Preparation method and application of hydrogen sulfide releasing agent

Abstract

This invention discloses a method for preparing a hydrogen sulfide releasing agent and its application, relating to the field of biopharmaceutical preparation technology. The method involves first reacting compound one and sulfur in dimethylacetamide under heating to synthesize compound two; then, using pyridine hydrochloride to demethylate compound two, forming compound three; dissolving hyaluronic acid in a mixed solvent one composed of N,N-dimethylformamide and double-distilled water to form a homogeneous solution, then adding EDCHCl and DMAP and reacting it with compound three to synthesize compound four; dissolving compound four in a mixed solvent two composed of EDCHCl and double-distilled water to form a DMF solution, then reacting it with the amino group of the short peptide SS31 to form a hydrogen sulfide releasing agent. The hydrogen sulfide releasing agent prepared by this invention can simultaneously achieve targeted enrichment at tumor sites, long-term slow release of hydrogen sulfide, and precise damage to the mitochondria of tumor cells, forming a triple synergistic anti-tumor effect, effectively inhibiting the proliferation, migration, and invasion of human esophageal squamous cell carcinoma cells.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical preparation technology, specifically relating to a method for preparing and applying a hydrogen sulfide release agent. Background Technology

[0002] Esophageal cancer is one of the deadliest and most common malignant tumors in the world. Esophageal cancer is divided into two main histological types: squamous cell carcinoma (ESCC) and adenocarcinoma (EAC). Early symptoms of esophageal squamous cell carcinoma are insidious, and most patients are diagnosed at an advanced stage. Surgical resection and traditional chemotherapy are prone to drug resistance, and the 5-year survival rate of patients is less than 15%. The development of new, highly effective, and targeted anti-esophageal squamous cell carcinoma drugs has become an urgent need in the current biomedical field. Hydrogen sulfide, as an important endogenous gaseous signaling molecule, works with nitric oxide and carbon monoxide to regulate cellular physiological and pathological processes, exhibiting a dual regulatory role in tumor treatment and becoming a hot topic in the development of novel anti-tumor drugs. Traditional small-molecule hydrogen sulfide donors suffer from poor water solubility, short in vivo circulation time, and lack of tissue targeting, making it difficult to achieve efficient and safe in vivo delivery and limiting their clinical translation. Hyaluronic acid (HA), as a natural high-molecular-weight polysaccharide, possesses excellent biocompatibility, degradability, and CD44 receptor targeting ability, making it an ideal drug delivery carrier. The short peptide SS31 can specifically target mitochondria, achieving efficient enrichment without relying on mitochondrial transmembrane potential, and can precisely act on the core sites of cellular energy metabolism. Combining hydrogen sulfide donors, hyaluronic acid carriers, and the short peptide SS31 targeting peptide can construct a novel hydrogen sulfide release system that combines targeting and bioactivity. Currently, there are no reports on the application of a combined release agent with tumor targeting, mitochondrial enrichment, and sustained hydrogen sulfide release functions, prepared by covalently coupling hydrogen sulfide donors, hyaluronic acid, and mitochondrial targeting peptides, in the treatment of esophageal squamous cell carcinoma. Existing hydrogen sulfide donor drugs cannot simultaneously achieve the synergistic anti-tumor effects of targeted enrichment, long-term release, and mitochondrial damage, and are therefore difficult to effectively inhibit the proliferation, migration, and invasion of esophageal squamous cell carcinoma cells. Summary of the Invention

[0003] The purpose of this invention is to provide a method for preparing a hydrogen sulfide release agent and its application. The hydrogen sulfide release agent prepared by this method can simultaneously achieve targeted enrichment, long-term release, and synergistic anti-tumor effects against mitochondrial damage, effectively inhibiting the proliferation, migration, and invasion of esophageal squamous cell carcinoma cells, thereby solving the problems mentioned in the background art.

[0004] To achieve the above objectives, the present invention adopts the following technical solution: A method for preparing a hydrogen sulfide releasing agent includes the following steps: S1. Compound 1 and sulfur were reacted in dimethylacetamide by heating to synthesize compound 2; S2. Compound II is demethylated using pyridine hydrochloride to form compound III; S3. Hyaluronic acid was dissolved in a mixed solvent consisting of N,N-dimethylformamide and double-distilled water to form a homogeneous solution. EDCHCl and DMAP were then added and reacted with compound three to synthesize compound four. S4. Compound 4 is dissolved in a mixed solvent 2 consisting of EDCHCl and double-distilled water to form a DMF solution, which then reacts with the amino group of short peptide SS31 to form a hydrogen sulfide release agent.

[0005] Preferably, in step S1, the first compound is anethole, and the molar ratio of the first compound to sulfur is 1:5-10.

[0006] Preferably, in the synthesis of compound two, compound one and sulfur are reacted in dimethylacetamide at 140-150°C. After the reaction is complete, the mixture is cooled to room temperature, water is added to the resulting solution, and the solution is extracted with diethyl ether. The combined organic phases are washed successively with water and brine, dried over anhydrous Na2SO4, and the solvent is removed under reduced pressure. Finally, the mixture is crystallized with ethyl acetate to obtain compound two.

[0007] Preferably, in step S2, during the demethylation reaction of compound two, pyridine hydrochloride and compound two are heated to 210-220°C under argon protection and melted for 15-30 minutes. After melting, the mixture is cooled to 100°C, water is added, and the mixture is filtered while hot. The filter cake is first washed with alkali, then dissolved in water, and the pH is adjusted to 2 using hydrochloric acid. The precipitate is then filtered, washed with water until neutral, and dried to obtain compound three.

[0008] Preferably, the molar ratio of compound two to pyridine hydrochloride is 1:5-10, and 10wt% NaOH is used for alkaline washing, while the hydrochloric acid is concentrated hydrochloric acid with a concentration of 36-38wt%.

[0009] Preferably, in step S3, the molar ratio of N,N-dimethylformamide and double-distilled water in the mixed solvent one is 1:1-2.5. When adding EDCHCl and DMAP to the homogeneous solution, the temperature is controlled at 0℃ for 50-70 minutes before adding compound three. The reaction is carried out at 0℃ for 25-40 minutes, and then at room temperature until the crude product is completely obtained. The crude product is then dialyzed, and the supernatant after dialyzing is filtered through a microporous membrane with a pore size of 0.45 μm and freeze-dried to obtain compound four.

[0010] Preferably, the molar ratio of hyaluronic acid, EDCHCl, DMAP, and the compound is 2-4:2-4:1:2-4. When dialyzing the crude product, the solution is dialyzed with DMF in a 3.5kDa dialysis bag for 5-10 hours, then with water / DMF at a volume ratio of 1:1 for 10-15 hours, and then with water in a 3.5kDa dialysis bag for 20-24 hours. The resulting supernatant is then filtered through a microporous membrane with a pore size of 0.45 μm.

[0011] Preferably, in step S4, when compound four is dissolved in mixed solvent two, it is stirred at 0°C for 1-4 hours to obtain a DMF solution. Then, short peptide SS31 is dissolved in the DMF solution, and the reaction is stirred at room temperature under argon protection for 5-12 hours. The reaction solution is then transferred to a 3.5 kDa dialysis bag and dialyzed with double-distilled water for 16-24 hours. The supernatant is filtered through a microporous membrane with a pore size of 0.45 μm and then freeze-dried to obtain a hydrogen sulfide releasing agent.

[0012] Preferably, the structural formula of the hydrogen sulfide releasing agent is: , Where p = 4 - 6, q = 16 - 22.

[0013] The present invention also provides an application of a hydrogen sulfide releasing agent, which is prepared by the above-described method for preparing a hydrogen sulfide releasing agent, and the hydrogen sulfide releasing agent is used in the preparation of a drug for treating human esophageal squamous cell carcinoma.

[0014] The present invention provides a method for preparing and applying a hydrogen sulfide releasing agent, which has the following advantages compared with the prior art: 1. This invention utilizes a hydrogen sulfide release agent prepared primarily from biocompatible materials, such as hyaluronic acid and short peptide SS31. Hyaluronic acid targets the CD44 receptor on the surface of tumor cells, achieving specific enrichment of esophageal squamous cell carcinoma tissue and reducing toxic side effects on normal tissues. The short peptide SS31 achieves mitochondrial targeting, precisely acting on the core sites of tumor cell energy metabolism, thereby enhancing the precision of anti-tumor effects. 2. This invention uses hyaluronic acid polymer as a carrier, which can avoid the rapid release and metabolism of hydrogen sulfide in the body, prolong the duration of action, improve drug utilization efficiency, and at the same time improve the defect of poor water solubility of traditional hydrogen sulfide donors, thus improving in vivo delivery efficiency. 3. The method of this invention achieves a synergistic anti-tumor effect by combining the anti-tumor activity, mitochondrial damaging effect and targeted delivery function of hydrogen sulfide. It inhibits the proliferation, migration and invasion of esophageal squamous cell carcinoma cells through multiple pathways. The prepared hydrogen sulfide release agent can be degraded in vivo, has no toxic side effects, has good biosafety, and is suitable for clinical translational application. Attached Figure Description

[0015] Figure 1 This is a flowchart of the preparation method of the present invention; Figure 2 This is a synthetic route diagram of the hydrogen sulfide releasing agent of the present invention; Figure 3 This invention relates to the effect of hydrogen sulfide releasing agent on the proliferation of human esophageal squamous cell carcinoma cells. Figure 3 A represents the survival rate of human esophageal squamous cell carcinoma detected by the MTT assay 24 hours after drug administration; Figure 3 The effect of SS31-HA-ADT on the proliferation of human esophageal squamous cell carcinoma cells when BC is detected by EDU method; Figure 4 The present invention relates to the effect of hydrogen sulfide releasing agent on the migration of human esophageal squamous cell carcinoma cells, wherein... Figure 4 AB is the scratch assay used to detect the effect of SS31-HA-ADT on the migration of human esophageal squamous cell carcinoma cells; Figure 5 This invention relates to the effect of hydrogen sulfide releasing agent on the migration and invasion of human esophageal squamous cell carcinoma cells, wherein... Figure 5 AD was used to detect the effects of SS31-HA-ADT on the migration and invasion of human esophageal squamous cell carcinoma cells using Transwell and Invasion assays. Figure 6 This invention relates to the effect of hydrogen sulfide releasing agent on the mitochondrial structure of human esophageal squamous cell carcinoma cells. Figure A shows the effect of SS31-HA-ADT on the structure of human esophageal squamous cell carcinoma cells when detected by the MitoTracker fluorescent probe. Figure 7 To illustrate the effect of the hydrogen sulfide release agent of this invention on mitochondrial function of human esophageal squamous cell carcinoma cells, Figures AC show the effect of JC-1 probe and ATP content detection on the function of SS31-HA-ADT on human esophageal squamous cell carcinoma cells. Figure 8 The effect of the hydrogen sulfide releasing agent of the present invention on ferroptosis in human esophageal squamous cell carcinoma cells, wherein... Figure 8 In section AB, the effect of lipid ROS on ferroptosis of SS31-HA-ADT on human esophageal squamous cell carcinoma cells was detected. Figure 9 The effect of SS31-HA-ADT on pyroptosis in human esophageal squamous cell carcinoma cells, among which Figure 9 In AB, the N-GSDMD immunofluorescence assay was used to detect the effect of SS31-HA-ADT on pyroptosis in human esophageal squamous cell carcinoma cells. Detailed Implementation

[0016] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The specific embodiments described herein are merely used to explain the present invention and are not intended to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0017] This invention provides, for example Figure 1 The method for preparing a hydrogen sulfide releasing agent, as shown, includes the following steps: S1. Compound 1 and sulfur were reacted in dimethylacetamide by heating to synthesize compound 2; Compound 1 is anethole. The molar ratio of compound 1 to sulfur is 1:5-10, which ensures the efficient and stable synthesis of compound 2 and improves the reaction conversion rate and product purity. In the synthesis of compound 2, compound 1 and sulfur are heated to 140-150°C in dimethylacetamide for reaction. After the reaction is complete, the mixture is cooled to room temperature, water is added to the reaction solution, and then the solution is extracted with diethyl ether. The combined organic phases are washed with water and brine in sequence, dried with anhydrous Na2SO4, and the solvent is removed under reduced pressure. Finally, the mixture is crystallized with ethyl acetate to obtain compound 2. Specifically, 8g of anethole and 96.93g of sulfur were added to 30mL of dimethylacetamide and reacted at 145℃ for 10h. The mixture was then cooled to room temperature, 100mL of H2O was added, and the product was extracted twice with diethyl ether, each time with 100mL of water. The combined organic phases were washed three times with water, first with 100mL of water and then with 100mL of brine. The mixture was then dried with Na2SO4, the solvent was removed under reduced pressure, and then crystallized with ethyl acetate to obtain compound two, namely 5-(p-methoxyphenyl)-3H-1,2-dithiacyclopentane-3-thionone, abbreviated as ADT. S2. Compound II is demethylated using pyridine hydrochloride to form compound III; During the demethylation reaction of compound 2, pyridine hydrochloride and compound 2 were heated to 210-220°C under argon protection and melted for 15-30 minutes. After melting, the mixture was cooled to 100°C, water was added, and the mixture was filtered while hot. The filter cake was first washed with alkali, then dissolved in water, and the pH was adjusted to 2 with hydrochloric acid. The precipitate was then filtered, washed with water until neutral, and dried to ensure the smooth progress of the demethylation reaction, thereby obtaining compound 3. The molar ratio of compound 2 to pyridine hydrochloride is 1:5-10. During alkaline washing, 10wt% NaOH is used for washing, and concentrated hydrochloric acid with a concentration of 36-38wt% is used. By precisely controlling the reaction ratio and reagent concentration, the selectivity of the demethylation reaction is improved, byproducts are reduced, and the purity of compound 3 is increased. Specifically, 32.74 g of pyridine hydrochloride and 11.33 g of ADT were added to a dry flask, mixed, and then heated to 215 °C for 20 min under argon protection. After cooling to 100 °C, 150 mL of warm water at 70 °C was added and filtered while hot. The filter cake was placed in a beaker and 150 mL of NaOH solution was added. The mixture was stirred for 4-8 h, filtered, and the filter cake was dissolved in 1 L of water. The pH was then adjusted to 2 with concentrated hydrochloric acid. The red precipitate was filtered, washed with water until neutral, and then dried in a vacuum desiccator to obtain compound triad, namely 5-(p-hydroxyphenyl)-3H-1,2-dithiacyclopentane-3-thione, abbreviated as ADT-OH. S3. Hyaluronic acid was dissolved in a mixed solvent consisting of N,N-dimethylformamide (DMF) and double-distilled water (ddH2O) to form a homogeneous solution. EDCHCl and DMAP were then added, and the solution was reacted with compound three to synthesize compound four. The molar ratio of N,N-dimethylformamide and double-distilled water in the mixed solvent is 1:1-2.5. When adding EDCHCl and DMAP to the homogeneous solution, the temperature is controlled at 0℃ for 50-70 min before adding compound 3. The reaction is carried out at 0℃ for 25-40 min, and then at room temperature until the crude product is completely obtained. The crude product is then dialyzed, and the supernatant after dialyzing is filtered through a microporous membrane with a pore size of 0.45 μm and freeze-dried to ensure efficient coupling between hyaluronic acid and compound 3, thereby obtaining the crude product of compound 4 and completing the purification. The molar ratio of hyaluronic acid, EDCHCl, DMAP and the compound used is 2-4:2-4:1:2-4. When dialyzing the crude product, it is dialyzed with DMF in a 3.5kDa dialysis bag for 5-10 hours, then dialyzed with water / DMF at a volume ratio of 1:1 for 10-15 hours, and then dialyzed with water in a 3.5kDa dialysis bag for 20-24 hours. The resulting supernatant is filtered through a microporous membrane with a pore size of 0.45 μm. Specifically, 1.50 g of hyaluronic acid (HA, molecular weight 8-10 kDa) was dissolved in DMF / ddH2O to form a homogeneous solution. Then, 0.76 g of EDCHCl and 0.16 g of DMAP were added, and the temperature was maintained at 0℃ for 1 h. After that, 0.60 g of ADT-OH in DMF solution was added, and the reaction was carried out at 0℃ for 30-60 min, and then at room temperature for 12 h. The crude product was dialyzed against DMF in a 3.5 kDa dialysis bag for 5-10 h, then against water / DMF (1:1-2.5, v / v) for 10-15 h, and then against water for 20-24 h. The supernatant was filtered through a microporous membrane with a pore size of 0.45 μm. The product was then frozen at -60℃ for 6 h and then vacuum dried at 20℃ for 24 h to obtain compound four, namely HA-ADT, with a yield of 79%.

[0018] S4. Compound IV is dissolved in a mixed solvent II consisting of EDCHCl and double-distilled water to form a DMF solution, which then reacts with the amino group of short peptide SS31 to form a hydrogen sulfide release agent. In mixed solvent 2, the molar ratio of EDCHCl to double-distilled water is 1:10, and the molar ratio of compound 4 to EDCHCl is 2-4:1. When compound 4 is dissolved in mixed solvent 2, it is stirred at 0°C for 1-4 h to obtain a DMF solution. Then, short peptide SS31 is dissolved in the DMF solution, and the reaction is stirred at room temperature under argon protection for 5-12 h. The reaction solution is then transferred to a 3.5 kDa dialysis bag and dialyzed with double-distilled water for 16-24 h. The supernatant is filtered through a microporous membrane with a pore size of 0.45 μm and then freeze-dried to obtain a hydrogen sulfide releasing agent. Specifically, 0.108 g of HA-ADT was dissolved in 1-2 mL of ddH2O, and 0.192 g of EDCHCl was added. The mixture was stirred at 0°C for 2-4 h to activate the carboxyl groups on HA-ADT, resulting in a DMF solution. Then, 0.016 g of the short peptide SS31 was slowly added dropwise to the DMF solution to ensure that the amino group reacted with the activated carboxyl group. Under argon protection, the mixture was stirred at room temperature for 4-8 h to form the SS31-HA-QT conjugate. The reaction solution was transferred to a 3.5 kDa dialysis bag and dialyzed with ddH2O for 20-24 h to remove unbound short peptide SS31 and small molecule impurities. The product was first frozen at -60°C for 6 h, and then vacuum dried at 20°C for 24 h to obtain the hydrogen sulfide release agent, namely SS31-HA-ADT.

[0019] The structural formula of the hydrogen sulfide releasing agent is: , Where p = 4-6, q = 16-22; the specific synthetic route for the hydrogen sulfide release agent is as follows: Figure 2 As shown; The hydrogen sulfide release agent prepared by the above method can achieve a synergistic anti-tumor effect of targeted enrichment, long-term release and mitochondrial damage when used in the preparation of drugs against human esophageal squamous cell carcinoma, effectively inhibiting the proliferation, migration and invasion of esophageal squamous cell carcinoma cells.

[0020] To investigate the effect of the hydrogen sulfide release agent prepared by the present invention on the proliferation of human esophageal squamous cell carcinoma cells, the MTT assay was used to determine the effect of SS31-HA-ADT on tumor cell survival and the EDU assay was used to determine the effect of SS31-HA-ADT on tumor cell proliferation. The specific process for determining the effect of SS31-HA-ADT on tumor cell survival using the MTT assay is as follows: Cells in the logarithmic growth phase were collected and resuspended in culture medium containing 10% fetal bovine serum to form a single-cell suspension. Cells were counted using a counting chamber, and the cell concentration was adjusted to the desired number at 5 x 10⁶ cells per well. 3 Cells were seeded at 100 μL / well in 96-well plates and incubated at 37°C in a CO2 incubator with 5% (v / v) concentration. After cell attachment, 100 μL of drug-containing medium was added per well (physiological saline for the negative control group, 200 mol / L LADT-OH for the positive control group ADT-OH, 200 mol / L HA-ADT for the positive control group HA-ADT, and 200 mol / L SSS31-HA-ADT for the SSS31-HA-ADT group). Each group was divided into 3 replicates. After culturing for 24 h, 10 LMT was added to each well, and the cells were incubated for 4 h. The supernatant was aspirated, and 100 LDMSO was added to each well. The cells were shaken at room temperature, and the absorbance (A value) of each well was measured at 490 nm using a microplate reader. Cell viability was calculated based on the absorbance values. Cell viability (%of control) = (Drug group A value - Zeroing well A value) / (Control well A value - Zeroing well A value) × 100% Experimental results are as follows Figure 3 As shown in Figure A, after SS31-HA-ADT was applied to human esophageal squamous cell carcinoma cells, the cell survival rate was significantly lower than that of tumor cells in the control group. It can be concluded that SS31-HA-ADT reduces the survival rate of tumor cells.

[0021] The specific process of determining the effect of SS31-HA-ADT on tumor cell proliferation using the EDU method is as follows: Log-phase human esophageal squamous cell carcinoma cells KYSE30 and KYSE450 were seeded at 4000 cells per well in 96-well plates and cultured until normal growth was achieved. Each group was treated with physiological saline, 200 mol / L LADT-OH, 200 mol / L HA-ADT, and 200 mol / L SS31-HA-ADT, respectively. Each group had three replicates.

[0022] EdU labeling (96-well plate operation): Dilute EdU solution (reagent A) with cell culture medium at a ratio of 1000:1 to prepare an appropriate amount of 50 MEdU medium; add 100 L of 50 MEdU medium to each well and incubate for 2 h, then discard the medium; wash cells twice with PBS for 5 min each time.

[0023] Cell fixation: Add 50 μL of cell fixation solution (i.e., PBS containing 4% paraformaldehyde) to each well and incubate at room temperature for 30 min, then discard the fixation solution; add 50 μL of 2 mg / mL glycine to each well and incubate on a shaker for 5 min to decolorize, then discard the glycine solution; add 100 μL of PBS to each well and wash once for 5 min, then discard the PBS; (enhanced) Add 100 μL of permeabilizer (PBS containing 0.5% Triton-X100) to each well and incubate on a shaker for 10 min to decolorize, then wash once with PBS for 5 min.

[0024] Apollo staining: Add 100L of 1Apollo staining reaction solution to each well (make sure to prepare in the correct order, prepare fresh for each use, and use within 30 minutes), incubate in the dark at room temperature on a decolorizing shaker for 30 minutes, and then discard the staining reaction solution. The preparation order of Apollo staining reaction solution is shown in Table 1 below: Table 1 Add 100L of penetrant (0.5% Triton-X100 in PBS) and wash twice on a shaker for 10 minutes each time, then discard the penetrant. (Enhanced) Add 100L of methanol to each well and wash 1-2 times for 5 minutes each time, then wash once with PBS for 5 minutes each time.

[0025] DNA staining: Dilute reagent F with deionized water at a ratio of 100:1 to prepare an appropriate amount of 1 Hoechst 33342 reaction solution, and store it in the dark; add 100 L of 1 Hoechst 33342 reaction solution to each well, and incubate in the dark at room temperature on a destaining shaker for 30 min, then discard the staining reaction solution; wash each well three times with 100 L of PBS for 5 min each time; store each well with 100 L of PBS, take pictures, and count the cell proliferation rate.

[0026] The results are as follows Figure 3 As shown in CD, after stimulating human esophageal squamous cell carcinoma cells with different drugs for 24 hours, the survival rate and proliferation rate of KYSE30 and KYSE450 cells in the SS331-HA-ADT group were much lower than those in the positive control group ADT-OH and the positive control group HA-ADT. This indicates that SS31-HA-ADT can inhibit the growth of tumor cells.

[0027] To investigate the effects of SS31-HA-ADT on tumor cell migration and invasion, the scratch assay was used to determine the effect of SS31-HA-ADT on tumor cell migration, and the Transwell and Invasion assays were used to determine the effects of SS31-HA-ADT on tumor cell migration and invasion. The specific process of determining the effect of SS31-HA-ADT on tumor cell migration using the scratch assay is as follows: Human esophageal squamous cell carcinoma cells KYSE30 and KYSE450 were digested and counted separately, and seeded into 6-well culture plates at a seeding density of 5102. 5 Cells were incubated at 37°C with 3 mL of culture medium per well. Once cells reached the logarithmic growth phase, a scratch assay was performed. After scratching, cells were washed three times with PBS. Different serum-free culture media were used for each group, with the control group receiving physiological saline, the positive control group (ADT-OH) receiving 200 mol / L LADT-OH, the positive control group (HA-ADT) receiving 200 mol / L HA-ADT, and the SS31-HA-ADT group receiving 200 mol / L SS31-HA-ADT. Cells were photographed under a 100-microscope at 0 h, 12 h, and 24 h. Cell migration rate was calculated. Cell migration rate (%) = (distance from scratch at 0h - distance from scratch at 24h) / distance from scratch at 0h × 100%.

[0028] The specific procedure for determining the effects of SS31-HA-ADT on tumor cell migration and invasion using Transwell and Invasion assays is as follows: Prepare complete culture medium with 20% serum. Add 800 μL of complete culture medium to each well of a 24-well plate. Place a 0.8 μm polycarbonate membrane chamber into the 24-well plate. Take KYSE30 and KYSE450 cells in logarithmic growth phase, resuspend the cells in serum-free culture medium, and vertically add 200 μL of a mixture of cells and drug to each well, ensuring 410 cells / well per well. 4 The cells were selected and the required drug concentration was determined. The 24-well plates were incubated in an incubator. After 24 hours, the chambers were removed, the culture medium was discarded, and the cells were fixed with 95% ethanol for 15 minutes. The cells were washed twice with PBS and then stained with crystal violet for 30 minutes. After staining, the surface crystal violet stain was washed away with running pure water, and the cells were air-dried before being photographed and the results recorded under a microscope. The Invasion assay procedure is largely the same as the Transwell assay procedure, except that a matrix gel is added to the well before adding the cell suspension to the upper layer of the chamber. Dilute 50 mg / L matrix gel 10-fold with serum-free medium, add 100 L of the diluted matrix gel to the upper layer of the chamber, allow it to solidify at room temperature for 30 min, then add 200 L of the cell-drug mixture. Subsequent steps are the same as the Transwell assay procedure.

[0029] Scratch migration results as follows Figure 4 As shown in Figures A and B, after stimulating human esophageal squamous cell carcinoma cells with different drugs for 24 hours, the migration rates of KYSE30 and KYSE450 cells in the SS31-HA-ADT group were significantly lower than those in the control group; migration and invasion results are as follows. Figure 5As shown in AD, after stimulating human esophageal squamous cell carcinoma cells with different drugs for 24 hours, the migration and invasion abilities of KYSE30 and KYSE450 cells in the SS31-HA-ADT group were significantly reduced compared with those in the control group.

[0030] To investigate the effects of SS31-HA-ADT on mitochondria of human esophageal squamous cell carcinoma cells, MitoTracker staining was used to determine the effect of SS31-HA-ADT on mitochondrial structure of tumor cells, JC-1 assay was used to determine the effect of SS31-HA-ADT on mitochondrial function of tumor cells, and ATP assay was used to determine the effect of SS31-HA-ADT on mitochondrial function of tumor cells. The specific procedure for determining the effect of SS31-HA-ADT on the mitochondrial structure of tumor cells using the MitoTracker staining method is as follows: Using a 20mm confocal culture dish, 1mL of complete culture medium was added to the dish and preheated in an incubator; KYSE30 and KYSE450 cells in logarithmic growth phase and in good condition were selected, digested and resuspended, and an appropriate amount was added to the confocal culture dish, mixed thoroughly, and then cultured in an incubator; when the cells reached 70%-80% confluence, the original culture medium was discarded, and different drugs were added for further culture for 24 hours; 2 After 4 hours, discard the drug and wash twice with PBS. Prepare Hoechst solution by diluting Hoechst (1000) staining solution 1000-fold with complete culture medium under light-protected conditions. Add 1 mL to each confocal culture dish and incubate for 10-15 minutes. Wash once with PBS. Prepare MitoTracker staining solution by diluting the MitoTracker fluorescent probe to 200 nM with complete culture medium under light-protected conditions. Add 1 mL of staining solution to each confocal culture dish and incubate for 15 minutes. Wash twice with PBS. Add complete culture medium and photograph the results promptly. Use a confocal microscope with 60x oil immersion to photograph the morphology of cell mitochondria.

[0031] The results are as follows Figure 6 As shown in Figure A, after stimulating human esophageal squamous cell carcinoma cells with different drugs for 24 hours, the mitochondria of KYSE30 and KYSE450 cells in the SS31-HA-ADT group showed a punctate or short fragmented distribution and decreased fluorescence intensity, indicating that SS31-HA-ADT damages the mitochondrial structure. Furthermore, structural damage was also observed in the mitochondria of the ADT-OH and HA-ADT groups, but the degree of damage was less severe than that in the SS31-HA-ADT group.

[0032] The specific procedure for determining the effect of SS31-HA-ADT on mitochondrial function in tumor cells using the JC-1 assay is as follows: 1 mL of complete culture medium was added to each well of a 6-well plate and the plate was preheated in an incubator; KYSE30 and KYSE450 cells in logarithmic growth phase were taken, counted using a cell counting chamber, and then resuspended in 1:10⁻⁶ cells. 6 Cell suspension was prepared by adding 1 mL of the cell suspension to each well of a 6-well plate and shaking well. The 6-well plate was then placed in an incubator for incubation. When the cell density reached 85%, different drugs were added and the plate was incubated for another 24 hours. 15 mL of JC-1 staining working solution was prepared, along with 75 mL of JC-1 (200), 12 mL of ultrapure water, and 3 mL of JC-1 buffer (5). The drugs were discarded, and the cells were washed once with PBS. Under light-protected conditions, 1 mL of complete culture medium and 1 mL of JC-1 staining working solution were added to each well, and the plates were placed in an incubator for 20 min. During the waiting period, 4 mL of JC-1 staining buffer (5) and 16 mL of ultrapure water were mixed to prepare 20 mL of staining buffer. The JC-1 staining solution was discarded, and the cells were washed twice with the staining buffer. 2 mL of complete culture medium was added to each well, and the results were photographed under a fluorescence microscope.

[0033] The results are as follows Figure 7 As shown in Figures A and B, after stimulating human esophageal squamous cell carcinoma cells with different drugs for 24 hours, the red fluorescence of KYSE30 and KYSE450 cells in the SS31-HA-ADT group was significantly reduced, while the green fluorescence was significantly enhanced, and the red-green fluorescence ratio was decreased. The ADT-OH and HA-ADT groups also showed a decreasing trend in membrane potential, but the degree of decrease was less than that in the SS31-HA-ADT group. These results indicate that SS31-HA-ADT can decrease the mitochondrial membrane potential of human esophageal squamous cell carcinoma cells, causing severe damage to the membrane potential.

[0034] The specific procedure for determining the effect of SS31-HA-ADT on mitochondrial function of tumor cells using the ATP assay is as follows: Human esophageal squamous cell carcinoma cells KYSE30 and KYSE450 were digested and counted, and seeded into 100 mm culture dishes. After the cells grew to the logarithmic growth phase, the medium was changed to serum-free medium for each group. PBS was added to the blank control group, and 200 mol / L LADT-OH, 200 mol / L HA-ADT, and 200 mol / L SS31-HA-ADT were added to the control groups, respectively. After culturing for 24 h, protein was extracted. Using a black 96-well plate, 100 μL of ATP detection working solution was added to each well and incubated at room temperature for 3-5 min. Then, 20 μL of sample and standard were added, mixed thoroughly with a pipette, and the RLU value was measured using a chemiluminescence analyzer. A standard curve was plotted based on the results, and the ATP concentration in the sample was calculated.

[0035] The results are as follows Figure 7As shown in Figure C, after stimulating human esophageal squamous cell carcinoma cells with different drugs for 24 hours, the ATP content of KYSE30 and KYSE450 cells in the SS31-HA-ADT group was significantly reduced, indicating a decrease in ATP synthesis capacity and impaired mitochondrial energy metabolism function.

[0036] To investigate the effects of SS31-HA-ADT on ferroptosis and pyroptosis in human esophageal squamous cell carcinoma cells, lipid ROS was used to determine the effect of SS31-HA-ADT on tumor cell ferroptosis and N-GSDMD immunofluorescence assay was used to determine the effect of SS31-HA-ADT on tumor cell pyroptosis. The specific process for determining the effect of SS31-HA-ADT on tumor cell ferroptosis using lipid ROS is as follows: Cells in the logarithmic growth phase were collected and prepared into a single-cell suspension using culture medium containing 10% fetal bovine serum. Cells were counted using a counting chamber, and the cell concentration was adjusted to the required number, with 510 cells per well. 3 Cells were seeded at 100 μL per well in 96-well plates and incubated at 37°C in a CO2 incubator containing 5% (v / v) medium. After cell attachment, 100 μL of drug-containing medium was added per well (PBS was added to the blank control group, and 200 mol / L LADT-OH, 200 mol / L HA-ADT, and 200 mol / L LSS31-HA-ADT were added to the drug-treated groups, with 3 replicates for each group). After culturing for 24 h, BODIPY581 / 591C11 staining solution was prepared using serum-free medium to a concentration of 2 M, and 100 μL of working solution was added to each well. The cells were incubated at room temperature for 30 min, followed by washing three times with PBS for 5 min each time. The working solution was discarded, and the cells were washed once with PBS. 100 μL of serum-free medium was then added. The results were observed and photographed under a fluorescence microscope.

[0037] The results are as follows Figure 8 As shown in Figure AB, after 24 hours of stimulation with different drugs on human esophageal squamous cell carcinoma cells, the green fluorescence intensity of KYSE30 and KYSE450 cells in the SS31-HA-ADT group gradually increased, while the red fluorescence intensity gradually decreased, and the green / red fluorescence ratio gradually increased. The number of red and green fluorescence cells in the ADT-OH and HA-ADT groups was less than that in the SS31-HA-ADT group. This result indicates that SS31-HA-ADT can promote ferroptosis in human esophageal squamous cell carcinoma cells.

[0038] The specific procedure for determining the effect of SS31-HA-ADT on tumor cell apoptosis using N-GSDMS immunofluorescence assay is as follows: Cell crawling slides were plated in 6-well plates, with 2-3 slides per well. Initial cell treatment was the same as the scratch assay, except that the cell crawling slides were added first, followed by the cell suspension and mixing. After 24 hours of drug treatment, the drug was discarded, and the cells were washed 2-3 times with PBS. 1 mL of 4% paraformaldehyde was added to each well for fixation for 30 minutes, followed by 2-3 washes with PBS. 1 mL of 0.5% Triton X-100 permeation buffer was added to each well for 20 minutes, followed by 2-3 washes with PBS. Primary antibody was prepared. Add an appropriate amount of primary antibody solution to each well to cover the surface of the cell slide, incubate at 4°C in the dark for at least 12 hours, recover the primary antibody, and wash with PBS 2-3 times. Add an appropriate amount of the corresponding fluorescent secondary antibody to the slide, incubate at room temperature in the dark for 1 hour, and wash with PBS 2-3 times. Add DAPI staining reagent (ready-to-use type) to the slide to completely cover it, incubate at room temperature in the dark for 10-15 minutes, and wash with PBS 2-3 times. Take a glass slide, add an appropriate amount of anti-fluorescence quencher to it, place the cell slide face down on the anti-fluorescence quencher, and fix the position of the slide with nail polish. Observe and photograph the results using a confocal microscope.

[0039] The results are as follows Figure 9 As shown in Figures A and B, after stimulating human esophageal squamous cell carcinoma cells with different drugs for 24 hours, the N-GSDMD fluorescence intensity of KYSE30 and KYSE450 cells in the SS31-HA-ADT group was significantly enhanced, and the fluorescence signal was concentrated at the cell membrane edge, appearing as a speckled aggregation, indicating pyroptosis. Cells in the ADT-OH and HA-ADT groups showed only a slight increase in N-GSDMD fluorescence, which was still mainly diffusely distributed in the cytoplasm, indicating a weaker degree of pyroptosis activation in the SS31-HA-ADT group.

[0040] In summary, MTT and EDU assays showed that the addition of 200 mol / L SS31-HA-ADT inhibited cell growth in human esophageal squamous cell carcinoma cells KYSE30 and KYSE450. Scratch, transwell, and invasion assays showed that SS31-HA-ADT inhibited cell migration. MitoTracker, JC-1, and ATP assays revealed that the addition of 200 M SS31-HA-ADT affected mitochondrial structure and function. Furthermore, the detection of ferroptosis and pyroptosis-related indicators showed that the addition of 200 M SS31-HA-ADT promoted ferroptosis and pyroptosis in human esophageal squamous cell carcinoma cells, thus inhibiting cell growth.

[0041] Finally, it should be noted that the above description is only 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 foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a hydrogen sulfide releasing agent, characterized in that: Includes the following steps: S1. Compound 1 and sulfur were reacted in dimethylacetamide by heating to synthesize compound 2; S2. Compound II is demethylated using pyridine hydrochloride to form compound III; S3. Hyaluronic acid was dissolved in a mixed solvent consisting of N,N-dimethylformamide and double-distilled water to form a homogeneous solution. EDCHCl and DMAP were then added and reacted with compound three to synthesize compound four. S4. Compound 4 is dissolved in a mixed solvent 2 consisting of EDCHCl and double-distilled water to form a DMF solution, which then reacts with the amino group of short peptide SS31 to form a hydrogen sulfide release agent.

2. The method for preparing a hydrogen sulfide releasing agent according to claim 1, characterized in that: In step S1, the first compound is anethole, and the molar ratio of the first compound to sulfur is 1:5-10.

3. The method for preparing a hydrogen sulfide releasing agent according to claim 2, characterized in that: In the synthesis of compound 2, compound 1 and sulfur were reacted in dimethylacetamide at 140-150°C. After the reaction was complete, the mixture was cooled to room temperature, water was added to the resulting solution, and the solution was extracted with diethyl ether. The combined organic phases were washed with water and brine in sequence, dried over anhydrous Na2SO4, and the solvent was removed under reduced pressure. Finally, the solution was crystallized with ethyl acetate to obtain compound 2.

4. The method for preparing a hydrogen sulfide releasing agent according to claim 1, characterized in that: In step S2, during the demethylation reaction of compound two, pyridine hydrochloride and compound two are heated to 210-220°C under argon protection and melted for 15-30 minutes. After melting, the mixture is cooled to 100°C, water is added, and the mixture is filtered while hot. The filter cake is first washed with alkali, then dissolved in water, and the pH is adjusted to 2 with hydrochloric acid. The precipitate is then filtered, washed with water until neutral, and dried to obtain compound three.

5. The method for preparing a hydrogen sulfide releasing agent according to claim 4, characterized in that: The molar ratio of compound 2 to pyridine hydrochloride is 1:5-10. During alkaline washing, 10wt% NaOH is used for washing, and the hydrochloric acid is concentrated hydrochloric acid with a concentration of 36-38wt%.

6. The method for preparing a hydrogen sulfide releasing agent according to claim 1, characterized in that: In step S3, the molar ratio of N,N-dimethylformamide and double-distilled water in the mixed solvent one is 1:1-2.

5. When adding EDCHCl and DMAP to the homogeneous solution, the temperature is controlled at 0℃ for 50-70 min before adding compound three. The reaction is carried out at 0℃ for 25-40 min, and then at room temperature until the crude product is completely obtained. The crude product is then dialyzed, and the supernatant after dialyzing is filtered through a microporous membrane with a pore size of 0.45 μm and freeze-dried to obtain compound four.

7. The method for preparing a hydrogen sulfide releasing agent according to claim 6, characterized in that: The molar ratio of hyaluronic acid, EDCHCl, DMAP, and the compound used is 2-4:2-4:1:2-4. When dialyzing the crude product, it is dialyzed with DMF in a 3.5kDa dialysis bag for 5-10 hours, then dialyzed with water / DMF at a volume ratio of 1:1 for 10-15 hours, and then dialyzed with water in a 3.5kDa dialysis bag for 20-24 hours. The resulting supernatant is filtered through a microporous membrane with a pore size of 0.45 μm.

8. The method for preparing a hydrogen sulfide releasing agent according to claim 1, characterized in that: In step S4, when compound four is dissolved in mixed solvent two, it is stirred at 0°C for 1-4 hours to obtain a DMF solution. Then, short peptide SS31 is dissolved in the DMF solution, and the reaction is stirred at room temperature for 5-12 hours under argon protection. The reaction solution is then transferred to a 3.5 kDa dialysis bag and dialyzed with double-distilled water for 16-24 hours. The supernatant is filtered through a microporous membrane with a pore size of 0.45 μm and then freeze-dried to obtain a hydrogen sulfide releasing agent.

9. The method for preparing a hydrogen sulfide releasing agent according to claim 1, characterized in that: The structural formula of the hydrogen sulfide releasing agent is: ， Where p = 4 - 6, q = 16 - 22.

10. The application of a hydrogen sulfide releasing agent, wherein the hydrogen sulfide releasing agent is prepared by the preparation method of a hydrogen sulfide releasing agent according to any one of claims 1-9, characterized in that: The hydrogen sulfide releaser is used in the preparation of drugs for treating human esophageal squamous cell carcinoma.

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