Ru.521-evs anti-radiation nanogel and preparation method and application thereof

Abstract

The application provides an RU.521-EVs anti-radiation nanogel and a preparation method and application thereof. The RU.521-EVs anti-radiation nanogel is obtained by loading microalgal-derived extracellular vesicles and a small molecule compound RU.521 on chitosan nanoparticles formed by cross-linking of chitosan. The RU.521-EVs anti-radiation nanogel has stronger adhesion and permeability in the lung and can effectively release RU.521 and EVs, and the RU.521 and EVs can synergistically exert anti-inflammatory and antioxidant effects, effectively start the local antioxidant and anti-inflammatory defense response of the lung, and exhibit excellent anti-radiation effect, and can be used for preventing and treating radiation-induced lung injury.

Description

TECHNICAL FIELD

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

[0002] Radiotherapy (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, which easily induces radiation-induced lung injury (RILI), causing cough, chest tightness, dyspnea, and even respiratory failure, ultimately endangering life. Glucocorticoids are currently the commonly used and effective drugs for treating RILI, but their side effects are obvious. Systemic use of glucocorticoids can easily cause serious side effects such as infection, gastric ulcer, and femoral head necrosis, greatly affecting the quality of life of patients, so glucocorticoids are not suitable for long-term treatment or for preventing RILI. At present, there is a lack of ideal treatment measures for RILI, and therefore, there is an urgent need to find new targets and new methods for preventing and treating RILI.

[0003] The cGAS (cyclic guanosine-phosphate adenosine synthetase)-STING (stimulator of interferon genes) signaling pathway is a newly identified important signal transduction pathway that regulates innate immune responses, and has gradually become a key mediator of inflammatory responses under conditions of infection, cell stress, and tissue damage. Studies have shown that the cGAS-STING signaling pathway plays a key role in the development of various inflammatory diseases.

[0004] Patent application with publication number US20220143035A1 discloses a method for treating an autoimmune or autoinflammatory disease, which comprises administering a cGAS inhibitor and / or a STING inhibitor, the cGAS inhibitor comprising 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 application 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 cell-released components. Studies have shown that plant EVs bind well with cell lipids, can penetrate cells, and are therefore fully taken up by cells.

[0006] Patent application with publication number CN118201625A discloses a composition containing extracellular vesicles (MEVs) from microalgae, wherein the microalgae are Chlorella, and the composition can be used as a vaccine, an anticancer therapeutic agent, a diagnostic agent, and the like.

[0007] Pulmonary airway instillation is a non-invasive method of drug delivery through the bronchial route, which can achieve good local drug delivery and minimize systemic side effects, and 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 lung mucosa to be absorbed. Moreover, the lung's own clearance capacity also makes the drug less effective in the lungs. Chitosan (CS) is a cationic polysaccharide formed by deacetylation of chitin, which has high biocompatibility, biodegradability, and 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 form a positively charged soluble polymer, and can interact with the negatively charged sialic acid in mucus, with stronger adhesion and stronger permeability in the lungs. SUMMARY

[0008] To provide a drug that can effectively treat RILI with fewer side effects, the present application provides a RU.521-EVs anti-radiation nanogel and a preparation method and application thereof. The RU.521-EVs anti-radiation nanogel has stronger adhesion and permeability in the lungs, can effectively release RU.521 and EVs, effectively initiate local antioxidant and anti-inflammatory defense responses in the lungs, exhibit excellent anti-radiation effects, and can be used for the prevention and treatment of radiation-induced lung injury.

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

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

[0011] Preferably, in the RU.521-EVs anti-radiation nanogel, the mass ratio of chitosan, protein in the microalgal-derived extracellular vesicles 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 the inflammatory response. The EVs of Chlorella inherit the functions of Chlorella and have antioxidant, anti-inflammatory and immunomodulatory effects. The RU.521-EVs anti-radiation nanogel can protect surrounding healthy cells by eliminating the production of ROS (reactive oxygen species), preventing ROS-induced mitochondrial damage and DNA damage. Since the RU.521-EVs anti-radiation nanogel is modified with CS, it has the ability to adhere to the lungs and penetrate the mucosa, allowing for local administration.

[0013] The present application provides a preparation method of the RU.521-EVs anti-radiation nanogel, which comprises the following steps:

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

[0015] (2) Extract the EVs of microalgae and disperse them in a buffer to prepare an EVs suspension, and add the EVs suspension to solution 1, stir uniformly to obtain solution 2;

[0016] (3) Prepare a crosslinking agent solution, add RU.521 to the crosslinking agent solution to obtain solution 3, add solution 3 to solution 2 to obtain solution 4, and stir and ultrasonic the solution 4 to obtain the RU.521-EVs anti-radiation nanogel.

[0017] Preferably, in step (1), the solvent is an aqueous acetic acid solution, and further preferably a 1wt% 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 3-10mg / mL.

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

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

[0021] Further preferably, the operating conditions of the ultracentrifugation method are 150000xg centrifugation at 4℃ for 1.5h.

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

[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, so that the EVs of microalgae and chitosan are pre-mixed together.

[0024] Preferably, in step (3), the crosslinking agent solution is a TPP aqueous solution, the concentration of the TPP aqueous solution is 1wt%-3wt%, RU.521 is dissolved in a solvent to prepare a RU.521 solution, then the RU.521 solution is added to the TPP aqueous solution, the concentration of the RU.521 solution is 5-20 μM, the solvent is preferably DMSO (dimethyl sulfoxide), and the volume ratio of solution 3 and 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 5-10 μM, and the volume ratio of solution 3 and solution 2 is 1:3-1:5, so that the stability of the prepared RU.521-EVs anti-radiation nanogel is better.

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

[0027] The application also provides an anti-radiation drug, and the active ingredient of the anti-radiation drug is the above-mentioned RU.521-EVs anti-radiation nanogel.

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

[0029] The application provides an RU.521-EVs anti-radiation nanogel and a preparation method and application thereof. The RU.521-EVs anti-radiation nanogel can effectively release RU.521 and EVs, and the RU.521 and EVs can synergistically play an anti-inflammatory and antioxidant role, can reduce mitochondrial damage, DNA breakage and the like of cells after irradiation, and can be used for treating radiation-induced lung injury after radiotherapy. In vitro, the RU.521-EVs anti-radiation nanogel can improve the inflammatory state of cells, reduce the lipid peroxidation level, and restore the proliferation activity of cells. In addition, the RU.521-EVs anti-radiation nanogel can significantly improve the survival rate of radiation-induced lung injury mice, reduce the inflammatory response of the lung lesion site, and finally plays a role in preventing and treating radiation-induced lung injury. Therefore, the RU.521-EVs anti-radiation nanogel is a very potential material for preventing and treating radiation-induced lung injury. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 Figure 1 is a characterization diagram of the RU.521-EVs anti-radiation nanogel. Wherein a diagram: transmission electron microscopy (TEM) diagrams of EVs, CS NPs and RU.521-EVs NPs; b diagram: photos of CS, CS NPs, EVs NPs, RU.521 NPs and RU.521-EVs NPs; c diagram: particle size diagrams of EVs, CS NPs, EVs NPs, RU.521 NPs and RU.521-EVs NPs; d diagram: potential diagrams of EVs, CS NPs, EVs NPs, RU.521 NPs and RU.521-EVs NPs; e diagram: photos of RU.521-EVs NPs before and after standing for 24 hours.

[0031] Figure 2 Figure 2 is an effect of the RU.521-EVs anti-radiation nanogel on the proliferation ability of irradiated cells. Wherein a diagram: cell death and live staining of the blank group, the irradiation group and the irradiation group+various treatment groups; b diagram: crystal violet staining diagram of cell colony formation experiment of different groups of cells; c diagram: CCK8 detection of cell survival rates of different treatment groups after being treated at 4, 6, 8Gy for 48h and 72h. * represents P<0.05, and ** represents P<0.01.

[0032] Figure 3 Figure 3 is an antioxidant capacity of the RU.521-EVs anti-radiation nanogel. Wherein a diagram: fluorescence diagrams of different treatment groups after DAPI and γ-H2A.X staining; b diagram: fluorescence diagrams of different treatment groups after BODIPY 581 / 591-C11 probe staining; c diagram: SOD activity of different treatment groups; d diagram: CAT activity of different treatment groups; e diagram: MDA detection of lipid peroxidation capacity of different treatment groups. ** represents P<0.01.

[0033] Figure 4 Figure 2 shows the in vivo radiation-resistant effect of RU.521-EVs nanogels at week 2. Panel a: H&E staining of each group; Panel b: MPO immunohistochemical staining of each group; Panel c: Quantitative analysis of IL-1β, IL-6, and TNF-α levels in lung homogenates from different treatment groups; Panel f: MDA assay for lipid peroxidation in different treatment groups; Panel g: SOD activity in different treatment groups; Panel h: CAT activity in different treatment groups. * indicates P < 0.05, ** indicates P < 0.01.

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

[0035] Figure 6 Figure 5: The in vitro and in vivo safety of RU.521-EVs radiation-resistant nanogels. Figure a: CCK8 assay for 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 major organs (heart, liver, spleen, lung, and kidney) in each group. DETAILED DESCRIPTION

[0036] Experimental Materials:

[0037] Chitosan was purchased from Sigma-Aldrich (USA). Trisodium phosphate (TPP) was purchased from Macklin. Chlorella vulgaris was purchased from Shanghai Guangyu Biotechnology Co., Ltd. Fetal bovine serum (FBS), DMEM medium were purchased from Gibco. The special medium for MH-S cells was purchased from Ponsay (Wuhan, China). Double distilled water (ddH2O) was from a 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 method) were purchased from Biyun Tian. BODIPY 581 / 591-C11 probe was purchased from Thermo Fisher Scientific (China). CCK-8 detection kit was purchased from Meilunbio. SOD detection kit, CAT detection kit and ELISA detection kit were purchased from Elabscience. MDA detection kit was purchased from Solabio.

[0038] Characterization instruments:

[0039] Dynamic light scattering (DLS) was used to measure the surface potential and particle size 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 anti-radiation nanogel comprises the following steps:

[0042] (1) Chitosan (CS) was dissolved in 1 wt% acetic acid aqueous solution, 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 centrifugation (4000 x g, 10 min) of the Chlorella vulgaris suspension, the supernatant was further centrifuged (10000 x g, 30 min), and the supernatant was further filtered with a 0.22 μm filter membrane and then subjected to ultracentrifugation (150000 x g, 90 min). The precipitate was then collected as EVs (extracellular vesicles of Chlorella vulgaris), and the EVs were resuspended in PBS (phosphate buffered saline) to obtain an EVs suspension.

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

[0045] (4) RU.521 was dissolved with DMSO (dimethyl sulfoxide) to make its concentration 10 mM, and then the RU.521 solution was diluted to 10 μM with normal saline.

[0046] (5) A 2 wt% TPP solution was configured with ddH2O, 10 μM RU.521 solution and 2 wt% TPP aqueous solution were mixed in a volume ratio of 1:1 to obtain solution 3. Solution 3 was added dropwise into the stirring solution 2 to obtain solution 4, wherein the volume ratio of solution 3 and solution 2 was 1:5. Then solution 4 was stirred at room temperature for 30 min, and then ultrasonic for 30 min, finally RU.521-EVs anti-radiation nanogel, referred to as RU.521-EVs NPs, was obtained.

[0047] Examples 2-14

[0048] The difference between Example 1 and Examples 2-14 is that some parameters of the preparation process of Examples 2-14 are adjusted, and the adjustment details are shown in Table 1.

[0049] Table 1

[0050]

[0051]

[0052] The results show that the stability of RU.521-EVs anti-radiation nanogel under different reaction conditions is different, and its stability can be adjusted by adjusting the above parameters.

[0053] Comparative Example 1

[0054] After the CS was crosslinked with TPP, chitosan nanogel, referred to as CS NPs, was obtained. The specific preparation process is as follows: CS was dissolved into 1 wt% acetic acid aqueous solution, stirred at room temperature for 12 h to obtain a CS solution with a concentration of 3 mg / mL. Then, 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. A 1 wt% TPP solution was configured with ddH2O, which was added dropwise into the stirring solution 1 to obtain solution 2, wherein the volume ratio of the TPP solution and solution 1 was 1:5. Then solution 2 was stirred at room temperature for 30 min, and then ultrasonic for 30 min, finally CS NPs was obtained.

[0055] Comparative Example 2

[0056] Only EVs were encapsulated on CS NPs to obtain EVs NPs. The specific preparation process is as follows: CS was dissolved in a 1wt% aqueous acetic acid 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 of the CS solution was adjusted to 5 with NaOH to obtain solution 1. According to the steps in Example 1, an EVs suspension was prepared and quantified. 500μL of EVs suspension was added to 5mL of solution 1 to make the protein concentration in the resulting solution 1mg / mL. The solution was stirred at 4°C for 30min to obtain solution 2. A 1wt% TPP solution was prepared with ddH2O and added dropwise to 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 sonicated for 30min to finally obtain EVs NPs.

[0057] Comparative Example 3

[0058] Only RU.521 was coated 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 added 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, and then ultrasonicated for 30min to finally obtain RU.521NPs.

[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 are a substance with a double-layer membrane structure and are 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 vacuoles appear in the nanogel. These vacuoles are the encapsulated EVs ( Figure 1 (a in the figure, where the red arrows point to vacuoles). When CS successfully cross-links with TPP to form nanogels, its color changes from transparent to milky white, and its viscosity increases ( Figure 1 b) in the above example.

[0060] The particle size and surface potential of CS NPs in Comparative Example 1, EVs NPs in Comparative Example 2, RU.521 NPs in Comparative Example 3, and EVs and RU.521-EVs NPs in Example 1 were measured by DLS, Figure 1 c in the table 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 are negatively charged, and after being encapsulated by CS, they become positively charged Figure 1 d in the table.

[0061] Detection Example 2

[0062] In order to detect the stability of the material, the RU.521-EVs anti-radiation nanogel synthesized in Examples 1-14 was observed for sedimentation after standing for 24 h. No precipitation is good, and precipitation is poor. The detection 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 h, indicating that it has good stability Figure 1 e in the table.

[0063] Detection Example 3

[0064] BEAS-2B cells (5x10 4 The cells were incubated at 37°C until the cell density reached 50%. Each cell was a group, and there were 5 groups in total. The BEAS-2B cells in the Control group were not irradiated and not treated with drugs, but only treated with PBS (phosphate buffered saline). The cells in the irradiation group (IR), the irradiation+RU.521 NPs group (IR+RU.521 NPs), the irradiation+EVs NPs group (IR+EVs NPs), and the irradiation+RU.521-EVs NPs group (IR+RU.521-EVs NPs) were treated with PBS, RU.521 NPs (prepared in Comparative Example 3), EVs NPs (prepared in Comparative Example 2), and RU.521-EVs NPs (prepared in Example 1), respectively. After 2 h, the cells in the four groups were irradiated in an X-ray irradiator (irradiation intensity was 8 Gy). After incubating the above five groups of cells at 37°C for 72 h, the cells were stained with a Calcein-AM / PI cell double staining kit. The results show that the IR+RU.521-EVs NPs group has the least red fluorescence among the groups treated by irradiation, indicating that the anti-irradiation effect of RU.521-EVs NPs is the best Figure 2 a in the table.

[0065] Detection Example 4

[0066] Cell Cloning Experiment: BEAS-2B cells (5 x 10 4 The cells were grouped and the drug treatment and irradiation steps of each group were the same as in Test Example 3, and after irradiation, the cells were incubated at 37°C for 24 h. Subsequently, the BEAS-2B cells were further digested and resuspended, counted and then plated (6-well plates) with 2000 cells per well, and incubated in a 37°C incubator for 7 days, then washed twice with PBS, stained with crystal violet dye for 30 min, and the dye was discarded, washed three times with PBS and photographed. The results showed that the cell colony number of the IR group was the least, while the cell colony number of the IR + RU.521-EVs NPs group increased significantly, which reflected the ability of RU.521-EVs anti-radiation nanogel to promote cell proliferation Figure 2 b) of the method in

[0067] Test Example 5

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

[0069] First, BEAS-2B cells (5 x 10 4 were planted in 96-well plates (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 by the protein amount of EVs), and the cells were treated, respectively. After 2 h, the cells in each group were irradiated with different irradiation doses (4, 6, 8 Gy) in an X-ray irradiator. After irradiation, the cells were incubated at 37°C. At 48 h and 72 h, CCK-8 reagent was added to the 96-well plates of the cells, and after incubation at 37°C for 1 h, the OD 450 was detected by a microplate reader, and the survival rate of the cells was calculated. The results showed that the cells treated with 1.25-10 μg / mL of RU.521-EVs NPs had significant anti-radiation ability under the above irradiation intensity and irradiation time Figure 2 c) of the method in

[0070] Test Example 6

[0071] Test the anti-radiation ability of RU.521-EVs NPs and the ability to promote cell proliferation.

[0072] First, BEAS-2B cells (5 x 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 the 72nd hour. After incubation at 37°C for 1 hour, OD 450 was detected with a microplate reader, and the cell survival rate was calculated to detect its ability to promote the proliferation of irradiated cells.

[0073] BEAS-2B cells (5×10 4 / mL) were seeded in a 96-well plate (100 μL per well) and incubated at 37°C until the cell density reached 80%. The RU.521-EVs NPs prepared in Example 1 were then diluted to different concentrations (0, 1.25, 2.5, 5, 10 μg / mL, calculated based on the protein amount of EVs) and treated with the cells respectively. The cells were incubated at 37°C for 24 hours, and then CCK-8 reagent was added to the 96-well plate of cells. After incubation at 37°C for 1 hour, OD 450 was detected using a microplate reader, 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 seeded in a 6-well plate (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, respectively (the source of the drug was the same as in Test Example 3). 2 hours later, the cells in each group were irradiated with 8Gy. The BEAS-2B cells in the Control group were not irradiated or treated with drugs, but only with PBS. The cells in each group were then incubated at 37°C for 72 hours, and then a DNA damage detection kit (γ-H2AX immunofluorescence assay, Beyotime) was used to detect the DNA breakage of the cells in each group. 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 showed the least green fluorescence, indicating the mildest DNA damage ( Figure 3 a) in the above.

[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, 10 μM 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, indicating the slightest lipid peroxidation ( Figure 3 The 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 the.

[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 Case 9

[0083] To further verify the anti-inflammatory and antioxidant effects of RU.521-EVs anti-radiation nanogels in vivo. First, C57 / BL mice (12 in each group) in IR group, IR+RU.521 NPs group, IR+EVs NPs group and IR+RU.521-EVs NPs group were anesthetized with Avertin (30 μL / g) and then the head and neck and the part below the chest of the mice were covered with a lead block, only the chest was exposed, and then 15 Gy of irradiation was given, which was recorded as D1, and then PBS (IR group), RU.521 NPs (IR+RU.521 NPs group), EVs NPs (IR+EVs NPs group), and RU.521-EVs NPs (IR+RU.521-EVs NPs group) were given on D1, D4, D7, D10, and D13, respectively. On D14, 6 mice from each group were euthanized, and 3 mice from each group were subjected to embedding, sectioning, and other treatments, and then H&E staining and myeloperoxidase (MPO) staining were performed. The lung homogenate of the other 3 mice from each group was prepared using PBS, grinding beads, and a tissue homogenizer, and then the supernatant of the lung homogenate was further tested using ELISA kits (IL-1β, IL-6, and TNF-α), MDA detection kit, SOD detection kit, and CAT detection kit.

[0084] The results of H&E staining showed that the IR group had obvious changes in lung tissue structure, including interalveolar space congestion, interstitial edema, and inflammatory cell infiltration. In contrast, 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 lighter than that of the other groups Figure 4

[0085] MPO is a heme peroxidase, and changes in its level and activity can be used as an indicator of neutrophil function and activation state, and provide insight into the degree of neutrophil infiltration in lung tissue.

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

[0087] The results of the MDA detection kit showed that the in vivo lipid antioxidant capacity of the IR+RU.521-EVs NPs group was the strongest Figure 4 ​​f) of FIG. 6. The results of SOD and CAT detection kits showed that IR+RU.521-EVs NPs group had the strongest antioxidant capacity in vivo Figure 4 g-h) of FIG. 6.

[0088] Example 10

[0089] Based on the experiment of Example 9, the remaining 6 mice in each group were euthanized on D30, and then the lung tissues of 3 mice in each group were subjected to H&E staining, Masson trichrome staining and MPO staining, and the lung tissues of another 3 mice in each group were made into lung homogenate for 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 pulmonary interstitial congestion, interstitial edema and inflammatory cell infiltration. In contrast, the lung injury of the IR+RU.521-EVs NPs group was the lightest, and the lung structure changed the least Figure 5 a) of FIG. 7.

[0091] After Masson trichrome staining, muscle fibers were red and collagen fibers were blue, which was mainly used to distinguish collagen fibers and muscle fibers. Masson trichrome staining was used to detect the level of pulmonary fibrosis. The results of Masson trichrome staining showed that the control group had very obvious blue fibers, i.e. collagen fibers, which represented the occurrence of pulmonary fibrosis. In contrast, the IR+RU.521-EVs NPs group hardly saw blue fibers, indicating that RU.521-EVs NPs had the potential to inhibit the occurrence and development of pulmonary fibrosis in the early stage Figure 5 b) of FIG. 7.

[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 c) of FIG. 7. In addition, the evaluation of the levels of IL-1β, IL-6 and TNF-α in lung tissues using ELISA also showed that the IR+RU.521-EVs NPs group had the least production of proinflammatory cytokines, indicating the lowest level of inflammation Figure 5 d-f) of FIG. 7.

[0093] The results of MDA detection kit showed that the IR+RU.521-EVs NPs group had the strongest lipid antioxidant capacity in vivo Figure 5 g) of FIG. 7. 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 h-i) of FIG. 7.

[0094] Example 11

[0095] To further facilitate the clinical application of RU.521-EVs anti-radiation nanogels, it is necessary to comprehensively evaluate its toxicity. The cytotoxicity of RU.521 NPs, EVs NPs and RU.521-EVs NPs was evaluated on BEAS-2B cells. First, BEAS-2B cells (5 x 10 4 Figure 6

[0096] Subsequently, we also carried out cell death and live staining detection. First, BEAS-2B cells (5 x 10 4 Figure 6

[0097] The long-term potential side effects of RU.521 NPs, EVs NPs and RU.521-EVs NPs were evaluated in healthy mice. The mice were respectively instilled with 50 μL of therapeutic dose of RU.521 NPs, EVs NPs and RU.521-EVs NPs (the drug source is the same as in Test Example 3) in the airway. On the 14th day, the heart, liver, spleen, lung and kidney of the mice were fixed, sectioned and H&E stained. The results showed that the tissue structure of these organs remained normal, and there was no obvious inflammation invasion symptoms, indicating that the in vivo biological safety of RU.521 NPs, EVs NPs and RU.521-EVs NPs was good Figure 6

[0098] ​​​​​The above results show that the RU.521-EVs anti-radiation nanogel has no obvious negative impact on the main organs of mice.

Claims

1. A RU.521-EVs anti-radiation nanogel, characterized in that, The preparation method of the RU.521-EVs anti-radiation nanogel comprises the following steps: (1) dissolving chitosan in a solvent to obtain a chitosan solution, adding a pH regulator to adjust the pH value of the chitosan solution to 3-7 to obtain 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, stirring uniformly to obtain solution 2; (3) preparing a crosslinking agent solution, adding RU.521 to the crosslinking agent 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 anti-radiation nanogel.

2. The RU.521-EVs anti-radiation nanogel according to claim 1, wherein, The crosslinking agent is sodium tripolyphosphate; and the microalgae are Chlorella vulgaris.

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

001.

4. The RU.521-EVs anti-radiation nanogel according to claim 1, wherein, In step (1), the solvent is an aqueous acetic acid solution; and the concentration of the chitosan solution is 3-10 mg / mL.

5. The RU.521-EVs anti-radiation nanogel according to claim 1, wherein, In step (2), the method for extracting extracellular vesicles of microalgae is ultracentrifugation, ultrafiltration, immunoaffinity, precipitation or microfluidic-based separation.

6. The RU.521-EVs anti-radiation nanogel according to claim 1, wherein, In step (2), the protein concentration of solution 2 is 0.5-1.5 mg / mL, and the volume ratio of the EVs suspension to solution 1 is 1:8-12.

7. The RU.521-EVs anti-radiation nanogel according to claim 1, wherein, In step (3), the crosslinking agent solution is an aqueous sodium tripolyphosphate solution, and the concentration of the aqueous sodium tripolyphosphate 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 aqueous sodium tripolyphosphate solution; and 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.

8. Use of the RU.521-EVs anti-radiation nanogel of any one of claims 1-7 in the preparation of an anti-radiation drug.

9. An anti-radiation medicament, characterized in that, The active ingredient of the anti-radiation drug is the RU.521-EVs anti-radiation nanogel of any one of claims 1-7.

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