Application of combined use of thymopentin, salinomycin and IR780 iodide in preparation of drugs for treating and inhibiting tumor dryness, and nano drug-loading platform for inhibiting tumor dryness

Abstract

The invention provides application of combined use of thymopentin, salinomycin and IR780 iodide in preparation of a medicine for treating and inhibiting tumor dryness. The invention also provides a pharmaceutical composition for inhibiting tumor dryness. The invention relates to an endoplasmic reticulum stress nano inducer (DTSS) based on salinomycin, which is used for synergistically inhibiting the dryness of cancer cells and effectively sensitizing cancer immunotherapy. The endoplasmic reticulum targeted phototherapy drug s-780 is prepared by coupling p-toluene sulfonamide and IR780 iodide (IR780), is assembled together with thymopentin and salinomycin, and is finally functionalized by biotin to obtain a delivery nano-platform (DTSS) at a specific site, so that systemic anti-tumor immune response can be effectively activated, metastasis and recurrence of cancers can be inhibited, and the application prospect is wide. And a promising solution is provided for sensitizing cancer immunotherapy by inhibiting cancer cell dryness.

Description

Technical Field

[0001] The invention relates to the use of thymopentin, salinomycin and IR780 iodide in combination in preparing a drug for treating and inhibiting tumor stemness, and a nano drug-carrying platform for inhibiting tumor stemness, belonging to the field of medicine. Background Art

[0002] Cancer stem cells (CSCs) are considered to be a key factor leading to the failure of tumor treatment due to their self-renewal, multidirectional differentiation potential, and important roles in tumorigenesis, development, drug resistance, and recurrence. Cancer immunotherapy is one of the most promising treatment modalities in the clinic, with the potential to improve treatment efficacy, reduce metastasis, and prevent recurrence. In particular, the clinically used immunomodulator thymopentin (thymopentin) effectively promotes the differentiation of dendritic cells and enhances the function of mature T cells, thereby enhancing anti-tumor immunity. However, due to the excellent water solubility and high IC50 of thymopentin, its release behavior is uncontrolled, and high loading efficiency is required to achieve high doses. In addition, a small fraction of cancer stem cells are insensitive to T cells, which weakens the effect of T cell-mediated immunotherapy and leads to the metastasis and recurrence of cancer. Therefore, inhibiting tumor stemness can be used as a potential therapeutic strategy to effectively sensitize cancer immunotherapy.

[0003] Due to the high water solubility and short half-life of thymopentin, its clinical application is limited. Some studies have co-assembled it with the chemotherapy drug doxorubicin (DOX) into a carrier-free, injectable chemotherapeutic immunotherapy nanogel. This nanogel achieves co-assembly through hydrogen bonding sites, ensuring a high drug loading of TP5 and DOX, and achieving site-specific and controlled release, thereby enhancing the cancer immune cycle (Ning Ding, Combating melanoma metastasis by enhancing the cancer immune cycle, carrier-free delivery of thymopentin-regulated injectable nanogels, DOI: 10.1016 / j.mtbio.2023.100645). This paper uses hydrogel as a carrier to achieve controlled release of thymopentin. However, it mainly relies on the tumor killing achieved by the traditional chemotherapy drug doxorubicin (DOX), and thymopentin is only used as a means to improve prognosis; although the clinical problem of thymopentin's easy hydrolysis is solved, it does not combine natural immune activation with specific immune enhancement to achieve a design of sensitized tumor immunotherapy. The hydrogel in this literature also does not address the targeting of doxorubicin and thymopentin to hidden lesions and metastases (it can only be controlled and released at the injection site of the hydrogel, and the targeting of distal lesions is poor); among the many small molecule inhibitors targeting tumor stemness, salinomycin is a carboxylate ion antibiotic that shows selective activity against multidrug-resistant (MDR) cancer cells. Mechanistically, salinomycin can effectively regulate the Wnt signaling pathway and target various stem cell-related transcription factors such as β-catenin and SOX2. Therefore, salinomycin selectively reduces the viability of breast cancer stemness, and its potency is almost 100 times that of the traditional chemotherapy drug paclitaxel. However, the poor water solubility and limited delivery efficiency of salinomycin significantly limit its clinical application. Therefore, it is urgent to develop a multifunctional nanoplatform to co-deliver thymopentin and salinomycin to improve their bioavailability and sensitize cancer immunotherapy.

[0004] Significantly, the ER is also considered to be a key organelle for inhibiting cancer cell stemness and regulating anti-tumor immune responses. On the one hand, under ER stress conditions, reduced calcium ion uptake inhibits the downstream Wnt signaling pathway, thereby inhibiting the maintenance of cancer cell stemness. On the other hand, ER stress can effectively phosphorylate programmed cell death ligand 1 (PD-L1) and degrade PD-L1 protein, thereby significantly reducing regulatory T cell (Treg)-mediated immunosuppression. In addition, this excessive ER stress has also been shown to play an important role in activating the cGAS-STING pathway, thereby enhancing anti-tumor immunity. Notably, salinomycin can promote the production of reactive oxygen species (ROS), thereby triggering ER stress.

[0005] IR780 iodide is a small molecule with strong optical properties and excellent photoconversion efficiency, especially under near-infrared (NIR) irradiation, which has attracted widespread attention in the field of cancer treatment and imaging. IR780 nanoparticles have been widely studied for their absorption and photothermal conversion capabilities in the near-infrared region, and they show great application prospects in cancer imaging and photothermal and photodynamic therapy. The application of IR780 nanoparticles in tumor photothermal therapy showed excellent photothermal effect and excellent selective toxicity, while also showing good biocompatibility and safety. The experimental results confirmed that IR780 has strong optical properties and excellent photoconversion efficiency under near-infrared irradiation, thereby inducing apoptosis of tumor cells. In in vitro experiments, the survival rate of tumor cells decreased significantly with the increase in the concentration of IR780 nanoparticles; in in vivo experiments, IR780 also significantly inhibited the growth of tumors in nude mice (Wu Yangjing, et al., Application of IR780 Nanoparticles in Tumor Photothermal Therapy and Its Medical Prospects, International Clinical Medicine: Vol. 5, No. 4, 2023) Both salinomycin and IR780 iodide are hydrophobic, which limits their clinical application. Some studies have encapsulated SAL and IR780 on the surface of upconversion nanoparticles (UCNPs) to form UCNPs (UISPs) loaded with SAL and IR780. This nanoplatform exhibits a strong photothermal effect under near-infrared light irradiation and can effectively inhibit the vitality and migration of liver cancer stem cells (LCSCs) (Salinomycin and upconversion nanoparticles loaded with IR780 affect the biological behavior of liver cancer stem cells by continuously activating the MAPK signaling pathway, DOI: 10.1016 / j.yexcr.2023.113865). This document focuses on the ability of salinomycin to inhibit stemness and the activation of the MAPK pathway by photothermal therapy. In fact, it is just a drug designed to enhance the destruction of "drug-resistant tumor stem cells" by salinomycin. The paper also uses upconversion nanoparticles (UCNPs, a new type of fluorescent probe material that can emit high-energy photons when excited by low-energy near-infrared light) as a carrier; and the authors claim that its targeting is achieved by the folic acid carried by the carrier, and distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG) only serves as an encapsulating agent.

[0006] Currently, the understanding of the involvement of the cGAS STING pathway in effective immunotherapy mainly revolves around the role of mitochondrial or nuclear regulation. Despite this, the role of endoplasmic reticulum (ER) stress in activating the cGAS STING mechanism to enhance immunity against tumors remains largely unexplored. In this study, it was demonstrated that ER stress can be used as a strategy to stimulate the cGAS-STING pathway, thereby enhancing the immune response to cancer (Zhang K, Qi C, Cai K. Manganese-Based TumorImmunotherapy. Adv Mater 2023;35:e2205409, 21. Lv M, Chen M, Zhang R, Zhang W,Wang C, Zhang Y, et al. Manganese is critical for antitumor immune responsesvia cGAS-STING and improves the efficacy of clinical immunotherapy. Cell Res2020;30:966-79, 22.Samson N, Ablasser A. The cGAS-STING pathway and cancer.Nat Cancer 2022;3:1452-63). To achieve this goal, ER-targeted p-toluenesulfonamide tailored IR780 (p-780) was synthesized and loaded into CaO2 nanoparticles, which were further functionalized with DSPE-PEG-biotin to form PEG / CaO2@p-780NPs. The disruption of calcium homeostasis, coupled with the p-780-mediated increase in reactive oxygen species (ROS) levels, and hyperthermia, collectively led to the amplification of endoplasmic reticulum (ER) stress. This cascade of events effectively triggered the cGAS STING pathway while promoting the degradation of PD-L1 protein. In addition, the oxygen released through CaO2 decomposition is expected to promote p-780-mediated phototherapy while reversing the hypoxia-associated immunosuppressive tumor microenvironment. In addition, DSPE-PEG-biotin promoted tumor site-specific drug delivery through biotin receptor-mediated active targeting. Overall, PEG / CaO2@p-780 nanoparticles successfully activated systemic antitumor immunity by enhancing ER stress. This approach provides an alternative strategy for activating the cGAS STING pathway and has the potential to revolutionize cancer immunotherapy.This document focuses on the study of endoplasmic reticulum stress and downstream immune activation events, and does not purposefully use specific immune-enhancing drugs and drugs targeting tumor stem cells. It relies on photothermal and photodynamic therapy triggered by near-infrared light and oxygen release to improve the hypoxic microenvironment. Therefore, it can only be used for superficial tumors that can be phototreated, such as breast cancer (before metastasis) and melanoma.

[0007] Currently, there is no report on the combined use of thymopentin, salinomycin, and IR780 iodide for inhibiting tumor stemness, let alone preparing them into a delivery nanoplatform. Summary of the invention

[0008] The present invention has developed a nano-inducer (DTSS NPs) based on salinomycin to induce endoplasmic reticulum stress, so as to synergistically inhibit the stem cell characteristics of cancer cells. In this way, the systemic immunity is significantly activated, the metastasis and recurrence of cancer are inhibited, and a promising solution is provided for improving the sensitivity of cancer immunotherapy.

[0009] The present invention provides the use of thymopentin, salinomycin and IR780 iodide in combination for preparing a drug for treating and inhibiting tumor stemness.

[0010] Wherein, the drug is a drug for sensitizing cancer immunotherapy.

[0011] Wherein, the mass ratio of thymopentin, salinomycin and IR780 iodide is: 2-6 parts of thymopentin, 0.5-1.5 parts of salinomycin, 1-3 parts of IR780 iodide.

[0012] Preferably, the mass ratio of thymopentin, salinomycin and IR780 iodide is: 4 parts of thymopentin, 1 part of salinomycin, and 2 parts of IR780 iodide.

[0013] The present invention also provides a pharmaceutical composition for inhibiting tumor stemness, which is a preparation containing thymopentin, salinomycin and IR780 iodide as active ingredients, and the mass ratio of thymopentin, salinomycin and IR780 iodide is: 2-6 parts of thymopentin, 0.5-1.5 parts of salinomycin, 1-3 parts of IR780 iodide.

[0014] Preferably, the dosage ratio of thymopentin, salinomycin and IR780 iodide is: 4 parts of thymopentin, 1 part of salinomycin, and 2 parts of IR780 iodide.

[0015] The pharmaceutical composition of the present invention is prepared into an endoplasmic reticulum-targeted phototherapy drug s-780 by coupling p-toluenesulfonamide and IR780 iodide (IR780), and is co-assembled with thymopentin and salinomycin, and finally coated with biotin to prepare a site-specific delivery nanoplatform (DTSS).

[0016] The present invention also provides a method for preparing the pharmaceutical composition for inhibiting tumor stemness. The preparation process of the delivery nanoplatform (DTSS) comprises the following steps: a. Synthesis of s-780: p-Toluenesulfonamide and IR780 iodide were dissolved in N,N-dimethylformamide (DMF); triethylamine (TEA) was added and stirred magnetically at 85°C for 4 hours while protecting from light and under a nitrogen atmosphere to ensure a stable reaction; thereafter, the mixture was cooled to room temperature and evaporated under reduced pressure to remove the solvent; b. Synthesis of DTSS: The s-780 prepared in step a is purified by chromatography on a column, and then dissolved together with salinomycin in anhydrous methanol; a saturated aqueous solution of thymopentin is added, and ultrasound is applied at the same time, and a pipette is used to continuously blow and dissolve the membrane to accelerate dissolution, and TSS nanoparticles are collected by centrifugation, and washed with double distilled water; the obtained TSS nanoparticles are suspended in double distilled water for later use; biotin is dissolved in anhydrous methanol, and an aqueous solution of TSS nanoparticles is added, and ultrasound is applied for at least 8 minutes. After the solvent is evaporated at low temperature in vacuum, the mixture is dispersed in deionized water using ultrasound to obtain a delivery nanoplatform (DTSS); The power of the ultrasonic dispersion is 100-200 W; a pulse mode is adopted, working for 2 seconds and pausing for 1 second.

[0017] Wherein, the molar ratio of p-toluenesulfonamide to IR780 in step a is 2:1; and the biotin in step b is DSPE-PEG2000-Biotin.

[0018] The present invention also provides the use of the pharmaceutical composition in preparing a drug for inhibiting tumor stemness.

[0019] The present invention proposes a salinomycin-based endoplasmic reticulum stress nanoinducer (DTSS) to synergistically inhibit cancer cell stemness and effectively sensitize cancer immunotherapy. The endoplasmic reticulum-targeted phototherapy drug s-780 is prepared by coupling p-toluenesulfonamide and IR780 iodide (IR780), and is co-assembled with thymopentin and salinomycin, and finally functionalized with biotin to obtain a site-specific delivery nanoplatform (DTSS).

[0020] The present invention utilizes the targeting of DSPE-PEG-Bio to release thymopentin, salinomycin, and s780 to the lesion, and achieves the best therapeutic effect with an extremely low dosage. The drug raw materials used are less toxic, and there is no need to strictly control the dosage like doxorubicin. The toxic and negative effects are almost zero, which is safe and reliable. The present invention uses salinomycin, thymopentin and IR780 derivatives (using p-toluenesulfonamide to achieve endoplasmic reticulum targeting, effectively enhancing photothermal photodynamics while inducing a series of downstream events through endoplasmic reticulum stress) to achieve self-assembly, and no additional carrier is required, which effectively improves the drug loading rate. The focus is on activating natural immunity (activating the cGAS-STING pathway, releasing contents to induce immunogenic death), and relieving immunosuppression (degrading immunosuppressive molecules such as PD-L1) after photothermal photodynamics kills most tumor cells; at the same time, thymopentin activates specific immunity and further sensitizes tumor immunotherapy. On the basis of tumor immunotherapy, salinomycin inhibits tumor stemness and cooperates with phototherapy to effectively eliminate residual tumor stem cells, serving as an effective supplement to immunotherapy to achieve the expectation of improving prognosis (inhibiting metastasis and recurrence).

[0021] The drug-carrying platform of the present application has the ability to sensitize immunotherapy and inhibit stem-like cells at the same time, and it also has a certain effect on tumor lesions that are difficult to phototherapy. This nanoplatform not only improves the bioavailability of thymopentin and salinomycin, but also ensures the control of drug release and reduces the side effects of therapeutic agents. It is worth noting that s-780-mediated phototherapy and salinomycin-mediated inhibition of cancer cell stemness synergize, accompanied by downregulation of programmed cell death 1 ligand 1 (PD-L1) expression and activation of the cGAS-STING pathway. In addition, thymopentin significantly promoted the proliferation and differentiation of dendritic cells (DCs) and T lymphocytes, thereby enhancing the response of immune-active cells to cancer cells. The results showed that the DTSS nanoparticles of the present invention effectively activated the systemic anti-tumor immune response, inhibited the metastasis and recurrence of cancer, and provided a promising solution for sensitizing cancer immunotherapy by inhibiting the stemness of cancer cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Preparation of s-780. a) Synthesis route of s-780. b) 1H NMR spectrum of s-780. c) Mass spectrometry analysis of s-780; Figure 2Preparation and characterization of DTSS (a) Size distribution and transmission electron microscopy images of TSS and b) DTSS. Scale bar: 50 nm; c) Zeta potential of TSS and DTSS (n=3); d) UV‒vis absorption spectra of TSS and DTSS; e) Infrared thermal imaging of different drugs after 1.0 W / cm^2 laser irradiation; f) Heating curve of DTSS (808 nm) in water after 1.0 W / cm^2 laser irradiation; g) Remaining amount of DPBF in each group after laser irradiation; h) Release behavior of s-780 at pH 7.4 or 5.0; i) The particle size of TSS and DTSS was observed for 7 days; jk) The uptake behavior of DTSS in 4T1 cells was evaluated by j) fluorescence imaging and k) flow cytometry analysis (n=3); l) Cellular uptake of different treatments in 4T1 cells after 4 hours of incubation period. Scale bar: 50 μm); Figure 3 Other characteristics of NPs (a) IR spectrum of s-780. b) UV-visible absorption spectra of IR780 and s-780. c) Standard curve of s-780. d) Temperature changes of s-780 and TSS after laser irradiation (1.0 W / cm2, 660 nm, 6 min). e) UV-visible absorption spectra of DPBF at different time intervals after laser irradiation in DTSS and aqueous solution. f) Tyndall effect of TSS and DTSS at designated time points); Figure 4 Cytotoxicity of DTSS and its inhibitory stemness (a) 4T1, b) MDA-MB-231, and c) MCF-7 cell survival after treatment with S-780, TSS, or DTSS and laser irradiation (n=3); d) HUVEC cell survival after treatment with 1 μM S-780, TSS, or DTSS; e) Fluorescence images of 4T1 cells subjected to various treatments in EdU assay (n=3). Scale bar: 100 μm; f) Colony formation images and statistical analysis of 4T1 cells treated with SAL, S-780, TSS, or DTSS (n=3); gh) Flow cytometric analysis of 4T1 cell apoptosis under laser irradiation conditions (n=3, 1.0 W / cm2, 660 nm, 30 s).

[0023] i) Detection of LDH levels in cell culture supernatants; jk) Detection of cell culture supernatants and intracellular ATP content; l) Flow cytometric analysis of the proportion of cancer stem cells (CD24- and CD44+) after different treatments; m) Microscope images and n) Statistical analysis of the relative lactosphere formation ratio of treated 4T1 cells (n=3). Scale bar: 100 μm; o) Immunoblotting analysis of stemness-related transcription factors in cells after different treatments; p) Immunoblotting analysis of the downstream Wnt signaling pathway. Differences with p values ​​*<0.05, **<0.01, and ***<0.001 were considered significant); Figure 5 In vivo antitumor immune response of DTSS (a) Schematic diagram showing the bilateral tumor treatment model; b) Body weight of mice in different groups at the indicated time points (n = 5); c) Corresponding images of distant tumors; d) Primary tumor volume at different time points in each group (n = 5); e) Weight of primary tumors in mice and percentage of tumor inhibition (n = 5); f) Volume of distant tumors in each group at different time points (n = 5); g) Weight of distant tumors in mice and percentage of tumor inhibition (n = 5); hi) Flow cytometry analysis; h) Changes in T lymphocytes (CD3+CD4+ / CD3+CD8+) in distant tumors; i) Changes in dendritic cells (CD11c+CD80+CD86+) in distant tumors; The above analysis results are data after the indicated treatments (n = 3); j) Immunohistochemistry was used to detect changes in PD-L1 expression after different treatments and statistical analysis was performed; k) Immunohistochemistry analysis of Ki67 and H&E in distant tumors after different treatments. Scale bar = 100 μm; lo) Quantitative analysis of T lymphocytes (lm), M1 (n), and M2 (o) in tumors (n = 3); pq) Quantitative analysis of T lymphocytes in spleens (n ​​= 3); Figure 6 . In vivo immune response (flow cytometry results and statistical analysis showed changes in a) M1 tumor-associated macrophages (M1 TAMs, CD11b+iNOS+F4 / 80+), b) M2 tumor-associated macrophages (M2TAMs, CD11b+CD206+F4 / 80+), and c) regulatory T cells (Tregs, CD4+CD25+Foxp3+) in tumors after designated treatment (n = 3). Flow cytometry results and statistical analysis showed changes in de) T lymphocytes (CD3+CD4+ / CD3+CD8+) and f) dendritic cells (DCs, CD11c+CD80+CD86+) in spleen. (NS, no significant difference; P<0.05; * P<0.01; *** P<0.001)); Figure 7. The FVB-MMTV-PyMT genetically engineered mouse model demonstrated the effect of inhibiting tumor growth and improving prognosisa) Design of the FVB-MMTV-PyMT genetically engineered mouse model for evaluating tumor inhibitory effects;b) Images showing the tumor inhibitory effects of intravenous injection of MIX (a simple mixture of TP5, s780, and SAL) and DTSS (n=4);c) Bar graph showing tumor weight statistics (n=4);de) Flow cytometric graphs showing changes in the ratio of CD4+ T cells to CD8+ T cells in tumors;fk) Bar graphs showing changes in the ratio of CD4+ T cells, CD8+ T cells, DCs, M1, M2, and Tregs after treatment with MIX and DTSS;l) Design of a relapse model based on the FVB-MMTV-PyMT genetically engineered mouse;m) Photos of relapsed tumors;n) Weight of relapsed tumors;op) Flow cytometric graphs and bar graphs showing changes in the ratio of memory T cells in relapsed tumors;q) Flow cytometric graphs showing changes in the ratio of CD4+ T cells in relapsed tumors;r) Bar graph showing changes in the proportion of CD4+ T cells in recurrent tumors after treatment with MIX and DTSS; s) Flow cytometry graph showing changes in the proportion of CD8+ T cells in recurrent tumors; t) Bar graph showing changes in the proportion of CD8+ T cells in recurrent tumors after treatment with MIX and DTSS; differences were considered significant at p values ​​*<0.05, **<0.01, and ***<0.001); Figure 8 Schematic diagram (a) Preparation of DTSS nanoparticles. bc) Mechanism diagram of DTSS nanoparticles mediating endoplasmic reticulum stress-induced tumor cell toxicity, regulating the stem cell characteristics of tumor cells by inhibiting transcription factor function, and sensitizing T cells). DETAILED DESCRIPTION

[0024] Experimental Example 1 Preparation of DTSS Nanoparticles of the Present Invention 1. Materials Dulbecco's Modified Eagle Medium (DMEM) and fetal bovine serum albumin (FBS) were provided by ThermoFisher Scientific. Table 1 in the Supporting Information lists the product codes and suppliers of all commercial flow cytometers used in this study. Table 2 lists the product codes and suppliers of all commercial antibodies. The antibody dilutions used for immunoblotting, immunofluorescence, and immunohistochemistry were 1:1000, 1:200, and 1:200, respectively. Tables 3 and 4 list the product codes and suppliers of all commercial detection kits and chemical reagents used in this study.

[0025] Table 1. Flow cytometry antibodies in the present invention Antibody Name Company Name and Item Number FITC anti-mouse CD3 Antibody Biolegend, 100203 APC anti-mouse CD4 Antibody Biolegend, 100412 PE anti-mouse CD8a Antibody Biolegend, 100708 APC anti-mouse CD80 Antibody Biolegend, 104714 PE anti-mouse CD86 Antibody Biolegend, 159204 FITC anti-mouse CD11c Antibody Biolegend, 117306 FITC anti-mouse F4 / 80 Antibody Biolegend, 157310 PE anti-iNOS Antibody Biolegend, 696806 PerCP / Cyanine5.5 anti-mouse CD86 Antibody Biolegend, 105028 PerCP / Cyanine5.5 anti-mouse CD206 Antibody Biolegend, 141716 FITC anti-mouse CD25 Antibody Biolegend, 102006 PE anti-mouse FOXP3 Antibody Biolegend, 126404 APC anti-mouse / human CD11b Antibody Biolegend, 101212 PE anti-mouse / human CD44 Antibody Biolegend, 103007 PerCP anti-mouse CD62L Antibody Biolegend, 104430 Table 2. Product codes and suppliers of commercial antibodies Antibody Name Company Name and Item Number CHOP Cell Signaling Technology, #2895 ATF4 Cell Signaling Technology, #11815 eIF2-α Cell Signaling Technology, #5324 p-eIF2-α Cell Signaling Technology, #3398 cGAS Cell Signaling Technology, #79978 STING Cell Signaling Technology, #13647 P-STING Cell Signaling Technology, #72971 IRF-3 Cell Signaling Technology, #4302 P-IRF-3 Cell Signaling Technology, #4947 PD-L1 Cell Signaling Technology, #13684 HMGB1 Cell Signaling Technology, #79823 Calreticulin Cell Signaling Technology, #72516 Ki67 Cell Signaling Technology, #9449 Actin Santa Cruz Biotechnology, sc-8432 Table 3 Detection kit Detection kit Company name and catalog number Reactive Oxygen Species Assay Kit Beyotime, S0033S LDH Cytotoxicity Assay Kit Beyotime, C0017 Fluo-4 Calcium Assay Kit Beyotime, S1061S GSH and GSSG Assay Kit Beyotime, S0053 BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 488 Beyotime, C0071S Annexin V-FITC / PI Apoptosis Detection Kit YEASEN, 40302ES50 ATP Assay Kit Beyotime, S0026 2'3'-cGAMP ELISA Kit AmyJet Scientific Inc, 501700-96S Mouse Perforin ELISA kit Wuhan Fine Biotech Co., Ltd., EM1308 Mouse Granzyme B ELISA Kit Reddot Biotech, RDR-GZMB-Mu Mouse TNF-α ELISA Kit Beyotime, PT512 ELISA MAX™ Deluxe Set Mouse IFN-γ Biolegend, 430804 ELISA MAX™ Deluxe Set Mouse TNF-α Biolegend, 430904 Table 4. Chemical reagents Chemical reagent Company name and catalog number DMSO Chronchem, 68-12-2 DMFSalinomycin Chronchem,67-68-5MedChemExpress,HY-15597 IR780 MACKLIN, I885055 p-Toluenesulfonamide Aladdin Reagent Co., Ltd. M130028 EDCI Aladdin Reagent Co., Ltd. E106172 Triethanolamine (TEA)DMAP Aladdin Reagent Co., Ltd. T431269Aladdin Reagent Co.,D109207 TP5 Meilunbio, MB1244 Tauroursodeoxycholic acid Aladdin Scientific Corp, T421687 ER-Tracker Green Beyotime, C1042S DSPE-PEG2000-Biotin Yuanye, S27129 2. Synthesis of s-780 p-Toluenesulfonamide (41 mg, 0.24 mmol) and IR780 (80 mg, 0.12 mmol) were dissolved in 5 ml of N,N-dimethylformamide (DMF). Subsequently, 67 μL of triethylamine (TEA) was added and magnetically stirred at 85 °C for 4 hours while protecting from light and under a nitrogen atmosphere to ensure the stability of the reaction. Afterwards, the mixture was cooled to room temperature and evaporated under reduced pressure to remove the solvent. The product s-780 was isolated and purified by silica gel column chromatography. The structure of the product was then elucidated using 1H NMR spectroscopy (Bruker Avance400 spectrometer), mass spectrometry (Thermo Scientific QE plus), infrared spectroscopy (Thermo Scientific NicoletiS20) and UV spectrophotometer (Thermo Scientific™ Evolution 60S). Figure 1 bc, Figure 3 ab).

[0026] 3. Preparation of DTSS drug delivery platform To prepare TSS nanoparticles, 8 mg of s-780 and 7.5 mg of salinomycin were completely dissolved in 5 mL of anhydrous methanol. They were evenly distributed by thin film dispersion in a round-bottom flask. Subsequently, an aqueous solution of thymopentin containing 40 mg of thymopentin was slowly added while applying ultrasound and using a pipette to continuously blow and dissolve the film to accelerate dissolution. Next, TSS nanoparticles were collected by centrifugation at 10,000 rpm and subsequently washed three times with double distilled water. The resulting product was then suspended in double distilled water for further use. DSPE-PEG2000-Biotin (10 mg) was completely dissolved in 2 mL of anhydrous methanol. Subsequently, an aqueous solution of TSS nanoparticles was slowly added and ultrasound was applied for at least 8 minutes. After evaporating the solvent in a vacuum at low temperature, the mixture was dispersed using ultrasound in 2 mL of deionized water.

[0027] DTSS was prepared by a self-assembly method. Initially, IR780 was sulfonated with p-toluenesulfonamide to form S-780 (see Figure 1 a), the reaction is carried out by 1H NMR, mass spectrometry, UV‒vis absorption spectroscopy, and infrared spectroscopy (see Figure 1 bc) for verification. Then, s-780 was dispersed with salinomycin by thin film hydration method and co-assembled with thymopentin to form TSS. Finally, a layer of DSPE-PEG2000-biotin was coated on the surface of TSS to obtain DTSS. DSPE-PEG-biotin effectively targeted integrins on the surface of tumor cells and enhanced the cellular uptake of drugs.

[0028] The morphology of TSS and DTSS was observed using a transmission electron microscope (TEM). In addition, the particle dispersibility and zeta potential of TSS and DTSS were determined using a Malvern zetasizer nanoanalyzer. The absorption spectra of TSS and DTSS were determined using a UV-vis spectrophotometer. The drug loading (DL) and encapsulation efficiency (EE) of s-780 and thymopentin were calculated by the following formula: DL % = (W total drug - W free drug) / WDTSS × 100% EE % = (W total drug - W free drug) / Wtotal drug × 100% Where Wtotaldrug and Wfreedrug represent the weight of drug in the initial solution and the weight of free drug in the supernatant, respectively. WDTSS represents the weight of DTSS.

[0029] 4. Characterization of DTSS Nanoparticles Transmission electron microscopy (TEM) images showed that DTSS nanoparticles were smooth spherical nanoparticles with a thick layer of DSPE-PEG2000-biotin on the surface. The particle sizes of TSS and DTSS were 101.67±1.13 nm and 129.27±4.38 nm, respectively ( Figure 2 ab), suitable for achieving enhanced permeation and retention (EPR) effect for passive targeted delivery. In addition, the zeta potential of TSS and DTSS shows a significant negative charge ( Figure 2 c), indicating that DTSS has high blood stability. UV‒vis absorption spectroscopy was used to confirm the synthesis of TSS and DTSS and the successful coating of DSPE-PEG2000-biotin ( Figure 2 d). The drug encapsulation efficiency of s-780 in DTSS is 78.46±1.11%. Figure 3 The standard curve is shown in c. Quantification of in vitro laser irradiation (DTSS at 808 nm, S-780 and TSS at 660 nm, 1 W / cm 2 )( Figure 2 ef and Figure 3 d) revealed that the temperature increase of DTSS was concentration-dependent. In addition, the photodynamic efficiency was evaluated using 1,3-diphenylisobenzofuran (DPBF) as a probe, indicating that DTSS has a high ROS-generating capacity ( Figure 2 g and Figure 3 e). These results indicate that DTSS can be used as an efficient phototherapy nanoplatform for cancer treatment. At the same time, s-780 can be effectively released from DTSS in the acidic tumor microenvironment ( Figure 2 h), indicating that DTSS has pH-responsive drug delivery. The stability of DTSS was evaluated by dynamic light scattering (DLS) over 7 days ( Figure 2 i), and also confirmed the persistence of the Tyndall effect ( Figure 3 f). In precision medicine targeting cancer cells, effective drug uptake and accumulation within cancer cells are crucial for cancer treatment (Gao T, Yuan S, Liang S, Huang X, Liu J, Gu P, et al. In SituHydrogel Modulates cDC1-Based Antigen Presentation and Cancer Stemness toEnhance Cancer Vaccine Efficiency. Adv Sci (Weinh) 2024:e2305832;Yu C, DingB, Zhang X, Deng X, Deng K, Cheng Z, et al. Targeted iron nanoparticles withplatinum-(IV) prodrugs and anti-EZH2 siRNA show great synergy in combatingdrug resistance in vitro and in vivo. Biomaterials 2018;155:112-23). ​​Fluorescence imaging and flow cytometry results showed that the nanoparticles were effectively internalized by cells within 4 hours ( Figure 2 jk), indicating that laser irradiation 4 hours after drug administration is suitable for subsequent in vitro studies. To further clarify the role of biotin receptors, competitive inhibition experiments of biotin were performed by fluorescence microscopy (as shown in the DPB group, 4T1 cells were pre-incubated with free biotin (10 mg / mL) for 2 hours to occupy biotin receptors, and then DTSS was added, which inhibited biotin-mediated active targeting uptake ( Figure 2l). These results support that DTSS can be effectively internalized through biotin receptor-mediated active targeted delivery. 5 mL of DTSS nanoparticle solution was injected into a pre-swollen dialysis bag with a molecular weight cutoff (MWCO) of 3000 Da. The dialysis bag was then immersed in two phosphate-buffered saline (PBS) solutions with different pH values: one with pH 5.0 and the other with pH 7.4. The device was then placed in a shaking incubator at 37 °C. At different time points (0, 1, 2, 4, 6, 8, 10, 12, 24, 36, 48 h), 1 mL samples of the solution surrounding the dialysis bag (called dialysate) were collected for analysis. This sample contained the substances that diffused out of the dialysis bag. After each sampling, the dialysate was immediately supplemented with an equal amount of fresh corresponding solution (1 mL) to maintain confluence conditions. Thymopentin and s-780 in all samples were detected using the BSA method and UV spectrophotometer, respectively.

[0030] The beneficial effects of the present invention are demonstrated by the following efficacy test.

[0031] Test Example 1 1. Experimental Methods 1. Breast cancer cell lines and mice Mouse breast cancer cell lines 4T1, 4T1-luciferase (4T1-luc), and EMT-6, human breast cancer cell lines MCF-7 and MDA-MB-231, and human umbilical vein endothelial cells (HUVEC) were provided by the American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM medium (MeilunBio) containing 10% FBS (HyClone, SH30088.03) and streptomycin (Invitrogen), 100 U / mL penicillin at 37°C and 5% CO2. Female BALB / c mice (6–8 weeks old) were purchased from SPF Biotechnology Co., Ltd. Spontaneous breast cancer mice (FVB-MMTV-PyMT) were purchased from GemPharmatech Co., Ltd. All in vitro and in vivo experiments were performed in accordance with the Regulations on Laboratory Animal Affairs of the People's Republic of China and the Animal Ethics Committee of Sichuan University.

[0032] 2. MTT assay 4T1, MCF-7, and MDA-MB-231 cells were seeded in 96-well plates at a density of approximately 3 x 10^3 cells per well and incubated for 24 hours. The cells were treated with different concentrations of s-780, TSS, and DTSS for 4 hours. After removing the supernatant and washing the cells with phosphate-buffered saline (PBS), the cells were exposed to laser irradiation (660 nm for s-780 and TSS, and 808 nm for DTSS; 1.0 W / cm^2) or no irradiation. After that, they were cultured for another 20 hours. Next, 10 μL of MTT solution (5 mg / mL) was added and incubated for 2 hours. After removing the culture medium, 150 μL of dimethyl sulfoxide (DMSO) was added, and the absorbance intensity at a wavelength of 570 nm was measured.

[0033] 3. Colony formation assay Long-term antiproliferative effects were evaluated using a colony formation method. 5 x 10^2 cells were seeded per well in a 24-well plate and incubated for nearly 3 days before being treated with salinomycin, IR780, s-780, TSS, and DTSS for 4 hours (concentrations were salinomycin: 3μg / mL, IR780 and s-780: 2μg / mL). After removing the supernatant and washing the cells with PBS, they were treated with laser irradiation (s-780 and TSS at 660 nm, DTSS at 808 nm; 1.0 W / cm^2) or without irradiation. The medium was changed every 3 days. After 10 days, the cells were stained with crystal violet for 30 minutes and rinsed three times. Pictures were taken after washing three times with ultrapure water.

[0034] 4. EdU labeling detection Cells were seeded in 96-well plates and treated as above. After removal of the supernatant, cells were irradiated with 660 nm laser (s-780 and TSS at 660 nm, DTSS at 808 nm; 1.0 W / cm^2) or not and cultured for another 20 hours. The proportion of continuously replicating cells was determined using a kit (Ribobio, C10310-3).

[0035] 5. Lactate dehydrogenase (LDH) and adenosine triphosphate (ATP) release detection These tumor cells were treated with salinomycin, IR780, s-780, TSS, or DTSS for 4 h (concentrations were salinomycin: 3 μg / mL, IR780 and s-780: 2 μg / mL), and then exposed to laser irradiation (s-780 and TSS at 660 nm, DTSS at 808 nm; 1.0 W / cm^2) for 10 s. Afterwards, the contents of LDH, ATP, and HMGB1 were measured using the relevant detection kits according to the manufacturer's instructions.

[0036] 6. Apoptosis Detection Briefly, cells were distributed into six-well plates with 5 × 10^4 cells per well and incubated for 24 hours. Next, they were exposed to thymopentin, salinomycin, s-780, TSS, and DTSS (at concentrations of thymopentin: 16 μg / mL, salinomycin: 3 μg / mL, and s-780: 2 μg / mL) for 4 hours. Subsequently, cells were irradiated with near-infrared light of the corresponding wavelength at an intensity of 1 W / cm^2, followed by another 20-hour incubation. Subsequently, cells were co-stained with Annexin V-FITC and PI for 15 minutes at 4°C. Apoptosis was detected using a flow cytometer according to the manufacturer's instructions.

[0037] 7. Western Blot Cells were distributed in six-well plates with 5 x 10^4 cells per well and cultured for 24 h. Next, cells were treated with the indicated treatments. Afterwards, cells were detached, rinsed with pre-chilled PBS, and lysed with radiofrequency immunoprecipitation assay (RIPA) lysis buffer. Lysis was accelerated by ultrasound. Lysates were processed for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with skim milk powder for 2 h at room temperature. After overnight incubation at 4°C with gentle shaking, primary antibodies were applied for further treatment, followed by secondary antibodies for an additional 2 h. Protein expression was assessed using a ChemiScope 6000 Touch instrument (Clinx, Shanghai) after incubation with Immobilon Western HRP substrate (Millipore, WBKLS0050).

[0038] 8. In vitro non-adherent mammosphere formation assay Mammosphere formation assay is used to study the biology of mammary stem cells and their role in breast cancer development and progression. Briefly, cells were distributed into six-well plates with 5 × 10^4 cells per well and incubated for 24 hours. Next, they were exposed to thymopentin, salinomycin, s-780, TSS, and DTSS (at concentrations of thymopentin: 16 μg / mL, salinomycin: 3 μg / mL, s-780: 2 μg / mL) for 4 hours. Subsequently, the cells were irradiated with near-infrared light of the corresponding wavelengths at an intensity of 1 W / cm^2. After further culturing for 20 hours, the cells were digested and suspended to a density of 2×10^3 cells per milliliter. Then, 0.7% agarose gel was mixed with 20% FBS + 2× salinomycin medium in a 1:1 ratio. For each cell group, 2 mL of 20% FBS + 2× salinomycin medium and 2 mL of 0.7% agarose gel were first prepared in a centrifuge tube and mixed thoroughly, and then placed in a 42°C water bath. Next, the cell suspension was added to the mixed solution, mixed thoroughly, and quickly dispensed into a 6-well plate, 1 mL per well. After the agarose solidified on the surface, the culture was continued for 14 days, and bright field images of tumor spheres were taken using an inverted fluorescence microscope. When counting the formation of non-adherent mammospheres, only cell clusters with a diameter of more than 50 μm were counted.

[0039] 9. In vitro ROS production detection Cells were seeded in 6-well plates and incubated for 24 hours. They were then treated with the indicated drugs for 24 hours (thymopentin: 16 μg / mL, salinomycin: 3 μg / mL, s-780: 2 μg / mL), then washed twice with PBS and treated with trypsin. The cells were then collected by centrifugation (4000 rpm, 3 minutes). The resulting pellet was then resuspended in serum-free medium and incubated with the DCFH-DA probe at 37°C for 30 minutes. Afterwards, the cells were washed with PBS three times. Fluorescence intensity was measured using a FACS Calibur flow cytometer. Relevant data analysis was performed using Flowjo VX software.

[0040] 10. Biosafety Analysis The liver, heart, spleen, lung, and kidney of the removed mice were fixed in 4% formaldehyde for 48 h and then examined by H&E staining. Serum biochemical analysis was used to evaluate liver and kidney function, including measurement of blood urea nitrogen and creatinine (BUN / CR) to evaluate renal function, and measurement of alanine aminotransferase and aspartate aminotransferase (ALT / AST) to evaluate liver function.

[0041] Distribution of DTSS in vivo 5x10^5 4T1 cells were injected into the mammary adipose tissue of female BALB / c mice to establish a breast cancer model. When the tumor volume reached 75 mm³, s-780 or DTSS was injected intravenously (100 μL, dose of 2 mg / kg). Subsequently, in vivo imaging was performed at the designated time points (1, 2, 4, 6, 8, 12, and 24 hours) after anesthesia. 24 hours after injection, the mice were sacrificed, and their major organs and tumors were removed for fluorescent physiological imaging.

[0042] 11. In vivo therapeutic effects of DTSS Orthotopic 4T1 tumors were cultured as described previously, and treatment was initiated when the average tumor size reached approximately 75 mm³. Thirty 8-week-old female BALB / c mice were randomly assigned to six groups of five mice each. Mice were injected with normal saline (NS), thymopentin, salinomycin, s-780, TSS, or DTSS via the tail vein (doses were thymopentin: 4 mg / kg, salinomycin: 1 mg / kg, and s-780: 2 mg / kg). Six hours after the tail vein injection of the formula, mice in the s-780, TSS, and DTSS treatment groups were exposed to 30 seconds of laser irradiation. The body weight of the mice was recorded daily. At the end of day 12, the mice were sacrificed, the tumors were removed, their weights were measured, and their histological analysis was performed. The mice were then monitored, and the tumor volume was calculated according to the following formula: Tumor volume on day n (mm³) = length x width² / 2. The inhibition ratio of tumor-bearing mice was calculated according to the following equation: Tumor inhibition ratio (%) = (WN - Ws) / WN x 100%, where WN is the average tumor weight of the normal control group and Ws is the average tumor weight of the treatment group.

[0043] 12. In vivo immune response study Cell suspensions from primary tumors, non-syngeneic tumors, and spleens were stained with antibodies to analyze the proportions of T lymphocytes, dendritic cells (DCs), and regulatory T cells (Tregs). Finally, these immune cells were detected by flow cytometry. The levels of cGAMP, perforin, cytokine B, TGFβ, INFγ, and TNFα were measured by enzyme-linked immunosorbent assay (ELISA).

[0044] Evaluation of in vivo tumor stemness inhibition efficiency 4T1 or EMT-6 cells were injected into mice, with three gradients of 10^4, 10^5, and 10^6 cells per treatment group, and 6 injection sites per gradient. Subsequently, mice were treated accordingly every day for the next seven days. Mice were sacrificed and the injection sites were examined for the presence of solid tumors, which were recorded and counted.

[0045] 13. Statistical analysis Statistical analysis and graphical representation were performed using GraphPad 9 software (GraphPad, La Jolla, CA, USA). Statistical differences were assessed using Student's t-test or one-way analysis of variance (ANOVA). Data are presented as mean and standard deviation (SD). Statistical values ​​of P < 0.05 were considered statistically significant.

[0046] 2. Experimental Results 1. Cytotoxicity and stemness inhibition mediated by the DTSS drug delivery platform of the present invention The present invention uses various breast cancer cells, including human breast cancer cell lines MCF-7 and MDA-MB-231 and mouse breast cancer cell line 4T1, to detect the in vitro cytotoxicity of DTSS. Figure 4 As shown in Figures ac, DTSS showed a significant tumor inhibition effect under laser irradiation. Figure 4 d and showed that free salinomycin and DTSS had only low side effects on normal cells, such as human umbilical vein endothelial cells (HUVEC). EdU and colony formation assays showed that the use of DTSS effectively inhibited the proliferation of tumor cells ( Figure 4 ef). The ability of DTSS to induce apoptosis in 4T1 cells was then evaluated. Flow cytometric analysis showed that the percentage of apoptotic cells in the s-780+L group was 10.50±3.51%, while that in the NS group was only 1.48±0.94% (p<0.001), indicating that s-780 has a pro-apoptotic effect ( Figure 4 gh). The apoptosis rate in the DTSS group showed that photodynamic therapy effectively promoted cell apoptosis. The percentage of apoptotic cells in the DTSS group was 42.7±0.97%, which was significantly higher than that in the s-780+L group, indicating that inhibiting cancer stem cells can enhance the effect of photodynamic therapy. In addition, Figure 4 As shown in Figure 5, DTSS stimulated the release of lactate dehydrogenase (LDH) and adenosine triphosphate (ATP) into the extracellular space, further indicating that DTSS can induce cell membrane rupture, which helps to expose antigens and activate immunity.

[0047] In the DTSS nanoplatform designed by the present invention, salinomycin can directly inhibit the stemness of tumor cells (29. Lu D, Choi MY, Yu J, Castro JE, Kipps TJ, Carson DA. Salinomycin inhibits Wnt signaling and selectively induces apoptosis in chronic lymphocyticleukemia cells. Proc Natl Acad Sci USA 2011; 108: 13253-7). Figure 4 As shown in Figure 1, the proportion of CD24-CD44+ breast cancer stem cells decreased significantly after treatment, especially in the DTSS group; soft agar 3D culture ( Figure 4 m) also showed an excellent stemness inhibition effect of DTSS. On the other hand, under the condition of endoplasmic reticulum stress, the downstream Wnt signaling pathway was inhibited, thereby inhibiting the maintenance of cancer cell stemness (14. Costa R, Peruzzo R, Bachmann M, Montà GD, Vicario M, Santinon G, et al. Impaired Mitochondrial ATP ProductionDownregulates Wnt Signaling via ER Stress Induction. Cell Rep 2019;28:1949-60.e6). DTSS further reduced the clone formation ability to 1.38±0.93, indicating its significant ability to inhibit cell proliferation at the 3D level. It is worth noting that DTSS further reduced the clone formation ability to 1.38±0.93, indicating a significant ability to inhibit cancer stemness by enhancing endoplasmic reticulum stress. In addition, salinomycin can also inhibit related transcription factors (30.QiD, Liu Y, Li J, Huang JH, Hu X, Wu E. Salinomycin as a potent anticancer stem cell agent: State of the art and future directions. Med Res Rev 2021;42:1037-63 31. Dewangan J, Srivastava S, Mishra S, Divakar A, Kumar S, Rath SK. Salinomycin inhibits breast cancer progression via targeting HIF-1alpha / VEGFmediated tumor angiogenesis in vitro and in vivo. BiochemPharmacol 2019;164:326-35). Therefore, immunoblotting was used to detect the expression levels of OCT4, KLF4, NANOG, SOX2 and the downstream Wnt signaling pathway in cells ( Figure 4 op). The expression levels of these transcription factors were significantly decreased after DTSS treatment.

[0048] 2. DTSS drug delivery platform enhances systemic anti-tumor immune response Using a bilateral tumor model, we performed experiments to further validate the ability of DTSS to activate specific immunity. Figure 5 As shown in a, the primary tumor is designated as the left subcutaneous tumor, while the distant tumor represents the tumor on the right side. All intratumoral injections and laser irradiation were applied only to the proximal tumor to ensure that the effect on the distant tumor was minimized. NS, thymopentin, salinomycin, s-780, TSS, and DTSS were injected intratumorally every two days for a total of 10 days. To ensure the safety of the drugs, the body weight of the mice was measured after each administration ( Figure 5 b). Subsequently, the primary tumor and distant tumor tissues were photographed and weighed to calculate the tumor inhibition rate ( Figure 5 cg). Encouragingly, the tumor inhibition rate of DTSS in distant tumors exceeded 90%, indicating that these DTSS formulations effectively activated the systemic immune response against cancer. At the same time, we also observed phenomena similar to orthotopic tumors during the treatment of bilateral tumor immunity models. Salinomycin treatment initially effectively inhibited the progression of distant tumors, but gradually lost control when the tumor volume exceeded a certain threshold. Thymopentin had weak inhibitory ability at the beginning, but after immune activation, tumor growth was significantly inhibited. Due to the slow immune activation, S-780 only showed a certain inhibitory effect in the later stage of the experiment. These results further illustrate the importance of integrating tumor stemness inhibition and immune activation in the same nanodrug delivery platform. In addition, this combination treatment can better compensate for the limitations of slow immune activation in photodynamic therapy.

[0049] Immunohistochemistry was used to evaluate the expression level of PD-L1 in tumor tissues. Figure 5 j), the results showed that the PD-L1 level of distant tumors was significantly reduced after treatment with the DTSS nanoformulation. This indicates that the tumor immunosuppressive microenvironment has been improved. After collecting distant tumors, flow cytometry analysis was performed on mouse tumor tissues and spleens ( Figure 5 Hi, Figure 6 af). The results showed that after DTSS treatment, the levels of T cells and dendritic cells in mouse tumors and immune organs increased, while Treg cells decreased significantly, accompanied by the repolarization of macrophages ( Figure 5 lq). Tissue analysis was performed using Ki-67 and H&E staining ( Figure 5k), minimal nuclear staining was observed in distant tumors after treatment, indicating maximal necrosis. Together, these results suggest that DTSS treatment is able to effectively activate the immune system, thereby improving the pre-existing tumor suppressive immune microenvironment within tumor tissue. Furthermore, the strategy of combining stemness suppression with immune activation successfully inhibited the progression of non-photodamaged (distal) tumors. This finding suggests that DTSS has significant potential in inhibiting tumor metastasis and recurrence.

[0050] 3. Spontaneous breast cancer model FVB-MMTV-PyMT verifies the therapeutic effect of DTSS drug delivery platform We used the FVB-MMTV-PyMT genetically engineered mouse model to more comprehensively evaluate DTSS-mediated cancer immunotherapy ( Figure 7 a). Figure 7 The tumor images and weights shown in bc indicate that DTSS effectively inhibited tumor growth. Flow cytometry analysis showed that compared with the MIX group (a simple mixture of TP5, s780, and SAL), the number of T cells ( Figure 7 dg)、dendritic cells( Figure 7 h) and M1 macrophages ( Figure 7 i) were significantly activated, while M2 macrophages and Treg cells were significantly inhibited ( Figure 7 jk). These results suggest that nanosized DTSS has a better antitumor effect than free drug mixtures, which can be attributed to the good targeting and pH-responsive delivery behavior of our designed nanoparticles. We also used this FVB-MMTV-PyMT genetically engineered mouse model to establish a postoperative recurrence model to study the antitumor immunity of DTSS ( Figure 7 l). Figure 7 As shown in Figure 2, DTSS treatment significantly inhibited tumor recurrence. In addition, DTSS effectively promoted the infiltration of immune memory T cells in tumor tissues, with the level being 2.0 times higher than that in the control group ( Figure 7 op). Flow cytometry analysis showed that the DTSS drug delivery system effectively promoted the proliferation and differentiation of T cells in recurrent tumors ( Figure 7 qt). These results strongly support the ability of DTSS nanoparticles to generate a highly effective immune memory response against cancer recurrence.

[0051] 3. Results Analysis Cancer cells with stem cell characteristics can effectively evade recognition and killing by immune-active cells (such as T cells), which is considered to be the key reason for the failure of immunotherapy. Compared with developing new drugs from scratch, "drug repurposing" greatly shortens the R&D cycle, reduces investment, and can effectively alleviate drug shortages for patients with drug-resistant tumors. Based on the drug repurposing strategy, the carboxylic acid antibiotic salinomycin shows a strong inhibitory effect on various cancer stem cells by regulating the Wnt signaling pathway and targeting various stem cell-related transcription factors (such as β-catenin and SOX2) (Hao C, Chen P, Setrerrahmane S, Xu H. A peptide-salinomycin conjugate with a bystander effect reduces the stemness characteristics of ovarian cancer cells and enhances drug sensitivity. Eur J Med Chem 2024;276:116701; Li B, Ming H, Qin S, Zhou L, Huang Z, Jin P, et al. HSPA8 activatesWnt / β-catenin signaling to facilitate BRAF V600E colorectal cancer progression by CMA-mediated CAV1 degradation. Adv Sci (Weinh) 2024;11:e2306535). Therefore, salinomycin shows great potential in specifically inhibiting cancer cell stemness and is expected to sensitize cancer immunotherapy. However, the poor water solubility and limited delivery efficiency of salinomycin significantly restrict its clinical application.

[0052] ER stress is an adaptive response of cells to the accumulation of misfolded proteins in the ER, which is not only related to the maintenance of normal cell function but also closely related to diseases. The ER membrane proteins IRE1α, PERK, and ATF6α sense the accumulation of unfolded and misfolded proteins. FOXK2 can inhibit ER stress and promote the stemness of ovarian cancer cells by activating IRE1α. In addition, ER stress can inhibit the stemness of breast cancer by regulating the IRE1α, PERK, and ATF6α signaling pathways. Therefore, the specific enhancement of ER stress can inhibit the stemness of cancer cells to sensitize chemotherapy-immunotherapy. This mechanism has not been fully elucidated and urgently needs to be studied.

[0053] Based on this, we rationally designed salinomycin-repurposed endoplasmic reticulum stress nanoinducers (DTSS) to synergistically inhibit cancer cell stemness and sensitize cancer immunotherapy (see Figure 8 ). Salinomycin was self-assembled with the clinically used immunomodulator thymopentin and the ER-targeting phototherapeutic agent s-780, and finally customized with biotin for targeted drug delivery. DTSS nanoparticles not only significantly improved the bioavailability of thymopentin and salinomycin, but also ensured controlled drug release and reduced side effects. Notably, s-780-mediated phototherapy synergized with salinomycin-mediated inhibition of cancer cell stemness, accompanied by downregulation of PD-L1 expression and activation of the cGAS-STING pathway. In addition, thymopentin significantly promoted the proliferation and differentiation of dendritic cells (DCs) and T lymphocytes, thereby enhancing the response of immune-competent cells to cancer cells. Therefore, DTSS nanoparticles effectively activated systemic antitumor immune responses against cancer metastasis and recurrence, providing a promising solution for sensitizing cancer immunotherapy by inhibiting cancer cell stemness.

Claims

1. Use of thymopentin, salinomycin and IR780 iodide in combination for the preparation of drugs for treating and inhibiting tumor stemness.

2. The use according to claim 1, characterized in that: The drug is a drug for sensitizing cancer immunotherapy.

3. The use according to claim 1 or 2, characterized in that: The mass ratio of the thymopentin, salinomycin and IR780 iodide is: 2-6 parts of thymopentin, 0.5-1.5 parts of salinomycin, 1-3 parts of IR780 iodide.

4. The use according to claim 3, characterized in that: The mass ratio of the thymopentin, salinomycin and IR780 iodide is: 4 parts of thymopentin, 1 part of salinomycin, and 2 parts of IR780 iodide.

5. A pharmaceutical composition for inhibiting tumor stemness, characterized in that: It is a preparation containing thymopentin, salinomycin and IR780 iodide as active ingredients, and the mass ratio of thymopentin, salinomycin and IR780 iodide is: 2-6 parts of thymopentin, 0.5-1.5 parts of salinomycin, 1-3 parts of IR780 iodide.

6. The pharmaceutical composition for inhibiting tumor stemness according to claim 5, characterized in that: The mass ratio of the thymopentin, salinomycin and IR780 iodide is: 4 parts of thymopentin, 1 part of salinomycin, and 2 parts of IR780 iodide.

7. The pharmaceutical composition for inhibiting tumor stemness according to claim 5 or 6, characterized in that: It is prepared by coupling p-toluenesulfonamide and IR780 iodide (IR780) to form the endoplasmic reticulum-targeted phototherapy drug s-780, which is then assembled with thymopentin and salinomycin, and finally coated with biotin to form a site-specific delivery nanoplatform (DTSS).

8. A method for preparing the pharmaceutical composition for inhibiting tumor stemness according to claim 7, characterized in that: The preparation process of the delivery nanoplatform (DTSS) comprises the following steps: a. Synthesis of s-780: p-Toluenesulfonamide and IR780 iodide were dissolved in N,N-dimethylformamide (DMF); triethylamine (TEA) was added and stirred magnetically at 85°C for 4 hours while protecting from light and under a nitrogen atmosphere to ensure a stable reaction; thereafter, the mixture was cooled to room temperature and evaporated under reduced pressure to remove the solvent; b. Synthesis of DTSS: The s-780 prepared in step a is purified by chromatography on a column, and then dissolved together with salinomycin in anhydrous methanol; a saturated aqueous solution of thymopentin is added, and ultrasound is applied at the same time, and a pipette is used to continuously blow and dissolve the membrane to accelerate dissolution, and TSS nanoparticles are collected by centrifugation, and washed with double distilled water; the obtained TSS nanoparticles are suspended in double distilled water for later use; biotin is dissolved in anhydrous methanol, and an aqueous solution of TSS nanoparticles is added, and ultrasound is applied for at least 8 minutes. After the solvent is evaporated at low temperature in vacuum, the mixture is dispersed in deionized water using ultrasound to obtain a delivery nanoplatform (DTSS); The power of the ultrasonic dispersion is 100-200 W; a pulse mode is adopted, working for 2 seconds and pausing for 1 second.

9. The method for preparing the pharmaceutical composition for inhibiting tumor stemness according to claim 1, characterized in that: The molar ratio of p-toluenesulfonamide to IR780 in step a is 2:1; the biotin in step b is DSPE-PEG2000-Biotin.

10. Use of the pharmaceutical composition according to any one of claims 5 to 7 in the preparation of a drug for inhibiting tumor stemness.

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