripping method. The hafnium diselenide nanosheet disclosed by the invention has good capabilities of removing active oxygen and active nitrogen and inhibiting ferroptosis, and can be used for treating colitis.

Description

Technical Field

[0001] The invention belongs to the technical field of nanomaterials, and specifically relates to a hafnium diselenide nanosheet and an application thereof in treating colitis. Background Art

[0002] Ulcerative colitis (UC) is a chronic nonspecific intestinal inflammatory disease with a high incidence and difficult to cure. Reactive oxygen species (ROS) are a class of oxygen-containing single-electron byproducts produced by oxygen molecules during cellular respiration and biological metabolism, including superoxide free radicals, hydroxyl free radicals, hydrogen peroxide, etc. Reactive nitrogen species (RNS) refers to a class of highly reactive nitrogen compounds, including nitrogen free radicals, NO, etc. Current studies have shown that the intestinal epithelium is exposed to a diverse and complex environment at the mucosal interface, and the destruction of the integrity of the epithelial barrier is related to inflammatory pathology. The large accumulation of ROS and RNS (hereinafter collectively referred to as RONS) at the inflammatory site will lead to oxidative stress and trigger cell death in multiple pathways. Cell death will destroy the mucosal barrier, induce further inflammation and bleeding, and secrete a series of proinflammatory cytokines, forming a vicious cycle. Many animal and clinical studies have confirmed that high levels of RONS lead to oxidative damage to proteins, lipids and DNA, which further leads to aggravation of mucosal damage, mucosal ulcers and intestinal inflammation.

[0003] Ferroptosis is a type of programmed cell death caused by iron-induced lipid peroxidation and is involved in many pathological conditions. Ferroptosis is a key regulatory mechanism for intestinal diseases. Relevant literature reports that the use of iron chelators can significantly reduce ROS in colon tissue of UC patients and improve clinical symptoms. Existing studies have found that selenium plays an indispensable role in the body and exists as an essential trace element for the body. The role of selenoprotein GPX4 in the regulatory pathway of ferroptosis is also irreplaceable. It can use glutathione to inhibit ferroptosis by detoxifying lipid hydroperoxides into non-toxic lipid alcohols, thereby further maintaining a healthy intestinal environment. Summary of the invention

[0004] Inspired by the mechanism of scavenging reactive oxygen and inhibiting ferroptosis, the present invention successfully developed a hafnium diselenide nanosheet with the ability to scavenge reactive oxygen and reactive nitrogen and inhibit ferroptosis, thereby solving the problem of increased reactive oxygen and reactive nitrogen and ferroptosis of intestinal cells during the onset of enteritis.

[0005] In order to achieve the above object, the present invention adopts the following technical scheme:

[0006] The present invention first discloses a hafnium diselenide nanosheet, which is prepared by a liquid phase exfoliation method using hafnium diselenide powder as a raw material. The preparation method comprises the following steps:

[0007] First, Pluronic F127 is dissolved in isopropanol, and then hafnium diselenide powder is added thereto, and ultrasonication is performed for 6 to 8 hours at a power of 250 to 350 W using a cell disruptor under ice bath conditions; after the ultrasonication is completed, the supernatant is centrifuged at 1500 to 2000 rpm for 10 to 15 minutes, and the supernatant is collected; the collected supernatant is centrifuged at 12000 to 15000 rpm for 15 to 20 minutes, and the obtained precipitate is washed by centrifugation with deionized water to obtain hafnium diselenide nanosheets, which are dispersed in deionized water and stored at 4°C; wherein the usage ratio of hafnium diselenide powder, Pluronic F127 and isopropanol is 30 mg: 120 to 150 mg: 25 to 30 mL.

[0008] The hafnium diselenide nanosheets prepared by the present invention have a particle size of 270-350 nm and a thickness of 1-1.5 nm, and can treat colitis by efficiently removing excessive ROS and RNS at the inflammatory site and inhibiting ferroptosis without hemolytic behavior.

[0009] The beneficial effects of the present invention are embodied in:

[0010] 1. The present invention prepares hafnium diselenide nanosheets by a liquid phase exfoliation method, which is simple and mild. The obtained nanosheets have uniform size and stable performance. Moreover, the nanosheets have good stability and dispersibility through modification with Pluronic F127.

[0011] 2. The hafnium diselenide nanosheets of the present invention improve the ability and types of removing active oxygen and active nitrogen through low-cost selenium, and can target a variety of inflammatory diseases.

[0012] 3. The hafnium diselenide nanosheets of the present invention have the ability to inhibit ferroptosis of intestinal cells and can be used to alleviate the occurrence of colitis.

[0013] 4. The hafnium diselenide nanosheets of the present invention have good biosafety, are conducive to clinical use, and can be used to treat colitis. In the DSS (dextran sulfate)-induced delayed colitis model, the occurrence of enteritis was significantly alleviated. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a transmission electron microscope image of the hafnium diselenide nanosheets prepared in Example 1.

[0015] Figure 2 This is an atomic force microscope image of the hafnium diselenide nanosheets prepared in Example 1.

[0016] Figure 3 The hafnium diselenide nanosheets prepared in Example 1 are effective in removing RONS, wherein (a) is the removal effect of DPPH, and (b) is the removal effect of hydrogen peroxide.

[0017] Figure 4 This is a graph showing the GPX4-like enzyme activity of the hafnium diselenide nanosheets prepared in Example 1.

[0018] Figure 5 This is a test graph of the hafnium diselenide nanosheets prepared in Example 1 inhibiting ferroptosis of Caco-2 cells.

[0019] Figure 6 This is a graph showing the in vitro hemolytic performance test of the hafnium diselenide nanosheets prepared in Example 1.

[0020] Figure 7 This is a graph showing the colon length recovery effect of mice before and after treatment with the hafnium diselenide nanosheets prepared in Example 1 and the control group.

[0021] Figure 8 This is a graph showing the MDA content in the colon of mice before and after treatment with the hafnium diselenide nanosheets prepared in Example 1 and the control group. DETAILED DESCRIPTION

[0022] In order to make the above-mentioned purposes, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail in combination with the embodiments below. The following content is only an example and explanation of the concept of the present invention. The technicians in the relevant technical field make various modifications or supplements to the specific embodiments described or replace them in a similar manner. As long as they do not deviate from the concept of the invention or exceed the scope defined by the claims, they should all fall within the protection scope of the present invention.

[0023] Example 1

[0024] In this embodiment, hafnium diselenide nanosheets are prepared as follows:

[0025] First, 120 mg of Pluronic F127 was dissolved in 25 mL of isopropanol, and then 30 mg of hafnium diselenide powder was added thereto, and ultrasonicated for 8 h with a cell disruptor at a power of ~300 W in an ice bath; after the ultrasonication was completed, the mixture was centrifuged at 1500 rpm for 15 min to remove larger hafnium diselenide nanosheets, and the supernatant was collected; the collected supernatant was centrifuged at 15000 rpm for 20 min, and the obtained precipitate was washed by centrifugation with deionized water to obtain hafnium diselenide nanosheets (denoted as HfSe2@F127 NSs), which were dispersed in deionized water and stored at 4°C.

[0026] Figure 1 This is a transmission electron microscope image of the hafnium diselenide nanosheet obtained in this example. From the image, it can be seen that the material presents a sheet-like structure, and the size of the nanosheet is 270-350 nm.

[0027] Figure 2 This is an atomic force microscope image of the hafnium diselenide nanosheet obtained in this example ( Figure 2(a) and thickness distribution diagram ( Figure 2 In (b), it can be seen that the thickness of the nanosheet is 1 to 1.5 nm.

[0028] Figure 3 This is a diagram showing the effect of the hafnium diselenide nanosheets obtained in this example on removing RONS. Figure 3 (a) is a diagram of the scavenging effect of DPPH. The characterization method is as follows: weigh 2 mg of DPPH and dissolve it in 40 mL of ethanol. The UV absorbance at 519 nm is measured at 1.2-1.3. Take 1 mL of DPPH solution and then add 500 μL of different concentrations of HfSe2 nanosheet aqueous dispersion (so that the final concentration of the material in the system is 2.5, 5, 10, 20 μg / mL). React at room temperature for 15 minutes. Set the DPPH group alone as a positive control and measure the UV absorbance of each group at 519 nm. It can be seen from the figure that the ability to scavenging DPPH can reach 95% even at a very low concentration (20 μg / mL). Figure 3 (b) is a hydrogen peroxide scavenging effect diagram, and its characterization method is: add 100 μL of different concentrations of hafnium diselenide nanosheet aqueous dispersion to 300 μL, 10 μM hydrogen peroxide solution (so that the final concentration of the material in the system is 20, 40, 80, 160 μg / mL), and react at 37°C for 2 hours. After the reaction is completed, add 300 μL of fluorescent red and 300 μL of catalase, react at room temperature and avoid light for 15 minutes, and finally measure the absorbance at 570nm with a fluorescence photometer. It can be seen from the figure that the ability to scavenging hydrogen peroxide reaches 95% at the highest concentration of 160 μg / mL. The above results show that hafnium diselenide nanosheets have good RONS scavenging ability.

[0029] Figure 4 The GPX4-like enzyme activity of the hafnium diselenide nanosheets obtained in this example is characterized by using 5,5'-dithio-2-nitrobenzoic acid (DTNB) as a substrate to detect the GPX4-like enzyme activity of HfSe2@F127. -1 ) was added to the aqueous dispersion of hafnium diselenide NSs (final concentration of 1 mM) and H2O2 (final concentration of 2 mM). After incubation for 1 hour, the reaction solution was mixed with 800 μL PBS buffer and DTNB (final concentration of 1 mM) was added. After incubation in the dark for 5 minutes, the supernatant was obtained by centrifugation (12000 rpm, 5 minutes), and the absorbance at 450 nm was finally measured. The sample without NSs or H2O2 was used as a control. The results show that hafnium diselenide nanosheets have good GPX4-like enzyme activity.

[0030] Figure 5The experimental figure of the hafnium diselenide nanosheets obtained in this example inhibiting the ferroptosis of colon adenocarcinoma cells in vitro is shown in Figure 1. The characterization method is as follows: 100 μL (1×10 5 ) were incubated with Caco-2 cells for 24 hours, the supernatant was discarded, and different concentrations of hafnium diselenide nanosheets prepared with DMEM (the final concentration after addition was 0, 6.25, 12.5, 25, 50 μg / mL) were added, and then the ferroptosis inducer RSL3 (the final concentration after addition was 30 μM) was added for a total incubation of 24 hours. After the incubation, 20 μL MTT (5 mg / mL) was added to each well and incubated in the incubator for 4 hours. Finally, the supernatant was discarded, and 150 μL DMSO was added to each well to terminate the culture. Incubate in the dark at 37°C for 10 minutes, and measure the absorbance at 490 nm. It can be seen from the figure that with the increase in the concentration of hafnium diselenide nanosheets, the cell survival rate increases accordingly, indicating that hafnium diselenide nanosheets have a good ability to inhibit ferroptosis of colon adenocarcinoma cells.

[0031] Figure 6 The in vitro hemolysis performance test diagram of the hafnium diselenide nanosheet obtained in this embodiment is characterized by: adding 0.5mL of fresh blood to 4.5mL of normal saline and centrifuging and washing 5 to 8 times, the centrifugal speed is 3000rpm, the time is 10min, washing until the supernatant is clear and transparent, discarding the supernatant, and diluting to 5mL with normal saline. 0.2mL of different concentrations of hafnium selenide nanosheet aqueous dispersions are mixed with 0.2mL of treated blood and 0.6mL of normal saline (the final concentration of the nanosheet is 6.25, 12.5, 25, 50, 100μg / mL), incubated at 37℃ for 4h, and positive and negative control groups are set. After that, centrifuge at 3000rpm for 10min, take the supernatant, measure the absorbance at 541nm, and calculate the hemolysis rate. It can be seen from the figure that there is no obvious hemolysis of hafnium diselenide nanosheets of different concentrations, indicating that the material has good biocompatibility.

[0032] In order to verify the therapeutic effect of the hafnium diselenide nanosheets prepared in this example on DSS-induced colitis, the following design was performed: 40 female C57BL / 6 mice were prepared and adapted to the environment for one week. The colitis modeling group was given 2.5% DSS on days 0 to 7, and the blank control group was given water. The mice were gavaged on days 5, 7, 9, and 11. On day 13, the mice were euthanized and their colon tissues were taken. The 40 C57BL / 6 mice were divided into 5 groups: (1) normal control group: PBS+water; (2) control group: PBS+2.5% DSS; (3) low concentration treatment group: hafnium diselenide nanosheets 10 mg / kg+2.5% DSS; (4) medium concentration treatment group: hafnium diselenide nanosheets 20 mg / kg+2.5% DSS; (5) high concentration treatment group: hafnium diselenide nanosheets 30 mg / kg+2.5% DSS. There were 8 mice in each group. PBS and hafnium diselenide nanosheets were intragastrically administered on days 5, 7, 9, and 11. On day 13, the colons of mice in each group were dissected and separated, and the colon lengths were recorded by taking photos. Figure 7 This is a statistical graph of the colon length of mice in each group after treatment. It can be seen from the figure that the colon length was effectively restored after treatment with hafnium diselenide nanosheets.

[0033] To further demonstrate the therapeutic effect of the hafnium diselenide nanosheets obtained in this example on DSS-induced colitis, the colons of each group of mice were dissected and separated, and the contents were removed. The mixture was homogenized with a homogenizer on ice at a ratio of 100 mg tissue + 2 mL PBS, centrifuged at 10,000 g-12,000 g for 10 minutes, and the supernatant was taken. The MDA content was then measured using an MDA detection kit. The results are shown in Figure 2. Figure 8 As shown in the figure, it can be seen that the MDA content in the colon tissue of mice was significantly reduced after treatment with hafnium diselenide nanosheets.

[0034] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A hafnium diselenide nanosheet, characterized in that: The hafnium diselenide nanosheet is prepared by a liquid phase exfoliation method using hafnium diselenide powder as a raw material.

2. The hafnium diselenide nanosheet according to claim 1, characterized in that: The particle size of the hafnium diselenide nanosheet is 270-350 nm, and the thickness is 1-1.5 nm.

3. A method for preparing the hafnium diselenide nanosheets according to claim 1 or 2, characterized in that: The following steps are involved: First, Pluronic F127 is dissolved in isopropanol, and then hafnium diselenide powder is added thereto, and ultrasonication is performed for 6 to 8 hours with a cell disruptor at a power of 250 to 350 W under ice bath conditions; after the ultrasonication is completed, the supernatant is centrifuged at 1500 to 2000 rpm for 10 to 15 minutes, and the supernatant is collected; the collected supernatant is centrifuged at 12000 to 15000 rpm for 15 to 20 minutes, and the obtained precipitate is washed by centrifugation with deionized water to obtain hafnium diselenide nanosheets, which are dispersed in deionized water and stored at 4°C; The usage ratio of hafnium diselenide powder, Pluronic F127 and isopropyl alcohol is 30 mg: 120-150 mg: 25-30 mL.

4. An application of the hafnium diselenide nanosheet according to claim 1 or 2, characterized in that: Used to prepare a medicament for treating colitis by inhibiting ferroptosis and scavenging reactive oxygen and reactive nitrogen.

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