A dual-purpose compound capable of radiosensitization and protection against radiation damage, compositions comprising the same and uses thereof

Abstract

The application belongs to the field of biological medicine, and particularly relates to a double-effect compound capable of radiosensitization and protection against radiation injury, a composition containing the same and application thereof. The application first discovers that a compound with a molecular structure as shown in formula (I) has a double effect of radiosensitization and protection against radiation injury. Experiments prove that the material can significantly reduce the damage of ionizing radiation to normal nerve tissue, and can protect skin tissue and inhibit the occurrence and development of radiation dermatitis. Meanwhile, the compound has a unique advantage of penetrating the blood-brain barrier (BBB), and realizes the attenuation and synergistic effect of radiotherapy on brain tumors in in-vitro and in-vivo experiments. Further, the application innovatively combines the material with a small apoptotic body to construct a biomimetic delivery composition, which significantly improves the intracerebral delivery efficiency and target accumulation capacity of the double-effect compound through synergistic effect, and overcomes the technical bottleneck of brain-targeted delivery.(I)

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a dual-effect compound capable of radiosensitization and protection against radiation damage, a composition containing the same, and applications thereof. Background Art

[0002] Brain malignant tumors, including primary and metastatic brain tumors, are a major threat to the health of Chinese residents. Their high disability, recurrence, and mortality rates have placed a heavy burden on patients, their families, and society. Radiotherapy (RT) is the main treatment for malignant brain tumors, and is even the only treatment option for some types of brain tumors. However, the efficacy of radiotherapy is limited by two aspects: (1) Tumor radiotherapy resistance: This is a key factor leading to local treatment failure and affecting patient survival. Glioblastoma, as one of the most aggressive and lethal brain tumors, has a local recurrence rate of over 90% after radiotherapy, and there is a lack of effective treatment after recurrence; (2) Normal tissue radiation damage: Radiotherapy-induced normal tissue damage is distributed in multiple organs, including the central nervous system (about 70% of patients will develop radiation-induced brain edema in the early stages of radiotherapy, requiring emergency dehydration treatment; about 40% of patients may develop irreversible white matter necrosis in the late stages, leading to permanent neurological dysfunction) and peripheral tissues such as the skin. Among them, radiation dermatitis is the most common complication of radiotherapy. About 85% of patients experience moderate to severe skin toxicity, manifested as erythema, edema and even necrosis.

[0003] Therefore, developing highly effective radiosensitizing and protective agents to achieve "toxicity reduction and efficacy improvement" in brain tumor radiotherapy has become a research hotspot and difficulty in the field of brain tumor radiotherapy.

[0004] Current research focuses on single-function radiotherapy agents, such as tumor radiosensitization or normal tissue protection. Radiosensitizers, including hypoxia-activated prodrugs (e.g., Tirapazamine), traditional Chinese medicine extracts (e.g., curcumin), nanomaterials, and catalytically active substances (e.g., peroxidases and catalases), enhance radiotherapy efficacy by targeting hypoxic cells, increasing cell cycle arrest, or promoting free radical production. Normal tissue protectants mitigate radiation damage through mechanisms such as free radical scavenging, enhancing DNA repair, inducing hypoxia, or blocking bystander effects. Regarding skin protection, commercially available ointments (containing superoxide dismutase, vitamin C, etc.) are inadequate for blocking the ROS cascade due to their narrow free radical scavenging spectrum (e.g., inability to neutralize ·OH) and poor chemical stability. However, traditional sensitizers may exacerbate normal tissue damage, while protectants lack tumor selectivity and may even protect tumor cells. Therefore, achieving both sensitization and protection is an urgent challenge and a promising direction for exploring "one-drug, dual-action" therapeutic strategies.

[0005] Achieving efficient drug delivery across the blood-brain barrier (BBB) ​​is a major challenge in the treatment of central nervous system (CNS) diseases and a significant bottleneck in neurological drug development. While protecting the brain from pathogens and toxins, the BBB also blocks nearly all large-molecule drugs and over 98% of small-molecule drugs, severely limiting the treatment of CNS diseases such as neurodegenerative diseases, brain tumors, brain infections, and stroke. For example, while temozolomide is a standard concurrent radiotherapy agent for glioblastoma, it does not protect against radiation damage. Amifostine, the only FDA-approved radioprotectant, cannot cross the BBB and therefore cannot effectively protect the CNS. Therefore, developing multifunctional agents that combine radiosensitization, multi-organ protection (especially BBB penetration combined with skin targeting), and broad-spectrum ROS scavenging capabilities is key to overcoming the efficacy-toxicity paradox in brain tumor radiotherapy. Summary of the Invention

[0006] To achieve a "one-drug, dual-action" therapeutic strategy for reducing toxicity and enhancing efficacy in brain tumor radiotherapy, the present invention discovered for the first time that a compound with the molecular structure represented by Formula (I) can both radiosensitize and protect against radiation damage. Furthermore, it can penetrate the blood-brain barrier (BBB), demonstrating both in vitro and in vivo efficacy in brain tumor radiotherapy and radioprotection of normal neural tissue and skin. Experimental studies have confirmed that this material significantly reduces ionizing radiation damage to normal tissues, and preclinical studies have demonstrated protective effects on skin tissue, inhibiting the development and progression of radiation dermatitis. Furthermore, this compound possesses the unique advantage of BBB penetration, demonstrating both in vitro and in vivo efficacy in brain tumor radiotherapy. To further enhance its bioavailability, promote the efficacy of this dual-action compound, and improve its efficient delivery within the brain, the present invention innovatively combines this material with small apoptotic bodies (vesicles secreted by dying cells) to construct a biomimetic delivery system. Small apoptotic bodies have excellent BBB penetration ability and immune escape properties, making them ideal brain-targeted delivery vehicles. The present invention has demonstrated that this system significantly improves the brain delivery and targeted accumulation of the dual-action compound through synergistic effects, overcoming the technical bottleneck of brain-targeted delivery.

[0007] The molecular structure shown in formula (I) is as follows:

[0008]

[0009] (I).

[0010] Specifically, the present invention provides a dual-action compound or a composition containing the same for use in preparing a drug for radiosensitization to treat tumors and / or for protecting against radiation damage, wherein the molecular structure of the dual-action compound is shown in formula (I).

[0011] As used herein, the preparation method of the dual-action compound having a molecular structure as shown in formula (I) can be prepared by a suitable method known in the art, for example, the method of the present invention can be used. Figure 1 Prepared according to the synthetic route shown in .

[0012] Furthermore, the dual-action compound having a molecular structure as shown in formula (I) has catalase (CAT) activity, peroxidase (POD) activity and superoxide dismutase (SOD) activity.

[0013] Furthermore, the composition comprises the dual-action compound encapsulated in small apoptotic bodies.

[0014] As used herein, "apoptotic bodies" refer to small vesicle-like structures containing intracellular components that are formed by the cell membrane when cells undergo apoptosis, a process of programmed cell death. "Small apoptotic bodies" refer to apoptotic bodies with a diameter of less than 1000 nm.

[0015] Furthermore, the tumor includes a brain tumor.

[0016] Furthermore, the brain tumor includes glioblastoma.

[0017] Furthermore, the radiation damage includes radiation brain damage and radiation dermatitis.

[0018] Furthermore, the small apoptotic bodies are prepared from melanoma cells.

[0019] The present invention also provides a composition for radiosensitization to treat tumors and / or for protecting against radiation damage, the composition comprising the dual-action compound as described herein encapsulated in small apoptotic bodies.

[0020] Furthermore, the small apoptotic bodies are prepared from melanoma cells.

[0021] Furthermore, the tumor includes a brain tumor.

[0022] Furthermore, the radiation damage includes radiation brain damage.

[0023] Furthermore, the radiation damage includes radiation dermatitis.

[0024] The present invention also provides a method for preparing a composition for radiosensitization for treating tumors and / or for protecting against radiation damage as described herein, the preparation method comprising: adding the dual-action compound as described herein to the culture supernatant of melanoma cells, inducing apoptosis of the melanoma cells by treating them with hydrogen peroxide and ultraviolet light, and extracting small apoptotic bodies encapsulating the dual-action compound from the culture supernatant after incubation for a period of time by gradient centrifugation.

[0025] Furthermore, the treatment concentration of the hydrogen peroxide is 200-600 nM.

[0026] Furthermore, the intensity of the ultraviolet rays in the ultraviolet treatment is 100-200 mJ / cm 2 , the processing time is 25-35min.

[0027] Furthermore, the incubation time is 12-48 hours.

[0028] Furthermore, the gradient centrifugation method includes: first centrifuging at 250-350g for 8-12 minutes and collecting the supernatant, then centrifuging the supernatant at 2500-3500g for 15-25 minutes and collecting the supernatant, and finally centrifuging the supernatant at 10000-15000g for 20-40 minutes and collecting the precipitate.

[0029] Advantageous Effects of the Invention

[0030] The present invention discovers for the first time that a compound having a molecular structure as shown in formula (I) (referred to herein as Ru(bda)[Me-bpy][4-SO3-Py]) has the dual effects of radiosensitization and protection against radiation damage, and is capable of penetrating the blood-brain barrier (BBB). It can achieve a toxicity-reducing and synergistic effect on brain tumor radiotherapy through intravenous injection without the need for intraventricular injection.

[0031] To further enhance the efficacy of this dual-action compound and improve its efficient delivery within the brain, the present invention also combined this material with small apoptotic bodies (vesicular structures secreted by dying cells) to construct a biomimetic delivery system (referred to herein as Ru(bda)[Me-bpy][4-SO3-Py]-sABs). Small apoptotic bodies exhibit excellent BBB penetration and immune evasion properties, making them ideal vehicles for brain-targeted delivery. The present invention used B16F10 (mouse melanoma) cells to prepare small apoptotic bodies. During apoptosis, B16F10 cells release apoptotic bodies (sABs), which possess inherent biocompatibility and immune evasion properties. The present invention added pre-prepared organic small molecule nanoparticles to the cell supernatant after apoptosis induction and found that the apoptotic bodies actively encapsulated these nanoparticles. This encapsulation process not only utilizes the natural membrane structure of the apoptotic bodies but also leverages their negative surface charge to enhance their adsorption and encapsulation capabilities for the nanoparticles. This system utilizes the natural blood-brain barrier (BBB) ​​crossing ability of apoptotic bodies to significantly improve the delivery efficiency of nanoparticles in the brain. Furthermore, the immune evasion properties of apoptotic bodies reduce the clearance of nanoparticles from the blood circulation, further enhancing their targeted accumulation in the brain. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1The synthetic route of Ru(bda)[Me-bpy][4-SO3-Py] is shown.

[0033] Figure 2 NMR spectra showing the molecular structure of Ru(bda)[Me-bpy][4-SO3-Py] and enzyme activity assays: (A) Molecular structure; (B) NMR spectra; (C) CAT enzyme activity assay; (D) SOD enzyme activity assay; (E) POD enzyme activity assay.

[0034] Figure 3 Transmission electron micrographs of small apoptotic bodies are shown. Panel A shows a normal apoptotic body, while panels B and C show the encapsulated apoptotic bodies. Magnification: 120,000x, scale bar: 100 nm.

[0035] Figure 4 The brain delivery effects of simple Ru(bda)[Me-bpy][4-SO3-Py] and Ru(bda)[Me-bpy][4-SO3-Py] + small apoptotic bodies (A) and their contents in isolated organs such as brain, heart, liver, spleen, lung, and kidney (B) are shown.

[0036] Figure 5 The results of in vitro anti-tumor sensitization experiments are shown: (A) Representative reactive oxygen species (ROS) images of GL261 cells receiving different treatments; (B) Representative γ-H2AX fluorescence staining; (C) Representative Calcein-AM / PI fluorescence staining; (D) Representative flow cytometry images of cell apoptosis.

[0037] Figure 6 The in vivo experimental results of anti-tumor sensitization are shown: (A) In vivo imaging of glioma in GL261-luc tumor-bearing mice on the 6th, 9th, 12th and 15th days from the start; (B) Experimental treatment flow chart; (C) Body weight change curves of GL261-luc tumor-bearing mice in different groups; (D) Survival analysis of GL261-luc tumor-bearing mice in different groups.

[0038] Figure 7 Figure 3 shows the results of in vitro experiments on protection against radiation-induced brain injury: (A) Representative ROS images of BV2 cells receiving different treatments; (B) Representative γ-H2AX fluorescence staining; (C) Representative Calcein-AM / PI fluorescence staining; (D) Representative flow cytometry images of cell apoptosis.

[0039] Figure 8In vivo experimental results of protecting against radioactive brain injury: (A) HE staining results of the coronal plane of the brain of a radioactive brain injury mouse model receiving different treatments; (B) Neun immunofluorescence images; (C) lba1 immunofluorescence images; (D) GFAP immunofluorescence images; (E) Neun / lba1 / GFAP / DAPI combined immunofluorescence images.

[0040] Figure 9 In vitro experimental results of protecting against radioactive dermatitis: (A) Representative Calcein-AM / PI fluorescence staining of HeCaT cells receiving different treatments; (B) Representative ROS images; (C) Representative reactive oxygen γ-H2AX fluorescence staining; (D) Representative flow cytometry detection of cell apoptosis images.

[0041] Figure 10 In vivo experimental results of protecting against radioactive dermatitis: (A) Photographs of the therapeutic effect of a radioactive brain dermatitis mouse model not receiving treatment; (B) Photographs of the therapeutic effect of a radioactive brain dermatitis mouse model receiving treatment. DETAILED DESCRIPTION

[0042] The present application will be further described in conjunction with specific embodiments, but the embodiments do not limit the present application in any form. Unless otherwise specified, the reagents, methods, and equipment used in the present application are conventional reagents, methods, and equipment in the technical field.

[0043] Example 1: Preparation of Ru(bda)[Me-bpy][4-SO3-Py]

[0044] The synthetic route is shown in Figure 1 .

[0045] 1. Weigh 2 g of RuCl3-xH2O and place it in a 100 mL flask, then add 30 mL of dimethyl sulfoxide (DMSO) and reflux for 5 minutes. After cooling to room temperature, distill under reduced pressure, and after leaving a small amount of solvent, filter, wash with excess acetone, and dry in a vacuum to collect a yellow powder. Ru(DMSO)4Cl2 is obtained.

[0046] 2. Weigh 242 mg of Ru(DMSO)4Cl2 and 122 mg of H2BDA and place them in a 50 mL two-necked flask. Add 20 mL of methanol and 0.4 mL of triethylamine and reflux for 12 hours. Wash with excess methanol, acetone, and diethyl ether, and dry in a vacuum to collect a red-brown powder of Ru(bda)(DMSO)2.

[0047] 3. Weigh 125 mg of Ru(bda)(DMSO)2 and 51.7 mg of [Me-bpy]Cl, place them in a 50-mL flask, add 10 mL of acetonitrile, heat under reflux for 1 h, filter, wash with excess acetonitrile and ether, and dry in vacuo to obtain a reddish-black solid Ru(bda)([Me-bpy]Cl)(DMSO)CH3CN.

[0048] 4. Weigh 159.17 mg of pyridine-4-sulfonic acid and 58.3 mg of Na2CO3 into a 50 mL flask. Add 15 mL of methanol and reflux for 1 h. Filter and collect the filtrate. Rotary evaporate to collect the white solid sodium pyridine-4-sulfonate.

[0049] 5. Weigh 114.3 mg of Ru(bda)([Me-bpy]Cl)(DMSO)CH3CN and 27.4 mg of sodium pyridine-4-sulfonate into a 50 mL flask, add 10 mL of ethanol, reflux for 3 h, filter, and wash with excess ethanol and ether to obtain a reddish-black solid Ru(bda)[Me-bpy][4-SO3-Py], whose molecular formula is as follows: Figure 2 As shown in A.

[0050] Example 2: Preparation of Ru(bda)[Me-bpy][4-SO3-Py]-sABs

[0051] The present invention induces apoptosis of B16F10 (mouse melanoma cells) by treating them with hydrogen peroxide and ultraviolet light. The dual-action compound described herein is added to the supernatant of the cells after apoptosis induction and incubated. The small apoptotic bodies encapsulating the dual-action compound in the culture supernatant are extracted by gradient centrifugation. In brief, 1×10 7 B16F10 cells were cultured in DMEM medium containing 10% FBS. Before induction of apoptosis, the medium was replaced with serum-free DMEM containing 50 μg / ml Ru(bda)[Me-bpy][4-SO3-Py]. The cells were then irradiated with UV at 150 mJ / cm 2 Irradiate for 30 min to induce apoptosis, and adjust the H2O2 concentration in the culture medium to 600 nM ， After incubation for 24 h, the cell supernatant was collected and centrifuged at 300 g for 10 min, the supernatant was collected, the supernatant was centrifuged at 3000 g for 20 min, and the supernatant was collected. Finally, the supernatant was centrifuged at 12000 g for 30 min and the precipitate was collected to obtain Ru(bda)[Me-bpy][4-SO3-Py]-sABs.

[0052] Example 3: Structure and enzyme-like activity characterization of Ru(bda)[Me-bpy][4-SO3-Py]

[0053] The structure and composition of Ru(bda)[Me-bpy][4-SO3-Py] were characterized in detail by nuclear magnetic resonance (NMR) technology. Figure 2 B) Shows the hydrogen atom signals in different chemical environments in the material. The molecular structure and composition of the material can be clarified through the chemical shift, peak area and coupling constant of these signals, which confirms the synthesis of Ru(bda)[Me-bpy][4-SO3-Py].

[0054] The guaiacol method was used to detect the catalase (CAT) activity and peroxidase (POD) activity of the materials; the xanthine oxidase method was used to determine the superoxide dismutase (SOD) activity. Figure 2 As shown in CE, Ru(bda)[Me-bpy][4-SO3-Py] has catalase (CAT) activity, peroxidase (POD) activity and superoxide dismutase (SOD) activity.

[0055] Figure 3 Shown is a transmission electron micrograph of small apoptotic bodies in the supernatant of B16F10 cells after induction of apoptosis, indicating that small apoptotic bodies are generated after apoptosis. Figure 3 A is a small apoptotic body with normal morphology. Figure 3 B and 3C are small apoptotic bodies after encapsulation of the material.

[0056] Figure 4 The fluorescence images of the in vivo distribution of small apoptotic bodies after encapsulation of Ru(bda)[Me-bpy][4-SO3-Py]-Cy5.5 and simple Ru(bda)[Me-bpy][4-SO3-Py]-Cy5.5 after tail vein injection in brain glioma model mice are shown. Figure 4 A is the brain distribution image of two groups of glioma model mice. Figure 4 B is the distribution characteristics of different tissue materials in vitro from the two groups of mice at different time points.

[0057] Example 4: Radiosensitization Effects of Ru(bda)[Me-bpy][4-SO3-Py] and Ru(bda)[Me-bpy][4-SO3-Py]-sABs in Vitro and in Vivo

[0058] Experimental Materials and Methods

[0059] 1. Cell line: GL261 cells were cultured using a 0.5 mg mL -1 DMEM (Gibco) medium containing penicillin-streptomycin (Gibco) and 10% certified fetal bovine serum (ZATA, Shanghai, China, Cat Z7185FBS-500).

[0060] 2. In vitro anti-tumor and sensitization experiments

[0061] 2.1 Detection of ROS production in cells after irradiation: GL261 cells were collected during the growth phase, counted using a cell counting plate, and seeded into 6-well plates (1×10 6 Cells were cultured overnight in PBS (100 μg / mL) containing 60 μg / mL of Ru(bda)[Me-bpy][4-SO3-Py) in the Ru(bda)[Me-bpy][4-SO3-Py] group, the Ru(bda)[Me-bpy][4-SO3-Py]-sABs group, the Ru(bda)[Me-bpy][4-SO3-Py] + radiotherapy group, and the Ru(bda)[Me-bpy][4-SO3-Py]-sABs + radiotherapy group. The control and radiotherapy groups each contained an equal amount of blank PBS. The original cell culture medium was removed from the cells in each group and incubated with the drug-containing medium for 4 hours. The drug-containing medium was then removed and fresh medium was added. The cells were then irradiated with 6 Gy of X-rays. The cells in the normal control group were sham-irradiated using a lead block as a shield. After irradiation, the original culture medium was removed and serum-free culture medium containing DCFH-DA prepared according to the instructions of the ROS detection kit was added (1 mL / well). The cells were placed in an incubator in the dark and incubated for 20 min. After washing the cells three times with serum-free culture medium, the ROS green fluorescence of each group of cells was observed using a fluorescence microscope.

[0062] 2.2 Live / dead staining Calcein (AM) / Propidium iodide (PI) staining: GL261 cells were collected in the growth phase and seeded in 6-well plates (1×10 6 Cells were cultured overnight in 4% paraformaldehyde (PNA) solution (per well). Cell dosing and irradiation procedures were the same as above. Live cells were stained with 1 µl of AM solution in detection buffer, and dead cells were stained with 1 µl of PI solution. The captured cells in each cell group were then observed and counted using a fluorescence microscope.

[0063] 2.3 DNA Damage: GL261 cells were harvested from the growing phase and seeded into 96-well plates (5000 cells / well) for overnight culture. Cell dosing and irradiation procedures were the same as above. After fixation with 4% paraformaldehyde, cells were washed three times with washing buffer (5 minutes each). Immunostaining blocking buffer was added for 20 minutes at room temperature. After blocking, rabbit monoclonal antibody against γ-H2AX was added, and cells were incubated overnight at 4°C. Cells were then washed three times with washing buffer (5 minutes each). Anti-rabbit 488 was added, and cells were incubated in the dark for 1 hour at room temperature. Cell nuclei were counterstained with DAPI at room temperature. Cells were washed with washing buffer and examined under a fluorescence microscope.

[0064] 2.4. Animal Model: Male C57 / 6J mice (6–8 weeks old) were purchased from GemPharmatech Co., Nanjing, China. All animal experimental procedures were performed in accordance with the Regulations on the Administration of Laboratory Animals approved by the State Council of the People's Republic of China. All mice were housed under specific pathogen-free (SPF) conditions (temperature ~22°C, humidity ~50%) with a 12 / 12 h dark / light cycle. An orthotopic glioma model was established by stereotaxic injection of fluorescently labeled GL261 cells. Tumor size was assessed by in vivo imaging starting on day 6 after injection. For the radiotherapy group, the Ru(bda)[Me-bpy][4-SO3-Py]+radiotherapy group, and the Ru(bda)[Me-bpy][4-SO3-Py]-sABs+radiotherapy group, tumors were observed when they grew to 0.2 mm. 3 At the same time, 3 Gy X-ray whole-brain precise radiotherapy was performed, once every three days, for a total of two times. Ru(bda)[Me-bpy][4-SO3-Py] or Ru(bda)[Me-bpy][4-SO3-Py]-sABs was injected into the tail vein 1 hour before each irradiation. For the Ru(bda)[Me-bpy][4-SO3-Py] group and the Ru(bda)[Me-bpy][4-SO3-Py]-sABs group, Ru(bda)[Me-bpy][4-SO3-Py] or Ru(bda)[Me-bpy][4-SO3-Py]-sABs group were injected into the tail vein without radiotherapy at the same time points. The experimental treatment process is as follows: Figure 6 B. Tumor growth assessment: Mouse body weight and survival status were recorded. Tumor size was assessed by in vivo imaging every 3 days after orthotopic injection.

[0065] Experimental results

[0066] 1. ROS Generation and DNA Damage

[0067] To investigate the radiosensitizing ability of Ru(bda)[Me-bpy][4-SO3-Py], we first examined the generation of reactive oxygen species (ROS) in GL261 cells (pH 6.8) after co-incubation and radiotherapy. Figure 5 As shown in A, no significant increase in ROS generation was observed in the radiotherapy group and the Ru(bda)[Me-bpy][4-SO3-Py] group compared with the control group. However, the combined treatment group (Ru(bda)[Me-bpy][4-SO3-Py]+radiotherapy group) showed significant ROS generation, indicating that the compound can effectively promote ROS generation under radiotherapy conditions.

[0068] In addition, we detected the expression of DNA damage marker γ-H2AX by immunofluorescence. Figure 5As shown in Figure 2B, a large number of γ-H2AX foci were formed in the combined treatment groups (Ru(bda)[Me-bpy][4-SO3-Py]+radiotherapy group and Ru(bda)[Me-bpy][4-SO3-Py]-sABs+radiotherapy group), indicating that this treatment significantly increased the number of DNA double-strand breaks, further confirming its radiosensitization ability.

[0069] 2. Cell Viability and Apoptosis

[0070] In order to evaluate the toxicity and radiosensitization effect of Ru(bda)[Me-bpy][4-SO3-Py] on GL261 cells, we used Calcein-AM / PI double staining to detect cell viability. Figure 5 As shown in Figure C, the proportion of dead cells in the combined treatment groups (Ru(bda)[Me-bpy][4-SO3-Py]+radiotherapy group and Ru(bda)[Me-bpy][4-SO3-Py]-sABs+radiotherapy group) increased significantly, while the number of live cells decreased, indicating that this treatment significantly enhanced the cell-killing effect of radiotherapy.

[0071] Furthermore, we detected the cell apoptosis rate by flow cytometry. Figure 5 As shown in Figure (D), the apoptotic rates in each group were: 2.04% in the control group, 1.49% in the Ru(bda)[Me-bpy][4-SO3-Py] group, 2.67% in the Ru(bda)[Me-bpy][4-SO3-Py]-sABs group, 12.92% in the radiotherapy group, 33.63% in the Ru(bda)[Me-bpy][4-SO3-Py]+radiotherapy group, and 36.86% in the Ru(bda)[Me-bpy][4-SO3-Py]-sABs+radiotherapy group. The apoptotic rate in the combined treatment group (Ru(bda)[Me-bpy][4-SO3-Py]+radiotherapy) increased by approximately 23% compared with the radiotherapy group alone, indicating that Ru(bda)[Me-bpy][4-SO3-Py] has a significant radiosensitizing effect. Furthermore, the small apoptotic bodies (sABs) did not exhibit significant cytotoxicity.

[0072] 3. In vivo radiosensitization experiments

[0073] To evaluate the in vivo radiosensitization anti-tumor efficacy of Ru(bda)[Me-bpy][4-SO3-Py], we established a GL261-luc right cerebral glioblastoma model in C57BL / 6J mice. Figure 6 The treatment flow chart shown in B was used for the experiment. Figure 6As shown in Figure A, after comparing and analyzing the tumor growth inhibition effects of each treatment group, it was found that the tumor inhibition effect of Ru(bda)[Me-bpy][4-SO3-Py] combined with X-ray radiation was better than that of radiotherapy alone, and the mice had less weight loss and higher survival rate, indicating that tail vein injection of Ru(bda)[Me-bpy][4-SO3-Py] can treat brain tumors and that Ru(bda)[Me-bpy][4-SO3-Py] can penetrate the BBB. It is worth noting that the inhibitory effect on tumor growth in the Ru(bda)[Me-bpy][4-SO3-Py]-sABs+radiotherapy combined treatment group was the most significant, indicating that the nanoformulation combined with the small apoptotic body delivery system can achieve further enhanced radiosensitization ability in vivo. At the same time, the weight loss of mice in the Ru(bda)[Me-bpy][4-SO3-Py]-sABs+radiotherapy group was less than that in other groups ( Figure 6 C), and the survival rate was the highest among all groups ( Figure 6 D).

[0074] Experimental Conclusion

[0075] Ru(bda)[Me-bpy][4-SO3-Py] exhibited significant radiosensitization in both in vitro and in vivo models. Synergistic combination with small apoptotic bodies (sABs) further enhanced its antitumor efficacy, suggesting a dual mechanism of action involving radiosensitization and apoptosis enhancement. These findings suggest that Ru(bda)[Me-bpy][4-SO3-Py] may be a promising new radiosensitizer for clinical use in glioma treatment.

[0076] Example 5: Neuroradiotherapy Protection Effects of Ru(bda)[Me-bpy][4-SO3-Py] and Ru(bda)[Me-bpy][4-SO3-Py]-sABs in Vitro and In Vivo

[0077] Experimental Materials and Methods

[0078] 1. Detection of ROS production in cells after irradiation: Microglial BV2 cells were collected during the growth phase, counted using a cell counting plate, and seeded into 6-well plates (1×10 5Cells were cultured overnight in a PBS solution (100 μg / mL) containing 60 μg / mL of Ru(bda)[Me-bpy][4-SO3-Py) in the Ru(bda)[Me-bpy][4-SO3-Py] group, the Ru(bda)[Me-bpy][4-SO3-Py]-sABs group, the Ru(bda)[Me-bpy][4-SO3-Py]+radiotherapy group, and the Ru(bda)[Me-bpy][4-SO3-Py]-sABs+radiotherapy group. The control and radiotherapy groups each contained an equal amount of blank PBS. The original cell culture medium was removed from the cells in each group and incubated with the drug-containing culture medium for 4 hours. The drug-containing culture medium was then aspirated and replaced with fresh culture medium. The cells were then irradiated with 8 Gy of X-rays. The cells in the normal control group were sham-irradiated using a lead block as a shield. The detection method was the same as above.

[0079] 2. Live / dead staining: Calcein (AM) / Propidium iodide (PI) staining: BV2 cells in the growth phase were collected and seeded in 6-well plates (1×10 5 Cells were cultured overnight in 4% paraformaldehyde (PNA) / well (μg / ml). Cell dosing and irradiation procedures were the same as above. Detection methods were the same as above.

[0080] 3. DNA Damage: BV2 cells were harvested from the growing phase and seeded into 96-well plates (5000 cells / well) for overnight culture. The cell administration and irradiation procedures were the same as above, and the detection method was the same as above.

[0081] 4. A mouse model of radiation-induced brain injury was established using the X-RAD SmART+ System 3D image-guided precision irradiation system. The specific method is as follows: Male SPF-grade C57BL / 6J mice, approximately 7-8 weeks old, were anesthetized with isoflurane inhalation and placed in a prone position on a dedicated holder. The head was adjusted so that the top of the skull was aligned horizontally with the center of the radiation field. Using the system's built-in cone-beam CT, the mouse brain (from the anterior to the posterior bregma area along the midline of the skull) was precisely located. The X-RAD SmART+ System was activated to confirm the dose distribution and irradiation field, ensuring full brain coverage. A single 225 kV X-ray fraction of 30 Gy was administered. One hour before irradiation, 60 μg of Ru(bda)[Me-bpy][4-SO3-Py] or Ru(bda)[Me-bpy][4-SO3-Py]-sABs containing 60 μg of Ru(bda)[Me-bpy][4-SO3-Py] were injected into the tail vein. The drugs were then administered every three days for 8 weeks. Histological examination of brain injury: Eight weeks after irradiation, brain tissues from mice in each group were fixed by intracardial perfusion with 0.9% sodium chloride solution (containing heparin) and 4% paraformaldehyde. Brain tissues were collected and evaluated for brain injury, including vacuolation, angiogenesis, and hemorrhage, by HE staining. Neuronal morphology and number were also observed. Golgi staining was used to examine the morphology and density of dendritic spines. Immunofluorescence was used to assess the expression of the neuron-specific nuclear protein NeuN to assess neuronal number. Immunofluorescence was used to assess the expression of lba1 and GFAP to assess microglial and astrocyte activation.

[0082] Experimental results

[0083] 1. In vitro radiotherapy protection experiment

[0084] In an in vitro experiment, Ru(bda)[Me-bpy][4-SO3-Py] was co-incubated with microglial cells BV2 (pH 8.0) and treated with X-rays (radiotherapy) for 24 hours. The experimental results showed that compared with the radiotherapy group, the Ru(bda)[Me-bpy][4-SO3-Py]+radiotherapy group significantly reduced the generation of reactive oxygen species (ROS) (by about 45%), effectively alleviating the cellular oxidative stress state ( Figure 7 A). Figure 7 As shown in C, Calcein-AM / PI fluorescence staining showed that the ratio of dead / live cells in the Ru(bda)[Me-bpy][4-SO3-Py]+radiotherapy group was reduced by about 40% compared with the radiotherapy group ( Figure 7 C), and the expression of DNA damage marker γ-H2AX decreased by about 55% ( Figure 7B). Flow cytometry detection of cell apoptosis rates showed that the apoptosis rates of the control group, Ru(bda)[Me-bpy][4-SO3-Py] monotherapy group, and Ru(bda)[Me-bpy][4-SO3-Py]-sABs group were 6.99%, 6.18%, and 6.01%, respectively, indicating that the nanoformulation and small apoptotic bodies (sABs) have excellent biocompatibility. It is worth noting that the apoptosis rate of microglia in the radiotherapy group was as high as 58.33%, while the apoptosis rates of the Ru(bda)[Me-bpy][4-SO3-Py]+radiotherapy group and the Ru(bda)[Me-bpy][4-SO3-Py]-sABs+radiotherapy group were reduced to 7.26% and 6.63%, respectively ( Figure 7 D). These data indicate that Ru(bda)[Me-bpy][4-SO3-Py] can significantly alleviate radiation-induced oxidative stress, cell apoptosis, and DNA damage, and the combined use of sABs delivery system further enhances its protective effect.

[0085] 2. In vivo radiotherapy protection experiments

[0086] By constructing a C57 / 6J mouse model of radiation-induced brain injury, the neuroprotective effects of Ru(bda)[Me-bpy][4-SO3-Py] and its combination with small apoptotic bodies (sABs) during radiotherapy were systematically evaluated. Figure 8 A's experimental results showed that mice in the radiotherapy group (radiotherapy) showed a significant increase in inflammatory cells, vacuolated cells, and neovascularization in the hippocampus, suggesting typical pathological features of radiation-induced brain injury. However, mice treated with Ru(bda)[Me-bpy][4-SO3-Py] combined with radiotherapy (Ru(bda)[Me-bpy][4-SO3-Py]+radiotherapy group) and mice further treated with small apoptotic bodies (Ru(bda)[Me-bpy][4-SO3-Py]-sABs+radiotherapy group) showed a significant protective effect, with a significant decrease in the number of inflammatory cells and vacuolated cells, and suppressed neovascularization.

[0087] In addition, NeuN immunofluorescence detection revealed that ( Figure 8 B) The neuroprotective effect of the Ru(bda)[Me-bpy][4-SO3-Py]-treated group was significantly enhanced, indicating that it has potential neuroprotective function against radiation-induced brain injury. Figure 8 C) and GFAP ( Figure 8 D) Immunofluorescence results further showed that the activation of microglia and astrocytes in the Ru(bda)[Me-bpy][4-SO3-Py] combined with radiotherapy group was significantly reduced compared with that in the radiotherapy alone group. Figure 8E is a combined NeuN / lba1 / GFAP / DAPI immunofluorescence image. This indicates that Ru(bda)[Me-bpy][4-SO3-Py] not only enhances the anti-tumor effect of radiotherapy but also reduces radiation-induced brain damage by inhibiting the overactivation of glial cells.

[0088] Experimental Conclusion

[0089] In summary, Ru(bda)[Me-bpy][4-SO3-Py] and its combination with small apoptotic bodies (sABs) exhibited significant neuroprotective effects in a model of radiation-induced brain injury. The mechanism may be related to inhibiting neuroinflammation, reducing glial activation, and protecting neuronal function. These findings provide important experimental evidence for the development of novel strategies for the prevention and treatment of radiation-induced brain injury and offer potential therapeutic targets for clinical application.

[0090] Example 6: Skin radiotherapy protection effect of Ru(bda)[Me-bpy][4-SO3-Py] in vitro and in vivo

[0091] Experimental Materials and Methods

[0092] 1. Detection of ROS Production in Irradiated Cells: Immortalized human skin keratinocytes were collected from the growth phase, counted using a cell counter, and seeded into 6-well plates (1×10⁵ / well) for overnight culture. Drug-containing cell culture media were prepared: the Ru(bda)[Me-bpy][4-SO⁃-Py] group and the Ru(bda)[Me-bpy][4-SO⁃-Py] + radiotherapy group each contained an equal volume of Ru(bda)[Me-bpy][4-SO⁃-Py] (60 μg / mL). The control and radiotherapy groups each contained an equal volume of blank PBS. The original cell culture medium was removed from the cells in each group and incubated with drug-containing culture medium for 4 h. Subsequently, the drug-containing culture medium was aspirated and replaced with fresh culture medium. The cells were then irradiated with 15 Gy X-rays. The cells in the normal control group were sham-irradiated using a lead block as a shield. The detection method was the same as above.

[0093] 2. Live / Dead Staining: Calcein (AM) / Propidium Iodide (PI) Staining: HeCaT cells were harvested from the growth phase and seeded into 6-well plates (1 × 105 cells / well) for overnight culture. Cell administration and irradiation procedures were the same as above. Detection methods were the same as above.

[0094] 3. DNA Damage: HeCaT cells were harvested from the growing phase and seeded into 96-well plates (5000 cells / well) for overnight culture. The cell administration and irradiation procedures were the same as above, and the detection method was the same as above.

[0095] 4. Male C57BL / 6J mice (6-8 weeks old) were purchased from GemPharmatech Co., Nanjing, China. All animal experimental procedures were performed in accordance with the Regulations on the Administration of Laboratory Animals approved by the State Council of the People's Republic of China. All mice were housed under specific pathogen-free (SPF) conditions (temperature ~22°C, humidity ~50%) with a 12 / 12 h dark / light cycle. First, mice were anesthetized with sodium pentobarbital. The leg skin of the mice was irradiated with X-rays (Bio X-ray irradiator, X-RAD 320) for 20 minutes at a dose of 20 Gy, and irradiated twice for a total dose of 40 Gy. Materials were applied to the legs of the mice, including saline (control group) and Ru(bda)[Me-bpy][4-SO3-Py] (experimental group). Subsequently, changes in the irradiated skin of each mouse were monitored daily. After 7 days of treatment, the mice were sacrificed, and the skin status was photographed and recorded.

[0096] Experimental results

[0097] 1. In vitro experiment on skin radiotherapy protection

[0098] In an in vitro experiment, Ru(bda)[Me-bpy][4-SO3-Py] was co-incubated with human keratinocytes HeCaT and treated with X-rays (radiotherapy) for 24 hours. The experimental results showed that compared with the radiotherapy group, the Ru(bda)[Me-bpy][4-SO3-Py]+radiotherapy group significantly reduced the generation of reactive oxygen species (ROS) and effectively alleviated the cellular oxidative stress state ( Figure 9 B). Calcein-AM / PI fluorescence staining showed that the ratio of dead / live cells in the Ru(bda)[Me-bpy][4-SO3-Py]+radiotherapy group was reduced by approximately 40% compared with the radiotherapy group ( Figure 9 A), and the expression of the DNA damage marker γ-H2AX decreased by about 55% ( Figure 9 C). Flow cytometry analysis of cell apoptosis revealed that the apoptosis rates in the control group, the Ru(bda)[Me-bpy][4-SO3-Py] monotherapy group, the radiotherapy group, and the Ru(bda)[Me-bpy][4-SO3-Py]+radiotherapy group were 3.74%, 2.77%, 23.43%, and 3.1%, respectively. Figure 9 D). The above data indicate that Ru(bda)[Me-bpy][4-SO3-Py] can significantly reduce radiation-induced oxidative stress, cell apoptosis, and DNA damage, and has a good protective effect on the skin.

[0099] 2. In vivo experiments on skin radiotherapy protection

[0100] By constructing a C57 / 6J mouse model of radiation dermatitis, the skin protective effect of Ru(bda)[Me-bpy][4-SO3-Py] during radiotherapy was systematically evaluated. Figure 10 The experimental results of A showed that the leg skin of mice in the radiotherapy group showed obvious redness, swelling, ulcers and other inflammatory symptoms, while the mice treated with Ru(bda)[Me-bpy][4-SO3-Py] combined with radiotherapy showed a significant protective effect, with the edema of the leg skin significantly subsiding, indicating that there was no obvious ulceration ( Figure 10 B).

[0101] In summary, Ru(bda)[Me-bpy][4-SO3-Py] exhibited significant skin-protective effects in a radiation-induced dermatitis model. The mechanism may be related to inhibiting skin inflammation, scavenging radiation-generated ROS, and reducing DNA damage. These findings provide important experimental evidence for the development of novel strategies for the prevention and treatment of radiation-induced skin damage and offer potential therapeutic targets for clinical application.

[0102] It should be noted that the preferred embodiments of the present invention are given in the specification and drawings of the present invention. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. These embodiments are not intended to be additional limitations on the content of the present invention. The purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive. In addition, the above-mentioned technical features can be combined with each other to form various embodiments not listed above, which are all considered to be within the scope of the description of the present invention. Furthermore, it is obvious to those skilled in the art that improvements or changes can be made based on the above description, and all such improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. Use of a dual-action compound or a composition comprising the same in the preparation of a drug for radiosensitization to treat tumors and / or for protecting against radiation damage, characterized in that: The molecular structure of the dual-action compound is shown in formula (I): (I)； The tumor is a brain tumor; The radiation injury is radiation brain injury and / or radiation dermatitis.

2. The use according to claim 1, characterized in that The dual-action compound has catalase activity, peroxidase activity and superoxide dismutase activity; The composition comprises the dual-action compound encapsulated in small apoptotic bodies.

3. The use according to claim 1, characterized in that The brain tumors include glioblastoma.

4. The use according to claim 2, characterized in that The small apoptotic bodies are prepared from melanoma cells.

5. A composition for radiosensitization for treating tumors and / or for protecting against radiation damage, characterized in that: The composition comprises a dual-action compound as defined in claim 1 encapsulated in small apoptotic bodies.

6. The composition according to claim 5, characterized in that The small apoptotic bodies are prepared from melanoma cells; The tumors include brain tumors; The radiation injuries include radiation brain injury and radiation dermatitis.

7. A method for preparing a composition for radiosensitization for treating tumors and / or for protecting against radiation damage according to claim 5 or 6, characterized in that: The preparation method comprises: adding the dual-action compound defined in claim 1 to the culture supernatant of melanoma cells, inducing apoptosis of the melanoma cells by treating with hydrogen peroxide and ultraviolet light, and extracting small apoptotic bodies encapsulating the dual-action compound in the culture supernatant after incubation for a period of time by gradient centrifugation.

8. The preparation method according to claim 7, characterized in that The treatment concentration of the hydrogen peroxide is 200-600nM; The intensity of ultraviolet light in the ultraviolet treatment is 100-200 mJ / cm 2 , processing time is 25-35min; The incubation time is 12-48h; The gradient centrifugation method includes: first centrifuging at 250-350g for 8-12 minutes and collecting the supernatant, then centrifuging the supernatant at 2500-3500g for 15-25 minutes and collecting the supernatant, and finally centrifuging the supernatant at 10000-15000g for 20-40 minutes and collecting the precipitate.

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