A peptide-based ferroptosis inducer for enhancing the radiotherapy efficacy of melanoma, its preparation method and application

Abstract

The present invention discloses a peptide-based ferroptosis inducer DYY-Pen-NO for enhancing the radiotherapy efficacy of melanoma, and its preparation method and application. The ferroptosis inducer is constructed by covalently coupling the tyrosinase substrate peptide DYY with a nitric oxide (NO) donor Pen-NO. Catalyzed and oxidized by tyrosinase highly expressed in melanoma, the inducer can form melanin-like aggregates in situ in tumor cells for long-term retention, accompanied by the generation of quinones and the controlled release of NO, effectively depleting glutathione (GSH). On the other hand, reactive oxygen species (ROS) generated by radiotherapy and NO can spontaneously generate peroxynitrite anion (ONOO ‑ ), inducing lipid peroxidation, triggering ferroptosis of tumor cells, enhancing the radiotherapy efficacy and promoting the release of immunogenic substances. In addition, the formed melanin-like aggregates can capture antigens through Michael addition reaction, promote the maturation of dendritic cells (DC), and further activate the systemic anti-tumor immune response.

Description

Technical Field

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

[0002] As an important means of tumor treatment, radiotherapy mainly kills tumor cells by inducing DNA damage and ROS accumulation through ionizing radiation. However, its efficacy is often limited by the inherent radioresistance of tumor cells. Recent studies have found that ferroptosis, an iron-dependent lipid peroxidation-driven programmed cell death mode, 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, most ROS species (such as superoxide anion) generated by radiotherapy can cause lipid peroxidation to a certain extent, but their effect is weak, resulting in a relatively limited level of lipid peroxidation and unable to break through the cell death threshold. In addition, the inherent oxidative defense system of tumor cells will inhibit the occurrence of ferroptosis. For example, GSH, as a key cofactor of GPX4, can effectively scavenge lipid peroxides to maintain the redox balance in tumor cells. Therefore, developing new ferroptosis inducers, through the organic synergy of targeting the depletion of GSH and generating strong oxidizing substances (such as reactive nitrogen species), is expected to increase the level of lipid peroxidation and enhance the anti-tumor effect of radiotherapy, 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 enhance the radiotherapy efficacy of melanoma. The ferroptosis inducer of the present invention has the following advantages: (1) The raw materials are economically available and the preparation process is simple; (2) Based on the characteristic of high expression of tyrosinase in melanoma, DYY-Pen-NO can be oxidized in situ in melanoma to form melanin-like aggregates, thus effectively remaining in the tumor site; (3) The quinone structure formed during the melanin-like generation process and the responsive release of NO can deplete intracellular GSH, and at the same time, the ROS generated by radiation reacts with NO to generate ONOO - can induce lipid peroxidation in tumor cells, and the two together cause ferroptosis of tumor cells, enhance the radiotherapy efficacy and promote the release of immunogenic substances; (4) The melanin-like aggregates efficiently capture immunogenic substances through their rich quinone structure to promote the efficiency of tumor antigen phagocytosis and presentation by DCs, activate the anti-tumor immune response of the body, and immunologically kill melanoma.

[0004] In order to achieve the object of the present invention, the technical solution of the present invention is as follows:

[0005] A peptide-based ferroptosis inducer DYY-Pen-NO for enhancing the efficacy of radiotherapy for melanoma, which is covalently coupled by a tyrosine-containing short peptide and a NO donor Pen-NO. The tyrosine-containing short peptides include, but are not limited to, DYY, DFY, EFY, EYY, FFY, YYY, etc. The structure of DYY-Pen-NO is shown as follows (taking DYY as an example):

[0006]

[0007] The present invention further discloses a preparation method of a peptide-based ferroptosis inducer for enhancing the efficacy of radiotherapy for melanoma, which is characterized in that the preparation steps are as follows:

[0008] (1) Weigh 50 - 100 mg of DYY-Pen and dissolve it in 1 - 2 mL of ultradry DMF, then place it in a pre-cooled environment at 0 °C for 30 minutes to obtain solution A;

[0009] (2) Take 100 μL of specific nitrite ester and dilute it 10 times with ultradry DMF to obtain solution B;

[0010] (3) Under nitrogen protection, slowly add solution B dropwise to solution A, and react at 0 °C in the dark for 3 hours;

[0011] (4) After the reaction is completed, slowly add the obtained reaction mixture dropwise to pre-cooled ice ether to precipitate a solid, then centrifuge at 12000 - 15000 rpm for 10 minutes, and freeze-dry to obtain the product DYY-Pen-NO.

[0012] The synthesis method of the described DYY-Pen is as follows (taking DYY as an example):

[0013] (1) Weigh 0.5 - 1 g of dichloride resin into a solid-phase synthesis tube, add 10 - 20 mL of dichloromethane (DCM) and soak for 5 - 10 minutes to fully swell the resin, and squeeze out the DCM in the synthesis tube with an ear syringe;

[0014] (2) Weigh Fmoc-S-Trityl-L-Penicillamine (0.5 - 1 mmol, 306 - 712 mg) into a vial, sequentially add 10 - 20 mL of DCM and the catalyst N,N-diisopropylethylamine (DIEA) (1 - 2 mmol, 200 - 400 μL), fully dissolve and then add it to the solid-phase synthesis tube, and react at room temperature for 2 - 4 hours;

[0015] (3) Squeeze out the reaction solution, wash it 5 times with DCM, add 10 - 20 mL of a blocking solution (DCM:CH3OH:DIEA = 14:2:1) and react for 0.5 - 1 hour to block the remaining active reaction sites;

[0016] (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, exposing the reactive amino group;

[0017] (5) Wash with DMF five times. Weigh Fmoc-Tyr(tBu)-OH (2 - 4 mmol, 918 - 1836 mg), coupling agent O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) (2 - 4 mmol, 758 - 1516 mg), and catalyst DIEA (4 - 8 mmol, 800 - 1600 μL) into a vial, fully dissolve with DMF and then add to the solid-phase synthesis column for reaction for 2 - 4 hours;

[0018] (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, catalysts, and coupling agents. Then add 95% trifluoroacetic acid (TFA) (H2O:TIS:TFA = 2.5:2.5:95) to cleave the peptide chain from the resin;

[0019] (7) Remove TFA by rotary evaporation. Add anhydrous ether to the resulting viscous liquid, and collect the precipitated solid to obtain the crude polypeptide. Finally, purify the pure DYY-Pen by high-performance liquid chromatography, and its chemical structure is shown in Structural Formula II

[0020]

[0021] The present invention also discloses the application of the peptide-based ferroptosis inducer DYY-Pen-NO for enhancing the efficacy of radiotherapy for melanoma in terms of enhancing the efficiency of melanoma radiotherapy. Experimental results show that DYY-Pen-NO combined with radiotherapy can deplete intracellular GSH in tumor cells and induce severe lipid peroxidation, leading to ferroptosis in melanoma cells and significantly enhancing the sensitivity of melanoma cells to radiotherapy. The colony formation assay shows that after pretreatment with DYY-Pen-NO and then irradiation with a dose of 6 Gy, compared with the single irradiation group, the number of colonies is significantly reduced. Calculating the radiotherapy sensitization ratio through the survival curve is 1.94, which is much higher than that of commercially available sodium glycididazole (1.17). In addition, the aggregate of DYY-Pen-NO after oxidation by tyrosinase has good antigen capture ability. After loading antigens and co-incubating with bone marrow-derived DCs, it can significantly promote the maturation of DCs, demonstrating its immune activation performance after antigen capture.

[0022] The present invention is described in more detail as follows:

[0023] The polypeptide derivative DYY-Pen was synthesized by the classical solid-phase synthesis method, and further reacted with tert-butyl nitrite through a nucleophilic substitution reaction to generate the NO-grafted ferroptosis inducer DYY-Pen-NO. DYY-Pen-NO was catalytically oxidized by tyrosinase highly expressed in melanoma to in-situ form melanin-like aggregates for long-term retention, accompanied by the generation of quinone structures and the controlled release of NO, effectively depleting GSH. Under the action of γ-rays, the ROS generated by radiation and NO spontaneously generated ONO - , specifically triggering ferroptosis of tumor cells through the lipid peroxidation pathway, significantly enhancing the radiotherapy efficacy and promoting the release of immunogenic substances, and activating the anti-tumor immune response.

[0024] A peptide-based ferroptosis inducer for enhancing the radiotherapy efficacy of melanoma, and the preparation steps are as follows:

[0025] (1) Weigh 50-100 mg of DYY-Pen and dissolve it in 1-2 mL of ultradry DMF, then place it in a pre-cooled 0 °C for 30 minutes to obtain solution A;

[0026] (2) Dilute 100 μL of specific nitrite ester 10 times with ultradry DMF to obtain solution B;

[0027] (3) Under nitrogen protection, add solution B dropwise to solution A, and react at 0 °C in the dark for 3 hours;

[0028] (4) After the reaction is completed, add the obtained reaction mixture dropwise to pre-cooled ice ether to precipitate a solid, then centrifuge at 12000-15000 rpm for 10 minutes, and freeze-dry to obtain the product DYY-Pen-NO.

[0029] The synthesis method of the above-mentioned DYY-Pen is as follows (taking DYY as an example):

[0030] (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 fully swell the resin, and squeeze out the DCM in the synthesis tube with an ear bulb;

[0031] (2) Weigh Fmoc-S-Trityl-L-Penicillamine (0.5-1 mmol, 306-712 mg) into a vial, sequentially add 10-20 mL of DCM and the catalyst DIEA (1-2 mmol, 200-400 μL), fully dissolve and then add it to the solid-phase synthesis tube, and react at room temperature for 2-4 hours;

[0032] (3) Extrude the reaction solution, wash it 5 times with DCM, add 10 - 20 mL of blocking solution (DCM:CH3OH:DIEA = 14:2:1), and react for 0.5 - 1 hour to block the remaining active reaction sites;

[0033] (4) Wash it 5 times with DCM and then 5 times with DMF successively. Add 20% piperidine (15 - 30 mL) to remove the Fmoc protecting group of Fmoc - S - Trityl - L - Penicillamine, exposing the active amino group;

[0034] (5) Wash it 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, fully dissolve them with DMF, and then add them into the solid - phase synthesis column for reaction for 2 - 4 hours;

[0035] (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 successively to remove the unreacted amino acids, catalyst, and coupling agent. Then add 95% TFA (H2O:TIS:TFA = 2.5:2.5:95) to cleave the peptide chain from the resin;

[0036] (7) Rotavapor to remove TFA. Add anhydrous ether to the obtained viscous liquid, collect the precipitated solid to obtain the crude polypeptide. Finally, purify it by high - performance liquid chromatography to obtain the pure DYY - Pen.

[0037] The raw materials or reagents involved in the present invention are all ordinary commercially available products, and the operations involved are all conventional operations in the art without special instructions.

[0038] The beneficial effects of the peptide - based ferroptosis inducer for enhancing the radiotherapy efficacy of melanoma and its preparation method and application disclosed in the present invention are as follows:

[0039] The present invention prepares a ferroptosis inducer based on self - assembled peptides and NO and uses it to enhance the radiotherapy efficacy of melanoma. It has the following advantages: (1) simple preparation and easy availability of raw materials; (2) achieving long - term retention of the ferroptosis inducer in situ in tumor cells based on the characteristic of high expression of tyrosinase in melanoma; (3) depleting GSH and generating ONOO -A dual strategy for inducing 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 efficiently captures immunogenic substances to promote the efficiency of tumor antigen phagocytosis and presentation by DCs, activating the anti-tumor immune response of the body, and is expected to provide a new strategy for enhancing the efficacy of radiotherapy for melanoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The accompanying drawings herein are incorporated into the specification and form a part of this specification, showing embodiments in accordance with the present invention, and are used together with the specification to explain the principles of the present invention;

[0041] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required for the description of the embodiments or the prior art. Obviously, for those of ordinary skill in the art, without creative efforts, other accompanying drawings can also be obtained based on these drawings;

[0042] Figure 1 In (A) and (B) are the high-resolution mass spectra of DYY-Pen and DYY-Pen-NO prepared in Example 1 and Example 2 of the present invention, respectively;

[0043] Figure 2 In (A) are the photos of DYY-Pen-NO before and after oxidation in Example 3 of the present invention; (B) is the ultraviolet absorption curve of DYY-Pen-NO before and after oxidation in Example 3 of the present invention; (C) is the transmission electron microscope image of DYY-Pen-NO before and after oxidation in Example 3 of the present invention;

[0044] Figure 3 Shows the results of depleting GSH in melanoma B16F10 cells (A), the flow cytometry results of intracellular ONOO- generation (B), and the results of inducing lipid peroxidation (C) in Example 4 of the present invention;

[0045] Figure 4 Shows the colony 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;

[0046] Figure 5 Shows the antigen capture effect (A) and the results of in vitro stimulating DC maturation (B) in Example 6 and Example 7 of the present invention;

[0047] Figure 6 Is a schematic diagram of the chemical structure and reaction process of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0048] In order to more clearly understand the above-mentioned objects, features, and advantages of the present invention, the solution of the present invention will be further described below.

[0049] In the following description, many specific details are set forth in order to provide a thorough understanding of the present invention. However, the present invention may be practiced in other ways different from those described herein. Obviously, the embodiments in the specification are only a part of the embodiments of the present invention, rather than all of the embodiments.

[0050] The preferred embodiments of the present invention will be described in detail below in conjunction with the embodiments. It should be understood that the following embodiments are given only for the purpose of illustration and are not used to limit the scope of the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from the spirit and scope of the present invention. The experimental methods used in the following embodiments are all conventional methods unless otherwise specified.

[0051] The materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified. Among them, reagents such as coupling agent HBTU, catalyst DIEA, dichlororesin, dichloromethane (DCM), N,N-dimethylformamide (DMF), piperidine, Fmoc-Tyr(tBu)-OH, Fmoc-S-Trityl-L-Penicillamine, Fmoc-Asp(OtBu)-OH, dichlororesin, Fmoc-S-Trityl-L-Penicillamine, etc. are all commercially available.

[0052] Example 1. Synthesis of DYY-Pen (taking DYY as an example):

[0053] (1) Weigh 1.1 g of dichlororesin into a solid-phase synthesis tube, add 15 mL of DCM and swell for 10 minutes to fully swell the resin, and squeeze out the DCM in the synthesis tube with an ear bulb;

[0054] (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. After fully dissolving, add it to the solid-phase synthesis tube and react at room temperature for 3 hours;

[0055] (3) Squeeze out the reaction solution, wash it 5 times with DCM, add 15 mL of blocking solution (DCM:CH3OH:DIEA = 14:2:1) and react for 0.5 hours to block the remaining active reaction sites;

[0056] (4) Wash it 5 times with DCM and DMF respectively, add 20% piperidine (20 mL) to remove the Fmoc protecting group of Fmoc-S-Trityl-L-Penicillamine, exposing the active amino group;

[0057] (5) Wash it 5 times with DMF. 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 then add them into the solid-phase synthesis tube for reaction for 3 hours;

[0058] (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, catalysts and coupling agents. Then add 95% TFA (H2O:TIS:TFA = 2.5:2.5:95) to cut the peptide chain from the resin;

[0059] (7) Rotavapor to remove TFA. Add anhydrous ether to the obtained viscous liquid, collect the precipitated solid to obtain the crude polypeptide. Finally, separate and purify it by high performance liquid chromatography to obtain the pure product of DYY-Pen.

[0060] Confirm the structure of DYY-Pen by high-resolution mass spectrometry, and the results are shown in Appendix Figure 1 A.

[0061] Example 2. Synthesis of DYY-Pen-NO

[0062] (1) Weigh 100 mg of DYY-Pen and dissolve it in 2 mL of ultra-dry DMF, then place it in a pre-cooled bath at 0 °C for 30 minutes to obtain solution A;

[0063] (2) Take 100 μL of nitrous acid ester and dilute it 10 times with ultra-dry DMF to obtain solution B;

[0064] (3) Dropwise add solution B to solution A under nitrogen protection, and react at 0 °C in the dark for 3 hours;

[0065] (4) After the reaction is completed, dropwise add the obtained reaction mixture to pre-cooled ice ether, precipitate a solid, then centrifuge at 15000 rpm for 10 minutes and lyophilize to obtain the product DYY-Pen-NO.

[0066] Confirm the structure of DYY-Pen-NO by high-resolution mass spectrometry, see Appendix Figure 1 B.

[0067] Example 3: Verification of the enzyme responsiveness of DYY-Pen-NO

[0068] Dissolve 10 mg of DYY-Pen-NO in 1 mL of PBS, adjust the pH to 7.0 with sodium carbonate, then add tyrosinase (0.2 mg / mL), and incubate at 37 °C. Take pictures and observe at different time points, detect the generation of melanin-like substances using ultraviolet-visible spectroscopy, and observe the morphological changes of DYY-Pen-NO before and after oxidation by tyrosinase using transmission electron microscopy.

[0069] See Appendix Figure 2 , as the oxidation time of DYY-Pen-NO extends, the color of the solution gradually deepens. At the same time, the ultraviolet spectrum shows that as the oxidation time extends, an absorption peak of melanin-like substances gradually appears at 400 nm, confirming the generation of melanin-like substances; the transmission electron microscopy results show that there is no obvious microscopic morphology of DYY-Pen-NO before oxidation, and fibrous substances can be clearly observed after 12 hours of oxidation, further confirming the formation of melanin-like aggregates.

[0070] Example 4: Verification of the induction of ferroptosis in tumor cells by DYY-Pen-NO

[0071] Verification of GSH depletion: Inoculate B16F10 cells in the logarithmic growth phase into a six-well plate (3 × 10 5 cells / dish), incubate at 37 °C for 24 hours, then discard the culture medium, and add fresh culture medium containing DYY-Pen-NO (0.5 mg / mL). Continue to incubate for 12 hours, then irradiate with γ-rays at a dose of 6 Gy, and continue to incubate for 12 hours after irradiation. Collect the cells and lyse the cells by repeated freezing and thawing, and take the supernatant after centrifugation to measure the GSH content in the cells using a GSH detection kit.

[0072] See Appendix Figure 3 A, after the action of DYY-Pen-NO, the intracellular GSH content decreased by about 50%. After further irradiation, due to the ROS generated by irradiation, the intracellular GSH level was further reduced, resulting in an extremely high level of oxidative stress in the cells.

[0073] ONOO - Verification of intracellular generation: Inoculate B16F10 cells in the logarithmic growth phase into a six-well plate (3 × 10 5 cells / dish), incubate at 37 °C for 24 hours, then discard the culture medium, and add fresh culture medium containing DYY-Pen-NO (0.5 mg / mL). Continue to incubate for 12 hours, then irradiate with γ-rays at a dose of 6 Gy, and immediately stain with the ONOO - probe after irradiation. Collect the cells and detect the intracellular ONOO in tumor cells by flow cytometry- Content. The results are shown in the appendix Figure 3 In B, when DYY-Pen-NO is combined with radiotherapy, it can significantly increase the content of ONOO - in melanoma cells, which is beneficial to the occurrence of ferroptosis in cells.

[0074] Detection of lipid peroxidation induced by DYY-Pen-NO in tumor cells: Inoculate B16F10 cells in the logarithmic growth phase into a six-well plate (3 × 10 5 cells / dish). After incubating at 37 °C for 24 hours, discard the culture medium, and add fresh culture medium containing DYY-Pen-NO (0.5 mg / mL). Continue to incubate for 12 hours, then irradiate with γ-rays at a dose of 6 Gy, and continue to incubate for 12 hours after irradiation. Collect the cells and stain them with BODIPY 581 / 591 C11 probe, and then detect them by flow cytometry after staining.

[0075] See the appendix Figure 3 In C, after the combined action of DYY-Pen-NO and radiotherapy, obvious lipid peroxidation occurred in the cells. This result may be caused by the synergistic effect of depleting GSH and generating ONOO - .

[0076] Example 5. Verification of the in vitro radiotherapy sensitization effect of DYY-Pen-NO

[0077] Colony formation assay: Take B16F10 cells in the logarithmic growth phase and inoculate them into a 12-well plate at a density of 500 cells / well. Continue to culture at 37 °C for 24 hours, then discard the culture medium. Add fresh culture medium containing DYY-Pen-NO (100 μg / mL) and incubate together. After 12 hours, remove the culture medium, wash once with PBS, add fresh culture medium, and irradiate with γ-rays at doses of 0, 2, 4, or 6 Gy. After irradiation, place the cells in a carbon dioxide incubator and continue to culture for 7 days. When the cell colonies grow to be visible to the naked eye (the number of cells in a single cell colony is greater than 50), carefully remove the original culture medium, slowly wash 2 times with PBS, add 300 μL of 0.25% crystal violet staining solution to each well, and stain for 30 minutes. Finally, discard the crystal violet staining solution, wash with water, dry, take pictures and count, and calculate the radiotherapy sensitization ratio of the material according to the number of cells in the cell colonies.

[0078] See the appendix Figure 4 , the number of clone colonies in the DYY-Pen-NO pretreatment group was significantly reduced, proving that DYY-Pen-NO exerted an efficient radiotherapy sensitization effect. And through the calculation of the survival curve, its radiotherapy sensitization ratio was 1.94, which was much higher than that of the commercially available radiotherapy sensitizer sodium glycididazole (1.17).

[0079] Example 6. Evaluation of the effect of capturing antigens

[0080] Protein gel electrophoresis: After irradiating B16F10 cells (1 × 10 7 cells) with a dose of 6 Gy and continuing to incubate 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 it was boiled at 100 °C for 5 minutes. Immediately, the supernatant was centrifuged and taken for protein gel electrophoresis. After electrophoresis, Coomassie brilliant blue staining was used to judge the antigen adsorption amount of different groups.

[0081] See Appendix Figure 5 A. The results showed that after DYY-Pen-NO was responsive to tyrosinase and co-incubated with the model antigen, a large amount of protein was adsorbed, proving its good potential for capturing antigens.

[0082] Example 7. In vitro stimulation of DC maturation

[0083] Bone marrow cells were extracted from the bone marrow of C57 mice under sterile conditions throughout the process, and then GM-CSF (20 ng / mL) and IL-4 (20 ng / mL) were added and induced for 7 - 8 days to make them bone marrow-derived dendritic cells (BMDC) for standby. After incubating B16F10 cells with DYY-Pen-NO for 12 hours, the fresh medium was changed, and after being irradiated with a dose of 6 Gy, they were continued to be incubated for 24 hours. Then, the supernatant was aspirated and co-incubated with BMDC for 24 hours. The BMDC were collected and stained with flow antibodies (PE-anti-CD11c, FITC-anti-CD80, Percp-anti-CD86), and then detected by machine. The data was analyzed by flowjo to investigate the effect of DYY-Pen-NO on stimulating DC maturation in vitro after capturing antigens.

[0084] See Appendix Figure 5 B. When the supernatant of irradiated cells pretreated with DYY-Pen-NO was co-incubated with BMDC for 12 hours, flow cytometry analysis found that the supernatant of the DYY-Pen-NO + irradiation group could significantly promote DC maturation, and the maturation ratio was as high as 37.2%. The above experiments preliminarily confirmed that DYY-Pen-NO could capture tumor-associated antigens after radiotherapy and enhance antigen presentation, effectively activating DC, which laid a foundation for its induction of a systemic immune response after radiotherapy.

[0085] The above are only specific embodiments of the present invention, enabling those skilled in the art to understand or implement the present invention. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can 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 these embodiments described herein, but rather will conform to the broadest scope consistent with the principles and novel features disclosed herein.

Claims

1. A peptide-based ferroptosis inducer DYY-Pen-NO for enhancing the radiotherapy efficacy of melanoma, characterized in that: Its structural formula is shown as Structural Formula I. The design of this ferroptosis inducer mainly includes two parts: the tyrosinase-specific substrate peptide DYY and the NO donor Pen-NO are covalently coupled: 。 2. The preparation method of the peptide-based ferroptosis inducer DYY-Pen-NO for enhancing the radiotherapy efficacy of melanoma according to claim 1, characterized in that It is carried out according to the following steps: (1) Weigh 50−100 mg of DYY-Pen and dissolve it in 1−2 mL of ultradry DMF, then place it in a pre-cooled bath at 0 °C for 30 minutes to obtain Solution A; (2) Dilute 100 μL of tert-butyl nitrite 10 times with ultradry DMF to obtain Solution B; (3) Under nitrogen protection, add Solution B dropwise to Solution A and react at 0 °C in the dark for 3 hours; (4) After the reaction is completed, add the obtained reaction mixture dropwise to pre-cooled ice ether to precipitate a solid, then centrifuge at 12000−15000 rpm for 10 minutes and lyophilize to obtain the product DYY-Pen-NO.

3. Use of the peptidyl ferroptosis inducer DYY-Pen-NO described in claim 1 for enhancing the efficacy of radiotherapy for melanoma in the preparation of a drug for radiotherapy sensitization.

4. Use of the peptidyl ferroptosis inducer DYY-Pen-NO described in claim 1 for enhancing the efficacy of radiotherapy for melanoma in the preparation of a drug for antigen capture.

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