A brassica napus exosome loaded cerium dioxide composite material and a preparation method thereof

Abstract

The application provides a brassica napus exosome loaded cerium dioxide composite material and a preparation method thereof. The application takes exosomes from brassica napus as a natural nanocarrier, takes cerium dioxide particles as a functional active component, and constructs a composite material with plant-derived biocompatibility and sustained antioxidant capacity through extraction and purification, surface combination or package combination, purification and separation and other steps.

Description

Technical Field

[0001] This invention relates to a composite material, and more particularly to a broccoli exosome-loaded cerium dioxide composite material and its preparation method. Background Technology

[0002] Exosomes are nanoscale vesicles secreted by cells and possessing a lipid bilayer structure. They can carry proteins, nucleic acids, lipids, and various small molecule bioactive substances. In recent years, plant-derived exosomes or exosome-like nanovesicles have attracted widespread attention in drug delivery, anti-inflammation, anti-oxidation, and tissue repair fields due to their natural origin, good biocompatibility, high stability, and combined functions of bioactive substance delivery and bioregulation.

[0003] Broccoli-derived exosomes are an important type of plant-derived exosomes. Because broccoli is rich in isothiocyanates, sulforaphane precursors, and other antioxidant-related components, its derived vesicles not only possess a typical nanovesicle membrane structure but may also retain some natural biological activities. Therefore, they show promising applications in antioxidation, inflammation regulation, and immune intervention. Existing research indicates that broccoli-derived vesicles can serve as natural nanocarriers to load active small molecules, enhancing their stability and utilization efficiency, and playing a role in regulating macrophage function and mitigating oxidative stress damage.

[0004] On the other hand, cerium dioxide nanomaterials are a representative class of inorganic nano-antioxidant materials, due to the presence of Ce on their surface. 3+ With Ce 4+ Due to its reversible oxidation and oxygen vacancy structure, cerium dioxide nanoparticles possess nanozyme activity similar to superoxide dismutase and catalase, enabling them to continuously scavenge reactive oxygen species and demonstrating high application value in inflammatory damage, oxidative stress, and tissue repair. However, cerium dioxide nanoparticles typically suffer from insufficient dispersibility, easy aggregation, limited biocompatibility, and limited delivery efficiency when used alone, thus restricting their further applications.

[0005] To address the aforementioned issues, existing technologies mainly fall into the following categories: The first category involves the extraction and application of plant-derived exosomes. Existing patents disclose methods for preparing plant-derived extracellular vesicles and their applications in anti-inflammation, repair, and delivery of active substances. Other patents disclose the use of broccoli exosomes in anti-oxidation, anti-aging, and skin barrier repair. However, this type of technology primarily involves broccoli exosomes themselves and does not involve their combination with cerium dioxide. The second category involves loading organic active small molecules onto broccoli-derived vesicles. For example, loading natural active ingredients such as sulforaphane into broccoli membrane vesicles can improve their stability and biological effects. However, the loading targets in this type of technology are mainly organic small molecules, not inorganic nanomaterials. The third category involves the combination of exosomes and cerium dioxide. Existing literature reports the construction of composite systems by combining animal cell-derived exosomes with cerium dioxide nanocrystals for anti-oxidation and tissue protection, demonstrating the feasibility of combining exosomes with cerium dioxide. However, since the exosomes are derived from animal cells, the preparation cost is high, limiting large-scale application, and this category does not involve broccoli-derived exosomes. Another technology discloses a composite system constructed by broccoli exosomes and other metal nanomaterials, but its metal component is not cerium dioxide and its system structure is more complex.

[0006] In summary, existing technologies disclose the extraction and application of broccoli exosomes, the loading of active ingredients onto exosomes, and the related ideas of exosomes combined with cerium dioxide. However, these technologies suffer from at least the following problems: 1. Broccoli exosomes have limited antioxidant capacity and sustained action when used alone; 2. Cerium dioxide has insufficient dispersibility, biocompatibility, and delivery performance when used alone; 3. Existing exosome-cerium dioxide composite systems mostly rely on exosomes derived from animal cells, resulting in high costs and hindering widespread adoption; 4. A simple and stable composite material preparation scheme based on broccoli exosomes loaded with cerium dioxide is lacking. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a method for preparing a broccoli exosome-loaded cerium dioxide composite material. This preparation method can give full play to the advantages of the natural source, biocompatibility and carrier function of broccoli exosomes, and combine the antioxidant activity of cerium dioxide nanomaterials to obtain a composite material with good stability, good dispersibility and both natural activity and nanoenzyme antioxidant properties.

[0008] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows: A method for preparing a broccoli exosome-supported cerium dioxide composite material includes the following steps: S1. Wash the broccoli raw material to remove mud, sand and surface attachments, crush it and add it to PBS buffer, then homogenize it for 5-10 minutes using a tissue homogenizer to obtain a homogenate. S2. Centrifuge the homogenate obtained in step S1 at low speed for 5-15 min to obtain supernatant one, centrifuge supernatant one at medium speed for 20-40 min to obtain supernatant two, centrifuge supernatant two at high speed for 80-100 min to obtain broccoli exosome precipitate, and resuspend the broccoli exosome precipitate with PBS buffer to obtain broccoli exosome dispersion. S3. Add cerium acetate to deionized water, adjust the pH of the system to 10-11 with alkali, stir for 10-12 h to obtain a mixture, centrifuge the mixture for 10-20 min to obtain coarse cerium dioxide precipitate, wash the coarse cerium dioxide precipitate with deionized water 3 times to obtain cerium dioxide particles, add the cerium dioxide particles to PBS buffer, mix evenly to obtain cerium dioxide dispersion. S4. Combine the broccoli exosome dispersion obtained in step S2 with the cerium dioxide dispersion obtained in step S3 to obtain a composite solution; S5. Centrifuge the composite solution obtained in step S4 at high speed for 20-40 min to obtain a precipitate. Resuspend the precipitate in PBS buffer to obtain a resuspension. Shake the resuspension for 5-30 min to obtain a dispersion of broccoli exosome-loaded cerium dioxide composite material.

[0009] Furthermore, in step S1 of the present invention, the broccoli raw material is one or more of the florets, buds, stems or leaves of fresh, non-rotten broccoli; the PBS buffer is a 1×PBS buffer with a pH of 7.2-7.4, and the ratio of the fresh weight of the broccoli raw material to the PBS buffer is 1g:(1-10)mL.

[0010] Further, in step S2 of this invention, the low-speed centrifugation speed is 500-3000 rpm, the medium-speed centrifugation speed is 3000-10000 rpm, and the centrifugal force of the ultracentrifugation is 150000g; the PBS buffer is a 1×PBS buffer with a pH of 7.2-7.4. During resuspension, the broccoli exosome precipitate is added to the PBS buffer at a ratio of 1g:(200-500)mL, allowed to stand for 1-10 minutes, and then pipetted 5-20 times before being transferred to an EP tube; the particle size of the broccoli exosomes in the broccoli exosome dispersion is 30-200nm. The low-speed centrifugation in step S2 can remove coarse fibers, cell debris, and unbroken tissue, while the medium-speed centrifugation can remove larger particulate impurities; step S2 can obtain a broccoli exosome carrier with a natural origin, intact membrane structure, and suitable particle size distribution, providing a basic structural platform for subsequent loading of cerium dioxide.

[0011] Further, in step S3 of this invention, the ratio of cerium acetate to deionized water is 1g:(20-100)mL; the alkali is hydrazine hydrate; the washing time for the crude cerium dioxide precipitate with deionized water is 5min each time; the particle size of the cerium dioxide particles is 3-20nm; the PBS buffer is 1×PBS buffer with a pH of 7.2-7.4; and the ratio of cerium dioxide particles to PBS buffer is (0.05-5)mg:1mL. Step S3 uses liquid-phase precipitation to obtain cerium dioxide particles with a particle size of 3-20nm. This particle size range is beneficial for maintaining a large specific surface area, good dispersibility, and high surface activity. Step S3 can obtain cerium dioxide particles with small particle size, uniform dispersion, and continuous antioxidant function, so that they can be subsequently loaded onto broccoli exosomes.

[0012] Furthermore, in step S4 of this invention, the composite method is a stirring incubation composite method, an ultrasonic-assisted loading method, or a membrane extrusion composite method. Step S4 stabilizes cerium dioxide particles on the surface and / or interior of broccoli exosomes, improving the dispersion stability, biocontact, and delivery capacity of cerium dioxide, while enhancing the overall antioxidant properties of the exosomes.

[0013] Furthermore, the process of the stirring incubation composite method described in this invention is as follows: the broccoli exosome dispersion obtained in step S2 is mixed with the cerium dioxide dispersion obtained in step S3 at a volume ratio of 1:(2-4), and the mixture is stirred and incubated at room temperature for 24 hours to obtain the composite solution. The stirring incubation composite method allows the broccoli exosome dispersion and the cerium dioxide dispersion to fully contact and form a stable composite. The incubation time is preferably 24 hours, which is consistent with the commonly used time for co-incubation of plant-derived vesicles, and can balance loading efficiency and vesicle stability.

[0014] Further, the ultrasound-assisted loading method of the present invention involves mixing the broccoli exosome dispersion obtained in step S2 with the cerium dioxide dispersion obtained in step S3 at a volume ratio of 1:(2-4). The mixture is then subjected to intermittent ultrasound treatment with a probe under ice bath conditions for 4-8 cycles. Each cycle has an ultrasound duration of 10-120 seconds, a cycle interval of 30-300 seconds, an ultrasound frequency of 20-40 kHz, an ultrasound power of 40-120 W, and an ultrasound amplitude of 15-25%. After ultrasound treatment, the mixture is allowed to stand at 20-37°C for 30-240 minutes to obtain the composite solution. The ultrasound-assisted loading method can induce transient reversible permeability of the exosome membrane, thereby promoting the entry of cerium dioxide particles into the exosome membrane surface, intermembranous structure, or vesicle interior. Ultrasound is performed under ice bath conditions, which reduces thermal damage. The subsequent standing after ultrasound treatment promotes the recovery of the exosome membrane structure.

[0015] Further, the membrane extrusion composite method of the present invention involves mixing the broccoli exosome dispersion obtained in step S2 with the cerium dioxide dispersion obtained in step S3 at a volume ratio of 1:(2-4) to obtain a mixed dispersion. The mixed dispersion is then extruded 10-20 times through an extrusion die with a pore size of 200-400 nm to obtain a composite liquid. In one embodiment, the mixture can be extruded first through an extrusion membrane with a pore size of 400 nm, and then through an extrusion membrane with a pore size of 200 nm. The membrane extrusion composite method involves repeatedly extruding the broccoli exosome dispersion and the cerium dioxide dispersion through an extrusion membrane with a certain pore size to form a relatively homogeneous composite system.

[0016] Furthermore, in step S5 of this invention, the centrifugal force for ultracentrifugation is 150,000 g; the PBS buffer is a 1×PBS buffer with a pH of 7.2-7.4; during resuspension, the precipitate is added to the PBS buffer at a ratio of 1 g: 400 mL, and after standing for 3-5 minutes, it is pipetted 5-20 times to obtain a resuspension; the oscillation temperature is 4°C, and the oscillation speed is 50-150 rpm. The ultracentrifugation in step S5 can remove unloaded free cerium dioxide particles, residual reaction solution, and impurities; step S5 can improve the purity of the composite material, reduce the content of free particles, and ensure the stability of the finished product and the safety of subsequent applications.

[0017] The broccoli exosome-supported cerium dioxide composite material prepared by this invention can be stored at low temperatures of 4℃, -20℃ or -80℃, which can maintain the integrity of the exosome membrane structure and the dispersion state of cerium dioxide particles, and extend the storage period of the composite material.

[0018] The broccoli exosome-loaded cerium dioxide composite material prepared by this invention can be applied to fields such as anti-oxidation, anti-inflammation, tissue damage repair, drug delivery, and preparation of biomedical materials.

[0019] Another technical problem to be solved by the present invention is to provide a broccoli exosome-loaded cerium dioxide composite material prepared by the above preparation method.

[0020] Compared with the prior art, the present invention has the following beneficial effects: 1) This invention constructs a composite material of broccoli exosomes loaded with cerium dioxide by using broccoli exosomes as a natural nanocarrier and cerium dioxide particles as a functional active component, thus achieving an organic combination of plant-derived exosomes and inorganic nano-antioxidant materials. On the one hand, this invention utilizes broccoli exosomes to improve the dispersibility, stability, and biocompatibility of cerium dioxide particles, reducing their tendency to aggregate and low utilization efficiency when used alone. On the other hand, it leverages the nanoenzyme-like antioxidant function of cerium dioxide to compensate for the limited antioxidant capacity and sustained action of broccoli exosomes when used alone. Therefore, this invention further enhances the antioxidant, anti-inflammatory, and damage repair potential of the composite material, achieving a synergistic effect between the natural carrier function and the inorganic nano-active function.

[0021] 2) The preparation method provided by this invention has a relatively clear process, is relatively simple to operate, and has a certain degree of reproducibility and application feasibility. Compared with existing compound schemes that rely on animal cell-derived exosomes, this invention uses plant-derived raw materials, which helps to simplify the raw material acquisition process and reduce application costs, thus having good practical application prospects. Attached Figure Description

[0022] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, do not constitute an undue limitation of the invention. In the drawings: Figure 1 The zeta potential test results of CeO2, broccoli exosomes prepared in Example 1 of the present invention, and broccoli exosomes loaded with cerium dioxide composite material are shown in the figure. Figure 2 The graph shows the antioxidant capacity test results of broccoli exosomes and broccoli exosome-loaded cerium dioxide composite materials prepared in Example 1 of this invention.

[0023] Figure 3 The graph shows the test results of the anti-inflammatory ability of CeO2, broccoli exosomes prepared in Example 1 of this invention, and broccoli exosome-supported cerium dioxide composite material. Detailed Implementation

[0024] The present invention will now be described in detail with reference to specific embodiments. The illustrative embodiments and descriptions of the present invention are used to explain the present invention, but are not intended to limit the present invention. Example

[0025] The following steps were followed to prepare a cerium dioxide-supported broccoli exosome composite material: S1. Wash the broccoli raw material to remove mud and sand and surface attachments, crush it and add it to 1×PBS buffer with pH 7.2. Then homogenize it for 8 minutes using a tissue homogenizer to obtain a homogenate. The broccoli raw material is fresh, non-rotten broccoli florets. The ratio of fresh weight of broccoli raw material to PBS buffer is 1g:5mL. S2. Centrifuge the homogenate obtained in step S1 at 1500 rpm for 10 min to obtain supernatant one. Centrifuge supernatant one at 5000 rpm for 30 min to obtain supernatant two. Centrifuge supernatant two at 150000 g for 90 min to obtain broccoli exosome precipitate. Resuspend the broccoli exosome precipitate in 1×PBS buffer with a pH of 7.2 to obtain broccoli exosome dispersion (when resuspending, add the broccoli exosome precipitate to PBS buffer at a ratio of 1 g: 400 mL, let stand for 5 min, and then pipette 10 times). Then transfer it to EP tube. The particle size of broccoli exosomes in the broccoli exosome dispersion is 30-200 nm. S3. Add cerium acetate to deionized water at a ratio of 1g:50mL, adjust the pH of the system to 10.5 using hydrazine hydrate, stir for 11h to obtain a mixture, centrifuge the mixture for 15min to obtain a coarse cerium dioxide precipitate, wash the coarse cerium dioxide precipitate three times with deionized water (5min each time) to obtain cerium dioxide particles with a particle size of 3-20nm, add the cerium dioxide particles to 7.2×PBS buffer at a ratio of 1mg:1mL, mix well to obtain a cerium dioxide dispersion; S4. Combine the broccoli exosome dispersion obtained in step S2 with the cerium dioxide dispersion obtained in step S3 to obtain a composite solution; mix the broccoli exosome dispersion obtained in step S2 with the cerium dioxide dispersion obtained in step S3 at a volume ratio of 1:3, stir and incubate at room temperature for 24 hours to obtain the composite solution. S5. The composite solution obtained in step S4 is centrifuged at 150,000g for 30 minutes to obtain a precipitate. The precipitate is resuspended in 1×PBS buffer with a pH of 7.2 to obtain a resuspension (when resuspending, the broccoli exosome precipitate is added to PBS buffer at a ratio of 1g:400mL, and after standing for 4 minutes, it is pipetted 10 times to obtain a resuspension). The resuspension is shaken at 4℃ and 100rpm for 15 minutes to obtain a dispersion of broccoli exosome-loaded cerium dioxide composite material.

[0026] Zeta potentials were measured for CeO2, the broccoli exosomes prepared in step S2, and the broccoli exosome-supported cerium dioxide composite material prepared in step S5. The test results are as follows: Figure 1 As shown. By Figure 1It can be seen that the dispersion stability of broccoli exosomes and broccoli exosome-supported cerium dioxide composites is better than that of CeO2, and the dispersion stability of broccoli exosome-supported cerium dioxide composites is better than that of broccoli exosomes.

[0027] The antioxidant capacity (CAT) of the broccoli exosomes obtained in step S2 and the broccoli exosome-supported cerium dioxide composite material obtained in step S5 were tested respectively. The test results are as follows: Figure 2 As shown. By Figure 2 It can be seen that the antioxidant capacity of the broccoli exosome-loaded cerium dioxide composite material is better than that of broccoli exosomes.

[0028] The anti-inflammatory capabilities of CeO2, the broccoli exosomes prepared in step S2, and the broccoli exosomes loaded with cerium dioxide composite material prepared in step S5 were tested. Cellular intervention was performed using these materials, specifically by loading RAW264.7 cells at a density of 1.5 × 10⁶ cells per well. 5 The cells were seeded at a density in 6-well plates and cultured statically in a cell culture incubator for 24 hours. Then, they were treated with CeO2, broccoli exosomes prepared in step S2, and broccoli exosomes loaded with cerium dioxide composite material prepared in step S5, and cultured for another 24 hours. 1 mL of cell culture medium from each group was pipetted into a 1.5 mL EP tube and centrifuged at 3000 rpm for 30 min to collect the supernatant. The expression level of IL-6 was detected using an ELISA kit. The specific steps are as follows: (1) Preparatory steps: Preheat the microplate reader 30 minutes in advance; take the kit (Vancouver mouse species) out of the refrigerator in advance and place it at room temperature for 20 minutes; (2) Reagent preparation: Prepare the required reagents according to the time recommended in the kit instructions; (3) Sample addition: Add 50 μL of diluent to the blank well, and add 10 μL of cell supernatant sample, 40 μL of sample diluent, and 50 μL of different concentrations of standard to the other wells respectively. After sealing the plate with sealing film, mix well and incubate in a constant temperature incubator at 37°C for 30 minutes; (4) Washing: Shake off the liquid in the well plate, add 300 μL of the prepared 1× washing solution to each well, repeat the washing 5 times, soak for 30 seconds each time, and pat dry on filter paper; (5) Add enzyme labeling reagent: Do not add to the blank well, add 50 μL of HRP to the other wells, seal the plate with sealing film, mix well, and incubate in a constant temperature incubator at 37°C for 30 minutes. min; (6) Wash plate: same as step (4); (7) Add 50 μL of colorimetric solution A, then add 50 μL of colorimetric solution B, seal the plate with sealing film, mix well, and incubate in a 37℃ incubator for 10 min; (8) Add 50 μL of stop solution to each well to stop the reaction; (9) After mixing, immediately measure the OD value of each well at a wavelength of 450 nm using an ELISA reader, and calculate. The test results are as follows. Figure 3 As shown, by Figure 3It can be seen that the anti-inflammatory ability of the broccoli exosome-loaded cerium dioxide composite material is better than that of the broccoli exosome. Example

[0029] The following steps were followed to prepare a cerium dioxide-supported broccoli exosome composite material: S1. Wash the broccoli raw material to remove mud and sand and surface attachments, crush it and add it to 1×PBS buffer with pH 7.3. Then homogenize it for 5 minutes using a tissue homogenizer to obtain a homogenate. The broccoli raw material is fresh, non-rotten broccoli buds. The ratio of fresh weight of broccoli raw material to PBS buffer is 1g:1mL. S2. Centrifuge the homogenate obtained in step S1 at 3000 rpm for 5 min to obtain supernatant one. Centrifuge supernatant one at 10000 rpm for 20 min to obtain supernatant two. Centrifuge supernatant two at 150000 g for 80 min to obtain broccoli exosome precipitate. Resuspend the broccoli exosome precipitate in 1×PBS buffer with pH 7.3 to obtain broccoli exosome dispersion (when resuspending, add broccoli exosome precipitate to PBS buffer at a ratio of 1 g: 200 mL, let stand for 1 min, and then pipette 5 times). Then transfer to EP tube. The particle size of broccoli exosomes in the broccoli exosome dispersion is 30-200 nm. S3. Add cerium acetate to deionized water at a ratio of 1g:20mL, adjust the pH of the system to 10 using hydrazine hydrate, stir for 10h to obtain a mixture, centrifuge the mixture for 10min to obtain a coarse cerium dioxide precipitate, wash the coarse cerium dioxide precipitate three times with deionized water (5min each time) to obtain cerium dioxide particles with a particle size of 3-20nm, add the cerium dioxide particles to 7.3×PBS buffer at a ratio of 0.05mg:1mL, mix well to obtain a cerium dioxide dispersion; S4. Combine the broccoli exosome dispersion obtained in step S2 with the cerium dioxide dispersion obtained in step S3 to obtain a composite solution; mix the broccoli exosome dispersion obtained in step S2 with the cerium dioxide dispersion obtained in step S3 at a volume ratio of 1:2, stir and incubate at room temperature for 24 hours to obtain the composite solution. S5. The composite solution obtained in step S4 is centrifuged at 150,000g for 20 minutes to obtain a precipitate. The precipitate is resuspended in 1×PBS buffer with a pH of 7.3 to obtain a resuspension (when resuspending, the broccoli exosome precipitate is added to PBS buffer at a ratio of 1g:400mL, and after standing for 3 minutes, it is pipetted 5 times to obtain a resuspension). The resuspension is shaken at 4℃ and 150rpm for 5 minutes to obtain a dispersion of broccoli exosome-loaded cerium dioxide composite material. Example

[0030] The following steps were followed to prepare a cerium dioxide-supported broccoli exosome composite material: S1. Wash the broccoli raw material to remove mud and sand and surface attachments, crush it and add it to 1×PBS buffer with pH 7.4. Then homogenize it for 10 min using a tissue homogenizer to obtain a homogenate. The broccoli raw material is fresh, non-rotten broccoli stems. The ratio of fresh weight of broccoli raw material to PBS buffer is 1g:10mL. S2. Centrifuge the homogenate obtained in step S1 at 500 rpm for 15 min to obtain supernatant one. Centrifuge supernatant one at 3000 rpm for 40 min to obtain supernatant two. Centrifuge supernatant two at 150000g for 100 min to obtain broccoli exosome precipitate. Resuspend the broccoli exosome precipitate in 1×PBS buffer with a pH of 7.4 to obtain broccoli exosome dispersion (when resuspending, add the broccoli exosome precipitate to PBS buffer at a ratio of 1g:500mL, let stand for 10 min, and then pipette 20 times). Then transfer it to EP tubes. The particle size of broccoli exosomes in the broccoli exosome dispersion is 30-200nm. S3. Add cerium acetate to deionized water at a ratio of 1g:100mL, adjust the pH of the system to 11 using hydrazine hydrate, stir for 12h to obtain a mixture, centrifuge the mixture for 20min to obtain a coarse cerium dioxide precipitate, wash the coarse cerium dioxide precipitate with deionized water 3 times (5min each time) to obtain cerium dioxide particles with a particle size of 3-20nm, add the cerium dioxide particles to 7.4×PBS buffer at a ratio of 5mg:1mL, mix well to obtain a cerium dioxide dispersion; S4. Combine the broccoli exosome dispersion obtained in step S2 with the cerium dioxide dispersion obtained in step S3 to obtain a composite solution; mix the broccoli exosome dispersion obtained in step S2 with the cerium dioxide dispersion obtained in step S3 at a volume ratio of 1:4, stir and incubate at room temperature for 24 hours to obtain the composite solution. S5. The composite solution obtained in step S4 is centrifuged at 150,000g for 40 minutes to obtain a precipitate. The precipitate is resuspended in 1×PBS buffer with a pH of 7.4 to obtain a resuspension (when resuspending, the broccoli exosome precipitate is added to PBS buffer at a ratio of 1g:400mL, and after standing for 5 minutes, it is pipetted 20 times to obtain a resuspension). The resuspension is shaken at 4℃ and 50rpm for 30 minutes to obtain a dispersion of broccoli exosome-loaded cerium dioxide composite material. Example

[0031] The difference from Example 1 is that the broccoli raw material used in step S1 is fresh, non-rotten broccoli leaves; step S4 is changed to: The broccoli exosome dispersion obtained in step S2 was mixed with the cerium dioxide dispersion obtained in step S3 at a volume ratio of 1:3. The mixture was subjected to intermittent ultrasonic treatment with a probe under ice bath conditions for 6 cycles. The ultrasonic time for each cycle was 60s, the cycle interval was 180s, the ultrasonic frequency was 30kHz, the ultrasonic power was 80W, and the ultrasonic amplitude was 20%. After ultrasonic treatment, the mixture was allowed to stand at 25℃ for 120min to obtain the composite solution. Example

[0032] The difference from Example 2 is that the broccoli used in step S1 is fresh, non-rotten broccoli florets; step S4 is changed to: The broccoli exosome dispersion obtained in step S2 was mixed with the cerium dioxide dispersion obtained in step S3 at a volume ratio of 1:2. The mixture was subjected to intermittent ultrasonic treatment with a probe under ice bath conditions for 4 cycles. The ultrasonic time for each cycle was 10s, the cycle interval was 30s, the ultrasonic frequency was 40kHz, the ultrasonic power was 120W, and the ultrasonic amplitude was 15%. After ultrasonic treatment, the mixture was allowed to stand at 20℃ for 30min to obtain the composite solution. Example

[0033] The difference from Example 3 is that the broccoli raw material used in step S1 is fresh, non-rotten broccoli buds; step S4 is changed to: The broccoli exosome dispersion obtained in step S2 was mixed with the cerium dioxide dispersion obtained in step S3 at a volume ratio of 1:4. The mixture was subjected to intermittent ultrasonic treatment with a probe under ice bath conditions for 8 cycles. The ultrasonic time for each cycle was 120s, the cycle interval was 300s, the ultrasonic frequency was 20kHz, the ultrasonic power was 40W, and the ultrasonic amplitude was 25%. After ultrasonic treatment, the mixture was allowed to stand at 37℃ for 240min to obtain the composite solution. Example

[0034] The difference from Example 1 is that the broccoli raw material used in step S1 is fresh, non-rotten broccoli stems; step S4 is changed to: The process of the ultrasonic-assisted loading method is as follows: the membrane extrusion composite method is as follows: the broccoli exosome dispersion obtained in step S2 and the cerium dioxide dispersion obtained in step S3 are mixed at a volume ratio of 1:3 to obtain a mixed dispersion. The mixed dispersion is first extruded 10 times through an extrusion membrane with a pore size of 400 nm, and then extruded 10 times through an extrusion membrane with a pore size of 200 nm to obtain a composite liquid. Example

[0035] The difference from Example 2 is that the broccoli raw material used in step S1 is fresh, non-rotten broccoli leaves; step S4 is changed to: The process of the ultrasonic-assisted loading method is as follows: the process of the membrane extrusion composite method is as follows: the broccoli exosome dispersion obtained in step S2 is mixed with the cerium dioxide dispersion obtained in step S3 at a volume ratio of 1:2 to obtain a mixed dispersion. The mixed dispersion is then extruded 10 times through an extrusion die with a pore size of 300 nm to obtain a composite liquid. Example

[0036] The difference from Example 3 is that the broccoli used in step S1 is fresh, non-rotten broccoli florets; step S4 is changed to: The process of the ultrasonic-assisted loading method is as follows: the process of the membrane extrusion composite method is as follows: the broccoli exosome dispersion obtained in step S2 is mixed with the cerium dioxide dispersion obtained in step S3 at a volume ratio of 1:4 to obtain a mixed dispersion. The mixed dispersion is then extruded 15 times through an extrusion die with a pore size of 200 nm to obtain a composite liquid.

[0037] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A method for preparing a cerium dioxide-supported composite material from broccoli exosomes, characterized in that: Includes the following steps: S1. Wash the broccoli raw material to remove mud, sand and surface attachments, crush it and add it to PBS buffer, then homogenize it for 5-10 minutes using a tissue homogenizer to obtain a homogenate. S2. Centrifuge the homogenate obtained in step S1 at low speed for 5-15 min to obtain supernatant one, centrifuge supernatant one at medium speed for 20-40 min to obtain supernatant two, centrifuge supernatant two at high speed for 80-100 min to obtain broccoli exosome precipitate, and resuspend the broccoli exosome precipitate with PBS buffer to obtain broccoli exosome dispersion. S3. Add cerium acetate to deionized water, adjust the pH of the system to 10-11 with alkali, stir for 10-12 h to obtain a mixture, centrifuge the mixture for 10-20 min to obtain coarse cerium dioxide precipitate, wash the coarse cerium dioxide precipitate with deionized water 3 times to obtain cerium dioxide particles, add the cerium dioxide particles to PBS buffer, mix evenly to obtain cerium dioxide dispersion. S4. Combine the broccoli exosome dispersion obtained in step S2 with the cerium dioxide dispersion obtained in step S3 to obtain a composite solution; S5. Centrifuge the composite solution obtained in step S4 at high speed for 20-40 min to obtain a precipitate. Resuspend the precipitate in PBS buffer to obtain a resuspension. Shake the resuspension for 5-30 min to obtain a dispersion of broccoli exosome-loaded cerium dioxide composite material.

2. The method for preparing a broccoli exosome-supported cerium dioxide composite material according to claim 1, characterized in that: In step S1, the broccoli raw material is one or more of the following: fresh, non-rotten broccoli florets, buds, stems or leaves; the PBS buffer is a 1×PBS buffer with a pH of 7.2-7.4, and the ratio of the fresh weight of the broccoli raw material to the PBS buffer is 1g:(1-10)mL.

3. The method for preparing a broccoli exosome-supported cerium dioxide composite material according to claim 1, characterized in that: In step S2, the speed of low-speed centrifugation is 500-3000 rpm, the speed of medium-speed centrifugation is 3000-10000 rpm, and the centrifugal force of ultracentrifugation is 150000g; the PBS buffer is 1×PBS buffer with a pH of 7.2-7.

4. When resuspending, the broccoli exosome precipitate is added to the PBS buffer at a ratio of 1g:(200-500)mL. After standing for 1-10 minutes, it is pipetted 5-20 times and then transferred to an EP tube; the particle size of the broccoli exosomes in the broccoli exosome dispersion is 30-200nm.

4. The method for preparing a broccoli exosome-supported cerium dioxide composite material according to claim 1, characterized in that: In step S3, the ratio of cerium acetate to deionized water is 1g:(20-100)mL; the alkali is hydrazine hydrate; the washing time for the coarse cerium dioxide precipitate with deionized water is 5min each time; the particle size of cerium dioxide particles is 3-20nm; the PBS buffer is 1×PBS buffer with a pH of 7.2-7.4; and the ratio of cerium dioxide particles to PBS buffer is (0.05-5)mg:1mL.

5. The method for preparing a broccoli exosome-supported cerium dioxide composite material according to claim 1, characterized in that: In step S4, the composite method is either stirring incubation composite method, ultrasonic-assisted loading method, or membrane extrusion composite method.

6. The method for preparing a broccoli exosome-supported cerium dioxide composite material according to claim 5, characterized in that: The process of the stirring incubation composite method is as follows: the broccoli exosome dispersion obtained in step S2 is mixed with the cerium dioxide dispersion obtained in step S3 at a volume ratio of 1:(2-4), and the mixture is stirred and incubated at room temperature for 24 hours to obtain the composite solution.

7. The method for preparing a broccoli exosome-supported cerium dioxide composite material according to claim 5, characterized in that: The process of the ultrasound-assisted loading method is as follows: the broccoli exosome dispersion obtained in step S2 is mixed with the cerium dioxide dispersion obtained in step S3 at a volume ratio of 1:(2-4). The mixture is subjected to intermittent ultrasound treatment with a probe under ice bath conditions for 4-8 cycles. The ultrasound time of each cycle is 10-120s, the cycle interval is 30-300s, the ultrasound frequency is 20-40kHz, the ultrasound power is 40-120W, and the ultrasound amplitude is 15-25%. After the ultrasound treatment, the mixture is allowed to stand at 20-37℃ for 30-240min to obtain the composite solution.

8. The method for preparing a broccoli exosome-supported cerium dioxide composite material according to claim 5, characterized in that: The process of the membrane extrusion composite method is as follows: the broccoli exosome dispersion obtained in step S2 is mixed with the cerium dioxide dispersion obtained in step S3 at a volume ratio of 1:(2-4) to obtain a mixed dispersion. The mixed dispersion is then extruded through an extrusion die with a pore size of 200-400nm 10-20 times to obtain a composite liquid.

9. The method for preparing a broccoli exosome-supported cerium dioxide composite material according to claim 1, characterized in that: In step S5, the centrifugal force of ultracentrifugation is 150,000 g; the PBS buffer is 1×PBS buffer with a pH of 7.2-7.

4. When resuspending, the precipitate is added to the PBS buffer at a ratio of 1 g: 400 mL. After standing for 3-5 minutes, the precipitate is pipetted 5-20 times to obtain a resuspension. The oscillation temperature is 4℃ and the oscillation speed is 50-150 rpm.

10. The broccoli exosome-supported cerium dioxide composite material prepared by the preparation method according to claims 1 to 9.

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