RU.521-EVs anti-radiation nanogel as well as preparation method and application thereof

Abstract

The invention provides RU.521-EVs anti-radiation nanogel as well as a preparation method and application of the RU.521-EVs anti-radiation nanogel. The RU.521-EVs anti-radiation nanogel is a nanogel obtained by loading microalgae-derived extracellular vesicles and a small molecule compound RU.521 on chitosan nanoparticles formed by cross-linking chitosan, and the extracellular vesicles and the small molecule compound RU.521 are derived from microalgae. The RU.521-EVs anti-radiation nanogel has higher adhesion and permeability in the lung and effectively releases RU.521 and EVs, the RU.521 and EVs can synergistically play anti-inflammatory and anti-oxidation roles, local anti-oxidation and anti-inflammatory defense reactions of the lung are effectively started, the excellent anti-radiation effect is shown, and the RU.521-EVs anti-radiation nanogel can be used for preventing and treating radiation-induced lung injury.

Description

Technical Field

[0001] The present invention relates to the technical field of anti-radiation drugs, and in particular to a RU.521-EVs anti-radiation nanogel and a preparation method and application thereof. Background Art

[0002] Radiation therapy (RT) is an effective treatment option for thoracic malignancies, but it exposes patients to controlled levels of ionizing radiation. Lung tissue is particularly sensitive to ionizing radiation, and radiation can easily induce radiation-induced lung injury (RILI), causing coughing, chest tightness, dyspnea, and even respiratory failure, which can ultimately be life-threatening. Glucocorticoids are currently a commonly used and effective drug for the treatment of RILI, but they have significant side effects. Systemic use of glucocorticoids can easily cause serious side effects such as infection, gastric ulcers, and femoral head necrosis, which greatly affects the patient's quality of life. Therefore, glucocorticoids are not suitable for long-term treatment or prevention of RILI. Currently, there is a lack of ideal treatment measures for RILI. Therefore, there is an urgent need to find new targets and methods for the prevention and treatment of RILI.

[0003] The cGAS (cyclic guanosine monophosphate-adenylate synthetase)-STING (stimulator of interferon genes) signaling pathway is an important signal transduction pathway that has been newly identified in recent years to regulate innate immune responses. It has gradually become a key mediator of inflammatory responses under conditions such as infection, cell stress and tissue damage. Studies have shown that the cGAS-STING signaling pathway plays a key role in the development of a variety of inflammatory diseases.

[0004] Patent application with publication number US20220143035A1 discloses a method for treating autoimmune diseases or autoinflammatory diseases, which comprises administering a cGAS inhibitor and / or a STING inhibitor, wherein the cGAS inhibitor comprises RU.521 (CAS: 2262452-06-0).

[0005] Microalgae are a class of naturally occurring edible algae with antioxidant, anti-inflammatory and immunomodulatory effects. However, their micron size limits their clinical applications in deep tissues in the body, while extracellular vesicles (EVs) are lipid bilayer nanovesicles that are released into the surrounding environment by most cells and contain components released by cells. Studies have shown that plant EVs bind well to cellular lipids and can penetrate cells, thereby being fully taken up by cells.

[0006] Patent application with publication number CN118201625A discloses a composition containing extracellular vesicles (MEVs) from microalgae, wherein the microalgae is Chlorella vulgaris. The composition can be used as a vaccine, an anti-cancer therapeutic agent, a diagnostic agent, etc.

[0007] Pulmonary airway instillation is a non-invasive method of drug administration through the bronchus. This therapy can achieve local drug administration well and minimize systemic side effects, which has great potential in the treatment of various lung diseases. However, when drugs are administered through pulmonary airway instillation, they must first penetrate the mucus layer covering the pulmonary mucosa before they can be absorbed. Moreover, the lung's own clearance ability also makes the drug ineffective in the lungs. Chitosan (CS) is a cationic polysaccharide formed by deacetylation of chitin. It has high biocompatibility and biodegradability, and also has antioxidant, antibacterial and mucosal adhesion capabilities. In addition, CS is weakly alkaline with a pKa of about 6.5, that is, when the pH is below 6.5, the primary amine of CS can be protonated to generate a positively charged soluble polymer, which can interact with the negatively charged sialic acid in the mucus, and has stronger adhesion and stronger permeability in the lungs. Summary of the invention

[0008] In order to provide a drug that can effectively treat RILI with little side effects, the present invention provides a RU.521-EVs anti-radiation nanogel and its preparation method and application. The RU.521-EVs anti-radiation nanogel has stronger adhesion and permeability in the lungs, can effectively release RU.521 and EVs, effectively activate local antioxidant and anti-inflammatory defense responses in the lungs, show excellent anti-radiation effects, and can be used to prevent and treat radiation lung injury.

[0009] The present invention provides a RU.521-EVs anti-radiation nanogel, wherein the RU.521-EVs anti-radiation nanogel is a nanogel obtained by loading extracellular vesicles (hereinafter referred to as EVs) derived from microalgae and a small molecule compound RU.521 on chitosan nanoparticles formed by cross-linking chitosan (CS).

[0010] Preferably, the chitosan nanoparticles are nanoparticles made of chitosan using sodium tripolyphosphate (TPP) as a cross-linking agent; and the microalgae are Chlorella vulgaris.

[0011] Preferably, in the RU.521-EVs radiation-resistant nanogel, the mass ratio of chitosan, protein in the extracellular vesicles derived from microalgae, and the small molecule compound RU.521 is 2.0-8.0: 0.3-1.4: 0.0001-0.001.

[0012] RU.521 is a cGAS inhibitor that can inhibit the cGAS-STING pathway and reduce inflammatory responses. Chlorella EVs inherit the functions of Chlorella and have antioxidant, anti-inflammatory and immunomodulatory effects. RU.521-EVs anti-radiation nanogel can protect surrounding healthy cells by eliminating the production of ROS (reactive oxygen species) and preventing ROS-induced mitochondrial damage and DNA damage. Because RU.521-EVs anti-radiation nanogel is modified with CS, it has the ability to adhere to the lungs and penetrate the mucosa, and can be administered locally.

[0013] The present invention provides a method for preparing RU.521-EVs radiation-resistant nanogel, which comprises the following steps:

[0014] (1) dissolving chitosan in a solvent to obtain a chitosan solution, adding a pH adjuster to adjust the pH value of the CS solution to 3-7, and obtaining a solution 1;

[0015] (2) extracting EVs from microalgae and dispersing them in a buffer to prepare an EVs suspension, which is then added to solution 1 and stirred to obtain solution 2;

[0016] (3) preparing a crosslinker solution, adding RU.521 to the crosslinker solution to obtain solution 3, adding solution 3 to solution 2 to obtain solution 4, stirring and ultrasonicating solution 4 to obtain the RU.521-EVs radiation-resistant nanogel.

[0017] Preferably, in step (1), the solvent is an aqueous acetic acid solution, more preferably a 1 wt% aqueous acetic acid solution; the pH adjuster is one or more of hydrochloric acid, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium hydroxide and potassium hydroxide.

[0018] Preferably, in step (1), the concentration of the chitosan solution is 3-10 mg / mL.

[0019] Preferably, in step (1), the pH value of the CS solution is adjusted to 5, so that the RU.521-EVs radiation-resistant nanogel prepared has better stability.

[0020] Preferably, in step (2), the method for extracting EVs from microalgae is ultracentrifugation, ultrafiltration, immunoaffinity, precipitation or microfluidics-based separation, see patent application CN118201625A.

[0021] Further preferably, the operating conditions of the ultracentrifugation method are centrifugation at 150,000×g for 1.5 h at 4°C.

[0022] Preferably, in step (2), the protein concentration of solution 2 is 0.5-1.5 mg / mL, more preferably 0.5-1 mg / mL, so that the RU.521-EVs radiation-resistant nanogel prepared has better stability.

[0023] Preferably, in step (2), the mixing volume ratio of the EVs suspension and solution 1 is 1:8-12, and further preferably, the mixing volume ratio of the EVs suspension and solution 1 is 1:10. In this step, the microalgae EVs and chitosan are pre-mixed together.

[0024] Preferably, in step (3), the cross-linking agent solution is a TPP aqueous solution, and the concentration of the TPP aqueous solution is 1wt%-3wt%; RU.521 is first dissolved in a solvent to prepare a RU.521 solution, and then the RU.521 solution is added to the TPP aqueous solution, and the concentration of the RU.521 solution is 5-20μM; the solvent is preferably DMSO (dimethyl sulfoxide); the volume ratio of solution 3 to solution 2 is 1:3-1:7.

[0025] Further preferably, in step (3), the concentration of the TPP aqueous solution is 1wt%-3wt%, the concentration of the RU.521 solution is 5-10μM, and the volume ratio of solution 3 to solution 2 is 1:3-1:5. The RU.521-EVs radiation-resistant nanogel prepared in this way has better stability.

[0026] The present invention also provides the use of the RU.521-EVs anti-radiation nanogel in the preparation of anti-radiation drugs.

[0027] The present invention also provides an anti-radiation drug, the active ingredient of which is the RU.521-EVs anti-radiation nanogel.

[0028] The beneficial effects of the present invention are as follows:

[0029] The present invention provides a RU.521-EVs anti-radiation nanogel and its preparation method and application. The RU.521-EVs anti-radiation nanogel can effectively release RU.521 and EVs. RU.521 and EVs can synergistically exert anti-inflammatory and antioxidant effects, can reduce mitochondrial damage, DNA breakage and the like of cells after irradiation, and can be used to treat radiation lung injury caused by radiotherapy. In vitro, the RU.521-EVs anti-radiation nanogel can improve the inflammatory state of cells, reduce the level of lipid peroxidation, and restore the proliferation activity of cells. In addition, the RU.521-EVs anti-radiation nanogel can significantly improve the survival rate of mice with radiation lung injury, reduce the inflammatory response of lung lesions, and ultimately play a role in preventing and treating radiation lung injury. Therefore, the RU.521-EVs anti-radiation nanogel of the present invention is a very potential material for preventing and treating radiation lung injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Characterization diagram of RU.521-EVs radiation-resistant nanogel. Figure a: Transmission electron microscopy (TEM) images of EVs, CS NPs and RU.521-EVsNPs; Figure b: Photos of CS, CS NPs, EVs NPs, RU.521NPs and RU.521-EVs NPs; Figure c: Particle size images of EVs, CS NPs, EVs NPs, RU.521NPs and RU.521-EVs NPs; Figure d: Potential images of EVs, CS NPs, EVsNPs, RU.521NPs and RU.521-EVs NPs; Figure e: Photos of RU.521-EVs NPs before and after standing for 24 hours.

[0031] Figure 2 The effect of RU.521-EVs anti-radiation nanogel on the proliferation ability of irradiated cells. Figure a: Cell death and viability staining of blank group, irradiation group and irradiation group + various treatment groups; Figure b: Crystal violet staining of cell clone formation experiment in different groups. Figure c: CCK8 detection of cell survival rate in different treatment groups after 48h and 72h treatment at 4, 6, and 8Gy. * indicates P<0.05, ** indicates P<0.01.

[0032] Figure 3 The antioxidant capacity of RU.521-EVs radiation-resistant nanogels. Figure a: Fluorescence images of different treatment groups after DAPI and γ-H2A.X staining; Figure b: Fluorescence images of different treatment groups after BODIPY 581 / 591-C11 probe staining; Figure c: SOD activity of different treatment groups; Figure d: CAT activity of different treatment groups; Figure e: MDA detection of lipid peroxidation capacity of different treatment groups. ** indicates P<0.01.

[0033] Figure 4 The in vivo anti-radiation effect of RU.521-EVs anti-radiation nanogel in the second week. Figure a: H&E staining of each group; Figure b: MPO immunohistochemical staining of each group; Figure ce: Quantitative analysis of IL-1β, IL-6, and TNF-α levels in lung homogenates of different treatment groups; Figure f: MDA detection of lipid peroxidation capacity of different treatment groups; Figure g: SOD activity of different treatment groups; Figure h: CAT activity of different treatment groups. * indicates P<0.05, and ** indicates P<0.01.

[0034] Figure 5 The in vivo anti-radiation effect of RU.521-EVs anti-radiation nanogel in the first month. Figure a: H&E staining of each group; Figure b: MPO immunohistochemical staining of each group; Figure c: Masson's trichrome staining of each group; Figure df: Quantitative analysis of IL-1β, IL-6, and TNF-α levels in lung homogenates of different treatment groups; Figure g: MDA detection of lipid peroxidation capacity of different treatment groups; Figure h: SOD activity of different treatment groups; Figure i: CAT activity of different treatment groups. * indicates P<0.05, and ** indicates P<0.01.

[0035] Figure 6 The in vivo and in vitro safety of RU.521-EVs anti-radiation nanogels. Figure a: CCK8 detection of cell survival in different treatment groups; Figure b: Fluorescence images of cell death and viability staining in different treatment groups; Figure c: H&E staining of the main organs (heart, liver, spleen, lung, and kidney) in each group. DETAILED DESCRIPTION

[0036] Experimental Materials:

[0037] Chitosan was purchased from Sigma-Aldrich (USA). Sodium tripolyphosphate (TPP) was purchased from MacLean. Chlorella vulgaris was purchased from Shanghai Guangyu Biotechnology Co., Ltd. Fetal bovine serum (FBS) and DMEM medium were purchased from Gibco. MH-S cell-specific medium was purchased from Pronocell (Wuhan, China). Double distilled water (ddH2O) was from Milli-Q purification system. C57 / BL mice were from Shanghai Biotechnology Co., Ltd. Calcein-AM / PI cell double staining kit, DCFH-DA kit, crystal violet dye, and DNA damage detection kit (γ-H2AX immunofluorescence assay) were purchased from Bio-Tech. BODIPY 581 / 591-C11 probe was purchased from Thermo Fisher Scientific (China). CCK-8 detection kit was purchased from Meilun Biotechnology. SOD detection kit, CAT detection kit, and ELISA detection kit were purchased from Elabscience. MDA detection kit was purchased from Solebo.

[0038] Characterization Instruments:

[0039] The surface potential and particle size were measured by dynamic light scattering (DLS) using a Malvern Zetasizer. Transmission electron microscopy (TEM) was used to observe the structure of these particles.

[0040] Example 1

[0041] A method for preparing RU.521-EVs radiation-resistant nanogel comprises the following steps:

[0042] (1) Chitosan (CS) was dissolved in a 1 wt% acetic acid aqueous solution and stirred at room temperature for 12 h to obtain a CS solution with a concentration of 3 mg / mL. Subsequently, the CS solution was filtered with a 0.22 μm filter membrane, and the pH value of the CS solution was adjusted to 5 with NaOH to obtain Solution 1.

[0043] (2) After the Chlorella suspension was centrifuged (4000×g, 10 min), the supernatant was taken and continued to be centrifuged (10000×g, 30 min), and the supernatant was taken again, filtered with a 0.22 μm filter membrane, and then ultracentrifuged (150000×g, 90 min). The precipitate was then collected, which was EVs (extracellular vesicles of Chlorella), and the EVs were resuspended in PBS (phosphate buffered saline) to obtain an EVs suspension.

[0044] (3) The protein content of EVs was detected by BCA (2,2'-bicinchoninic acid, CAS: 1245-13-2) for quantification. 500 μL of EVs was added to 5 mL of solution 1 to make the protein concentration in the resulting solution 1 mg / mL, and stirred at 4°C for 30 min to obtain solution 2.

[0045] (4) RU.521 was dissolved in DMSO (dimethyl sulfoxide) to a concentration of 10 mM, and then the RU.521 solution was diluted to 10 μM with physiological saline.

[0046] (5) 2 wt% TPP solution was prepared with ddH2O, and 10 μM RU.521 solution and 2 wt% TPP aqueous solution were mixed at a volume ratio of 1:1 to obtain solution 3. Solution 3 was dripped dropwise into the stirring solution 2 to obtain solution 4, wherein the volume ratio of solution 3 to solution 2 was 1:5. Solution 4 was then stirred at room temperature for 30 min, and then ultrasonicated for 30 min to finally obtain RU.521-EVs radiation-resistant nanogels, referred to as RU.521-EVs NPs.

[0047] Example 2-Example 14

[0048] The difference from Example 1 is that some parameters of the preparation process of Example 2-Example 14 are adjusted, and the adjusted contents are detailed in Table 1.

[0049] Table 1

[0050]

[0051]

[0052] The results showed that the stability of RU.521-EVs anti-radiation nanogels was different under different reaction conditions, and its stability could be adjusted by adjusting the above parameters.

[0053] Comparative Example 1

[0054] Chitosan nanogels, referred to as CS NPs, were obtained by crosslinking CS with TPP. The specific preparation process is as follows: CS was dissolved in a 1wt% acetic acid aqueous solution and stirred at room temperature for 12h to obtain a CS solution with a concentration of 3mg / mL. Subsequently, the CS solution was filtered with a 0.22μm filter membrane, and the pH value of the CS solution was adjusted to 5 with NaOH to obtain solution 1. A 1wt% TPP solution was prepared with ddH2O, and it was dripped dropwise into the stirring solution 1 to obtain solution 2, wherein the volume ratio of TPP solution to solution 1 was 1:5. Then solution 2 was stirred at room temperature for 30min, and then ultrasonicated for 30min, and finally CS NPs were obtained.

[0055] Comparative Example 2

[0056] Only EVs were wrapped on CS NPs to obtain EVs NPs. The specific preparation process is as follows: CS was dissolved in a 1wt% acetic acid aqueous solution and stirred at room temperature for 12h to obtain a CS solution with a concentration of 3mg / mL. Subsequently, the CS solution was filtered with a 0.22μm filter membrane, and the pH value of the CS solution was adjusted to 5 with NaOH to obtain solution 1. According to the steps in Example 1, EVs suspension was prepared and quantified, and 500μL EVs suspension was added to 5mL solution 1 so that the protein concentration in the resulting solution was 1mg / mL, and stirred at 4°C for 30min to obtain solution 2. A 1wt% TPP solution was prepared with ddH2O, and it was dripped dropwise into solution 2 to obtain solution 3, wherein the volume ratio of TPP solution to solution 2 was 1:5. Solution 3 was then stirred at room temperature for 30min, and then ultrasonicated for 30min to finally obtain EVs NPs.

[0057] Comparative Example 3

[0058] Only RU.521 was wrapped on CS NPs to obtain RU.521NPs. The specific preparation process is as follows: CS was dissolved in a 1wt% acetic acid aqueous solution and stirred at room temperature for 12h to obtain a CS solution with a concentration of 3mg / mL. Subsequently, the CS solution was filtered with a 0.22μm filter membrane, and the pH value of the CS solution was adjusted to 5 with NaOH to obtain solution 1. A 10μM RU.521 solution was prepared according to the steps in Example 1. A 2wt% TPP solution was prepared with ddH2O, and the above RU.521 solution and a 2wt% TPP aqueous solution were mixed in a volume ratio of 1:1 to obtain solution 2. Solution 2 was dripped dropwise into the stirring solution 1 to obtain solution 3, wherein the volume ratio of solution 2 to solution 1 was 1:5. Solution 3 was then stirred at room temperature for 30min, then ultrasonicated for 30min, and finally RU.521NPs were obtained.

[0059] Test Example 1 The morphology of the CS NPs in Comparative Example 1 and the EVs suspension and RU.521-EVs NPs in Example 1 was observed by TEM. EVs is a substance with a double-layer membrane structure and is uneven in size. CS NPs are spherical polymers of uniform size, but after successfully loading EVs, the shape of RU.521-EVs NPs becomes irregular, and cavitation appears in the nanogel, which is the EVs ( Figure 1 When CS successfully cross-links with TPP to form nanogel, its color changes from transparent to milky white, and its viscosity increases ( Figure 1 b) in the above.

[0060] The particle sizes and surface potentials of the CS NPs in Comparative Example 1, the EVs NPs in Comparative Example 2, the RU.521 NPs in Comparative Example 3, and the EVs and RU.521-EVs NPs in Example 1 were measured by DLS. Figure 1 Figure c shows that the particle size of RU.521-EVs NPs is significantly larger than that of EVs and CS NPs, indicating that we have successfully encapsulated EVs. The surface potential results show that EVs have a negative potential, but after being encapsulated by CS, they become positively charged ( Figure 1 d) in.

[0061] Test Example 2

[0062] In order to detect the stability of the material, the RU.521-EVs anti-radiation nanogels synthesized in Examples 1 to 14 were left to stand for 24 hours and then observed for sedimentation. No precipitation was good, and precipitation was bad. The test results are shown in Table 1. Taking Example 1 as an example, the RU.521-EVs anti-radiation nanogel synthesized in Example 1 did not precipitate after standing for 24 hours, indicating that it has good stability ( Figure 1 e) in.

[0063] Test Example 3

[0064] BEAS-2B cells (5×10 4 / mL) were planted in confocal dishes (5 dishes, 1mL per dish) and incubated at 37°C until the cell density reached 50%. Each dish of cells was a group, for a total of 5 groups. The BEAS-2B cells in the Control group were not irradiated or treated with drugs, but were only treated with PBS (phosphate buffered saline). The irradiation group (IR), irradiation + RU.521NPs group (IR+RU.521NPs), irradiation + EVs NPs group (IR+EVs NPs), and irradiation + RU.521-EVs NPs group (IR+RU.521-EVsNPs) were treated with PBS, RU.521NPs (prepared in Comparative Example 3), EVs NPs (prepared in Comparative Example 2) and RU.521-EVs NPs (prepared in Example 1), respectively. After 2 hours, the four groups of cells were irradiated under an X-ray irradiator (radiation intensity of 8Gy). After incubating the above five groups of cells at 37°C for 72 hours, the cells were stained for live and dead using the Calcein-AM / PI cell double staining kit. The results showed that the IR+RU.521-EVs NPs group had the least red fluorescence among the irradiated groups, indicating that RU.521-EVs NPs had the best anti-irradiation effect ( Figure 2 a) in the figure.

[0065] Test Example 4

[0066] Cell cloning experiment: BEAS-2B cells (5×10 4 / mL) were planted in a 6-well plate (1mL per well) and incubated at 37°C until the cell density reached 50%. The cell grouping, drug treatment and irradiation steps of each group of cells were the same as those in Test Example 3, and the cells were incubated at 37°C for 24 hours after irradiation. Subsequently, the BEAS-2B cells were further digested and resuspended, counted and then plated (6-well plates), with 2000 cells in each well. After incubation in a 37°C incubator for 7 days, they were washed twice with PBS, stained with crystal violet dye for 30 minutes, the dye was discarded, and the cells were washed three times with PBS and photographed. The results showed that the number of cell clones in the IR group was the least, while the number of cell clones in the IR+RU.521-EVs NPs group increased significantly, reflecting the ability of RU.521-EVs anti-radiation nanogel to promote cell proliferation ( Figure 2 b) in the above.

[0067] Test Example 5

[0068] In order to further clarify the effects of irradiation intensity, irradiation time and RU.521-EVs NPs concentration on cell proliferation, the cells were quantitatively analyzed using the CCK-8 kit.

[0069] First, BEAS-2B cells (5×10 4 / mL) were planted in a 96-well plate (100 μL per well) and incubated at 37°C until the cell density reached 50%. Subsequently, the RU.521-EVs NPs prepared in Example 1 were diluted to different concentrations (0, 1.25, 2.5, 5, 10 μg / mL, calculated based on the protein amount of EVs), and the cells were treated separately. After 2 hours, each group of cells was irradiated with different irradiation doses under an X-ray irradiator (4, 6, 8Gy). After irradiation, the cells were incubated at 37°C. CCK-8 reagent was added to the 96-well plate of cells at 48h and 72h, respectively. After incubation at 37°C for 1h, OD 450 was detected with an enzyme marker, and the cell survival rate was calculated. The results showed that cells treated with 1.25-10μg / mL RU.521-EVs NPs had significant radiation resistance at the above-mentioned irradiation intensities and irradiation times ( Figure 2 c) in.

[0070] Test Example 6

[0071] The proliferation-promoting ability and cytotoxicity of RU.521-EVs NPs were detected.

[0072] First, BEAS-2B cells (5×10 4 / mL) were planted in a 96-well plate (100 μL per well) and incubated at 37°C until the cell density reached 50%. The RU.521-EVs NPs prepared in Example 1 were then diluted with PBS solution to different concentrations (0, 1.25, 2.5, 5, 10 μg / mL, calculated based on the protein amount of EVs), and the cells were treated separately. After 2 hours, each group of cells was irradiated with different irradiation doses under an X-ray irradiator (4, 6, 8Gy). After irradiation, each group of cells was incubated at 37°C, and CCK-8 reagent was added to each group of cells at 72h. After incubation at 37°C for 1h, OD 450 was detected with an enzyme marker, and the survival rate of the cells was calculated to detect its ability to promote the proliferation of irradiated cells.

[0073] BEAS-2B cells (5×10 4 / mL) were planted in a 96-well plate (100 μL per well) and incubated at 37°C until the cell density reached 80%. Subsequently, the RU.521-EVs NPs prepared in Example 1 were diluted to different concentrations (0, 1.25, 2.5, 5, 10 μg / mL, calculated based on the protein amount of EVs), and the cells were treated separately. After the cells were incubated at 37°C for 24 hours, CCK-8 reagent was added to the 96-well plate of the cells. After incubation at 37°C for 1 hour, OD 450 was detected with an enzyme marker, and the cell survival rate was calculated to detect its cytotoxicity.

[0074] The results are shown in Table 2, indicating that the higher the concentration of RU.521-EVs NPs, the stronger the anti-radiation effect, but at the same time the higher the cytotoxicity. Therefore, it is necessary to select an appropriate concentration of RU.521-EVs NPs to balance the relationship between the ability to promote radiation cell proliferation and cytotoxicity.

[0075] Table 2

[0076]

[0077] Test Example 7

[0078] BEAS-2B cells (5×10 4 / mL) were planted in 6-well plates (1mL per well) and incubated at 37°C until the cell density reached 50%. BEAS-2B cells in the IR group, IR+RU.521NPs group, IR+EVs NPs group and IR+RU.521-EVs NPs group were treated with PBS, RU.521NPs, EVs NPs and RU.521-EVs NPs (the source of the drug was the same as in Test Example 3), and 8Gy of irradiation was given to each group of cells 2 hours later. The BEAS-2B cells in the Control group were not irradiated or treated with drugs, but only treated with PBS. Then, each group of cells was incubated at 37°C for 72 hours, and then a DNA damage detection kit (γ-H2AX immunofluorescence method, Beyotime) was used to detect the DNA breakage of each group of cells. The stronger the green fluorescence, the more severe the DNA damage. The experimental results showed that the IR group had strong green fluorescence, indicating severe DNA damage. In contrast, the IR+RU.521-EVs NPs group had the least green fluorescence, indicating the slightest DNA damage ( Figure 3 a) in the figure.

[0079] Test Example 8

[0080] In order to further detect the anti-lipid peroxidation effect of RU.521-EVs radiation-resistant nanogel, the material processing and irradiation conditions were the same as those in test example 7, and then each group of cells was incubated at 37°C for 72 hours, and then verified with BODIPY 581 / 591-C11 probe and MDA detection kit respectively. First, 10μM BODIPY 581 / 591-C11 probe working solution and each group of BEAS-2B cells were incubated at 37°C for 30 minutes, then washed three times with PBS and observed under a laser confocal microscope. When lipid peroxidation occurs, the probe will change from red fluorescence to green fluorescence. The results showed that the green fluorescence of the IR+RU.521-EVsNPs group was the weakest, showing the slightest lipid peroxidation ( Figure 3 b). The test results of the MDA detection kit also showed that the IR+RU.521-EVs NPs group had the strongest anti-lipid peroxidation ability ( Figure 3 e) in.

[0081] Similarly, we used SOD detection kit and CAT kit to detect the antioxidant capacity of each group, and the results showed that the IR+RU.521-EVs NPs group had the strongest antioxidant capacity ( Figure 3 c, d).

[0082] Test Example 9

[0083] To further verify the anti-inflammatory and antioxidant effects of RU.521-EVs radiation-resistant nanogels in vivo. First, C57 / BL mice (12 mice in each group) in the IR group, IR+RU.521NPs group, IR+EVs NPs group, and IR+RU.521-EVs NPs group were intraperitoneally anesthetized with avertin (30 μL / g), and the head, neck, and lower chest of the mice were covered with lead blocks, leaving only the chest exposed. Then, 15 Gy of irradiation was given, and the day was recorded as D1. Subsequently, PBS (IR group), RU.521NPs (IR+RU.521NPs group), EVs NPs (IR+EVs NPs group), and RU.521-EVs NPs (IR+RU.521-EVsNPs group) were given on D1, D4, D7, D10, and D13, respectively. On D14, 6 mice in each group were euthanized, and the lung tissues of 3 mice in each group were embedded, sliced, and then stained with H&E and myeloperoxidase (MPO). The lung tissues of the other 3 mice in each group were made into lung homogenate using PBS, grinding beads and a tissue homogenizer, and then the supernatant of the lung homogenate was further tested using ELISA test kits (IL-1β, IL-6 and TNF-α), MDA test kits, SOD test kits, and CAT test kits.

[0084] The results of H&E staining showed that the IR group had obvious changes in lung tissue structure, including alveolar congestion, interstitial edema, and inflammatory cell infiltration. In comparison, the alveolar tissue structure of the IR+RU.521-EVs NPs group remained relatively clear, with only a small amount of edema and focal inflammatory cell infiltration in the alveolar cavity, and the degree of lung injury was significantly milder than that of the other groups ( Figure 4 a) in the figure.

[0085] MPO is a heme peroxidase, and changes in its level and activity can serve as an indicator of neutrophil function and activation status and provide insights into the extent of neutrophil infiltration in lung tissue.

[0086] Immunohistochemistry (IHC) analysis showed that the IR+RU.521-EVs NPs group had the lowest MPO expression, indicating minimal neutrophil infiltration ( Figure 4 b). In addition, ELISA was used to evaluate the levels of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) in lung tissue. The results showed that the IR+RU.521-EVs NPs group had the least production of pro-inflammatory cytokines, indicating the lowest level of inflammation ( Figure 4 ce in the.

[0087] The results of the MDA detection kit showed that the IR+RU.521-EVs NPs group had the strongest lipid antioxidant capacity in vivo ( Figure 4f). The results of SOD and CAT detection kits showed that the IR+RU.521-EVs NPs group had the strongest antioxidant capacity in vivo ( Figure 4 in gh).

[0088] Test Example 10

[0089] Based on the experiment of Test Example 9, the remaining 6 mice in each group were euthanized on D30, and the lung tissues of 3 mice in each group were then subjected to H&E staining, Masson trichrome staining and MPO staining, and the lung tissues of the other 3 mice in each group were made into lung homogenates and then subjected to ELISA detection, MDA detection, SOD detection and CAT detection.

[0090] The results of H&E staining showed that the IR group still had obvious changes in lung tissue structure after one month, including alveolar congestion, interstitial edema, and inflammatory cell infiltration. In contrast, the IR+RU.521-EVs NPs group had the mildest lung injury and the least lung structure changes ( Figure 5 a) in the figure.

[0091] After Masson's trichrome staining, muscle fibers appear red and collagen fibers appear blue, which is mainly used to distinguish collagen fibers from muscle fibers. Masson's trichrome staining is used to detect the level of lung tissue fibrosis. The results of Masson's trichrome staining showed that the control group had obvious blue fibers, namely collagen fibers, representing pulmonary fibrosis. On the contrary, almost no blue fibers were seen in the IR+RU.521-EVs NPs group, indicating that RU.521-EVs NPs have the potential to inhibit the occurrence and development of pulmonary fibrosis in the early stages ( Figure 5 b) in the above.

[0092] MPO staining analysis showed that the IR+RU.521-EVs NPs group had the lowest MPO expression in the first month, indicating the least neutrophil infiltration ( Figure 5 In addition, the levels of IL-1β, IL-6, and TNF-α in lung tissues evaluated by ELISA also showed that the IR+RU.521-EVs NPs group had the least production of pro-inflammatory cytokines, indicating the lowest level of inflammation ( Figure 5 df in .

[0093] The results of the MDA detection kit showed that the IR+RU.521-EVs NPs group had the strongest lipid antioxidant capacity in vivo ( Figure 5 g in the figure). The results of SOD and CAT detection kits showed that the IR+RU.521-EVs NPs group had the strongest antioxidant capacity in vivo ( Figure 5 in the text box).

[0094] Test Example 11

[0095] In order to further promote the clinical application of RU.521-EVs anti-radiation nanogels, a comprehensive evaluation of their toxicity is needed. The cytotoxicity of RU.521NPs, EVs NPs, and RU.521-EVs NPs was evaluated on BEAS-2B cells. 4 / mL) were planted in a 96-well plate and incubated at 37°C until the cell density reached 80%. Subsequently, different concentrations of RU.521NPs, EVs NPs and RU.521-EVs NPs (0, 1.25, 2.5, 5, 10μg / mL, calculated based on the protein amount of EVs, the drug source is the same as in Test Example 3) were given to treat the cells. After incubation at 37°C for 24h, CCK-8 reagent was added. After further incubation at 37°C for 1h, OD 450 was detected by a microplate reader and the cell survival rate was calculated. The CCK-8 results showed that RU.521NPs, EVsNPs and RU.521-EVs NPs did not show obvious cytotoxicity even at 20μg / mL (calculated based on the protein amount of EVs) Figure 6 a) in the figure.

[0096] We then performed cell death and viability staining tests. First, BEAS-2B cells (5×10 4 / mL) were planted in a confocal dish and incubated at 37°C until the cell density reached 80%. Subsequently, the cells were treated with 10μg / mL (calculated based on the protein amount of EVs) of RU.521NPs, EVs NPs and RU.521-EVs NPs (the source of the drug was the same as in Test Example 3), incubated at 37°C for 24h, and then stained with a cell death and viability staining kit (Calcein-AM / PI live cell / dead cell double staining kit) and observed under laser confocal. The cell death and viability staining experiment also confirmed that no obvious cell death was caused at the therapeutic dose ( Figure 6 b) in the above.

[0097] The long-term potential side effects of RU.521NPs, EVs NPs, and RU.521-EVs NPs were evaluated in healthy mice. A 50 μL therapeutic dose of RU.521NPs, EVs NPs, and RU.521-EVs NPs (drug sources were the same as those in Test Example 3) was instilled into the airways of mice. On the 14th day, the hearts, livers, spleens, lungs, and kidneys of the mice were fixed, sliced, and stained with H&E. The results showed that the tissue structure of these organs remained normal, with no obvious signs of inflammatory invasion, indicating that RU.521NPs, EVs NPs, and RU.521-EVs NPs had good in vivo biosafety ( Figure 6 c) in.

[0098] The above results indicate that RU.521-EVs anti-radiation nanogel has no obvious negative effects on the major organs of mice.

Claims

1. A RU.521-EVs radiation-resistant nanogel, characterized in that: The RU.521-EVs radiation-resistant nanogel is a nanogel obtained by loading extracellular vesicles from microalgae and a small molecule compound RU.521 on chitosan nanoparticles formed by cross-linking chitosan.

2. The RU.521-EVs radiation-resistant nanogel according to claim 1, characterized in that: The chitosan nanoparticles are nanoparticles made of chitosan using sodium tripolyphosphate as a cross-linking agent; and the microalgae are Chlorella vulgaris.

3. The RU.521-EVs radiation-resistant nanogel according to claim 1, characterized in that: In the RU.521-EVs radiation-resistant nanogel, the mass ratio of chitosan, protein in the extracellular vesicles derived from microalgae, and the small molecule compound RU.521 is 2.0-8.0: 0.3-1.4: 0.0001-0.

001.

4. The method for preparing the RU.521-EVs radiation-resistant nanogel according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) dissolving chitosan in a solvent to obtain a chitosan solution, adding a pH adjuster to adjust the pH value of the chitosan solution to 3-7, and obtaining a solution 1; (2) extracting extracellular vesicles of microalgae and dispersing them in a buffer to prepare an EVs suspension, adding the EVs suspension to solution 1, and stirring to obtain solution 2; (3) preparing a crosslinker solution, adding RU.521 to the crosslinker solution to obtain solution 3, adding solution 3 to solution 2 to obtain solution 4, stirring and ultrasonicating solution 4 to obtain the RU.521-EVs radiation-resistant nanogel.

5. The method for preparing the RU.521-EVs radiation-resistant nanogel according to claim 4, characterized in that: In step (1), the solvent is an acetic acid aqueous solution; and the concentration of the chitosan solution is 3-10 mg / mL.

6. The method for preparing the RU.521-EVs radiation-resistant nanogel according to claim 4, characterized in that: In step (2), the method for extracting the extracellular vesicles of the microalgae is ultracentrifugation, ultrafiltration, immunoaffinity, precipitation or microfluidics-based separation.

7. The method for preparing RU.521-EVs radiation-resistant nanogel according to claim 4, characterized in that: In step (2), the protein concentration of solution 2 is 0.5-1.5 mg / mL, and the mixing volume ratio of EVs suspension and solution 1 is 1:8-12.

8. The method for preparing RU.521-EVs radiation-resistant nanogel according to claim 4, characterized in that: In step (3), the cross-linking agent solution is a sodium tripolyphosphate aqueous solution, and the concentration of the sodium tripolyphosphate aqueous solution is 1wt%-3wt%; RU.521 is first dissolved in a solvent to prepare a RU.521 solution, and then the RU.521 solution is added to the sodium tripolyphosphate aqueous solution, wherein the concentration of the RU.521 solution is 5-20 μM; In step (3), the volume ratio of solution 3 to solution 2 is 1:3-1:

7.

9. Use of the RU.521-EVs anti-radiation nanogel described in any one of claims 1 to 3 in the preparation of anti-radiation drugs.

10. An anti-radiation drug, characterized in that: The active ingredient of the anti-radiation drug is the RU.521-EVs anti-radiation nanogel described in any one of claims 1 to 3.

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