Fusion nanovesicle and preparation method and application thereof

Abstract

This invention relates to the field of biomedical technology, and more particularly to a fused nanovesicle, its preparation method, and its application. The fused nanovesicle is formed by fusing tea leaf exosome nanovesicles with exosome nanovesicles from tea twig and citrus peel. The fused nanovesicle is a spherical nanovesicle with a phospholipid bilayer structure. It features uniform morphology and structural stability, and allows the fused nanovesicle to integrate multiple beneficial components, enabling its application in the preparation of nanomedicines for treating inflammation and tumors.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a fused nanovesicle, its preparation method, and its application. Background Technology

[0002] Inflammatory bowel disease (IBD) is an idiopathic inflammatory bowel disease affecting the ileum, rectum, and colon, primarily including ulcerative colitis (UC) and Crohn's disease (CD). In recent years, with changes in lifestyle, the incidence of IBD has been increasing annually. Its clinical manifestations mainly include diarrhea, abdominal pain, and even bloody stools. The pathogenic factors and mechanisms of IBD are complex and not yet fully understood. Conventional treatments mainly include immunosuppressants, systemic corticosteroids, and 5-aminosalicylic acid. However, due to side effects and drug resistance, these traditional treatments only aim to reduce inflammation and slow disease progression, but cannot cure IBD. Clinical practice has shown that even after symptom relief, the intestines of IBD patients continue to have an inflammatory response, leading to irreversible changes in intestinal structure and ultimately inducing various complications such as cancer, severely impacting patients' quality of life. Therefore, finding a safe and curative treatment for IBD is urgently needed.

[0003] Extracellular vesicles containing bioactive lipids, proteins, RNA, and other pharmacologically active molecules represent a class of highly promising natural nanomedicines, possessing unique morphological and compositional characteristics as natural nanocarriers. Compared to animal extracellular vesicles, edible plant-derived exosome-like nanovesicles (ELNVs), as natural products of plant cells, are more readily available, easier to prepare in large quantities, and exhibit low immunogenicity, low toxicity, and relative safety, showing promising application prospects in IBD targeted therapy.

[0004] Tea is one of the world's most popular and widely consumed beverages, possessing unique flavor qualities and potential health benefits. The beneficial compounds in tea, such as polyphenols, flavonoids, lipids, and polysaccharides, endow it with various biological activities, including anti-inflammatory, antioxidant, anti-tumor, and lipid-lowering effects.

[0005] Tea-derived ELNVs have been reported to reduce the production of reactive oxygen species in RAW 264.7 mouse macrophages, inhibit the expression of pro-inflammatory cytokines secreted by macrophages, and increase the content of the anti-inflammatory cytokine IL-10. Simultaneously, oral administration of tea-derived ELNVs can effectively inhibit intestinal inflammatory responses and prevent or alleviate IBD and IBD-related colon cancer. Furthermore, the peel of the tea-branch mandarin orange is rich in hesperidin, nobiletin, and other polymethoxyflavonoids, which can inhibit tumor cell proliferation. When dried, it becomes the traditional Chinese medicine Chenpi (dried tangerine peel), a high-quality product with significant medicinal value. Therefore, ELNVs derived from the peel of the tea-branch mandarin orange also have broad application prospects in the treatment of IBD and IBD-related cancers. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a fused nanovesicle, its preparation method and application, which has the characteristics of uniform morphology and stable structure, and enables the fused nanovesicle to integrate multiple beneficial components, which can work synergistically to enhance anti-inflammatory, antioxidant and anti-tumor activities.

[0007] The present invention solves the above-mentioned technical problems through the following technical means:

[0008] In a first aspect, this application provides a fused nanovesicle, which is formed by fusing tea exosome nanovesicles with exosome nanovesicles from tea branch citrus peel, and the fused nanovesicle is a spherical nanovesicle with a phospholipid bilayer structure.

[0009] Based on the above-mentioned technical means, nanovesicles in tea leaves are fused with nanovesicles in the peel of citrus twigs to form spherical nanovesicles with a phospholipid bilayer structure. This can integrate multiple functional components such as tea polyphenols (catechins), hesperidin, tangeretin, nodosum, and quercetin, and synergistically enhance anti-inflammatory, antioxidant, and anti-tumor activities, thus laying a theoretical foundation for the improvement of IBD by fusing nanovesicles.

[0010] In conjunction with the first aspect, as a preferred embodiment, the hydrated nanovesicles have a hydration particle size of 122.4±0.8 nm and a zeta potential of -17.2±0.8 mV.

[0011] Secondly, this application provides a method for preparing fused nanovesicles, characterized by comprising the following steps:

[0012] S1. Collect tea leaves and tea branch citrus peel, wash them clean, and then place them in pre-cooled phosphate buffer solution. Through homogenization, obtain the broken tea leaves and tea branch citrus peel separately.

[0013] S2. The broken tea leaves and tea branch citrus peel from step S1 are separated by centrifugation to obtain preliminarily separated tea leaf supernatant and tea branch citrus peel supernatant. The preliminarily separated tea leaf supernatant and tea branch citrus peel supernatant are then separated by ultra-high speed centrifugation, and the precipitates in the preliminarily separated tea leaf supernatant and tea branch citrus peel supernatant are collected respectively.

[0014] S3. The precipitates in the tea supernatant and the tea branch citrus peel supernatant from step S2 were resuspended in phosphate buffer, and then sucrose solutions of different concentration gradients were added. They were then separated by ultra-high speed centrifugation to obtain tea exosome nanovesicles and tea branch citrus peel exosome nanovesicles, respectively.

[0015] S4. After mixing the tea exosome nanovesicles from step S3 with the exosome nanovesicles from the peel of the tea branch citrus fruit, the lipids and water-soluble functional components are separated by solvent. The lipids and water-soluble functional components are then used to prepare recombinant liposomes by thin-film hydration. The recombinant liposomes are then extruded through a liposome extruder to obtain fused nanovesicles.

[0016] Based on the aforementioned technical methods, nanovesicles are first extracted from tea leaves and from the peel of citrus twigs, and then purified to obtain two pure nanovesicles. The purified nanovesicles are then separated into organic and aqueous phases to separate beneficial components such as tea polyphenols (catechins), hesperidin, tangeretin, nodosum, and quercetin. These nanovesicles are then recombined using a thin-film hydration method to prepare new nanovesicles that can re-load these beneficial components. This improves the cellular uptake of the effective active ingredients carried by the nanovesicles and synergistically enhances their anti-inflammatory, antioxidant, and anti-tumor activities. Furthermore, the recombined nanovesicles have a uniform morphology and more stable structure, ensuring their integrity during passage through the gastrointestinal tract. This lays a theoretical foundation for the in vivo improvement of IBD by nanovesicles.

[0017] In conjunction with the second aspect, as a preferred embodiment, in step S1, the tea leaves are fresh leaves and the peel of the tea branch citrus fruit is fresh peel. The tea leaves and the peel of the tea branch citrus fruit are first washed clean, then chopped, and then placed in a pre-cooled phosphate buffer solution. The weight ratio of the phosphate buffer solution to the tea leaves and the peel of the tea branch citrus fruit is (1-10):1.

[0018] In conjunction with the second aspect, as a preferred embodiment, in step S2, when centrifuging the broken pieces of tea leaves and tea branch citrus peel, the broken pieces of tea leaves and tea branch citrus peel are centrifuged multiple times. Each time, the supernatant is taken and centrifuged again, and this process is repeated.

[0019] In conjunction with the second aspect, as a preferred embodiment, in step S3, the sucrose solutions with different concentration gradients are specifically: 8-80% sucrose solutions prepared with 0-100mM Tris-HCl.

[0020] In conjunction with the second aspect, as a preferred embodiment, during the ultra-high speed centrifugation, equal volumes of 8-80% sucrose solution are added to the resuspended phosphate buffer solution, followed by ultra-high speed centrifugation. The mixtures containing 45% and 60% sucrose solutions are collected, and then the mixtures containing 45% and 60% sucrose solutions are washed with PBS to obtain pure tea leaf exosome nanovesicles and exosome nanovesicles from tea branch citrus peel, respectively.

[0021] In conjunction with the second aspect, as a preferred embodiment, in step S4, when separating the lipids and water-soluble functional components of the mixed tea exosome nanovesicles and the exosome nanovesicles in the tea branch and citrus peel using a solvent, methanol and chloroform are first added and vortexed, then chloroform and deionized water are added and vortexed again. After vortexing, the lipids and water-soluble functional components are obtained by centrifugation.

[0022] In conjunction with the second aspect, as a preferred embodiment, in step S4, when preparing recombinant liposomes by the thin-film hydration method, the lipids are first sonicated, then added to a round-bottom flask, and heated and evaporated under reduced pressure on a rotary evaporator to obtain a uniform thin film. Then, the centrifuged water-soluble functional component is immediately added, and the mixture is heated and rotated under normal pressure for 1-5 minutes to obtain recombinant liposomes.

[0023] Thirdly, this application also provides an application of fused nanovesicles in the preparation of nanomedicines for treating inflammation and tumors.

[0024] The present application, employing the above-described scheme, has the following beneficial effects:

[0025] 1. This application is the first to use membrane fusion technology to recombine ELNVs derived from tea leaves and tea branches, forming fused nanovesicles that integrate multiple active ingredients such as tea polyphenols (catechins), hesperidin, tangeretin, nodosum, and quercetin. This ensures safety while giving the fused nanovesicles stronger anti-inflammatory, antioxidant, and anti-tumor activities.

[0026] 2. The fused nanovesicles prepared in this application have the characteristics of uniform morphology and stable structure, and can ensure their integrity during the passage of gastrointestinal tract. At the same time, they improve the cellular uptake of the effective active ingredients they carry, laying a theoretical foundation for the in vivo improvement of IBD by fused nanovesicles. Attached Figure Description

[0027] This application can be further illustrated by the non-limiting embodiments given in the accompanying drawings;

[0028] Figure 1 This is a flowchart illustrating the preparation process of the fused nanovesicles described in this application;

[0029] Figure 2 These are electron micrographs and hydrodynamic diameters of ELNVs derived from fresh tea leaves, fresh citrus peels derived from tea branches, and fused nanovesicles.

[0030] Figure 3 This is an analysis diagram of the ELNVs derived from fresh tea leaves, ELNVs derived from fresh citrus peel, and the fused nanovesicles containing tea polyphenols and flavonoids.

[0031] Figure 4This is a graph showing the average fluorescence intensity in CT26 cells after incubation with different fluorescently labeled nanovesicles in this application;

[0032] Figure 5 This is a graph showing the effect of different concentrations of nanovesicles on the proliferation of RAW 264.7 mouse macrophages in (a) of this application;

[0033] (b) Average fluorescence intensity of ROS (DCFH-DA) in RAW 264.7 mouse macrophages after treatment with different nanovesicles;

[0034] Figure 6 This is a graph showing the in vitro anti-inflammatory activity of ELNVs derived from fresh tea leaves, ELNVs derived from fresh tea peel, and fused nanovesicles at different time points in this application.

[0035] Figure 7 This is a diagram showing the in vitro antitumor activity of ELNVs derived from fresh tea leaves, ELNVs derived from fresh peel of citrus tangerine peel, and fused nanovesicles in this application. Detailed Implementation

[0036] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can understand the advantages and effects of the present invention from the content disclosed in this specification:

[0037] Example 1: Preparation of fused nanovesicles

[0038] S1. Collect fresh tea leaves and fresh peels of tea branches and tangerines. Wash the fresh tea leaves and fresh peels of tea branches and tangerines with clean water. Then, cut the washed fresh tea leaves and fresh peels of tea branches and tangerines into small pieces with scissors. Place the cut fresh tea leaves and fresh peels of tea branches and tangerines into homogenizers. Add phosphate buffer (PBS, pH=7.2) to the fresh tea leaves and fresh peels of tea branches and tangerines in a weight ratio of 1:1. Homogenize and break down the fresh tea leaves and fresh peels of tea branches and tangerines into small pieces, respectively.

[0039] S2. The crushed tea leaves and tea branch citrus peels were centrifuged separately. For the first centrifugation, the centrifugation force was 1000g for 20 minutes. The supernatant from the first centrifugation was collected and centrifuged a second time. For the second centrifugation, the centrifugation force was 5000g for 40 minutes. The supernatant from the second centrifugation was collected and centrifuged a third time. For the third centrifugation, the centrifugation force was 10000g for 60 minutes to obtain the supernatant of the preliminarily separated tea leaves and tea branch citrus peels. The supernatant from the third centrifugation of the tea leaves and tea branch citrus peels was then centrifuged a second time using an ultra-high-speed centrifuge at a centrifugation force of 100000g for 120 minutes to obtain the precipitates of the supernatant after four centrifugations of the tea leaves and tea branch citrus peels.

[0040] S3. Prepare 8%, 30%, 45% and 60% sucrose solutions respectively using 20mM Tris-HCl with pH=7.2;

[0041] The precipitates from the supernatants of fresh tea leaves and fresh peels of citrus tangerines obtained in step S2 were resuspended in phosphate buffered saline (PBS, pH = 7.2), and then purified by adding 8%, 30%, 45%, and 60% sucrose solutions, respectively. The mixtures were centrifuged at 100,000 g for 120 min using an ultracentrifuge, and the mixtures containing 45% and 60% sucrose solutions were collected. The mixtures containing 45% and 60% sucrose solutions were then washed 3-5 times with PBS to obtain exosome nanovesicles (ELNVs) from fresh tea leaves and exosome nanovesicles (ELNVs) from fresh peels of citrus tangerines, respectively.

[0042] S4. Exosome nanovesicles (ELNVs) derived from fresh tea leaves and exosome nanovesicles (ELNVs) derived from fresh tea twig peel were mixed at a mass ratio of 1:1. 3.75 mL of a MeOH:CHCl3 mixture (MeOH:CHCl3 volume ratio 2:1) was added to the mixed nanovesicles (1 mL) and vortexed. CHCl3 (1.25 mL) and ddH2O (1.25 mL) were then added and vortexed. After vortexing, the mixture was centrifuged. Lipids (organic phase) and water-soluble functional components (aqueous phase) from ELNVs derived from tea leaves and tea branches were obtained at 2000 rpm for 10 min. The lipids (organic phase) were then sonicated for 5 min using a washing ultrasonic cleaner. After sonication, the lipids (organic phase) were added to a round-bottom flask and evaporated to dryness under reduced pressure using a rotary evaporator. A uniform thin film was obtained on the inner wall of the bottom of the flask. Immediately afterwards, the water-soluble functional components (aqueous phase) were added, and the mixture was heated and rotated at atmospheric pressure for 1-5 min to obtain recombinant liposomes.

[0043] Finally, the recombinant liposomes were passed through 200nm and 100nm filter membranes sequentially using an Avanti liposome extruder, and extruded 11-21 times each to obtain fused nanovesicles.

[0044] The entire preparation of fused nanovesicles is as follows Figure 1 As shown, where, Figure 1 In the figure, 'a' represents the extraction process of exosome nanovesicles (ELNVs) from fresh tea leaves and exosome nanovesicles (ELNVs) from fresh peel of tea branches and citrus fruits. Figure 1 In the figure, b represents the preparation process of fused nanovesicles.

[0045] The prepared ELNVs derived from fresh tea leaves, ELNVs derived from fresh tea branch and citrus peel, and fused nanovesicles were tested and characterized, such as... Figure 2 As shown, Figure 2 In the figure, scale bar = 50 μm, mean ± SD, n = 3; among them, representative transmission electron microscope images of ELNVs from fresh tea leaves are shown in the figure. Figure 2 As shown in a, the hydrodynamic diameter is as follows: Figure 2 As shown in b; a representative transmission electron microscope image of ELNVs derived from fresh peel of the tea branch mandarin orange is shown in Figure 1. Figure 2 As shown in c, the hydrodynamic diameter is as follows Figure 2 As shown in d; a representative transmission electron microscope image of the fused nanovesicles is shown in Figure d. Figure 2 As shown in e, the hydrodynamic diameter is as follows Figure 2 As shown in f in the figure.

[0046] from Figure 2 As can be seen from the data, the morphological characteristics of the ELNVs derived from fresh tea leaves, the ELNVs derived from fresh tea branch and citrus peel, and the fused nanovesicles are all spherical nanovesicles with a phospholipid bilayer structure. The hydrated particle sizes of the ELNVs derived from fresh tea leaves, the ELNVs derived from fresh tea branch and citrus peel, and the fused nanovesicles are 78.1±1.3nm, 115.4±1.9nm, and 122.4±0.8nm, respectively, and the PDI values ​​are relatively small, indicating good dispersibility.

[0047] The ELNVs derived from fresh tea leaves, ELNVs derived from fresh citrus peels, and fused nanovesicles were then placed in 2 mM PBS, and the zeta potentials were measured. The results are shown in the table below:

[0048] Group 1 2 3 Zeta(mV) -9.35±0.3 -23.6±1.7 -17.2±0.8

[0049] Group 1 represents ELNVs derived from fresh tea leaves; Group 2 represents ELNVs derived from fresh citrus peels from tea branches; and Group 3 represents fused nanovesicles, with an average value of ±SD and n=3.

[0050] The data above shows that the potential of the fused nanovesicles is lower than that of ELNVs derived from fresh tea leaves but higher than that of ELNVs derived from fresh citrus peels from tea branches, indicating that the two have been successfully fused.

[0051] The contents of tea polyphenols and flavonoids in fresh tea leaf ELNVs, fresh tea branch peel ELNVs, and fused nanovesicles were analyzed by LC-MS. The results are as follows: Figure 3 As shown;

[0052] The results showed that the fused nanovesicles integrate the active ingredients from tea / tea branch citrus ELNVs, including a variety of tea polyphenols / flavonoids, such as catechins (297.642 g / kg), hesperidin (218.553 g / kg), citrus peelin (17.638 g / kg), nobiletin (12.289 g / kg), quercetin (0.004 g / kg), etc., which provides the prerequisite for enhancing their anti-inflammatory and anti-tumor activities.

[0053] Where the error bars represent the mean ± SD, n = 3. Figure 3 In the figure, a, b, c, d, e, and f represent: the analysis results of tea polyphenols (a) and flavonoids (b) in ELNVs derived from fresh tea leaves; the analysis results of tea polyphenols (c) and flavonoids (d) in ELNVs derived from fresh peel of tea branches and citrus fruits; and the analysis results of tea polyphenols (e) and flavonoids (f) in fused nanovesicles.

[0054] Example 2: Simulated gastric juice stability and cellular uptake experiment of nanovesicles

[0055] Fluorescently labeled fused nanovesicles

[0056] Lipophilic DiO fluorescent dye solution (1-10 mM) was mixed with the fused nanovesicles (1 mg / mL) from Example 1 and incubated at 37°C for 0-30 min. Then, the mixture was centrifuged at 100,000 g for 1-2 h to remove the free DiO dye solution. The precipitate was collected and stored in the dark to obtain DiO-labeled fused nanovesicles.

[0057] Preparation of simulated gastric juice: Take 16.4 mL of 1 mol / L dilute hydrochloric acid, add 800 mL of water and 20 g of pepsin, shake, and then dilute with water to 1000 mL.

[0058] Stability and cellular uptake experiments

[0059] ELNVs derived from fresh tea leaves, ELNVs derived from fresh peel of Citrus aurantium, and fused nanovesicles were placed in simulated gastric fluid. The changes in particle size and zeta potential of ELNVs derived from fresh tea leaves, ELNVs derived from fresh peel of Citrus aurantium, and fused nanovesicles in simulated gastric fluid at 37℃ with incubation time were detected (mean ± SD, n = 3), as shown in the table below:

[0060]

[0061] The data above show that the particle size of ELNVs derived from fresh tea leaves, ELNVs derived from fresh citrus peels, and fused nanovesicles remained relatively stable within 4 hours of incubation in simulated gastric juice, and the PDI value and zeta potential also changed little. This indicates that they can maintain their integrity during the passage of gastrointestinal tract, which is expected to lay a theoretical foundation for their ability to improve IBD in vivo.

[0062] Lipophilic DiO fluorescent dye was used to fluorescently label ELNVs from fresh tea leaves and ELNVs from fresh peel of citrus tangerine peel, respectively. The fluorescent labeling method was the same as that used for fluorescent labeling fused nanovesicles with lipophilic DiO fluorescent dye.

[0063] ELNVs derived from fresh tea leaves (20 μg / mL), ELNVs derived from fresh citrus peel of tea branches, and fused nanovesicles were co-incubated with mouse colon cancer CT26 cells at 5% CO2 and 37℃ for different times. The fluorescence intensity of the nanoparticles taken up by the cells was detected by flow cytometry (FL1 detection channel). The results are as follows: Figure 4 As shown (error bars represent the average value ±SD, n=3, *P<0.05).

[0064] from Figure 4 The results showed that the fluorescence intensity of all incubated nanovesicle groups increased continuously with time, reaching its peak at 5 hours, indicating that the uptake of these nanovesicles by mouse colon cancer CT26 cells increased over time. This suggests that nanovesicles with a phospholipid bilayer outer membrane can fuse with the cell membrane, greatly promoting cellular uptake of nanovesicles, thereby enhancing the accumulation of anti-inflammatory and anti-tumor active ingredients within the cells, and ultimately improving the therapeutic effects of anti-inflammatory and anti-tumor treatment.

[0065] Example 3: Experimental study on the antioxidant, anti-inflammatory and antitumor activities of fused nanovesicles.

[0066] Effects on cells

[0067] RAW 264.7 cells were immersed in solutions containing ELNVs derived from fresh tea leaves, ELNVs derived from fresh citrus peels of tea branches, and fused nanovesicles at concentrations of 0, 10, 50, 100, and 250 μg / mL, respectively. The effects of different concentrations of these ELNVs on RAW 264.7 cells were observed, and the results are as follows: Figure 5 As shown in Figure a;

[0068] from Figure 5 As shown in Figure a, when the concentrations of ELNVs from fresh tea leaves, ELNVs from fresh citrus peels, and fused nanovesicles were within the range of 0-250 μg / mL, they had no cytotoxic effect on RAW 264.7 cells within 24 h, but stimulated cell proliferation of RAW 264.7 cells at a concentration of 250 μg / mL.

[0069] Intracellular antioxidant activity of nanovesicles

[0070] RAW 264.7 cells were co-incubated with 100 μg / mL ELNVs derived from fresh tea leaves, 250 μg / mL ELNVs derived from tea branches and citrus fruits, and 100 μg / mL fused nanovesicles at 5% CO2 and 37℃ for 24 h and 48 h, respectively. The cells were then treated with lipopolysaccharide (LPS, 1 μg / mL) for 4 h, followed by co-incubation with the DCFH-DA (10 μM) fluorescent probe for 20 min. Finally, the cells were washed three times with PBS, and the average fluorescence intensity of DCF was detected by flow cytometry. Results are as follows: Figure 5 As shown in Figure b, where (error bars represent mean ± SD, n = 3, **P < 0.01, ***P < 0.001), the mean fluorescence intensity of DCF represents the ROS signal within RAW 264.7 cells. Cells treated with LPS but not with nanovesicles served as the positive control group; cells treated with neither LPS nor nanovesicles served as the negative control group.

[0071] Excessive production or insufficient clearance of ROS can cause uncontrolled oxidative stress, which in turn leads to damage to lipids, proteins and nucleic acids. Oxidative stress in colon tissue can alter immune balance, causing mucosal damage and potentially inducing IBD.

[0072] from Figure 5 As shown in Figure b, the flow cytometry results indicate that compared with the positive control cells treated with LPS but not with nanovesicles, the ROS level in macrophages pretreated with nanovesicles was significantly reduced. This suggests that ELNVs from fresh tea leaves, ELNVs from fresh citrus peels, and fused nanovesicles can all effectively reduce LPS-induced ROS in activated macrophages RAW264.7, while fused nanovesicles exhibited the strongest antioxidant activity.

[0073] Anti-inflammatory experiment

[0074] RAW 264.7 macrophages were induced at a rate of 1×10⁻⁶. 5 Cells were seeded at a density in 24-well plates and co-incubated for 24 h and 48 h with 100 μg / mL ELNVs derived from fresh tea leaves, 250 μg / mL ELNVs derived from fresh tea branch and citrus peel, and 100 μg / mL fused nanovesicles, respectively. Cells were then stimulated with 1 μg / mL LPS for 4 h. The supernatant was subsequently collected, and the concentrations of various inflammatory factors (e.g., TNF-α, IL-6, IL-12, and IL-10) were quantified using the corresponding enzyme-linked immunosorbent assay (ELISA) kits. RAW 264.7 macrophages were used as a negative control group in the absence of nanovesicles and LPS, while macrophages stimulated only with LPS served as a positive control group.

[0075] In IBD lesions, typical pro-inflammatory cytokines such as TNF-α, IL-6, and IL-12 are mainly produced and secreted by macrophages. Therefore, an ELISA kit can be used to determine the effect of nanovesicles on the secretion of pro-inflammatory cytokines by RAW 264.7 macrophages.

[0076] The results are as follows Figure 6 As shown in Figure ac, the levels of pro-inflammatory cytokines in the supernatant of LPS-treated cells (positive control) were significantly higher than those in untreated cells (negative control). Furthermore, the expression levels of TNF-α, IL-6, and IL-12 were significantly downregulated in all nanovesicle treatment groups, demonstrating the anti-inflammatory activity of the nanovesicles.

[0077] Furthermore, the anti-inflammatory potential of nanovesicles was assessed by quantifying the expression of the anti-inflammatory cytokine IL-10. Figure 6 As shown in Figure d, compared with the negative and positive control groups, the levels of the anti-inflammatory cytokine IL-10 secreted by macrophages treated with nanovesicles were significantly increased. With prolonged time, the nanovesicles further reduced pro-inflammatory cytokines and increased the levels of anti-inflammatory cytokines. Furthermore, at any given time point, the fusion nanovesicle treatment group exhibited the best anti-inflammatory activity, indicating that these nanovesicles may provide a long-term therapeutic platform for preventing or alleviating inflammation, laying a theoretical foundation for the in vivo improvement of IBD using fusion nanovesicles.

[0078] Anti-tumor experiment

[0079] ELNVs derived from fresh tea leaves, ELNVs derived from fresh citrus peels, and fused nanovesicles were co-incubated with mouse colon cancer CT26 cells for 48 h (5% CO2, 37℃), and then cytotoxicity was tested using a CCK-8 assay kit.

[0080] The results are as follows Figure 7 As shown in the figure (where error bars represent mean ± SD, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001), the IC50 values ​​of ELNVs derived from fresh tea leaves, ELNVs derived from fresh peel of citrus tangerine peel, and fused nanovesicles were 500.1 μg / mL, 173.4 μg / mL, and 102.8 μg / mL, respectively. Furthermore, when the nanovesicle concentration was 200 μg / mL, the cell viability of ELNVs derived from fresh tea leaves, ELNVs derived from fresh peel of citrus tangerine peel, and fused nanovesicles were 65%, 49.3%, and 33%, respectively, with the fused nanovesicles exhibiting the lowest cell viability. This indicates that the fused nanovesicles possess the strongest antitumor activity.

[0081] In summary, ELNVs derived from fresh tea leaves, ELNVs derived from fresh peel of Citrus aurantium, and fused nanovesicles all exhibit antioxidant, anti-inflammatory, and antitumor activities. Fusion nanovesicles, however, demonstrate stronger antioxidant, anti-inflammatory, and antitumor activities and can be used in the preparation of nanomedicines for the treatment of inflammation and tumors. This lays a theoretical foundation for the in vivo improvement of IBD by ELNVs derived from fresh tea leaves, ELNVs derived from fresh peel of Citrus aurantium, and fused nanovesicles.

[0082] The foregoing has provided a detailed description of a fused nanovesicle, its preparation method, and its application. The specific embodiments described are merely illustrative of the method and its core concepts. It should be noted that those skilled in the art can make various improvements and modifications to the invention without departing from its principles, and these improvements and modifications also fall within the scope of protection of the claims.

[0083] It should be noted that: for experimental steps or conditions not specified in the examples, the procedures and conditions described in conventional experimental procedures in the literature of this art can be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0084] The above examples are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by anyone under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the scope of protection of the present invention.

Claims

1. A fused nanovesicle, characterized in that, The fused nanovesicles are formed by fusing tea leaf exosome nanovesicles with exosome nanovesicles from tea branch citrus peel. The fused nanovesicles are spherical nanovesicles with a phospholipid bilayer structure. The hydrated particle size of the fused nanovesicles is 122.4±0.8nm, and the zeta potential is -17.2±0.8mV.

2. A method for preparing fused nanovesicles as described in claim 1, characterized in that, Includes the following steps: S1. Collect tea leaves and tea branch citrus peel, wash them clean, and then place them in pre-cooled phosphate buffer solution. Through homogenization, obtain the broken tea leaves and tea branch citrus peel separately. S2. The broken tea leaves and tea branch citrus peel from step S1 are separated by centrifugation to obtain preliminarily separated tea leaf supernatant and tea branch citrus peel supernatant. The preliminarily separated tea leaf supernatant and tea branch citrus peel supernatant are then separated by ultra-high speed centrifugation, and the precipitates in the preliminarily separated tea leaf supernatant and tea branch citrus peel supernatant are collected respectively. S3. The precipitates in the tea supernatant and the tea branch citrus peel supernatant from step S2 were resuspended in phosphate buffer, and then sucrose solutions of different concentration gradients were added. They were then separated by ultra-high speed centrifugation to obtain tea exosome nanovesicles and tea branch citrus peel exosome nanovesicles, respectively. S4. After mixing the tea exosome nanovesicles from step S3 with the exosome nanovesicles from the peel of the tea branch citrus fruit, the lipids and water-soluble functional components are separated by solvent. The lipids and water-soluble functional components are then used to prepare recombinant liposomes by thin-film hydration. The recombinant liposomes are then extruded through a liposome extruder to obtain fused nanovesicles.

3. The method for preparing fused nanovesicles according to claim 2, characterized in that, In step S1, the tea leaves are fresh leaves and the peel of the tea branch citrus fruit is fresh peel. The tea leaves and the peel of the tea branch citrus fruit are first washed clean, then chopped, and then placed in pre-cooled phosphate buffer solution. The weight ratio of the phosphate buffer solution to the tea leaves and the peel of the tea branch citrus fruit is (1-10):

1.

4. The method for preparing fused nanovesicles according to claim 2 or 3, characterized in that, In step S2, when centrifuging the broken pieces of tea leaves and tea branch citrus peel, the broken pieces of tea leaves and tea branch citrus peel are centrifuged multiple times. Each time the pieces are centrifuged, the supernatant is taken and then centrifuged again, and the process is repeated.

5. The method for preparing fused nanovesicles according to claim 2, characterized in that, In step S3, the sucrose solutions with different concentration gradients are specifically: 8-80% sucrose solutions prepared with 0-100 mM Tris-HCl.

6. The method for preparing fused nanovesicles according to claim 5, characterized in that, During the ultra-high speed centrifugation, equal volumes of 8-80% sucrose solution were added to the resuspended phosphate buffer solution, and then ultra-high speed centrifugation was performed. The mixtures containing 45% and 60% sucrose solutions were collected, and then the mixtures containing 45% and 60% sucrose solutions were washed with PBS to obtain pure tea leaf exosome nanovesicles and exosome nanovesicles from tea branch citrus peel.

7. The method for preparing fused nanovesicles according to claim 2, characterized in that, In step S4, when separating the lipids and water-soluble functional components of the mixed tea exosome nanovesicles and the exosome nanovesicles in the peel of tea branches and citrus fruits using a solvent, methanol and chloroform are first added and vortexed, then chloroform and deionized water are added and vortexed again. After vortexing, the lipids and water-soluble functional components are obtained by centrifugation.

8. The method for preparing fused nanovesicles according to claim 7, characterized in that, In step S4, when preparing recombinant liposomes by the thin-film hydration method, the lipids are first sonicated, then added to a round-bottom flask and heated and evaporated under reduced pressure on a rotary evaporator to obtain a uniform thin film. Then, the centrifuged water-soluble functional components are immediately added, and the mixture is heated and rotated at normal pressure for 1-5 minutes to obtain recombinant liposomes.

9. An application of fused nanovesicles, characterized in that, The application of the fused nanovesicles in the preparation of nanomedicines for the treatment of inflammation and colon cancer.

Images