Application of thiolated carbon dots in the preparation of drugs for inhibiting ferroptosis or treating acute liver injury

Abstract

The present invention relates to the specific uses or application technologies of nanostructures, and more particularly to the use of thiolated carbon dots in the preparation of drugs for inhibiting ferroptosis or treating acute liver injury. When used in the preparation of drugs for inhibiting ferroptosis, the thiolated carbon dots can stably and long-term inhibit RSL3-induced ferroptosis. When used in the preparation of drugs for treating acute liver injury, the thiolated carbon dots have a significant alleviating effect on acetaminophen-induced acute liver injury in mice. Furthermore, the thiolated carbon dots exhibit a sustained, stable, and long-term inhibitory effect on acetaminophen-induced ferroptosis, with the inhibitory effect lasting at least 72 hours.

Description

Technical Field

[0001] The present invention relates to the field of specific uses or application technologies of nanostructures, and in particular to the use of thiolated carbon dots in the preparation of drugs for inhibiting ferroptosis or treating acute liver injury. Background Art

[0002] Ferroptosis is a regulated cell death mechanism that has been shown to be closely related to a variety of diseases, including kidney disease, heart disease, and liver disease. However, the widely used inhibitors currently designed for the ferroptosis pathway are limited by various factors. For example, Ferrostatin-1 is expensive and has a short half-life in the body and is not effective in inhibiting ferroptosis induced by certain drugs; the iron chelators DFO and DFP have low bioavailability; and free radical scavengers have unknown toxic side effects.

[0003] Acute liver injury is the rapid damage of liver cells caused by various causes in patients without chronic liver disease. Acetaminophen (APAP) is a clinical drug for the treatment of diseases such as fever, and its excessive accumulation often leads to acute liver injury. The occurrence and development of acetaminophen-induced acute liver injury is closely related to ferroptosis. Carbon dot nanoparticles have attracted widespread attention due to their excellent properties and are also widely used in the biomedical field, such as bioimaging, disease diagnosis, drug delivery, and cancer treatment. Recently, a large number of studies have found that carbon dot nanomaterials can promote or inhibit ferroptosis and are potential clinical preparations for the treatment of ferroptosis-related diseases. However, at present, few studies have reported that carbon dot nanoparticles can alleviate APAP-induced acute liver injury by inhibiting ferroptosis. Summary of the Invention

[0004] The purpose of the present invention is to provide the use of thiolated carbon dots in the preparation of drugs for inhibiting ferroptosis or treating acute liver injury, so as to overcome the defects or shortcomings of existing nanomaterials in their poor inhibitory effect on ferroptosis.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides the use of thioated carbon dots in preparing drugs for inhibiting cell ferroptosis.

[0007] Optionally, the thio-substituted carbon dots inhibit RSL3-induced cell ferroptosis.

[0008] Optionally, the method for preparing the thio-substituted carbon dots comprises the following steps:

[0009] Cystamine hydrochloride, m-phenylenediamine and water are mixed and subjected to a hydrothermal reaction. After the reaction is completed, the supernatant is collected by centrifugation to obtain thiocarbon dots.

[0010] Optionally, the dosage ratio of cystamine hydrochloride, m-phenylenediamine and water is 100 mg:50 mg:5-15 mL;

[0011] The temperature of the hydrothermal reaction is 150-200° C., and the time is 5-12 hours.

[0012] Optionally, the usage amount of the thio-substituted carbon dots is 5 μg / L or more.

[0013] The present invention also provides the use of thiocarbon dots in the preparation of drugs for treating acute liver injury.

[0014] Optionally, the acute liver injury is acetaminophen-induced acute liver injury.

[0015] Optionally, the method for preparing the thio-substituted carbon dots comprises the following steps:

[0016] Cystamine hydrochloride, m-phenylenediamine and water are mixed and subjected to a hydrothermal reaction. After the reaction is completed, the supernatant is collected by centrifugation to obtain thiocarbon dots.

[0017] Optionally, the dosage ratio of cystamine hydrochloride, m-phenylenediamine and water is 100 mg:50 mg:5-15 mL;

[0018] The temperature of the hydrothermal reaction is 150-200° C., and the time is 5-12 hours.

[0019] Optionally, the usage amount of the thio-substituted carbon dots is 0.3-1 mg / kg.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] When the present invention applies thiocarbon dots to the preparation of drugs that inhibit ferroptosis, it is found that the thiocarbon dots can significantly inhibit RSL3-induced hepatocyte ferroptosis; at the same time, when it is used to prepare drugs for treating acetaminophen (APAP)-induced acute hepatitis, the results of detecting ferroptosis-related indicators in liver pathological sections, HE staining, and measuring the AST and ALT levels in mouse serum show that the thiocarbon dots alleviate APAP-induced acute liver injury in mice by inhibiting ferroptosis, and have a sustained and stable long-term inhibitory effect on APAP-induced cell ferroptosis, and its inhibitory effect can last for at least 72 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 TEM image of the thio-substituted carbon dots in Preparation Example 1;

[0023] Figure 2 This is the UV absorption graph of the sulfur-substituted carbon dots in Preparation Example 1;

[0024] Figure 3 This is the fluorescence spectrum of the thio-substituted carbon dots in Preparation Example 1;

[0025] Figure 4 This is the infrared spectrum of the thio-substituted carbon dots in Preparation Example 1;

[0026] Figure 5 Figure 2 shows the inhibition of RSL3-induced hepatocyte ferroptosis by S-CDs at different concentrations.

[0027] Figure 6 Figure 2 shows the inhibition of APAP-induced hepatocyte ferroptosis by S-CDs at different concentrations.

[0028] Figure 7 This is a test diagram of the sustained inhibition of APAP-induced hepatocyte ferroptosis by S-CDs;

[0029] Figure 8 8-OHdG immunofluorescence and 4-HNE immunohistochemistry images of paraffin sections of mice with acute liver injury induced by acetaminophen were shown by S-CDs;

[0030] Figure 9 HE staining of paraffin sections of mice with acute liver injury induced by acetaminophen by S-CDs;

[0031] Figure 10 The graph shows the levels of AST and ALT in the serum of mice with acute liver injury induced by acetaminophen using S-CDs. DETAILED DESCRIPTION

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0034] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0035] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the present invention. The present description and examples are intended to be illustrative only.

[0036] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0037] Unless otherwise specified, the "room temperature" and "normal temperature" mentioned in the present invention are all calculated as 25±2°C.

[0038] The raw materials used in the following preparation examples and embodiments of the present invention are all commercially available.

[0039] The present invention provides the use of thioated carbon dots in preparing drugs for inhibiting cell ferroptosis.

[0040] In the present invention, the thio-substituted carbon dots inhibit RSL3-induced cell ferroptosis.

[0041] In the present invention, the method for preparing the thiolated carbon dots comprises the following steps:

[0042] Cystamine hydrochloride, m-phenylenediamine and water are mixed and subjected to a hydrothermal reaction. After the reaction is completed, the supernatant is collected by centrifugation to obtain thiocarbon dots.

[0043] In the present invention, the supernatant also needs to be purified by passing through a 0.22 micron filter membrane.

[0044] In the present invention, the particle size of the thio-substituted carbon dots is 1 to 7 nm, preferably 2 to 6 nm, and more preferably 4 to 5 nm.

[0045] In the present invention, the usage ratio of cystamine hydrochloride, m-phenylenediamine and water is 100 mg:50 mg:5-15 mL, preferably 100 mg:50 mg:6-14 mL, more preferably 100 mg:50 mg:8-12 mL, and even more preferably 100 mg:50 mg:10 mL;

[0046] The temperature of the hydrothermal reaction is 150-200° C., preferably 160-190° C., more preferably 170-180° C., and the time is 5-12 h, preferably 6-11 h, more preferably 8-10 h.

[0047] In the present invention, the centrifugal speed is 10,000 to 15,000 rpm, preferably 11,000 to 14,000 rpm, more preferably 13,000 rpm, and the centrifugation time is 0.5 to 3 h, preferably 1 to 2 h.

[0048] In the present invention, polyacrylic acid can also be used to modify the surface of thiolated carbon dots. The specific method is as follows: cystamine hydrochloride, m-phenylenediamine, and water are mixed and subjected to a hydrothermal reaction. After the reaction is completed, the supernatant is centrifuged and collected. The supernatant is mixed with a polyacrylic acid solution and purified to obtain thiolated carbon dots.

[0049] The purification is as follows: dialyzing for 24 to 36 hours using a 2500 to 3500 kDa dialysis bag;

[0050] The concentration of the polyacrylic acid solution is 0.1 to 0.5 g / mL, preferably 0.2 to 0.4 g / mL, and more preferably 0.25 to 3 g / mL.

[0051] In the present invention, the polyacrylic acid solution is obtained by dispersing polyacrylic acid in double distilled water.

[0052] In the present invention, the temperature for mixing the supernatant and the polyacrylic acid solution is 30 to 50° C., preferably 35 to 45° C., more preferably 40° C., the mixing speed is 100 to 300 rpm, preferably 150 to 250 rpm, more preferably 200 rpm, and the mixing time is 10 to 15 h, preferably 11 to 14 h, more preferably 12 to 13 h.

[0053] In the present invention, the usage amount of the thiolated carbon dots is 5 μg / L or more, preferably 5 to 80 μg / L, more preferably 10 to 40 μg / L, and even more preferably 20 to 30 μg / L.

[0054] The present invention also provides the use of thiocarbon dots in the preparation of drugs for treating acute liver injury.

[0055] In the present invention, the acute liver injury is acute liver injury induced by acetaminophen.

[0056] In the present invention, the method for preparing the thiolated carbon dots comprises the following steps:

[0057] Cystamine hydrochloride, m-phenylenediamine and water are mixed and subjected to a hydrothermal reaction. After the reaction is completed, the supernatant is collected by centrifugation to obtain thiocarbon dots.

[0058] In the present invention, the supernatant also needs to be purified by passing through a 0.22 micron filter membrane.

[0059] In the present invention, the particle size of the thio-substituted carbon dots is 1 to 7 nm, preferably 2 to 6 nm, and more preferably 4 to 5 nm.

[0060] In the present invention, the usage ratio of cystamine hydrochloride, m-phenylenediamine and water is 100 mg:50 mg:5-15 mL, preferably 100 mg:50 mg:6-14 mL, more preferably 100 mg:50 mg:8-12 mL, and even more preferably 100 mg:50 mg:10 mL;

[0061] The temperature of the hydrothermal reaction is 150-200° C., preferably 160-190° C., more preferably 170-180° C., and the time is 5-12 h, preferably 6-11 h, more preferably 8-10 h.

[0062] In the present invention, the centrifugal speed is 10,000 to 15,000 rpm, preferably 11,000 to 14,000 rpm, more preferably 13,000 rpm, and the centrifugation time is 0.5 to 3 h, preferably 1 to 2 h.

[0063] In the present invention, polyacrylic acid can also be used to modify the surface of thiolated carbon dots. The specific method is as follows: cystamine hydrochloride, m-phenylenediamine, and water are mixed and subjected to a hydrothermal reaction. After the reaction is completed, the supernatant is centrifuged and collected. The supernatant is mixed with a polyacrylic acid solution and purified to obtain thiolated carbon dots.

[0064] The purification is as follows: dialyzing for 24 to 36 hours using a 2500 to 3500 kDa dialysis bag;

[0065] The concentration of the polyacrylic acid solution is 0.1 to 0.5 g / mL, preferably 0.2 to 0.4 g / mL, and more preferably 0.25 to 3 g / mL.

[0066] In the present invention, the polyacrylic acid solution is obtained by dispersing polyacrylic acid in double distilled water.

[0067] In the present invention, the temperature for mixing the supernatant and the polyacrylic acid solution is 30 to 50° C., preferably 35 to 45° C., more preferably 40° C., the mixing speed is 100 to 300 rpm, preferably 150 to 250 rpm, more preferably 200 rpm, and the mixing time is 10 to 15 h, preferably 11 to 14 h, more preferably 12 to 13 h.

[0068] In the present invention, the usage of the thio-substituted carbon dots is 0.3 to 1 mg / kg, preferably 0.4 to 0.8 mg / kg, and more preferably 0.5 to 0.6 mg / kg.

[0069] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0070] Preparation Example 1

[0071] Preparation of sulfur-substituted carbon dots (S-CDs)

[0072] Dissolve 100 mg of cystamine hydrochloride and 50 mg of m-phenylenediamine in 10 mL of deionized water, stir until completely dissolved, react at 180°C for 10 h, centrifuge at 13,000 rpm for 1 h, collect the supernatant, and purify it through a 0.22 μm filter membrane to obtain sulfo-carbon dots (S-CDs);

[0073] Figure 1 The TEM image shows that the surface is spherical, the average particle size is 1 to 7 nm, and the potential is +31.27±0.84Mv. Figure 2 The UV absorption showed an absorption peak between 350 and 475 nm. The embodiment was scanned in 3D using a fluorescence spectrophotometer. Figure 3 It shows that when S-CDs are irradiated with 430 nm excitation light, they emit the strongest fluorescence at 520 nm. Figure 4 It shows that the material was successfully doped with sulfur.

[0074] Preparation Example 2

[0075] Preparation of sulfur-substituted carbon dots (S-CDs@PAA)

[0076] 100 mg of cystamine hydrochloride and 50 mg of m-phenylenediamine were dissolved in 10 mL of deionized water, stirred until completely dissolved, reacted at 180°C for 10 h, centrifuged at 13,000 rpm for 1 h, and the supernatant was collected. 2 g of polyacrylic acid was weighed and dissolved in 10 ml of double-distilled water. The collected supernatant and the polyacrylic acid solution were mixed and stirred at 40°C and 200 rpm for 12 h. The solution was dialyzed using a 3500 kDa dialysis bag for 36 h, with the water changed every 12 h to obtain sulfocarbon dots (S-CDs@PAA).

[0077] Example 1

[0078] Thio-carbon dots inhibit RSL3-induced cell ferroptosis test. The specific test method is as follows:

[0079] S1: Take L02 cells in the logarithmic growth phase, digest them with trypsin, and plate them at 2.5×10 cells per well. 5 (500 μL per well) were inoculated into 24-well plates and cultured for 24 h;

[0080] S2: Discard the culture medium and replace it with the following different drug-containing media;

[0081] RSL3 group: RSL3 (ferroptosis inducer) was diluted to 5 μM in cell culture medium;

[0082] Ferrostatin-1 group: Ferrostatin-1 (a commercial ferroptosis inhibitor) was diluted to 10 μM in cell culture medium;

[0083] RSL3+S-CDs group: The thio-carbon dots prepared in Example 1 were diluted with cell culture medium to 80 μg / L, 40 μg / L, 20 μg / L, 10 μg / L, and 5 μg / L solutions, respectively, and 5 μM RSL3 was added;

[0084] S3: After 8 h of treatment, the drug-containing medium was discarded, and 100 μL of PI dye solution (diluted with PBS) with a final concentration of 4 μM was added to each well. The results were then observed under a fluorescence microscope (PI dye stains dead cells, and red fluorescent spots represent dead cells);

[0085] Among them, the above cell culture medium is DMEM containing 1% FBS and 1% double antibody.

[0086] Test results such as Figure 5 As shown by Figure 5 It can be seen that RSL3 can induce massive cell death in L02 cells, while sulfhydryl carbon dots (S-CDs) can effectively inhibit RSL3-induced cell ferroptosis, and the inhibitory effect is basically equivalent to that of the commercial ferroptosis inhibitor Ferrostatin-1, which fully demonstrates that the sulfhydryl carbon dots of the present invention have an excellent inhibitory effect on RSL3-induced cell ferroptosis.

[0087] Example 2

[0088] Thio-carbon dots inhibit APAP-induced cell ferroptosis test. The specific test method is as follows:

[0089] S1: Take L02 cells in the logarithmic growth phase, digest them with trypsin, and plate them at 2.5×10 cells per well. 5 (500 μL per well) were inoculated into 24-well plates and cultured for 24 h;

[0090] S2: Discard the culture medium and replace it with the following different drug-containing media;

[0091] APAP group: APAP (acetaminophen) was diluted to 5 mM in cell culture medium;

[0092] Ferrostatin-1 group: Ferrostatin-1 (a commercial ferroptosis inhibitor) was diluted to 10 μM in cell culture medium;

[0093] RSL3+S-CDs group: The thio-carbon dots prepared in Example 1 were diluted with cell culture medium to 400 μg / L, 200 μg / L, 100 μg / L, and 50 μg / L solutions, respectively, and 5 mM APAP was added;

[0094] S3: After 8 h of treatment, the drug-containing medium was discarded, and 100 μL of PI dye solution (diluted with PBS) with a final concentration of 4 μM was added to each well. The results were then observed under a fluorescence microscope (PI dye stains dead cells, and red fluorescent spots represent dead cells);

[0095] Among them, the above cell culture medium is DMEM containing 1% FBS and 1% double antibody.

[0096] Test results such as Figure 6 As shown by Figure 6 It can be seen that APAP can induce massive cell death in L02 cells, while Ferrostatin-1 and sulfide-substituted carbon dots (S-CDs) can effectively inhibit APAP-induced cell death. The inhibitory effect of sulfide-substituted carbon dots (S-CDs) is basically equivalent to that of the commercial ferroptosis inhibitor Ferrostatin-1, which fully demonstrates that the sulfide-substituted carbon dots of the present invention have an excellent inhibitory effect on APAP-induced cell ferroptosis.

[0097] Example 3

[0098] Sustained inhibition of APAP-induced ferroptosis by sulfhydryl carbon dots (S-CDs) was tested using the following methods:

[0099] S1: Take L02 cells in the logarithmic growth phase, digest them with trypsin, and plate them at 2.5×10 cells per well. 5 (500 μL per well) were inoculated into 24-well plates and cultured for 24 h;

[0100] S2: Discard the culture medium and replace it with the following drug-containing culture medium;

[0101] APAP group: APAP (acetaminophen) was diluted to 5 mM in cell culture medium;

[0102] APAP+S-CDs group: the sulfo-carbon dots (S-CDs) prepared in Example 1 were diluted to a 100 μg / L solution using cell culture medium and 5 mM APAP was added;

[0103] S3: L02 cells were treated with the above culture medium for 24 h, 48 h, and 72 h, respectively. The drug-containing culture medium was discarded, and 100 μL of PI dye solution (diluted with PBS) with a final concentration of 4 μM was added to each well. The results were then observed under a fluorescence microscope (PI dye stains dead cells, and red fluorescent spots represent dead cells);

[0104] Among them, the above cell culture medium is DMEM containing 1% FBS and 1% double antibody.

[0105] Test results such as Figure 7 As shown by Figure 7It can be seen that S-CDs has a sustained, stable and long-term inhibitory effect on APAP-induced cell ferroptosis, and its inhibitory effect can last for at least 72 hours.

[0106] Example 4

[0107] The therapeutic effect of sulfo-carbon dots (S-CDs) on acetaminophen (APAP)-induced acute kidney injury in mice was tested. The specific testing method is as follows:

[0108] (1) Twenty-four 8- to 10-week-old C57BL / 6J male mice were randomly divided into the following four groups (6 mice in each group):

[0109] Control group: intraperitoneal injection of normal saline;

[0110] S-CDs group: S-CDs 0.5 mg / kg injected into tail vein;

[0111] APAP group: intraperitoneal injection of APAP 200 mg / kg, approximately 400 μl / mouse;

[0112] APAP+S-CDs group: intraperitoneal injection of APAP 200 mg / kg, about 400 μl / mouse + tail vein injection of S-CDs 0.5 mg / kg (conducted simultaneously).

[0113] (2) Acute liver injury was induced by APAP. Male C57BL / 6J mice (8-10 weeks old, 18-20 g) were fasted 15 h before the experiment but were allowed free access to water. After the injection, all mice were allowed free access to water and food.

[0114] (3) On the first day, all injections were completed 72 hours after the mice were killed to obtain kidney and liver tissues. The samples were fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned. HE, 8-OHdG, and 4-HNE staining were performed for liver function histological analysis and ferroptosis index detection. The test results were as follows: Figure 8 、 9 As shown. The serum of mice in all the experimental groups was taken for liver function test analysis. The test results are shown as follows Figure 10 shown.

[0115] Depend on Figure 8 It can be seen that compared with the APAP treatment group, the accumulation of necrotic tissue around the liver sinusoids in the APAP+S-CDs treatment group was significantly reduced; Figure 9 It was found that compared with the APAP treatment group, the APAP+S-CDs treatment group had significantly reduced ferroptosis oxidation products 8-OHdG and 4-HNE. Figure 10It was found that compared with the APAP-treated group, the AST and ALT levels in the serum of mice in the APAP+S-CDs-treated group were significantly reduced, which fully demonstrated that thio-carbon dots can alleviate acetaminophen-induced acute liver injury in mice by inhibiting ferroptosis.

[0116] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Application of thiolated carbon dots in the preparation of drugs for treating acute liver injury, characterized in that: The acute liver injury is acute hepatitis induced by acetaminophen; the preparation method of the thiolated carbon dots comprises the following steps: Cystamine hydrochloride, m-phenylenediamine and water are mixed and subjected to a hydrothermal reaction. After the reaction is completed, the supernatant is collected by centrifugation to obtain thiocarbon dots.

2. The use of the thiolated carbon dots in the preparation of a drug for treating acute liver injury according to claim 1, characterized in that: The dosage ratio of cystamine hydrochloride, m-phenylenediamine and water is 100 mg:50 mg:5-15 mL; The temperature of the hydrothermal reaction is 150-200°C and the time is 5-12 hours.

3. The use of the thiolated carbon dots in the preparation of a drug for treating acute liver injury according to claim 1, characterized in that: The usage of the thiocarbon dots is 0.3-1 mg / kg.

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