Synergistic anticancer application of redox-responsive condensate delivery of jujubein and 3-methyladenine

Abstract

This invention relates to the synergistic anticancer application of redox-responsive condensates (FFSSFF) for delivering jujube extract and 3-methyladenine. Specifically, this invention relates to the application of redox-responsive condensates in the preparation of targeted drug delivery products, wherein the targeted drug delivery products release drugs in response to glutathione. The active ingredients in the targeted drug delivery products are jujube extract and 3-methyladenine. This invention utilizes an FFSSFF carrier designed based on the LLPS principle, integrating redox-sensitive disulfide bond linkage modules to achieve spatiotemporally controlled drug delivery within a tumor tissue-specific microenvironment with high glutathione concentrations.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and biomaterials technology, specifically relating to novel nanomedicine delivery systems and precision tumor treatment technologies, and more specifically to the synergistic anticancer application of redox-responsive condensate delivery of apigenin and 3-methyladenine. Background Technology

[0002] Cancer remains one of the most serious public health challenges globally, with its incidence and mortality rates continuing to rise, placing a huge burden on human health and social healthcare systems. Although traditional treatments, including surgery, radiotherapy, and chemotherapy, have made continuous progress in recent years, these methods still have many limitations in clinical application. For example, chemotherapy drugs generally have significant toxic side effects, insufficient tumor selectivity, and drug resistance due to tumor cell heterogeneity, especially in patients with advanced or metastatic tumors, where treatment efficacy often falls short of expectations. Therefore, developing safer, more efficient, and targeted anti-tumor treatment strategies has become an important direction in current cancer treatment research.

[0003] In recent years, phytochemicals derived from natural plants have attracted widespread attention due to their unique biological activities. Plant-derived compounds typically exhibit significant structural diversity, multi-target mechanisms of action, and relatively low toxicity, demonstrating enormous application potential in disease prevention and treatment. Numerous studies have shown that these naturally occurring bioactive molecules can exert a wide range of biological effects by regulating multiple key cellular signaling pathways, including antioxidant, anti-inflammatory, antibacterial, antiviral, and antitumor effects, while also maintaining physiological homeostasis and participating in immune regulation. Therefore, phytochemicals are considered a valuable source of natural bioactive molecules, providing important candidate molecules and a research foundation for the development of novel drugs.

[0004] Among numerous natural phytochemicals, apigenin is a natural flavonoid compound widely found in propolis, honey, and various plants, and has been shown in recent years to possess significant anti-tumor potential. Studies have demonstrated that apigenin exerts its anti-cancer effects through multiple mechanisms, including inducing apoptosis, regulating the cell cycle, inhibiting tumor cell proliferation, and interfering with tumor-related signaling pathways. However, apigenin still faces significant challenges in practical applications due to its poor water solubility, low bioavailability, difficulty in effectively penetrating cell membranes to reach target cells, and its tendency to be non-specifically absorbed by normal tissue cells. These factors significantly limit its effective accumulation and targeting in vivo, thereby greatly weakening its potential anti-tumor efficacy.

[0005] Therefore, developing efficient drug delivery systems that can improve the solubility of apigenin, enhance its tumor-targeting delivery capability, and improve its bioavailability in vivo is of great significance for fully leveraging its natural anticancer activity and expanding its clinical application value. Summary of the Invention

[0006] This invention develops a tumor-targeted delivery system in which guar gum (CH) and 3-methyladenine (3-MA) ​​are co-assembled within a GSH-reactive FFSSFF condensate (CH / 3-MA@FFSSFF) through isomorphic interactions. This invention breaks through the construction of a synergistic therapeutic platform based on phase transition condensates. This invention also proposes an innovative therapeutic strategy of "autophagy stress cascade amplification" and constructs a drug delivery system based on condensates, CH / 3-MA@FFSSFF, to achieve a synergistic anti-tumor effect. The system has a dual mechanism of action: (1) At the molecular regulation level, guar gum enhances autophagy pressure by activating unfolded protein (UPR) and, in conjunction with 3-methyladenine, blocks autophagosome maturation, forming a vicious cycle of "autophagy flow obstruction - stress signal cascade amplification"; (2) At the delivery system level, the FFSSFF carrier, designed based on the principle of liquid-liquid phase separation (LLPS), integrates a redox-sensitive disulfide bond linking module to achieve spatiotemporal controlled drug delivery in a tumor tissue-specific high-concentration glutathione microenvironment.

[0007] Specifically, the present invention provides the following technical solutions.

[0008] On the one hand, the present invention provides formula I

[0009]

[0010] The illustrated delivery carrier is used in the preparation of a targeted drug delivery product that releases the drug in response to glutathione.

[0011] In some embodiments, the targeted drug delivery product includes an active ingredient and excipients, which are encapsulated in a delivery carrier as shown in Formula I.

[0012] In some implementations, the active ingredient is an antitumor drug.

[0013] In some embodiments, the active ingredient is selected from vincristine, camptothecin, taxanes, artemisinin and its derivatives, triptolide, lentinan, ganoderma lucidum polysaccharide, apigenin, quercetin, platinum-based drugs (e.g., carboplatin, cisplatin, oxaliplatin), antibodies, 3-methyladenine, and melitin.

[0014] In some embodiments, the active ingredients are apigenin and 3-methyladenine.

[0015] On the other hand, the present invention provides a pharmaceutical preparation comprising, as shown in Formula I

[0016]

[0017] The delivery carrier shown, along with apigenin and 3-methyladenine, are encapsulated in a delivery carrier as shown in Formula I.

[0018] In some embodiments, the pharmaceutical formulation may also include a pharmaceutically acceptable carrier.

[0019] In some embodiments, the pharmaceutically acceptable carrier is selected from excipients, diluents, dispersants, emulsifiers, buffers, stabilizers, binders, disintegrants, and solvents.

[0020] On the other hand, the present invention provides formula I

[0021]

[0022] The illustrated delivery carrier is used in the preparation of a medicament for treating tumors, which also includes apigenin and 3-methyladenine.

[0023] In some implementations, the tumor is selected from colorectal cancer, lung cancer, nasopharyngeal carcinoma, gastric cancer, pancreatic cancer, prostate cancer, esophageal cancer, and breast cancer.

[0024] On the other hand, the present invention provides a method for preparing a pharmaceutical formulation, the method comprising:

[0025] a. Dissolve guarnin and 3-methyladenine in a buffer solution to form a homogeneous first solution;

[0026] b. Slowly add as shown in Formula I

[0027]

[0028] The second solution of the delivery carrier shown is obtained by vortex mixing to ensure that the first and second solutions are fully mixed, thereby obtaining a drug formulation in which succinin and 3-methyladenine are encapsulated in the delivery carrier shown in Formula I.

[0029] On the other hand, the present invention provides a method for treating tumors, which includes administering the pharmaceutical preparation described above to a subject in need.

[0030] Beneficial effects

[0031] (1) This invention innovatively constructs an intelligent drug delivery carrier based on the principle of liquid-liquid phase separation (LLPS). Through dynamic biointerface engineering, it achieves precise control over the drug loading and release process, thereby effectively overcoming the technical bottlenecks of traditional nanomedicines in terms of low drug loading efficiency and insufficient targeting (the drug loading capacity of conventional carriers is 5%-10%, while the drug loading capacity of the FFSSFF condensate of this invention corresponds to an encapsulation efficiency of 56.7% for apigenin and 56.4% for 3-methyladenine. The delivery carrier of this invention is GSH-responsive, enabling targeted drug release in tumor cells with high GSH expression). This strategy combines the physicochemical properties of phase-separated condensates with a drug delivery system, providing a new technical paradigm for tumor targeted therapy and constructing a delivery platform with controllable drug loading and adjustable response characteristics. It also provides an important theoretical foundation and technical reserve for the development of multi-mechanism synergistic anti-tumor therapy systems under the background of precision medicine.

[0032] (2) Furthermore, the delivery system of this invention exhibited significant and stable antitumor activity in various tumor models, demonstrating potential for broad-spectrum anticancer applications. Notably, thanks to its excellent lung-targeting enrichment ability (considering that CH / 3-MA@FFSSFF can form micron-sized aggregates through fusion, and that micron-sized particles easily form size-dependent aggregates in pulmonary capillaries, the inventors further investigated its tissue distribution. After intravenous injection of IR775-CH / 3-MA@FFSSFF, lung accumulation was monitored using IVIS, and it was found that it could accumulate sufficiently in the lungs), this system showed particularly outstanding antitumor effects in the orthotopic lung cancer model, not only significantly inhibiting tumor growth but also effectively prolonging the survival of experimental animals, further highlighting the application potential of this platform in the treatment of solid tumors. In addition, the system of this invention also showed outstanding antitumor effects in the colorectal cancer model. Attached Figure Description

[0033] Figure 1 A flowchart of the method of the present invention is shown.

[0034] Figure 2 Characterization and features of FFSSFF are shown. (a) Synthetic route of FFSSFF. (b, c) Fusion of FFSSFF cosolvent droplets (1.0 mg / mL, pH 7) after incubation for 0 and 30 min. (d) pH-triggered phase transition of FFSSFF solution (0.5 mg / mL) by turbidity monitoring. (e) Concentration-triggered phase transition of FFSSFF solution (pH 7) by turbidity monitoring. (f) Phase transition of FFSSFF (1 mg / mL) solution after GSH-mediated reduction and GSSG-mediated oxidation, by turbidity monitoring.

[0035] Figure 3 The results of in vitro antitumor effects and mechanisms of CH / 3-MAFFSSFF are shown. G1-G6: PBS control, FFSSFF, CH / 3-MA, 3-MA@FFSSFF, CH@FFSSFF, and CH / 3-MA@FFSSFF. (a) Cell viability (CT26) after co-incubation with different treatments for 24 h (viability: FFSSFF: 93.36%, CH / 3-MA: 80.22%, 3-MA@FFSSFF: 47.76%, CH@FFSSFF: 38.54%, CH / 3-MA@FFSSFF: 5.95%). (b) Calcein-AM / PI double staining for apoptosis assessment. Calcein-AM: green; PI: red. (c) Western blot analysis of key endoplasmic reticulum stress markers (GRP78 / PERK) and downstream signaling proteins (Akt / mTOR), as well as autophagy-related proteins LC3 and p62. (d) Schematic diagram of the molecular mechanism of the antitumor effect of CH / 3-MA@FFSSFF. (e) Fluorescent images of CT26 cells stained with JC-1 after 24 h of incubation under different treatments.

[0036] Figure 4 The results of the in vivo antitumor effect study of CH / 3-MA@FFSSFF in a mouse model of subcutaneous colorectal cancer are shown. (a) Treatment strategy. (b) Mean tumor growth curves of different treatment groups. (c) Photographs of ex vivo tumors. Scale bar: 1 cm. (d) Tumor weight of mice at the end of the experiment (ex vivo tumor weights of PBS, FFSSFF, CH / 3-MA and CH / 3-MA@FFSSFF treatment groups were 0.524 g, 0.324 g, 0.248 g and 0.0352 g, respectively). (e) Tumor growth inhibition rate (TGI) of different treatment groups (tumor inhibition rates of PBS, FFSSFF, CH / 3-MA and CH / 3-MA@FFSSFF treatment groups were 8.39%, 38.16%, 52.67% and 93.28%, respectively). (f) Survival rates of CT26 tumor-bearing mice after different treatments (the survival rates of mice treated with PBS, FFSSFF, and CH / 3-MA were all 0 at days 30, 33, and 37, respectively, while the survival rate of CH / 3-MA@FFSSFF remained 100% at day 50). (g) Microscopic images of tumor sections after H&E, TUNEL, and ROS staining.

[0037] Figure 5The results of in vivo antitumor activity studies of CH / 3-MA@FFSSFF in a mouse model of orthotopic lung cancer are shown. G1-G4: PBS, FFSSFF, CH / 3-MA, and CH / 3-MA@FFSSFF. (a) In vivo bioluminescence imaging of LLC / LUC lung tumors at the indicated time points under different treatments. (b) Growth of mouse lung tumors under different treatments (mean fluorescence intensity at the end of the treatment cycle for the PBS, FFSSFF, CH / 3-MA, and CH / 3-MA@FFSSFF treatment groups was 6.845, 5.329, 3.697, 0.367 (10⁻⁶)). 7 p / s / cm 2 (c) Mean body weight of mice in each treatment group during the experiment (the body weight of mice in the PBS, FFSSFF, CH / 3-MA, and CH / 3-MA@FFSSFF treatment groups at the end of the treatment cycle were 19.05g, 18.65g, 19.35g, and 20.10g, respectively). (d) Survival curves of mice under different treatments. (e) H&E staining of mouse lung tissue under different treatments. (f) Laser confocal images of mouse lung tissue sections stained with CRT and HMGB1. (g) Mature DCs and CD8+ in tumor tissues of different treatment groups. + Cell proportion flow cytometry analysis of T cells, M2 TAMs and M1 TAMs.

[0038] Figure 6 The results of the CH / 3-MA@FFSSFF biosafety study are shown. (a) HE staining of organs from different treatment groups. (b) Complete blood count and liver and kidney function tests from different treatment groups.

[0039] Figure 7 The UV-Vis absorption standard curves and absorbance analysis of CH and 3-MA are shown. (a, b) UV-Vis absorption standard curves of CH and 3-MA. (c) Absorbance of unencapsulated CH and 3-MA in vitro. Detailed Implementation

[0040] The embodiments of this application will be clearly and completely described below. The technical solutions in the embodiments described below are exemplary and only possible technical solutions of this application, not all possible technical solutions. Those skilled in the art can combine the embodiments of this application to obtain other embodiments without creative effort, and these embodiments are also within the protection scope of this application.

[0041] In this invention, the term "Boc" is an abbreviation for tert-butyloxycarbonyl, which is the most commonly used amino protecting group in chemical synthesis, especially in peptide synthesis. It is very easy to remove under acidic conditions (such as trifluoroacetic acid or dilute hydrochloric acid) without damaging the peptide chain itself.

[0042] In this invention, the term "disulfide bond (SS)" refers to a chemical bond formed by the oxidative linkage of the thiol groups of two cysteine ​​residues. Under oxidative conditions, the two thiol groups undergo dehydrogenation and linkage; under reducing conditions, the bond breaks and reverts to thiol groups.

[0043] In this invention, the term "cystamine hydrochloride (CDC)" is formed by two cystamine molecules linked by a disulfide bond and is usually present in the form of hydrochloride to increase water solubility.

[0044] In this invention, the term "DCC" refers to N,N'-dicyclohexylcarbodiimide, a classic condensing agent used to link amino acids to form peptide bonds.

[0045] In this invention, the term "DCM" refers to dichloromethane, an aprotic polar solvent.

[0046] In this invention, the term "PERK / AKT signaling axis" refers to a key pathway that connects the unfolded protein response (UPR) with cell survival / apoptosis decisions when cells respond to endoplasmic reticulum stress.

[0047] In this invention, the term "ICD" refers to immunogenic cell death, a specific type of cell death. It not only kills tumor cells, but more importantly, it awakens the immune system, enabling immune cells to recognize and attack the tumor.

[0048] In this invention, the term "MTT assay" refers to a classic method for detecting cell proliferation and toxicity. The color intensity is measured using a microplate reader at a wavelength of 570 nm. A darker color indicates a higher concentration of viable cells.

[0049] In this invention, the term "CT26 cells" is commonly used in colorectal cancer research and belongs to undifferentiated colonic adenocarcinoma.

[0050] In this invention, the term "CD206" refers to a key receptor on the surface of macrophages, whose main function is to recognize and remove waste products (such as senescent cells and pathogens) from the body. In the tumor microenvironment, macrophages (TAMs) with high CD206 expression can help tumor growth and metastasis, and suppress the immune system's attack on cancer cells. In this invention, the proportions of CD206 cells in the PBS, FFSSFF, CH / 3-MA, and CH / 3-MA@FFSSFF treatment groups were 45.6%, 44.7%, 33.0%, and 24.4%, respectively. This fully demonstrates that the CH / 3-MA@FFSSFF drug delivery system constructed in this invention can overcome the immune evasion of tumor cells and achieve good anti-tumor effects.

[0051] In this invention, the term "CD86" refers to an activation switch on the surface of immune cells (primarily antigen-presenting cells). CD86 binds to CD28 on the surface of T cells, promoting T cell proliferation and differentiation into cytotoxic cells to eliminate pathogens. In this invention, the proportions of CD86 cells in the PBS, FFSSFF, CH / 3-MA, and CH / 3-MA@FFSSFF treatment groups were 1.6%, 2.5%, 10.5%, and 60.5%, respectively. This fully demonstrates that the CH / 3-MA@FFSSFF drug delivery system constructed in this invention can promote CD86 expression, thereby enabling T cells to proliferate and differentiate into cytotoxic cells, thus eliminating pathogens.

[0052] In this invention, the term "CD8" refers to a marker molecule on the surface of cytotoxic T cells (CTLs), and CD8+ T cells are the core force in clearing tumors and toxins. In tumor immunotherapy, the activity of CD8+ T cells directly determines the therapeutic effect. In this invention, the proportions of CD8 cells in the PBS, FFSSFF, CH / 3-MA, and CH / 3-MA@FFSSFF treatment groups were 7.2%, 7.4%, 8.2%, and 23.0%, respectively, which fully demonstrates that the CH / 3-MA@FFSSFF drug delivery system constructed in this invention has excellent anti-tumor effects.

[0053] In this invention, the term "F4 / 80" refers to the most classic surface marker of mouse macrophages. F4 / 80 is highly expressed almost exclusively on mature resident macrophages in mice, while its expression is extremely low on monocytes or other immune cells. In animal experiments, detecting F4 / 80 can determine the degree of macrophage infiltration in tissues (such as tumors or sites of inflammation), making it an indispensable indicator for studying macrophage function.

[0054] In this invention, the term "Foxp3" is a specific marker for regulatory T cells (Tregs). In the tumor microenvironment, tumor cells recruit Foxp3+ Treg cells to suppress anti-tumor immunity, helping the tumor evade immune attacks. Therefore, inhibiting Treg function is an important strategy in cancer immunotherapy.

[0055] In this invention, the term "CD80" refers to an activation switch on the surface of immune cells (mainly antigen-presenting cells), which, together with CD86, is responsible for initiating the attack program of T cells. CD86 binds to CD28 on the surface of T cells, promoting T cell proliferation and differentiation into killer cells to eliminate pathogens.

[0056] In this invention, the term "Calcein-AM / PI staining detection method" refers to a dual-color fluorescence detection technique specifically designed to distinguish between live and dead cells. Live cells are represented by green fluorescence, while dead cells are represented by red fluorescence. A higher concentration of red fluorescence indicates a greater number of dead cells.

[0057] In this invention, the term "JC-1 staining" is specifically used to detect changes in mitochondrial membrane potential in cells; healthy cells emit red fluorescence, while apoptotic cells emit green fluorescence.

[0058] In this invention, the term "LLC-LUC cells" refers to a lung cancer cell line, specifically a lung adenocarcinoma cell line that stably expresses luciferase.

[0059] In this invention, the term "IR775" refers to a near-infrared anthocyanin dye that exhibits strong fluorescence emission in the near-infrared region and can be used as a bioimaging probe to track the distribution of drugs in vivo.

[0060] In this invention, the term "D-luciferin potassium salt" refers to the substrate of luciferase, which is oxidized in the presence of ATP and oxygen and emits blue-green light (wavelength 560 nm).

[0061] In this invention, the active ingredients are not limited, and can be derived from natural products, synthetic drugs, and biological agents, including alkaloids, terpenes, polysaccharides, etc. Examples include vinblastines, camptothecins, taxanes, artemisinin and its derivatives, triptolide, lentinan, ganoderma lucidum polysaccharides, apigenin, platinum-based drugs (e.g., carboplatin, cisplatin, oxaliplatin), antibodies, 3-methyladenine, melilotinib, etc.

[0062] In this invention, the term "FFSSFF" refers to phenylalanine-phenylalanine-disulfide bond-phenylalanine-phenylalanine, whose structural formula is as follows:

[0063]

[0064] Formula I

[0065] In this invention, the term "apigenin (CH)" refers to a natural flavonoid compound, mainly found in propolis and passion fruit, which is often used as an anti-inflammatory and antioxidant active ingredient. CN117462536A discloses the use of apigenin in the preparation of a medicament for treating osteoarthritis. CN119632975A discloses the use of apigenin in the preparation of a medicament for treating NFAT2-related diseases in mammals. CN119564665A discloses the use of apigenin in the preparation of a medicament for treating alcoholic liver disease. CN119074717A discloses the use of apigenin-sensitized cisplatin in the treatment of esophageal squamous cell carcinoma. CN119033764A discloses the use of apigenin-sensitized VS4718 or ponatinib in the treatment of esophageal squamous cell carcinoma. CN117717544A discloses the use of apigenin in the preparation of a medicament for treating rheumatoid arthritis. CN115778937A discloses the use of apigenin in the preparation of drugs that inhibit retinal neovascularization. CN110652508A discloses the use of apigenin in the preparation of streptococcal hemolysin inhibitors. CN110025612A discloses the use of apigenin in the preparation of drugs for treating methicillin-resistant Staphylococcus aureus (MRSA) pneumonia. This invention demonstrates that a delivery system containing apigenin can treat tumors.

[0066] In this invention, the term "3-methyladenine (3-MA)" is a selective PI3K inhibitor that permanently inhibits type I PI3Kγ and temporarily inhibits type III PI3K vps34, involving biological functions such as cell proliferation, apoptosis, and autophagy. CN115919869A discloses the use of 3-methyladenine as a sensitizer for paclitaxel in the preparation of drugs for treating nasopharyngeal carcinoma. CN116919970A discloses the use of 3-methyladenine in the preparation of drugs for preventing or treating postoperative tissue adhesions. CN114224895A discloses the use of 3-methyladenine in the eradication of agalactococci in bovine mammary epithelial cells. CN113134004A discloses the use of 3-methyladenine in the preparation of drugs for preventing Pseudomonas aeruginosa-induced acute pneumonia. CN108159051A discloses the use of 3-methyladenine in the preparation of drugs for treating subretinal fibrosis. CN103948601A discloses the use of 3-methyladenine in the preparation of medicaments for treating atherosclerosis and related diseases. CN101433540A discloses the use of 3-methyladenine in the preparation of medicaments for treating neurodegenerative diseases. This invention demonstrates that a delivery system containing 3-methyladenine can treat tumors.

[0067] In this invention, the inventors discovered that by using a simple vortex, FFSSFF can encapsulate CH and 3-MA within it using hydrophobic forces, thus constructing a CH / 3-MA@FFSSFF delivery system.

[0068] In this invention, the inventors determined the IC50 values ​​of CH and 3-MA anti-CT26 cells to be 12.7 μg / mL (30 μM) and 224 μg / mL (1 mM), respectively. The inventors selected drug concentrations below the IC50 for subsequent experiments and also measured the toxicity of different concentrations of FFSSFF (0 / 80 / 100 / 150 / 200 / 250 μg / mL). At concentrations above 250 μg / mL, cell survival was below 80%, so the inventors selected a concentration of 200 μg / mL for subsequent experiments.

[0069] Therefore, in some implementations, the concentration of apigenin is not higher than 12.7 μg / mL, the concentration of 3-methyladenine is not higher than 224 μg / mL, and the concentration of the delivery carrier FFSSFF is not higher than 250 μg / mL.

[0070] In some embodiments, the concentration of the succinin is 1-12.7 μg / mL, 1-11 μg / mL, 1-10 μg / mL, 1-9 μg / mL, 1-8 μg / mL, 1-7 μg / mL, 1-6 μg / mL, 1-5 μg / mL, 1-4 μg / mL, 1-3 μg / mL, or 1-2 μg / mL.

[0071] In some embodiments, the concentration of the succinin is 12.7 μg / mL, 12 μg / mL, 11 μg / mL, 10 μg / mL, 9 μg / mL, 8 μg / mL, 7 μg / mL, 6 μg / mL, 5 μg / mL, 4 μg / mL, 3 μg / mL, 2 μg / mL, or 1 μg / mL.

[0072] In some embodiments, the concentration of 3-methyladenine is 1-224 μg / mL, 1-220 μg / mL, 1-210 μg / mL, 1-200 μg / mL, 1-190 μg / mL, 1-180 μg / mL, 1-170 μg / mL, 1-160 μg / mL, 1-150 μg / mL, 1-140 μg / mL, 1-130 μg / mL, 1-120 μg / mL, 1-110 μg / mL, 1-100 μg / mL, 1-90 μg / mL, 1-80 μg / mL, 1-70 μg / mL, 1-60 μg / mL, 1-50 μg / mL, 1-40 μg / mL, 1-30 μg / mL, 1-20 μg / mL, or 1-10 μg / mL.

[0073] In some embodiments, the concentration of the 3-methyladenine is 224 μg / mL, 220 μg / mL, 210 μg / mL, 200 μg / mL, 190 μg / mL, 180 μg / mL, 170 μg / mL, 160 μg / mL, 150 μg / mL, 140 μg / mL, 130 μg / mL, 120 μg / mL, 110 μg / mL, 100 μg / mL, 90 μg / mL, 80 μg / mL, 70 μg / mL, 60 μg / mL, 50 μg / mL, 40 μg / mL, 30 μg / mL, 20 μg / mL, or 10 μg / mL.

[0074] In some embodiments, the concentration of the FFSSFF is 1-250 μg / mL, 1-240 μg / mL, 1-230 μg / mL, 1-220 μg / mL, 1-210 μg / mL, 1-200 μg / mL, 1-190 μg / mL, 1-180 μg / mL, 1-170 μg / mL, 1-160 μg / mL, 1-150 μg / mL. 1-140μg / mL, 1-130μg / mL, 1-120μg / mL, 1-110μg / mL, 1-100μg / mL, 1-90μg / mL, 1-80μg / mL, 1-70μg / mL, 1-60μg / mL, 1-50μg / mL, 1-40μg / mL, 1-30μg / mL, 1-20μg / mL or 1-10μg / mL.

[0075] In some embodiments, the concentration of the FFSSFF is 250 μg / mL, 240 μg / mL, 230 μg / mL, 220 μg / mL, 210 μg / mL, 200 μg / mL, 190 μg / mL, 180 μg / mL, 170 μg / mL, 160 μg / mL, 150 μg / mL, 140 μg / mL, 130 μg / mL, 120 μg / mL, 110 μg / mL, 100 μg / mL, 90 μg / mL, 80 μg / mL, 70 μg / mL, 60 μg / mL, 50 μg / mL, 40 μg / mL, 30 μg / mL, 20 μg / mL, or 10 μg / mL.

[0076] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0077] Example 1: Preparation of FFSSFF

[0078] The first stage involved a condensation reaction between Boc-Phe-OH (Annegi, 15480) and cystamine hydrochloride (CDC) (Annegi, A010072) under DCC catalysis to construct a disulfide-bridged Boc-F-SS-F-Boc precursor. The second stage employed a TFA / DCM (1:1, v / v) system for deprotection treatment, yielding F-SS-F structural units containing free amino groups. The third stage utilized an iterative assembly strategy (which involves multiple rounds of stepwise nanostructure construction to achieve high drug loading and controllable multifunctionality) to couple the aforementioned units via amide bonds to form a Boc-FF-SS-FF-Boc tetramer. This tetramer was then subjected to secondary acid hydrolysis to obtain the target product FF-SS-FF. Purification was achieved through vacuum filtration combined with ether washing, followed by vacuum lyophilization to obtain a white crystalline solid with a yield of 82.3%.

[0079] The specific preparation steps are as follows (see...). Figure 2 a):

[0080] Synthesis of Boc-F-SS-F-Boc

[0081] Boc-Phe-OH (398 mg, 1.55 mmol), HBTU (O-benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, 557 mg, 1.47 mmol), and HoBT (1-hydroxybenzotriazole, 199 mg, 1.47 mmol) were dissolved in DMF (N,N-dimethylformamide) in a round-bottom flask and stirred with a magnetic stirrer until homogeneous. DIPEA (N,N-diisopropylethylamine, 1050 μL, 6.2 mmol) and CDC (158 mg, 0.7 mmol) were added every minute, and the reaction was stirred at room temperature for 24 hours. After the reaction was complete, the reaction solution was poured into 50 mL of ultrapure water, and the white precipitate was collected by suction filtration. The precipitate was washed with ultrapure water until the filtrate was clear and colorless, and the crude product was dried in a desiccator.

[0082] Synthesis of F-SS-F

[0083] The crude product Boc-F-SS-F-Boc (327 mg, 0.51 mmol) obtained in the first step was dissolved in a 1:5 trifluoroacetic acid / dichloromethane mixed solvent (3 mL) for deprotection reaction over 3 hours. After the reaction was complete, the product was rotary evaporated to a viscous oily state, and then diethyl ether was added to a round-bottom flask, where a film-like substance was observed to form. The white precipitate (F-SS-F) was collected by vacuum filtration, washed with diethyl ether, and finally dried.

[0084] Synthesis of Boc-FF-SS-FF-Boc

[0085] Boc-Phe-OH (297 mg, 1.12 mmol), HBTU (402 mg, 1.06 mmol), and HoBT (143 mg, 1.06 mmol) were dissolved in DMF (N,N-dimethylformamide) in a round-bottom flask and stirred with a magnetic stirrer. DIPEA (780 μL, 6.2 mmol) and F-SS-F (263 mg, 0.51 mmol) were added every minute, and the reaction was stirred at room temperature for 24 hours. After the reaction was complete, the reaction mixture was poured into 50 mL of ultrapure water, and a white paste-like intermediate was collected by vacuum filtration. The precipitate was washed with anhydrous ethanol until the filtrate was clear and colorless, and the crude product was dried in a desiccator.

[0086] FF-SS-FF Synthesis

[0087] The crude product Boc-FF-SS-FF-Boc obtained in step 3 (359 mg, 0.29 mmol, or 198.5 mg, 0.16 mmol) was dissolved in a 1:5 trifluoroacetic acid / dichloromethane (3 mL) for deprotection reaction over 3 hours. After the reaction was complete, the product was rotary evaporated to a viscous oily consistency, and then diethyl ether was added to a round-bottom flask, where a film-like substance was observed to form. The white precipitate (FF-SS-FF) was collected by suction filtration, washed with diethyl ether, and finally dried. 1 H NMR, 13 CNMR and LC-MS confirmed that the obtained product was FFSSFF.

[0088] Example 2: Characterization criteria of FFSSFF and construction of CH / 3-MA@FFSSFF

[0089] (1) Characteristic criteria of FFSSFF: In order to monitor the reversible redox process of the coprecipitate, we evaluate the redox behavior of FFSSFF solution by observing the change in its turbidity.

[0090] A 0.2 mg / mL FFSSFF aqueous solution was prepared and vortexed (2000 rpm, 3 min) to form a homogeneous system. The addition of PBS buffer triggered liquid-liquid phase separation, resulting in a significant increase in turbidity.

[0091] Reduction process analysis: Glutathione (GSH, 1.0 M, 10 μL) was introduced into the FFSSFF condensate system and magnetically stirred (500 rpm) at 25 °C. Real-time monitoring showed that the turbidity decreased to baseline level within 5 min, indicating that the breaking of disulfide bonds led to the dissociation of intermolecular forces.

[0092] Oxidation process verification: After adding oxidized glutathione (GSSG, 1.0 M, 10 μL), the turbidity of the system recovered to the initial value within 5 min, confirming the oxidation-induced molecular rearrangement.

[0093] This indicates that the redox reaction of FFSSFF solution is reversible, and that the oxidizing and reducing agents can regulate its liquid-liquid phase separation state.

[0094] Data Acquisition: The absorbance of the solution in the 210-600 nm wavelength range was measured using a visible spectrophotometer. Absorbance values ​​were recorded whenever significant changes in turbidity occurred to quantitatively analyze the transformations experienced by the coprecipitate during the redox process. The reversible GSH responsiveness of FFSSFF was confirmed through GSH reduction and GSSG oxidation. GSH cleaved the disulfide bonds in FFSSFF, generating FFSH and forming a clear solution, which was subsequently oxidized by GSSG to regenerate FFSSFF, thus re-coagulating. Figure 2 ef).

[0095] (2) Observation under an optical microscope

[0096] Bright-field imaging analysis was performed using an inverted fluorescence microscope: 30 μL of condensed FFSSFF solution (0.2 mg / mL, pH 7.0) was precisely pipetted into a 96-well plate, and the original morphology of the droplets was captured at the initial time point (0 min). After 30 min of continuous monitoring, imaging was performed again to track droplet fusion dynamics. The time-dependent morphological evolution characteristics of FFSSFF aggregates were evaluated by comparing the changes in the equivalent diameter of the droplets in the time-series images (analyzed using ImageJ software) and the interface fusion frequency. The results showed that FFSSFF exhibited a hollow spherical structure, and at different times, FFSSFF showed different sizes, reflecting its delayed fusion characteristics. Specifically, during the condensation process, FFSSFF self-assembled into a hollow spherical structure driven by hydrophobic interactions, and gradually fused into larger aggregates over time. Figure 2 bc).

[0097] (3) Construction of co-drug delivery system

[0098] The CH / 3-MA@FFSSFF complex was prepared by dissolving jujube extract (Aladdin, C110078, 254 μg / mL, 30 μL) and 3-methyladenine (Aladdin, M129496, 2.98 mg / mL, 50 μL) in PBS buffer (1 mL) to form a homogeneous solution. Subsequently, an aqueous solution of FFSSFF (4 mg / mL, 50 μL) was slowly added dropwise, and the two solutions were thoroughly mixed by gentle vortexing. This process facilitates the interaction between the FFSSFF molecules and jujube extract and 3-MA, thereby promoting drug encapsulation.

[0099] Example 3: In vitro anticancer activity study of CH / 3-MA@FFSSFF

[0100] MTT assay: CT26 cells (8000 cells / well) were seeded in 96-well cell culture plates and incubated overnight in an incubator (37℃, 5% CO2) to allow for complete adhesion. Each well in the 96-well plate was treated with different solutions: PBS, FFSSFF, CH / 3-MA, CH@FFSSFF, 3-MA@FFSSFF, and CH / 3-MA@FFSSFF (CH: 254 μg / mL, 30 μL; 3-MA: 2.98 mg / mL, 50 μL; FFSSFF: 4 mg / mL, 50 μL; DMEM was added to bring the total volume to 1 mL). The treatments were incubated for a total of 24 h. Cell viability was assessed by adding MTT working solution (1 mg / mL, 100 μL / well) and incubating for 4 h. After incubation, the original solution was replaced with DMSO (120 μL / well). The OD value at 570 nm was measured. Based on the absorbance difference measured by colorimetric method, the relative cell proliferation activity could be deduced from the optical density ratio of the experimental group to the control group. The activity of cancer cells was found to be below 10%, indicating that CH / 3-MA@FFSSFF possesses significant anti-cancer properties. The in vitro cytotoxicity of CH / 3-MA@FFSSFF was determined using the MTT assay. After co-incubation for 24 hours, CH / 3-MA@FFSSFF exhibited significant anti-tumor activity in various tumor cell lines, with its tumor activity being only 5.3% of the control group. Figure 3 a).

[0101] Example 4: Study on the in vitro anticancer activity and mechanism of CH / 3-MA@FFSSFF

[0102] (1) Study on the in vitro anticancer activity of CH / 3-MA@FFSSFF

[0103] Calcein-AM / PI staining assay

[0104] Cell Culture and Treatment: CT26 cells (Pronosai, CL-0071) (8000 cells / well) were seeded in 96-well cell culture plates and incubated overnight in an incubator (37℃, 5% CO2) to allow for complete adhesion. Each well in the 96-well plate was treated with different solutions: PBS, FFSSFF, CH / 3-MA, CH@FFSSFF, 3-MA@FFSSFF, and CH / 3-MA@FFSSFF (CH: 254 μg / mL, 30 μL; 3-MA: 2.98 mg / mL, 50 μL; FFSSFF: 4 mg / mL, 50 μL; DMEM was added to each well to bring the total volume to 1 mL). The treatment was continued for 24 h. The original medium was then replaced with Calcein AM / PI detection working solution (100 μL / well), and the plates were incubated for 30 min. After incubation, cells were washed three times with PBS to remove background fluorescence, and multi-channel imaging was performed using an inverted fluorescence microscope. CH / 3-MA@FFSSFF showed significant red fluorescence. Calcein-AM / PI was used to stain live cells (green) and dead cells (red), respectively. Strong red fluorescence and a weak green signal were observed in the CH / 3-MA@FFSSFF group, while other groups showed predominantly green fluorescence, confirming the strong anticancer effect of CH / 3-MA@FFSSFF. Figure 3 b).

[0105] (2) Validate the relevant protein pathways

[0106] Western blotting was used to detect the expression levels of key proteins (GRP78-PERK, AKT / mTor, p62, LC3; LC3 was divided into type I and type II, with the lower band in the figure representing type II) under different treatment conditions. First, CT26 cells (1×10⁻⁶) were... 6Cells were seeded at 1000 cells / well in 6-well plates and cultured for 24 h to allow for proper adhesion and growth. Subsequently, different treatment groups were added for co-incubation, including FFSSFF, CH / 3-MA, CH@FFSSFF, 3-MA@FFSSFF, and CH / 3-MA@FFSSFF (CH: 254 μg / mL, 45 μL; 3-MA: 2.98 mg / mL, 75 μL; FFSSFF: 4 mg / mL, 75 μL, with DMEM added to bring the total volume to 1.5 mL). Each treatment group was incubated for 24 h. After treatment, the culture medium was discarded, and 200 μL of RIPA lysis buffer (containing 1% PMSF and 2% phosphatase inhibitor) was added to lyse the cells. The cells were incubated at 4 °C for 20 min. The lysate was then centrifuged at 12000 rpm for 15 min at 4 °C, and the supernatant was collected as the total protein sample. Protein concentration was determined using a BCA protein quantification kit. Equal volumes of protein samples were added to loading buffer and denatured at 100 °C for 10 min. The samples were then separated by electrophoresis on 8%, 10%, or 12% SDS-PAGE gels, and the proteins were electrotransferred to a 0.45 μm PVDF membrane in transfer buffer. The PVDF membrane was then blocked in blocking buffer at room temperature for 1 h to block non-specific binding sites, followed by overnight incubation with the corresponding primary antibody at 4 °C. After incubation with secondary antibody, chemiluminescence was used to detect signals and analyze the expression levels of related proteins. Western blotting results showed that CH / 3-MA@FFSSFF treatment could activate the PERK / GRP78 signaling pathway via CH, thereby inducing endoplasmic reticulum stress response; simultaneously, this system inhibited the AKT / mTOR signaling pathway, promoting the initiation of autophagy-related processes. However, due to the presence of the autophagy inhibitor 3-MA, the expression level of the autophagy marker protein LC3-II decreased, while the expression of P62 significantly increased, suggesting that autophagy flux was blocked. The above results indicate that CH / 3-MA@FFSSFF can induce programmed cell death by inducing endoplasmic reticulum stress and interfering with autophagy.

[0107] (3) Mitochondrial function assessment

[0108] CT26 cells (2×10) 5Cells (10 cells / well) were seeded in 12-well cell culture plates and incubated overnight at 37°C, 5% CO2 until fully adhered. The original culture medium was removed, and the cells were gently washed twice with PBS. Six differential treatments were set up: PBS, FFSSFF, CH / 3-MA, CH@FFSSFF, 3-MA@FFSSFF, and CH / 3-MA@FFSSFF (CH: 30 μM, 3-MA: 1 mM, FFSSFF: 200 μg / mL), and incubated for 24 h. After the intervention, the treatment solution was discarded, and the cells were washed twice with PBS in a gradient to ensure the removal of unbound drugs. JC-1 staining working solution (1 mL / well) was added to 6-well plates to ensure complete coverage of the cell monolayer. The cells were transferred to an incubator and incubated for 20 min. After terminating the staining procedure, the staining solution was removed sequentially, and the cells were washed with 4°C staining buffer to effectively remove non-specifically bound mitochondrial probes. Images were captured using an inverted fluorescence microscope equipped with a dual-channel imaging system. The results showed that, compared with the CH / 3-MA, 3-MA@FFSSFF and CH@FFSSFF groups, the CH / 3-MA@FFSSFF treatment exhibited significant green fluorescence, indicating that it led to a significant loss of mitochondrial membrane potential. Figure 3 e). This indicates that it disrupts the energy metabolism homeostasis within tumor cells, thereby exerting a synergistic anti-tumor effect.

[0109] Example 5: Study on the in vivo anticancer activity mechanism of CH / 3-MA@FFSSFF

[0110] (1) In the subcutaneous colorectal cancer model: CT26 cells (3×10⁻⁶) were used. 6 A subcutaneous colorectal cancer model was established by subcutaneously inoculating BALB / c mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) with 100 cells per 5 weeks. The tumor volume reached approximately 100 mm². 3 Mice were randomly divided into four groups (PBS, FFSSFF, CH / 3-MA, and CH / 3-MA@FFSSFF) (CH: 2 mg / kg, 3-MA: 10 mg / kg, FFSSFF: 20 mg / kg). Each group received intratumoral injection of the appropriate treatment on days 0, 2, 4, 6, 8, and 10. Body weight and tumor size were measured during treatment, and excised tumors were weighed after treatment. Tumor sections were stained using H&E, ROS, and TUNEL assay kits. Final images of the stained tissue sections were captured using an inverted laboratory microscope. All results indicated that CH / 3-MA@FFSSFF possessed good anticancer activity. The treatment regimen is as follows: Figure 4 As shown in figure a, the changes in tumor volume during treatment were recorded. Figure 4 b). After treatment, the excised tumor was photographed and weighed. Figure 4c, d). Tumor growth inhibition rate was calculated based on tumor weight. The results showed that the tumor weight in the CH / 3-MA@FFSSFF treatment group was only 9.6% (p < 0.0001) and 4.6% (p < 0.001) of the PBS group and the CH / 3-MA treatment group, respectively. Figure 4 (e) This demonstrated the excellent antitumor effect of CH / 3-MA@FFSSFF. A 50-day survival study was also conducted. Figure 4 As shown in f, the survival rate of mice in the CH / 3-MA@FFSSFF treatment group was 100% on day 50, while the survival rate of the other groups was 0% on day 50. H&E staining of the CH / 3-MA@FFSSFF treatment group showed nuclear atrophy, fragmentation, and loss of cell outline. TUNEL staining showed the most prominent positive signal (red fluorescence), and the ROS (red fluorescence) positive signal in the treatment group was also strong, indicating that CH / 3-MA@FFSSFF disrupts the oxidative stress level of tumor cells, thus exhibiting a significant anti-tumor effect.

[0111] (2) For the orthotopic lung cancer model, BALB / c mice were intravenously injected with 8×10 5 LLC-LUC cells (Cyber-Clark, iCell-0078a). Five days later, mice were divided into four groups (PBS, FFSSFF, CH / 3-MA, CH / 3-MA@FFSSFF) (CH: 10 mg / kg, 3-MA: 20 mg / kg, FFSSFF: 20 mg / kg). Each group received the corresponding drug intravenously on days 2, 5, 8, and 11. Mice were anesthetized by inhalation of 2% isoflurane and intraperitoneally injected with D-fluorescein potassium salt (150 mg / kg) (Annegi, E011306). Bioluminescence imaging (BLI) was performed using an IVIS system 15 minutes after injection on days 3, 6, 9, and 12. Mice were sacrificed on day 15, and lung tissue was harvested for tumor metastasis and H&E staining. In a separate survival study, mice received the same treatment, and survival rates were recorded. Lungs, lymph nodes, and spleen were fixed with paraformaldehyde for HMGB1 and CRT immunofluorescence analysis. Flow cytometry was used to analyze tumor-infiltrating immune cells to identify mature dendritic cells (DCs) and CD8+. + The ratio of T cells to TAMs. CD8 +T cells were stained with anti-CD3-FITC, anti-FOXP3-APC, and anti-CD8-PC5.5, respectively. DC cells were stained with anti-CD11c-APC, anti-CD80-FITC, and anti-CD86-PC5.5, respectively. TAM cells were stained with anti-CD11b-PC5.5, anti-F4 / 80-FITC, anti-CD86-APC, and anti-CD206-APC, respectively. Studies on CRT, HMGB1, and related immune cells indicate that CH / 3-MA@FFSSFF achieves a highly effective anti-tumor effect by inducing ICD production in tumor cells. Figure 5 As shown in f, the CH / 3-MA@FFSSFF treatment group exhibited the strongest red fluorescence, indicating a significant increase in CRT exposure. Simultaneously, HMGB1 (green fluorescence) release was also enhanced, suggesting that dying tumor cells could effectively launch DAMPs. Since CRT exposure and HMGB1 release are hallmarks of ICD, we further evaluated immune-related cell populations in tumor tissue. To assess immune activation, we collected tumor tissue after treatment to detect DC maturation and cytotoxic T lymphocyte infiltration. Flow cytometry analysis showed significantly enhanced DC maturation in CH / 3-MA@FFSSFF treated mice. Consistent with this, tumor-invasive CD8 cells... + T cells were most abundant in the CH / 3-MA@FFSSFF group, increasing by 3.19-fold, 3.10-fold, and 2.70-fold compared to the PBS, FFSSFF, and CH / 3-MA groups, respectively. In addition to promoting the infiltration of immune effector cells, we also assessed phenotypic and functional changes in TAMs. Flow cytometry analysis showed that CH / 3-MA@FFSSFF treatment significantly reduced the proportion of M2-type TAMs while significantly increasing the proportion of M1-type TAMs, indicating that it effectively reprogrammed the immunosuppressive microenvironment and enhanced anti-tumor immunity. Specifically, CD86 / CD80 represents mature dendritic cells, CD206 / F480 represents M2-type macrophages, and CD86 / F480 represents M1-type macrophages. CD8 / Foxp3 represents CD8+. + T cells. The percentages of mature DCs treated with PBS, FFSSFF, CH / 3-MA, and CH / 3-MA@FFSSFF were 1.6%, 2.5%, 10.5%, and 60.5%; CD8+ + The proportions of T cells were 7.2%, 7.4%, 8.2%, and 23.0% in M2 macrophages, 45.6%, 44.7%, 33.0%, and 24.4% in M1 macrophages, and 22.1%, 25.0%, 53.8%, and 70.6% in M1 macrophages. Mature dendritic cells (DCs) and CD8+... +The increase in T cells and the decrease in the proportion of M2 macrophages and the increase in the proportion of M1 macrophages both indicate that CH / 3-MA@FFSSFF induces an immune response to tumors, thereby achieving an effective tumor-killing effect. Figure 5 g).

[0112] Example 6: Safety Study of CH / 3-MA@FFSSFF

[0113] (1) The entire treatment cycle was completed in a colorectal cancer model. After the mice were sacrificed, specimens of key organs such as the heart, liver, spleen, lungs, and kidneys were systematically collected. After fixation with 4% formaldehyde for 48 h, the mice were sequentially dehydrated with ethanol and embedded in paraffin. Hematoxylin-eosin double staining (H&E) was used to perform microscopic morphological analysis on 5 μm serial sections, focusing on detecting inflammatory infiltration, fibrosis, and abnormal cell structure, and systematically screening for potential treatment-related organ toxicity. H&E staining of tissue sections of major organs (including heart, liver, spleen, lungs, and kidneys) showed that the tissue structure of each organ was intact, the cell morphology was normal, and there was no obvious inflammatory infiltration, necrosis, or structural abnormality, indicating that CH / 3-MA@FFSSFF did not cause significant organ toxicity. Figure 6 a).

[0114] (2) Whole blood component analysis: White blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), and platelets (PLT) were measured using an automated blood analyzer to assess hematopoietic system function; Liver and kidney function spectrum detection: Alanine aminotransferase / aspartate aminotransferase (ALT / AST), ceruloplasmin (CER), and blood urea nitrogen (BUN) were quantified using a biochemical analyzer to characterize hepatocellular damage and glomerular filtration efficiency, respectively. The results all indicated that CH / 3-MA@FFSSFF has good biocompatibility. There were no significant differences in WBC, RBC, HGB, PLT, ALT / AST, CER, and BUN levels between CH / 3-MA@FFSSFF and the control group (p>0.5). This indicates that CH / 3-MA@FFSSFF has good biocompatibility. Figure 6 b).

[0115] This invention uses the mouse colorectal cancer cell line (CT26) as a model. The effects of the delivery system of this invention on tumor cell growth were investigated using the MTT assay and colony formation assay. Calcein / PI double staining was used to detect its ability to induce apoptosis. Simultaneously, Western blot was used to systematically assess the dynamic changes in the expression levels of key effector proteins in the apoptosis pathway. Western blot was used to detect the expression abundance of endoplasmic reticulum stress markers, key autophagy regulatory proteins, and migration-related effector factors. CH / 3-MA@FFSSFF upregulated p-PERK and GRP78, confirming that endoplasmic reticulum stress is activated through the GRP78-perk axis. Simultaneously, downregulation of p-AKT and p-mTOR indicated that autophagy was induced by inhibiting AKT / mTOR. Increased p62 levels and decreased LC3-II levels suggested that 3-MA effectively blocked autophagy flux. Mitochondrial staining was used to explore the interference effect of CH / 3-MA@FFSSFF on cancer cell energy metabolism. The results showed that CH / 3-MA@FFSSFF synergistically inhibited the malignant behavior of colorectal cancer cells through a dual regulatory mechanism: on the one hand, CH activated the PERK / AKT signaling axis to enhance autophagy activity, and on the other hand, 3-MA inhibited the formation of autophagosomes. These two mechanisms synergistically triggered autophagic flux disorder, ultimately inhibiting tumor progression. Under the dual effects of "enhanced endoplasmic reticulum stress and blocked autophagic flux," mitochondrial metabolism in tumor cells was impaired, and the imbalanced oxidative stress level ultimately induced apoptosis. In in vivo experiments, a BALB / c mouse subcutaneous xenograft model was first constructed, and the effects of CH / 3-MA@FFSSFF on tumor volume, tumor weight, and 50-day survival were systematically evaluated. Based on tumor weight, tumor growth inhibition was calculated, and the tumor weight in the CH / 3-MA@FFSSFF treatment group was only 9.6% (p < 0.0001) and 4.6% (p < 0.001) of the PBS group and the CH / 3-MA treatment group, respectively. Furthermore, this treatment group successfully ablated one CT26 tumor. Meanwhile, the survival rate of mice in the CH / 3-MA@FFSSFF treatment group was 100% on day 50, while all mice in other treatment groups died on day 50, further demonstrating the significant anti-cancer effect and excellent survival prolongation capability of this treatment regimen. Combined with the results of TUNEL and ROS staining of tumor tissue (… Figure 4Three staining assays (HE, TUNEL, and ROS) confirmed that the system significantly inhibited colorectal cancer growth and induced tumor cell apoptosis in vivo. Simultaneously, monitoring mouse body weight changes, HE staining of major organs, and blood routine and liver and kidney function indicators confirmed the good safety of the delivery system with no obvious toxic side effects. Further validation of its anti-tumor effect was performed in a BALB / c mouse orthotopic lung cancer xenograft model. Results showed that CH / 3-MA@FFSSFF significantly inhibited tumor growth and prolonged median survival to 30 days (in the PBS group, lung fluorescence intensity gradually increased, indicating sustained tumor growth, while the CH / 3-MA@FFSSFF group showed the least fluorescence enhancement, indicating effective inhibition of tumor proliferation). Immunological mechanism studies revealed that the system can induce immunogenic cell death (ICD) in tumor cells: by promoting CRT exposure and HMGB1 release, enhancing dendritic cell maturation and cytotoxic T cell infiltration, while simultaneously driving the repolarization of tumor-associated macrophages from a pro-tumor M2 phenotype to an anti-tumor M1 phenotype. Similarly, tumor-invasive CD8... + T cells were most abundant in the CH / 3-MA@FFSSFF group, and CD8 were significantly higher compared to the PBS, FFSSFF, and CH / 3-MA groups. + T cells increased by 3.19, 3.10, and 2.70-fold, respectively. In addition to promoting immune effector cell infiltration, we further evaluated the phenotypic and functional changes of TAMs. Flow cytometry analysis showed that CH / 3-MA@FFSSFF treatment significantly reduced the proportion of M2 TAMs while significantly increasing the proportion of M1 TAMs, indicating effective reprogramming of the immunosuppressive microenvironment and enhancement of antitumor immunity, thereby reshaping the antitumor immune microenvironment. This demonstrates that CH / 3-MA@FFSSFF possesses excellent broad-spectrum anticancer activity.

[0116] Other bioaggregates, such as WWSSWW (W representing tryptophan), are difficult to effectively accumulate and sustain at tumor sites due to poor stability, uncontrollable drug loading and release, and insufficient targeting, thus limiting their anti-tumor efficacy.

[0117] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. As shown in Formula I The application of the delivery carrier shown is characterized in that, The targeted drug delivery product releases the drug in response to glutathione.

2. The application according to claim 1, characterized in that, The targeted drug delivery product includes an active ingredient and excipients, which are encapsulated in a delivery carrier as shown in Formula I.

3. The application according to claim 1 or 2, characterized in that, The active ingredient is an anti-tumor drug.

4. The application according to claim 1 or 2, characterized in that, The active ingredient is selected from vincristine, camptothecin, taxanes, artemisinin and its derivatives, triptolide, lentinan, ganoderma lucidum polysaccharide, apigenin, platinum drugs (e.g., carboplatin, cisplatin, oxaliplatin), antibodies, 3-methyladenine and melitin, optionally, the active ingredient is apigenin and 3-methyladenine.

5. A pharmaceutical preparation, characterized in that, The pharmaceutical preparation includes, as shown in Formula I The delivery carrier shown, along with apigenin and 3-methyladenine, are encapsulated in a delivery carrier as shown in Formula I.

6. The pharmaceutical preparation according to claim 5, characterized in that, The pharmaceutical preparation also includes a pharmaceutically acceptable carrier.

7. The pharmaceutical preparation according to claim 6, characterized in that, The pharmaceutically acceptable carrier is selected from excipients, diluents, dispersants, emulsifiers, buffers, stabilizers, binders, disintegrants, and solvents.

8. As in formula I The illustrated delivery carrier is used in the preparation of a medicament for treating tumors, which also includes apigenin and 3-methyladenine.

9. The use according to claim 8, characterized in that, The tumors are selected from colorectal cancer, lung cancer, nasopharyngeal carcinoma, gastric cancer, pancreatic cancer, prostate cancer, esophageal cancer, and breast cancer.

10. A method for preparing a pharmaceutical formulation, characterized in that, The method includes: a. Dissolve guarnin and 3-methyladenine in a buffer solution to form a homogeneous first solution; b. Slowly add as shown in Formula I The second solution of the delivery carrier shown is obtained by vortex mixing to ensure that the first and second solutions are fully mixed, thereby obtaining a drug formulation in which succinin and 3-methyladenine are encapsulated in the delivery carrier shown in Formula I.

Images