Exosome for treating radioactive oral mucositis and preparation method thereof

Abstract

This invention relates to the field of biomedical technology, specifically disclosing an exosome for treating radiation-induced oral mucositis and its preparation method. The exosomes are prepared from human umbilical cord mesenchymal stem cells pretreated with FC-tagged IL-11, and thalidomide is encapsulated within the exosome lumen through incubation to form HUCMSCs. FI -exo-THD. This exosome exhibits excellent biocompatibility and targeting, significantly improving the delivery efficiency and therapeutic effect of thalidomide in the treatment of radiation-induced oral mucositis. Experimental results show that, compared with free thalidomide and other single-treatment exosomes, HUCMSCs... FI -exo-THD exhibits more significant effects in restoring cell viability and inhibiting apoptosis in X-ray-induced damage to human oral mucosal epithelial cells, increasing cell viability by more than 30% and reducing the apoptosis rate to below 10%. The preparation method of this invention is simple, reproducible, and suitable for large-scale production, providing a new strategy for the clinical treatment of radiation-induced oral mucositis.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to an exosome for treating radiation-induced oral mucositis and its preparation method. Background Technology

[0002] Radiation-induced oral mucositis (RIOM) is a common complication in patients with head and neck tumors undergoing radiotherapy. It is characterized by inflammation, ulceration, pain, and functional impairment of the oral mucosa, severely impacting patients' quality of life and treatment adherence. The pathogenesis of RIOM involves direct radiation damage to oral mucosal epithelial cells, leading to apoptosis, oxidative stress, and exacerbated inflammation. Current clinical treatments mainly include analgesics, oral moisturizers, growth factors, and anti-inflammatory drugs. However, these methods have limitations in efficacy, significant side effects, and poor targeting. For example, while free drugs such as thalidomide have anti-inflammatory and immunomodulatory effects, systemic administration easily causes adverse reactions such as neurotoxicity, and local concentrations are often insufficient.

[0003] Exosomes, as nanoscale vesicles secreted by cells, possess excellent biocompatibility, low immunogenicity, and natural targeting properties, making them a research hotspot in drug delivery systems. Exosomes derived from umbilical cord mesenchymal stem cells (HUCMSC-exo) show potential in tissue repair and anti-inflammatory therapy due to their ease of acquisition, strong expansion capacity, and rich bioactive substances. However, efficiently loading hydrophobic drugs (such as thalidomide) and improving their targeted delivery efficiency remain challenges in current technologies.

[0004] FC-tagged IL-11 is a fusion protein whose Fc fragment can prolong the half-life of IL-11 and enhance its stability. Pretreatment of human umbilical cord mesenchymal stem cells with it can activate intracellular repair-related signaling pathways (such as STAT3), promote the secretion of exosomes with anti-inflammatory and mucosal repair potential, thereby enhancing the targeting and therapeutic efficacy of exosomes on radiation-damaged tissues.

[0005] This invention addresses the aforementioned problems by encapsulating thalidomide within the exocavity of HUCMSCs using an incubation technique, constructing a novel loaded exosome. This method not only improves drug loading rate and stability but also leverages the natural homing effect of exosomes to enhance targeting of damaged mucosa, providing an innovative solution for RIOM treatment. Summary of the Invention

[0006] This invention first provides a method for preparing exosomes for treating radiation-induced oral mucositis, comprising the following steps: extracting empty exosomes from human umbilical cord mesenchymal stem cells pretreated with FC tagged IL-11, mixing the exosomes with a thalidomide solution, incubating to encapsulate the thalidomide within the exosome cavity, and purifying by centrifugation to obtain loaded exosomes.

[0007] In some embodiments, the human umbilical cord mesenchymal stem cells are primary cells that are passaged to the fifth generation before being used for exosome extraction.

[0008] In some embodiments, the extraction of empty exosomes includes cell culture, centrifugation to remove impurities, and ultracentrifugation purification steps, wherein the ultracentrifugation conditions are 110,000×g for 90 minutes.

[0009] In some embodiments, the incubation conditions are 37°C for 2 hours to promote thalidomide encapsulation.

[0010] In some embodiments, the thalidomide and exosomes are mixed in a ratio of 1:3 (v / v), the exosome concentration is 100 μg / mL, and the thalidomide concentration is 10 μg / mL.

[0011] The present invention also provides thalidomide-loaded umbilical cord mesenchymal stem cell exosomes prepared by the above method.

[0012] The present invention also provides a pharmaceutical composition comprising the above-described thalidomide-loaded umbilical cord mesenchymal stem cell exosomes and a pharmaceutically acceptable carrier.

[0013] The present invention also provides the use of the above-described pharmaceutical composition in the preparation of a medicament for treating radiation-induced oral mucositis.

[0014] In some embodiments, the drug is used to inhibit X-ray-induced apoptosis of oral mucosal epithelial cells and promote cell viability recovery.

[0015] Compared with the prior art, the present invention has at least the following beneficial effects: (1) Synergistic therapeutic effect: Exosomes and thalidomide work together to significantly inhibit X-ray-induced apoptosis and promote cell viability recovery.

[0016] (2) High biosafety: Based on exosomes derived from autologous cells, it has low immunogenicity and reduces the side effects of systemic administration.

[0017] (3) Simple and controllable process: The preparation process does not require complex chemical modification, is suitable for standardized production, and has good repeatability.

[0018] (4) FC tagged IL-11 pretreatment further optimized the bioactivity of exosomes, and synergistically enhanced the protective effect on oral mucosal cells by enhancing their homing ability and targeted delivery of loaded drugs. Attached Figure Description

[0019] Figure 1 Western Blot images of different exosomes Figure 2 Proliferation curves of HOMEpiC cells after X-ray irradiation with different pretreatments.

[0020] Figure 3 A graph showing the apoptosis rate of HOMEpiC cells after X-ray irradiation with different pretreatments. Detailed Implementation

[0021] This invention provides optimized umbilical cord mesenchymal stem cell exosomes (HUCMSCs) loaded with thalidomide. FI This paper describes a method for preparing exosomes (-exo-THD) and their application in the treatment of radiation-induced oral mucositis (RIOM). The exosomes are prepared from HUCMSCs cells pretreated with FCtagged IL-11 and loaded with thalidomide (THD). Compared with free THD or other single-treatment exosomes, HUCMSCs... FI -exo-THD showed significantly higher viability recovery and apoptosis inhibition effects on X-ray induced damage to human oral mucosal epithelial cells (HOMEpiC).

[0022] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0023] Experimental materials Table 1 Cell Source

[0024] Example 1: FC-tagged IL-11 pretreatment of human umbilical cord mesenchymal cells Primary HUCMSCs were cultured in DMEM containing 10% serum substitute, 100 U / mL streptomycin, and 100 U / mL penicillin at 37°C in a 5% CO2 incubator. When cell confluence reached 70%-80%, the cells were passaged at a 1:3 ratio to the fifth generation to ensure cell stability and exosome quality. CD90 levels were measured by flow cytometry. + / CD44 +The purity of HUCMSCs was confirmed to be >90%. When the fifth-generation cells reached 70%-80% confluence, the culture medium was discarded and the cells were washed twice with PBS. The culture medium was then replaced with FC tagged IL-11 exosome-free medium containing 200 ng / ml and cultured for another 48 h. The control group's culture medium was not supplemented with any other substances.

[0025] Example 2: Preparation of exosomes from umbilical cord mesenchymal stem cells The culture supernatant from Example 1 was collected and centrifuged at 500×g for 5 min to remove cells, followed by centrifugation at 1500×g for 15 min to remove cell debris, and then centrifugation at 20,000×g for 30 min to remove large particulate impurities. The supernatant was filtered through a 0.22 μm filter membrane and then centrifuged at 110,000×g for 90 min to obtain two types of umbilical cord stem cell exosomes (HUCMSCs). FI Exosomes (-exo and HUCMSCs-exo) were collected, the supernatant was discarded, and the exosomes were resuspended in PBS. The marker proteins CD9, CD81, and TSG101 were identified by Western blotting. The total protein content of the exosomes was determined according to the BCA method, and the exosome concentration was adjusted to 100 μg / ml for subsequent experiments.

[0026] Example 3: Preparation of umbilical cord stem cell exosomes loaded with thalidomide The exosomes obtained in Example 2 were mixed with 10 μg / mL thalidomide (10% DMSO aqueous solution) at a ratio of 1:3 (v / v), and the mixture was incubated at 37 °C for 2 h to promote thalidomide encapsulation. The thalidomide-loaded umbilical cord mesenchymal stem cell exosomes (HUCMSCs) were then purified by centrifugation at 100,000 × g for 90 min. FI -exo-THD and HUCMSCs-exo-THD. Control group: Exosomes obtained in Example 2 were mixed with 10% DMSO aqueous solution without thalidomide at a ratio of 1:3 (v / v), and the mixture was incubated at 37 °C for 2 h. After separation and purification by centrifugation at 100,000 × g for 90 min, empty-load umbilical cord mesenchymal stem cell exosomes HUCMSCs were obtained. FI -exo and HUCMSCs-exo were identified by Western blotting as marker proteins CD9, CD81, and TSG101. The thalidomide loading in exosomes was quantified using high-performance liquid chromatography (HPLC). HUCMSCs exosomes in this batch... FIThe loading amounts of -exo and HUCMSCs-exo (Example 2 untreated group) were 21 ng / μg and 25 ng / μg (thalidomide / exosomes), respectively. The total protein content of different exosomes was determined by the BCA method, and the exosome concentration was adjusted to 100 μg / ml for subsequent experiments.

[0027] Example 4: Establishment and grouping of a radiation-induced oral mucositis cell model Third-generation oral mucosal epithelial cells were seeded in DMEM containing 10% serum substitute, 100 U / mL streptomycin, and 100 U / mL penicillin. After incubation at 37 °C in a 5% CO2 incubator for 72 h, cells were digested with trypsin, counted, and the concentration was adjusted to 5 × 10⁻⁶ cells / mL. 4 Cells / mL: 100 μl (5 × 10³ cells) of cell suspension was added to 96-well plates and incubated in an incubator at 37°C and 5% CO2. Different groups were set up according to different treatment methods: Control (blank control, no treatment); THD (free THD treatment); HUCMSCs-exo treatment; HUCMSCs FI -exo processing; HUCMSCs-exo-THD processing; HUCMSCs FI -exo-THD treatment. Cells in each group were pretreated for 24 h and then irradiated with 4 Gy X-rays for 48 h to simulate radiation-induced oral mucositis in vitro. The THD content in the THD treatment group was 0.5 μg. The exosomes in the different exosome treatment groups were derived from Example 3, and the amount used was 200 μL (20 μg exosomes).

[0028] After irradiation, the culture medium was replaced with fresh medium and cultured for another 48 hours. The medium was then removed, and 100 μL of CCK-8 working solution (CCK-8 stock solution: DMEM = 1:9) was added. The OD value of each well at 450 nm was measured at different time points to compare the effects of different treatments on the viability of human oral mucosal epithelial cells after radiation irradiation. The results are as follows: Figure 2 As shown in the figure, X-ray irradiation significantly inhibited cell proliferation and caused persistent damage compared to the control group, indicating the successful establishment of the RIOM cell damage model. Oral mucosal epithelial cells pretreated with thalidomide-loaded exosomes exhibited the best radiation damage repair effect, demonstrating a significant synergistic effect between exosomes and thalidomide.

[0029] In addition, each group of cells was gently treated with 1×10 6Cells were resuspended at a density of / mL and stained with Annexin V-FITC and Annexin V-PI. The proportion of apoptotic cells was determined by flow cytometry to compare the effects of different treatments on apoptosis of human oral mucosal epithelial cells after radiation irradiation. The results are as follows: Figure 3 As shown.

[0030] via HUCMSCs FI -exo-THD pretreatment of oral mucosal epithelial cells resulted in the lowest apoptosis rate after irradiation, with a significantly better apoptosis inhibition effect than free drugs and other single-treatment exosomes.

[0031] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing exosomes for treating radiation-induced oral mucositis, characterized in that, Includes the following steps: Empty exosomes were extracted from human umbilical cord mesenchymal stem cells pretreated with FC tagged IL-11. The exosomes were mixed with thalidomide solution and incubated to encapsulate thalidomide within the exosome lumen. The loaded exosomes were then purified by centrifugation.

2. The preparation method according to claim 1, characterized in that, The human umbilical cord mesenchymal stem cells were primary cells that were passaged to the fifth generation and then pretreated with FC-tagged IL-11 for 48 hours before being used for exosome extraction.

3. The preparation method according to claim 1, characterized in that, The extraction of empty exosomes includes cell culture, centrifugation to remove impurities, and ultracentrifugation purification steps, wherein the ultracentrifugation conditions are 110,000×g for 90 minutes.

4. The preparation method according to claim 1, characterized in that, The incubation conditions were 37°C for 2 hours to promote thalidomide encapsulation.

5. The preparation method according to claim 1, characterized in that, The mixing ratio of thalidomide to exosomes was 1:3 (v / v), the exosome concentration was 100 μg / ml, and the thalidomide concentration was 10 μg / ml.

6. An umbilical cord mesenchymal stem cell exosome loaded with thalidomide, prepared by the method of any one of claims 1-5.

7. A pharmaceutical composition, characterized in that, The umbilical cord mesenchymal stem cell exosomes containing thalidomide as described in claim 6, and a pharmaceutically acceptable carrier.

8. Use of the pharmaceutical composition of claim 7 in the preparation of a medicament for treating radiation-induced oral mucositis.

9. The application as described in claim 8, characterized in that, The drug is used to inhibit X-ray-induced apoptosis of oral mucosal epithelial cells and promote the recovery of cell viability.

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