Peptidyl ferroptosis inducer for improving radiotherapy curative effect of melanoma as well as preparation method and application of peptidyl ferroptosis inducer

Abstract

The invention discloses a peptidyl ferroptosis inducer DYY-Pen-NO for improving the radiotherapy curative effect of melanoma as well as a preparation method and application of the peptidyl ferroptosis inducer DYY-Pen-NO. The ferroptosis inducer is constructed by covalent coupling of a tyrosinase substrate peptide DYY and a nitric oxide (NO) donor Pen-NO. The inducer is catalyzed and oxidized by tyrosinase highly expressed by melanoma, melanin-like aggregates can be formed in situ in tumor cells to realize long-acting retention, and glutathione (GSH) is effectively exhausted along with quinone generation and NO controllable release. On the other hand, reactive oxygen species (ROS) generated by radiotherapy and NO can automatically generate peroxynitrite anions (ONOO-), lipid peroxidation is induced, tumor cell ferroptosis is triggered, the radiotherapy curative effect is enhanced, and release of immunogenic substances is promoted. In addition, the formed melanin-like aggregate can capture an antigen through a Michael addition reaction, and promote the maturation of dendritic cells (DC), thereby activating systematic anti-tumor immune response.

Description

Technical Field

[0001] The present invention relates to the field of nano-biomedical materials, and more particularly to a method for preparing a peptide-based ferroptosis inducer and its application in improving the efficacy of radiotherapy for melanoma. Background Art

[0002] Radiotherapy, as an important means of tumor treatment, mainly kills tumor cells by inducing DNA damage and ROS accumulation through ionizing radiation, but its efficacy is often limited by the inherent radioresistance of tumor cells. Recent studies have found that ferroptosis, a type of programmed cell death driven by iron-dependent lipid peroxidation, has a potential synergistic effect with radiotherapy: radiation can cause lipid peroxidation through ROS accumulation, induce ferroptosis in tumor cells, and promote the release of immunogenic substances and the activation of anti-tumor immune responses. However, although most ROS species (such as superoxide anions) produced by radiotherapy can cause lipid peroxidation to a certain extent, their effect is weak, resulting in a relatively limited level of lipid peroxidation that cannot break through the cell death threshold. In addition, the inherent oxidative defense system of tumor cells inhibits ferroptosis. For example, GSH, as a key cofactor of GPX4, can effectively remove lipid peroxides and maintain the redox balance in tumor cells. Therefore, the development of new ferroptosis inducers is expected to increase the level of lipid peroxidation and enhance the anti-tumor effect of radiotherapy through the organic synergy of targeted depletion of GSH and the production of strong oxidizing substances (such as reactive nitrogen species), which has important clinical translation value. Summary of the invention

[0003] The present invention aims to develop a peptide-based ferroptosis inducer DYY-Pen-NO, and use it to improve the efficacy of radiotherapy for melanoma. The ferroptosis inducer of the present invention has the following advantages: (1) The raw materials are economical and easily available, and the preparation process is simple; (2) Based on the characteristics of high expression of tyrosinase in melanoma, DYY-Pen-NO can be oxidized in situ in melanoma to generate melanin-like aggregates, thereby effectively retaining them in the tumor site; (3) The quinone structure formed during the generation of melanin-like aggregates and the responsive release of NO can deplete intracellular GSH, and the ROS generated by radiation react with NO to generate ONOO - It can induce lipid peroxidation in tumor cells, and the two together cause ferroptosis of tumor cells, enhance the efficacy of radiotherapy and promote the release of immunogenic substances; (4) Melanin aggregates can efficiently capture immunogenic substances through their rich quinone structure to promote the efficiency of tumor antigen phagocytosis and presentation by DCs, activate the body's anti-tumor immune response, and kill melanoma cells immune-wise.

[0004] In order to achieve the purpose of the present invention, the technical solution of the present invention is as follows: A peptide-based ferroptosis inducer DYY-Pen-NO for improving the efficacy of radiotherapy for melanoma is covalently coupled with a tyrosine-containing short peptide and an NO donor Pen-NO, wherein the tyrosine-containing short peptide includes but is not limited to DYY, DFY, EFY, EYY, FFY, YYY, etc. The structure of DYY-Pen-NO is shown below (taking DYY as an example):

[0005] The present invention further discloses a method for preparing a peptide-based ferroptosis inducing agent for improving the efficacy of radiotherapy for melanoma, which is characterized in that the preparation steps are as follows: (1) Weigh 50-100 mg of DYY-Pen and dissolve it in 1-2 mL of ultra-dry DMF, then precool it at 0 °C for 30 minutes to obtain solution A; (2) Take 100 μL of specific nitrite and dilute it 10 times with super dry DMF to obtain solution B; (3) Add solution B dropwise into solution A under nitrogen protection and react at 0 °C in the dark for 3 hours; (4) After the reaction is completed, the resulting reaction mixture is added dropwise into pre-cooled ice ether to precipitate a solid, which is then centrifuged at 12,000-15,000 rpm for 10 minutes and freeze-dried to obtain the product DYY-Pen-NO.

[0006] The synthesis method of the DYY-Pen is as follows (taking DYY as an example): (1) Weigh 0.5-1 g of dichlororesin into a solid phase synthesis tube, add 10-20 mL of dichloromethane (DCM) and soak for 5-10 minutes to allow the resin to fully swell, and squeeze out the DCM in the synthesis tube with an ear bulb; (2) Weigh Fmoc-S-Trityl-L-Penicillamine (0.5-1 mmol, 306-712 mg) into a vial, add 10-20 mL DCM and catalyst N,N-diisopropylethylamine (DIEA) (1-2 mmol, 200-400 μL) in sequence, add to the solid phase synthesis tube after fully dissolving, and react at room temperature for 2-4 hours; (3) Squeeze out the reaction solution, wash with DCM 5 times, and add 10-20 mL of blocking solution (DCM:CH 3 OH:DIEA=14:2:1) for 0.5-1 hour to block the remaining active reaction sites; (4) Wash with DCM and DMF five times each, add 20% piperidine (15-30 mL) to remove the Fmoc protecting group of Fmoc-S-Trityl-L-Penicillamine to expose the active amine group; (5) Wash with DMF 5 times, weigh Fmoc-Tyr(tBu)-OH (2-4 mmol, 918-1836 mg), coupling agent O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU) (2-4 mmol, 758-1516 mg) and catalyst DIEA (4-8 mmol, 800-1600 μL) into a vial, fully dissolve them with DMF and add them to the solid phase synthesis tube for reaction for 2-4 hours; (6) Repeat the above steps (4) and (5) to couple Fmoc-Tyr(tBu)-OH and Fmoc-Asp(OtBu)-OH in sequence. Use DMF and DCM to wash and remove the unreacted amino acid, catalyst and coupling agent, and then add 95% trifluoroacetic acid (TFA) (H 2 O:TIS:TFA=2.5:2.5:95) to cleave the peptide chain from the resin; (7) Remove TFA by rotary evaporation, add anhydrous ether to the obtained viscous liquid, collect the precipitate to obtain the crude polypeptide. Finally, separate and purify by high performance liquid chromatography to obtain the pure product of DYY-Pen, whose chemical structure is shown in Structural Formula II

[0007] The present invention also discloses the application of peptide-based ferroptosis inducer DYY-Pen-NO for improving the efficacy of melanoma radiotherapy in enhancing the efficacy of melanoma radiotherapy. The experimental results show that DYY-Pen-NO combined with radiotherapy can deplete GSH in tumor cells and induce severe lipid peroxidation, leading to ferroptosis of melanoma cells, and significantly enhancing the sensitivity of melanoma cells to radiotherapy. The clone formation experiment shows that after DYY-Pen-NO pretreatment and then irradiation with a dose of 6 Gy, the number of clones is significantly reduced compared with the simple irradiation group. The radiosensitization ratio calculated by the survival curve is 1.94, which is much higher than the commercially available sodium glycidyl bisazole (1.17). In addition, the aggregates of DYY-Pen-NO after tyrosinase oxidation have good antigen capture ability. After loading antigens, co-incubation with bone marrow-derived DC can significantly promote DC maturation, proving that it has immune activation performance after capturing antigens.

[0008] The present invention is described in more detail as follows: The peptide derivative DYY-Pen was synthesized by the classic solid-phase synthesis method, and then further reacted with tert-butyl nitrite through nucleophilic substitution to generate NO-grafted ferroptosis inducer DYY-Pen-NO. DYY-Pen-NO was oxidized by tyrosinase, which is highly expressed in melanoma, to form melanin-like aggregates in situ to achieve long-term retention, accompanied by the generation of quinone structure and controlled release of NO, effectively depleting GSH. Under the action of γ-rays, the ROS generated by radiation and NO spontaneously generate ONOO- It specifically triggers tumor cell ferroptosis through the lipid peroxidation pathway, significantly enhances the efficacy of radiotherapy, promotes the release of immunogenic substances, and activates anti-tumor immune responses.

[0009] A peptide-based ferroptosis inducing agent for improving the efficacy of radiotherapy for melanoma, the preparation steps are as follows: (1) Weigh 50-100 mg of DYY-Pen and dissolve it in 1-2 mL of ultra-dry DMF, then precool it at 0 °C for 30 minutes to obtain solution A; (2) Take 100 μL of specific nitrite and dilute it 10 times with super dry DMF to obtain solution B; (3) Add solution B dropwise into solution A under nitrogen protection and react at 0 °C in the dark for 3 hours; (4) After the reaction is completed, the resulting reaction mixture is added dropwise into pre-cooled ice ether to precipitate a solid, which is then centrifuged at 12,000-15,000 rpm for 10 minutes and freeze-dried to obtain the product DYY-Pen-NO.

[0010] The synthesis method of the DYY-Pen is as follows (taking DYY as an example): (1) Weigh 0.5-1 g of dichlororesin into a solid phase synthesis tube, add 10-20 mL of DCM and soak for 5-10 minutes to allow the resin to fully swell, and squeeze out the DCM in the synthesis tube with an ear bulb; (2) Weigh Fmoc-S-Trityl-L-Penicillamine (0.5-1 mmol, 306-712 mg) into a vial, add 10-20 mL DCM and catalyst DIEA (1-2 mmol, 200-400 μL) in sequence, add to the solid phase synthesis tube after fully dissolving, and react at room temperature for 2-4 hours; (3) Squeeze out the reaction solution, wash with DCM 5 times, and add 10-20 mL of blocking solution (DCM:CH 3 OH:DIEA=14:2:1) for 0.5-1 hour to block the remaining active reaction sites; (4) Wash with DCM and DMF five times each, add 20% piperidine (15-30 mL) to remove the Fmoc protecting group of Fmoc-S-Trityl-L-Penicillamine to expose the active amine group; (5) Wash 5 times with DMF. Weigh Fmoc-Tyr(tBu)-OH (2 - 4 mmol, 918 - 1836 mg), coupling agent HBTU (2 - 4 mmol, 758 - 1516 mg), and catalyst DIEA (4 - 8 mmol, 800 - 1600 μL) into a vial. After fully dissolving with DMF, add it to a solid-phase synthesis tube and react for 2 - 4 hours; (6) Repeat the above steps (4) and (5) to couple Fmoc-Tyr(tBu)-OH and Fmoc-Asp(OtBu)-OH in sequence. Wash with DMF and DCM in sequence to remove unreacted amino acids, catalyst, and coupling agent. Then add 95% TFA (H 2 O:TIS:TFA = 2.5:2.5:95) to cleave the peptide chain from the resin; (7) Rotavapor to remove TFA. Add anhydrous ether to the obtained viscous liquid, and collect the precipitated solid to obtain the crude polypeptide. Finally, purify it by high-performance liquid chromatography to obtain the pure DYY-Pen.

[0011] All raw materials or reagents involved in the present invention are ordinary commercially available products, and all operations involved are conventional operations in the art without special instructions.

[0012] The beneficial effects of the peptide-based ferroptosis inducer for enhancing the efficacy of melanoma radiotherapy, its preparation method and application disclosed in the present invention are as follows: The present invention prepares a ferroptosis inducer based on self-assembled peptides and NO and uses it to enhance the efficacy of melanoma radiotherapy. It has the following advantages: (1) Simple preparation and easily available raw materials; (2) Based on the characteristic of high expression of tyrosinase in melanoma, the ferroptosis inducer can achieve long-term retention in tumor cells in situ; (3) Through the dual strategies of depleting GSH and generating ONOO - to induce lipid peroxidation to achieve radiotherapy-induced ferroptosis and promote the release of immunogenic substances; (4) The quinone structure formed by the oxidation of tyrosine by tyrosinase can efficiently capture immunogenic substances to promote the efficiency of tumor antigen phagocytosis and presentation by DCs, activate the anti-tumor immune response of the body, and is expected to provide a new strategy for enhancing the efficacy of melanoma radiotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The accompanying drawings here are incorporated into the specification and form a part of this specification, showing embodiments consistent with the present invention, and are used together with the specification to explain the principles of the present invention; To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, for those of ordinary skill in the art, other drawings can also be obtained based on these drawings without creative efforts; Figure 1 A and B are high-resolution mass spectra of DYY-Pen and DYY-Pen-NO prepared in Examples 1 and 2 of the present invention, respectively; Figure 2 (A) is a photograph of DYY-Pen-NO before and after oxidation in Example 3 of the present invention; (B) is an ultraviolet absorption curve of DYY-Pen-NO before and after oxidation in Example 3 of the present invention; (C) is a transmission electron microscope image of DYY-Pen-NO before and after oxidation in Example 3 of the present invention; Figure 3 The results of depleting GSH in melanoma B16F10 cells in Example 4 of the present invention (A), the flow cytometry results of ONOO- generated in the cells (B), and the results of inducing lipid peroxidation (C); Figure 4 The clone formation images (A) and cell survival curves (B) of melanoma B16F10 cells co-incubated with DYY-Pen-NO or PBS and irradiated with different doses in Example 5 of the present invention; Figure 5 The antigen capture effect (A) and the result of in vitro DC maturation stimulation (B) in Example 6 and Example 7 of the present invention; Figure 6 It is a schematic diagram of the chemical structure and reaction process of the present invention. DETAILED DESCRIPTION

[0014] In order to more clearly understand the above-mentioned objects, features and advantages of the present invention, the scheme of the present invention will be further described below.

[0015] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein; it is obvious that the embodiments in the specification are only part of the embodiments of the present invention, rather than all of the embodiments.

[0016] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0017] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources, among which the coupling agent HBTU, the catalyst DIEA, the dichloro resin, dichloromethane (DCM), N,N-dimethylformamide (DMF), piperidine, Fmoc-Tyr(tBu)-OH, Fmoc-S-Trityl-L-Penicillamine, Fmoc-Asp(OtBu)-OH, the dichloro resin, the Fmoc-S-Trityl-L-Penicillamine and other reagents are all commercially available.

[0018] Example 1, Synthesis of DYY-Pen (taking DYY as an example): (1) Weigh 1.1 g of dichlororesin into a solid phase synthesis tube, add 15 mL of DCM and swell for 10 minutes to allow the resin to swell fully. Use an ear bulb to squeeze out the DCM in the synthesis tube. (2) Weigh Fmoc-S-Trityl-L-Penicillamine (1 mmol, 712 mg) into a vial, add 15 mL of DCM and catalyst DIEA (2 mmol, 400 μL) in sequence, add to the solid phase synthesis tube after fully dissolving, and react at room temperature for 3 hours; (3) Squeeze out the reaction solution, wash with DCM 5 times, and add 15 mL of blocking solution (DCM:CH 3 OH:DIEA=14:2:1) for 0.5 h to block the remaining active reaction sites; (4) Wash with DCM and DMF five times each, add 20% piperidine (20 mL) to remove the Fmoc protecting group of Fmoc-S-Trityl-L-Penicillamine, exposing the active amine group; (5) Wash with DMF 5 times, weigh Fmoc-Tyr(tBu)-OH (4 mmol, 1836 mg), coupling agent HBTU (4 mmol, 1516 mg) and catalyst DIEA (8 mmol, 1600 μL) into a vial, fully dissolve them with DMF and add them to the solid phase synthesis tube for reaction for 3 hours; (6) Repeat the above steps (4) and (5) to couple Fmoc-Tyr(tBu)-OH and Fmoc-Asp(OtBu)-OH in sequence. Use DMF and DCM to wash and remove the unreacted amino acid, catalyst and coupling agent, and then add 95% TFA (H 2 O:TIS:TFA=2.5:2.5:95) to cleave the peptide chain from the resin; (7) TFA was removed by rotary evaporation, anhydrous ether was added to the obtained viscous liquid, and the precipitate was collected to obtain the crude polypeptide. Finally, the pure DYY-Pen was obtained by separation and purification by high performance liquid chromatography.

[0019] The structure of DYY-Pen was confirmed by high-resolution mass spectrometry. The results are shown in the attached Figure 1 Middle A.

[0020] Example 2: Synthesis of DYY-Pen-NO (1) Weigh 100 mg of DYY-Pen and dissolve it in 2 mL of ultra-dry DMF, then precool it at 0 °C for 30 minutes to obtain solution A; (2) Take 100 μL of specific nitrite and dilute it 10 times with super dry DMF to obtain solution B; (3) Add solution B dropwise into solution A under nitrogen protection and react at 0 °C in the dark for 3 hours; (4) After the reaction is completed, the resulting reaction mixture is added dropwise into pre-cooled ice ether to precipitate a solid, which is then centrifuged at 15,000 rpm for 10 minutes and freeze-dried to obtain the product DYY-Pen-NO.

[0021] The structure of DYY-Pen-NO was confirmed by high-resolution mass spectrometry. Figure 1 Middle B.

[0022] Example 3: Enzyme responsiveness verification of DYY-Pen-NO: 10 mg of DYY-Pen-NO was dissolved in 1 mL of PBS, and the pH was adjusted to 7.0 with sodium carbonate. Then, tyrosinase (0.2 mg / mL) was added and incubated at 37 °C. Photos were taken at different time points, and the generation of melanin was detected by UV-visible spectroscopy. The microscopic morphology of DYY-Pen-NO before and after tyrosinase oxidation was observed using transmission electron microscopy.

[0023] See attached Figure 2 As the oxidation time of DYY-Pen-NO increased, the color of the solution gradually deepened. At the same time, the UV spectrum showed that as the oxidation time increased, a melanin-like absorption peak gradually appeared at 400 nm, confirming the formation of melanin-like pigments. The transmission electron microscopy results showed that DYY-Pen-NO had no obvious microscopic morphology before oxidation, and the generation of crude fibrous substances could be clearly observed after 12 hours of oxidation, further confirming the formation of melanin-like pigment aggregates.

[0024] Example 4: Verification of DYY-Pen-NO-induced ferroptosis in tumor cells GSH depletion verification: B16F10 cells in the logarithmic growth phase were seeded in six-well plates (3 × 10 5After 24 hours of incubation at 37 °C, the culture medium was discarded and fresh culture medium containing DYY-Pen-NO (0.5 mg / mL) was added. After 12 hours of incubation, the cells were irradiated with γ-rays at a dose of 6 Gy and incubated for 12 hours. The cells were collected and lysed by repeated freezing and thawing. After centrifugation, the supernatant was taken and the GSH content in the cells was determined using a GSH detection kit.

[0025] See attached Figure 3 In Figure A, after the action of DYY-Pen-NO, the intracellular GSH content decreased by about 50%. After further irradiation, the ROS generated by irradiation further reduced the intracellular GSH level, resulting in extremely high levels of oxidative stress in the cells.

[0026] ONOO - Intracellular production verification: B16F10 cells in the logarithmic growth phase were seeded in six-well plates (3 × 10 5 After incubation at 37 °C for 24 hours, the culture medium was discarded and fresh culture medium containing DYY-Pen-NO (0.5 mg / mL) was added. After incubation for another 12 hours, the cells were irradiated with γ-rays at a dose of 6 Gy. - The cells were collected and flow cytometry was used to detect ONOO in tumor cells. - The results are shown in the attached Figure 3 Middle B, DYY-Pen-NO combined with radiotherapy can significantly increase ONOO in melanoma cells - content, which is conducive to cell ferroptosis.

[0027] DYY-Pen-NO induced lipid peroxidation in tumor cells: B16F10 cells in the logarithmic growth phase were seeded in six-well plates (3 × 10 5 After 24 hours of incubation at 37 °C, the culture medium was discarded and fresh culture medium containing DYY-Pen-NO (0.5 mg / mL) was added. After 12 hours of incubation, the cells were irradiated with γ-rays at a dose of 6 Gy and incubated for another 12 hours. The cells were collected and stained with BODIPY 581 / 591 C11 probe, and then detected by flow cytometry.

[0028] See attached Figure 3 C, after the combined action of DYY-Pen-NO and radiotherapy, significant lipid peroxidation occurred in the cells, which may be caused by the depletion of GSH and the generation of ONOO - caused by the synergistic effect.

[0029] Example 5: Verification of the in vitro radiotherapy sensitization effect of DYY-Pen-NO Colony formation assay: B16F10 cells in the logarithmic growth phase were seeded in a 12-well plate at a density of 500 cells / well and cultured at 37 °C for 24 hours. Then the medium was discarded. Fresh medium containing DYY-Pen-NO (100 μg / mL) was added and co-incubated. After 12 hours, the medium was removed, the cells were washed once with PBS, fresh medium was added, and the cells were irradiated with γ-rays at doses of 0, 2, 4, or 6 Gy. After irradiation, the cells were placed in a carbon dioxide incubator and cultured for 7 days. When the cell clusters grew to be visible to the naked eye (the number of cells in a single cell cluster was greater than 50), the original medium was carefully removed, the cells were slowly washed twice with PBS, 300 μL of 0.25% crystal violet staining solution was added to each well, and stained for 30 minutes. Finally, the crystal violet staining solution was discarded, the cells were washed with water, air-dried, photographed and counted, and the radiotherapy sensitization ratio of the material was calculated according to the number of cells in the cell clusters.

[0030] See Appendix Figure 4 , the number of clone clusters in the DYY-Pen-NO pretreatment group was significantly reduced, proving that DYY-Pen-NO exerted an efficient radiotherapy sensitization effect, and its radiotherapy sensitization ratio calculated by the survival curve was 1.94, which was much higher than that of the commercially available radiotherapy sensitizer sodium glycididazole (1.17).

[0031] Example 6, Evaluation of antigen capture effect Protein gel electrophoresis: B16F10 cells (1 × 10 7 cells) were irradiated with a dose of 6 Gy and then incubated for 24 hours. The cells were collected and lysed with RIPA lysis buffer to obtain a protein solution as a model antigen for standby. 100 μL of DYY-Pen-NO (2 mg / mL) was added to an equal volume of the model antigen and TYR (0.1 mg / mL) was added and incubated for 12 hours. The mixture solution was centrifuged at 12,000 rpm for 10 minutes, the supernatant was discarded, and then the precipitate was resuspended with 50 μL of PBS and 100 μL of protein loading buffer was added and boiled at 100 °C for 5 minutes. Immediately, the supernatant was centrifuged and taken for protein gel electrophoresis. After electrophoresis, the antigen adsorption amount of different groups was judged by Coomassie brilliant blue staining.

[0032] See Appendix Figure 5 A in it. The results showed that DYY-Pen-NO adsorbed a large amount of protein after being responsive to tyrosinase and co-incubated with the model antigen, proving its good potential for antigen capture.

[0033] Example 7, In vitro stimulation of DC maturation Bone marrow cells were extracted from the bone marrow of C57 mice in a sterile manner, and then GM-CSF (20 ng / mL) and IL-4 (20 ng / mL) were added to induce for 7-8 days to make them bone marrow-derived dendritic cells (BMDC) for use. B16F10 cells were incubated with DYY-Pen-NO for 12 hours, and then fresh culture medium was replaced. After receiving 6 Gy dose of irradiation, the cells were incubated for 24 hours. The supernatant was then aspirated and co-incubated with BMDC for 24 hours. BMDC were collected and stained with flow cytometry antibodies (PE-anti-CD11c, FITC-anti-CD80, Percp-anti-CD86), and tested on the machine. The data were analyzed by flowjo to investigate the effect of DYY-Pen-NO in stimulating DC maturation in vitro after capturing antigens.

[0034] See attached Figure 5 In Figure B, the supernatant of cells pretreated with DYY-Pen-NO and irradiated was co-incubated with BMDC for 12 hours. Flow cytometry analysis showed that the supernatant of the DYY-Pen-NO+irradiation group could significantly promote DC maturation, with a maturation rate of up to 37.2%. The above experiments preliminarily confirmed that DYY-Pen-NO can capture tumor-associated antigens and enhance antigen presentation after radiotherapy, effectively activating DC, which laid the foundation for its systemic immune response after radiotherapy.

[0035] The above description is only a specific embodiment of the present invention, so that those skilled in the art can understand or implement the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments described herein, but should conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A peptide-based ferroptosis inducer DYY-Pen-NO for improving the efficacy of radiotherapy for melanoma, characterized in that: Its structural formula is shown in Structural Formula I. The design of the ferroptosis inducer mainly includes two parts, tyrosinase-specific substrate peptide DYY and NO donor Pen-NO are covalently coupled: .

2. The peptide-based ferroptosis inducing agent DYY-Pen-NO for improving the efficacy of radiotherapy for melanoma according to claim 1, characterized in that: The tyrosinase-specific substrate peptide DYY specifically refers to a short peptide containing tyrosine, including but not limited to DYY, DFY, EFY, EYY, FFY, and YYY 2-10 peptides containing tyrosine.

3. The method for preparing the peptide-based ferroptosis inducing agent DYY-Pen-NO for improving the efficacy of melanoma radiotherapy according to claim 1, characterized in that Proceed as follows: (1) Weigh 50-100 mg of DYY-Pen and dissolve it in 1-2 mL of ultra-dry DMF, then precool it at 0 °C for 30 minutes to obtain solution A; (2) Take 100 μL of tert-butyl nitrite and dilute it 10 times with super dry DMF to obtain solution B; (3) Add solution B dropwise into solution A under nitrogen protection and react at 0 °C in the dark for 3 hours; (4) After the reaction is completed, the resulting reaction mixture is added dropwise into pre-cooled ice ether to precipitate a solid, which is then centrifuged at 12,000-15,000 rpm for 10 minutes and freeze-dried to obtain the product DYY-Pen-NO.

4. Use of the peptide-based ferroptosis inducer DYY-Pen-NO for improving the radiotherapy efficacy of melanoma as claimed in claim 1 in the preparation of radiotherapy sensitization drugs.

5. Use of the peptide-based ferroptosis inducer DYY-Pen-NO for improving the efficacy of melanoma radiotherapy as claimed in claim 1 in the preparation of therapeutic antigen capture drugs.

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