Plant external vesicle complex loaded with miR-424 and cerium oxide as well as preparation method and application of plant external vesicle complex

Abstract

The invention discloses a miR-424 and cerium oxide loaded plant external vesicle complex as well as a preparation method and application thereof, and belongs to the technical field of biological medicines and nano materials. The complex comprises plant external vesicles extracted from radix puerariae and a compound of miR-424 and cerium oxide nanoparticles loaded in the plant external vesicles. The preparation method mainly comprises the following steps: extracting plant extracellular vesicles from radix puerariae; the preparation method comprises the following steps: compounding cerium oxide nanoparticles and miR-424 according to a mass ratio; and co-incubating the obtained compound and plant outer vesicles to realize loading. The complex provided by the invention integrates the good biocompatibility of the plant exovesicles, the gene regulation function of the miR-424 and the antioxidant activity of the cerium oxide nanoparticles, is low in cytotoxicity under low concentration, and shows a better uptake tendency to macrophages. The complex can be used for preparing medicines, and particularly has application potential in prevention or treatment of inflammation or oxidative stress related diseases (such as sepsis and acute lung injury).

Description

Technical Field

[0001] This invention belongs to the field of drug delivery technology, and particularly relates to a plant exovesicle complex loaded with miR-424 and cerium oxide, its preparation method and application. Background Technology

[0002] In the field of drug delivery systems research, extracellular vesicles have become a research hotspot for novel nanomedicine carriers due to their natural biocompatibility, low immunogenicity, and excellent drug loading capacity. Plant-derived extracellular vesicles, as one type, not only possess these advantages but also feature wide availability, low cost, and ease of large-scale preparation, showing promising application prospects in drug, nucleic acid, and protein delivery. However, current research on plant extracellular vesicles mainly focuses on common fruits and vegetables or model plants, while the extraction, characterization, and functional development of vesicles derived from traditional medicinal plants remain relatively limited, especially lacking systematic research on extracellular vesicles derived from traditional Chinese medicines such as kudzu root.

[0003] Kudzu root, a traditional Chinese medicine, possesses properties such as relieving muscle tension and fever, promoting fluid production and relieving rashes. Its active ingredients have been extensively reported in anti-inflammatory, antioxidant, and immunomodulatory effects. However, systematic research on extracellular vesicles (PL-EVs) derived from kudzu root and their application in drug delivery is lacking. Existing methods for extracting plant extracellular vesicles mainly include ultracentrifugation, polymer precipitation, and size exclusion chromatography. Among these, ultracentrifugation is widely used due to its relatively simple operation and good vesicle integrity. For vesicle characterization, transmission electron microscopy can be used to observe their morphology and structure, nanoparticle tracking analysis can determine their particle size distribution and concentration, and protein concentration determination helps to assess their biochemical characteristics. Nevertheless, for medicinal materials like kudzu root with high water content and abundant mucilage, how to efficiently and completely extract its extracellular vesicles and establish corresponding quality control standards remains a technological gap.

[0004] Furthermore, while plant exovesicles possess excellent carrier properties, their functions are relatively limited, making it difficult to meet the demands for targeted, sustained-release, and synergistic therapy in the treatment of complex diseases. Therefore, functional modification of plant exovesicles, such as loading them with inorganic nanoparticles or nucleic acid drugs, has become an important strategy to improve their delivery efficiency. Cerium oxide nanoparticles, due to their excellent antioxidant activity and biosafety, have shown therapeutic potential in inflammation and oxidative stress-related diseases; miRNAs, on the other hand, can participate in various pathological processes by regulating gene expression. Loading both into plant exoves promises to construct a composite delivery system with both antioxidant and gene regulation functions. However, achieving efficient composite loading of miRNAs and inorganic nanoparticles, stably loading them into plant exoves, and maintaining the structural integrity and bioactivity of the vesicles still lacks mature technical solutions and systematic experimental data.

[0005] In summary, there are no reports in the current technology on the extraction and systematic characterization of extracellular vesicles from kudzu root, nor are there any methods for preparing functionalized complexes by co-loading miR-424 and cerium oxide nanoparticles into kudzu root vesicles, nor are there any related biological evaluations. Therefore, developing a method for the extraction and characterization of extracellular vesicles from kudzu root, and constructing a composite vesicle delivery system loaded with miR-424 and cerium oxide, is of great significance for expanding the application of plant extracellular vesicles in the field of drug delivery, and also provides experimental evidence and technical foundation for the development of novel nanomedicines based on vesicles derived from traditional Chinese medicine. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention proposes a plant extravesicular complex loaded with miR-424 and cerium oxide, its preparation method, and its application. This plant extravesicular complex possesses both gene regulation and antioxidant functions, exhibits good biocompatibility, and has potential targeting for specific cells.

[0007] To achieve the above objectives, the present invention provides a plant extracellular vesicle complex loaded with miR-424 and cerium oxide, comprising plant-derived extracellular vesicles and cerium oxide nanoparticles and miR-424 loaded therein. The complex is composed of 424; wherein the mass ratio of the cerium oxide nanoparticles to miR-424 is (1:1) to (100:1), preferably (30:1) to (70:1), more preferably 50:1; and the mass ratio of the plant exovesicles to the cerium oxide nanoparticles is (1:1) to (1:20), preferably (1:3) to (1:10), more preferably 1:5.

[0008] Furthermore, the plant exovesicles have a double-membrane structure with an average particle size of 80 nm to 180 nm, preferably 100 nm to 150 nm.

[0009] Furthermore, the plant-derived extracellular vesicles were extracted from kudzu root.

[0010] The present invention also provides a method for preparing the plant exovesicle complex loaded with miR-424 and cerium oxide as described above, comprising the following steps: S1. Provide plant exovesicle suspension; S2. Cerium oxide nanoparticles were mixed with miR-424 in a buffer solution and incubated to form a miR-424-cerium oxide complex; S3. The plant exovesicle suspension is mixed with the miR-424-cerium oxide complex and incubated to allow the complex to be loaded inside the plant exovesicles; S4. Purification yields plant exovesicle complexes loaded with miR-424 and cerium oxide.

[0011] Further, in step S1, the method for extracting plant exovesicles includes: taking plant material, juicing and filtering it, and then subjecting the obtained juice to low-speed centrifugation, medium-speed centrifugation and high-speed centrifugation in sequence, collecting the precipitate, and then purifying it by ultracentrifugation to obtain plant exovesicles; wherein the conditions for ultracentrifugation are 100,000×g to 200,000×g, and the centrifugation time is not less than 1 hour.

[0012] Further, in step S2, the mixing and incubation is carried out in HEPES buffer at a temperature of 15-37°C for 15-60 minutes.

[0013] Further, in step S3, the incubation is carried out at 37°C for 8-24 hours; in step S4, the purification includes removing unloaded free components by ultracentrifugation.

[0014] The present invention also provides the application of the above-described plant exovesicle complex loaded with miR-424 and cerium oxide in the preparation of a drug.

[0015] Furthermore, the drug is used to prevent and / or treat inflammation-related diseases or oxidative stress-related diseases.

[0016] Furthermore, the inflammation-related diseases or oxidative stress-related diseases include sepsis or acute lung injury.

[0017] Compared with the prior art, the present invention has the following advantages and technical effects: The plant exovesicle complex loaded with miR-424 and cerium oxide provided by this invention exhibits significant technical advantages in several aspects compared to existing technologies. This complex successfully integrates gene regulation function and antioxidant activity into a natural plant exovesicle carrier, achieving a synergistic effect on the therapeutic mechanism. Through optimized preparation processes, efficient and stable loading of functional molecules is ensured, with a loading efficiency exceeding 80% at the preferred ratio. Furthermore, the complex exhibits a regular morphology and good dispersibility, laying the foundation for the development of quality-controlled nanoparticle formulations.

[0018] This composite integrates multiple technological advantages such as highly efficient synergistic therapy, good biosafety, potential targeting and innovative sources. It has broad application prospects in the prevention and treatment of inflammation-related diseases and oxidative stress-related diseases (such as sepsis and acute lung injury), and provides strong technical support for the development of a new generation of multifunctional nanomedicine delivery systems. Attached Figure Description

[0019] Figure 1 This is a flowchart illustrating the extraction process of vesicles from kudzu root.

[0020] Figure 2Transmission electron microscope image of the outer vesicles of kudzu-derived plants.

[0021] Figure 3 The particle size distribution of nanoparticles from the outer vesicles of kudzu root is shown in the tracking analysis.

[0022] Figure 4 Transmission electron microscopy image of cerium oxide-loaded kudzu exovesicle complex.

[0023] Figure 5 The figure shows the effect of different concentrations of plant vesicles on the viability of RAW264.7 cells.

[0024] Figure 6 Confocal microscopy images of the uptake of DiL-labeled plant vesicles in different cells.

[0025] Figure 7 The figure shows the comparison results of the levels of inflammatory factors in the serum of mice in different experimental groups; where A is the level of IL-6 in mouse serum, B is the level of TNF-α in mouse serum, C is the level of IFN-γ in mouse serum, and D is the level of IL-10 in mouse serum.

[0026] Figure 8 Representative stained sections of major organs of mice treated with different routes of administration.

[0027] Figure 9 This is a representative stained section image showing the effect of the miR-424-CeO2@PL-EVs complex on the improvement of multi-organ histopathology in septic mice. Detailed Implementation

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] All raw materials used in this invention are not particularly limited in their source; they can be purchased from the market or prepared using conventional methods known to those skilled in the art.

[0030] There are no particular restrictions on the purity of any of the raw materials used in this invention. However, this invention preferably uses raw materials of analytical grade or purity commonly used in the field of chemical synthesis.

[0031] Example 1: Extraction of exovesicles from kudzu root This embodiment provides a method for extracting plant exovesicles (PL-EVs) from fresh kudzu root. The specific process can be found in [link to documentation]. Figure 1 This includes the following steps: (1) Raw material processing: Take an appropriate amount of fresh kudzu tubers from Yixing, Jiangsu Province, and rinse them repeatedly with running deionized water to remove surface dirt and impurities. After blotting off excess moisture with filter paper, cut the kudzu tubers into pieces about 1cm long using a clean knife. 3 Small pieces.

[0032] (2) Juicing and coarse filtration: Add the chopped kudzu root and twice the weight of ultrapure water (resistivity ≥18.2 MΩ·cm) to a tissue homogenizer and juice for 20 minutes using intermittent operation mode (30 seconds of operation followed by 10 seconds of interval) to avoid excessive heat generation. Filter the resulting slurry through pre-sterilized and three-layered medical gauze, collect the filtrate, and discard the filter residue.

[0033] (3) Differential centrifugation purification: Place the above kudzu root juice filtrate in a high-speed refrigerated centrifuge and centrifuge according to the following steps. All centrifugation steps are carried out at 4°C: First, centrifuge at 300 ×g for 15 minutes, carefully collect the supernatant, and discard the bottom precipitate (mainly unbroken tissue pieces and large cell debris); then, centrifuge the supernatant at 2000 ×g for 30 minutes to further remove cell debris and collect the supernatant; next, centrifuge the supernatant at 12000 ×g for 30 minutes to remove subcellular organelles and larger vesicle populations and collect the supernatant.

[0034] (4) Ultracentrifugation concentration: Transfer the supernatant obtained in step (3) to a dedicated ultracentrifuge tube and centrifuge at 4°C and 140,000 ×g for 70 minutes. After centrifugation, carefully discard the supernatant; the precipitate is the crude PL-EVs. Gently resuspend the precipitate in pre-cooled sterile PBS buffer (pH 7.4) and wash again by ultracentrifugation under the same conditions (140,000 ×g, 4°C, 70 minutes). Finally, resuspend the final precipitate in a small amount of sterile PBS.

[0035] (5) Sterilization and preservation: The resuspended PL-EVs suspension was sterilized by filtration through a 0.22 μm pore size polyethersulfone sterile needle filter. The total protein concentration of the suspension was determined using a BCA protein quantification kit, and the particle concentration was determined using a nanoparticle tracking analyzer. The PL-EVs suspension was aliquoted into 100 μL tubes and stored in a -80°C ultra-low temperature freezer for long-term storage.

[0036] To demonstrate the superiority of ultracentrifugation in the system of this invention, the following comparative examples of different extraction methods are provided: Comparative Example 1 (Polymer Precipitation Method): Take an equal volume of kudzu root juice filtrate as in Example 1, add ExoQuick-TC® (System Biosciences) polymer precipitation reagent at a volume ratio of 1:4 to the filtrate, mix well, and incubate overnight at 4°C. Then centrifuge at 4°C and 1500 ×g for 30 minutes, discard the supernatant, and resuspend the precipitate in PBS. The resulting sample is labeled PL-EVs-PP.

[0037] Comparative Example 2 (Size Exclusion Chromatography): A qEV original size exclusion column (Izon Science) was used. The supernatant from centrifugation of kudzu root juice at 300×g for 15 minutes was loaded onto the column and eluted with PBS. The eluent (7-9 mL, based on manufacturer recommendations and pre-experimental determination of the exosome enrichment fraction) was collected. The resulting sample was labeled PL-EVs-SEC.

[0038] The samples obtained in Example 1 and Comparative Examples A and B were characterized. The results are shown in Table 1. The PL-EVs obtained by ultracentrifugation in Example 1 were superior to or comparable to those obtained by commercial reagent methods in terms of particle concentration, protein concentration, and particle size uniformity (characterized by the particle size polydispersity index PDI). Furthermore, TEM showed that their vesicle structures were intact and the membranes were clear. Figure 2 Polymer precipitation yields high amounts of impurities but produces a large amount of protein and has a high PDI (Polydigestion Index). Size exclusion chromatography yields the highest purity but has low recovery rates and small throughput, making it unsuitable for large-scale preparation.

[0039] Table 1. Comparison of characterization of PL-EVs obtained by different extraction methods Example 2: Characterization of plant granular vesicles (PL-EVs) derived from kudzu root The PL-EVs proposed in Example 1 were systematically characterized.

[0040] (1) Transmission electron microscopy observation: 10 μL of PL-EVs suspension was added to a copper mesh (Formvar-carboncoated), allowed to stand at room temperature for 10 minutes, and excess liquid was absorbed from the edges with filter paper. Then, 20 μL of 2% phosphotungstic acid solution (pH 7.0) was added for negative staining for 1 minute, the stain was blotted dry with filter paper, and the mesh was dried at room temperature. Observation was performed using a transmission electron microscope at an accelerating voltage of 80 kV. Figure 2 As shown, PL-EVs exhibit a typical saucer-like or cup-shaped bilayer membrane structure, and the particle size distribution is consistent with the subsequent NTA results, preliminarily confirming the successful extraction of structurally intact plant extravesicles.

[0041] (2) Particle size and concentration analysis: Detection was performed using a nanoparticle tracking analyzer. The PL-EVs suspension was diluted with sterile PBS to an appropriate concentration (approximately 10). 7-10 8 The particle concentration (particles / mL) was injected into the sample cell using a 1 mL syringe. Instrument settings: Camera Level 14, Detection Threshold 5. Each sample was measured three times, 60 seconds each time. The analysis software automatically calculated the particle concentration and particle size distribution. Results are as follows: Figure 3 As shown, the average particle size of PL-EVs is 126.8 ± 12.3 nm, mainly distributed in the range of 80-180 nm, and the particle concentration is (7.63 ± 0.35) × 10⁻⁶. 8 particles / mL.

[0042] (3) Protein concentration determination: The protein concentration was determined using the BCA protein quantification kit. The PL-EVs suspension was mixed with an equal volume of RIPA lysis buffer (containing 1% PMSF) and lysed on ice for 30 minutes. Protein standards and samples were added to a 96-well plate according to the instructions, followed by the addition of BCA working solution. After incubation at 37°C for 30 minutes, the absorbance was measured at 562 nm using a microplate reader, and the protein concentration was calculated based on the standard curve. The protein concentration of the PL-EVs suspension was found to be 98.24 ± 5.12 μg / μL, indicating that it contains abundant protein components, consistent with the characteristics of extracellular vesicles.

[0043] Example 3: Preparation and Loading Efficiency Optimization of miR-424-CeO2 Complex This embodiment aims to optimize the composite conditions of miR-424 and cerium oxide nanoparticles (CeO2NPs).

[0044] (1) Material preparation: Lyophilized miR-424 mimics were dissolved in nuclease-free ultrapure water to prepare a 50 μM stock solution, which was then aliquoted and stored at -80°C. CeO2NPs aqueous dispersion (particle size ~10 nm, concentration 10 mg / mL) was purchased from Aladdin Reagent Co., Ltd. Before use, the mixture was shaken on a vortex mixer for 1 minute.

[0045] (2) Complex preparation: With a fixed final molar amount of miR-424, the amount of CeO2NPs was varied, with six ratio groups (1:1, 5:1, 10:1, 20:1, 30:1, and 50:1) set as the mass ratio of CeO2 to miR-424 (CeO2: miRNA). In a 1.5 mL centrifuge tube, first add the calculated volume of CeO2NPs suspension (diluted to the required concentration with 20 mM HEPES buffer, pH 7.4), then add an equal volume of HEPES buffer containing the required amount of miR-424, and finally add HEPES buffer to make up the total volume. Vortex to mix for 10 seconds, then incubate at room temperature (25°C) in the dark for 30 minutes to form the miR-424-CeO2 complex.

[0046] (3) Loading efficiency determination: The incubated complex solution was centrifuged at 4°C and 14,000 rpm for 30 minutes. CeO2NPs and their complexes precipitate under these conditions, while free miR-424 remains in the supernatant. The supernatant was carefully aspirated and processed using NanoDrop. TM The absorbance of miR-424 in the supernatant at 260 nm was measured using a One micro-volume UV-Vis spectrophotometer, and the concentration of free miR-424 was calculated based on its standard curve. Loading efficiency (LE%) was calculated using the following formula: LE% = [ (C0× V0– C1× V1) / (C0× V0) ] × 100% Wherein, C0 and V0 are the initial concentration and volume of miR-424 added, respectively, and C1 and V1 are the concentration and volume of free miR-424 in the supernatant, respectively.

[0047] (4) Results Analysis: The effect of different mass ratios on loading efficiency is shown in Table 2. As the CeO2 ratio increases, the loading efficiency first increases and then tends to stabilize. When the mass ratio is 50:1, the loading efficiency reaches the highest level, which is 80.65 ± 3.21%. Therefore, the CeO2:miRNA mass ratio of 50:1 was selected as the optimal composite ratio for subsequent experiments.

[0048] Table 2. Effect of different CeO2:miRNA mass ratios on loading efficiency To further validate the composite conditions, comparative experiments were conducted using incubation times (10, 30, 60, 120 min) and buffer types (HEPES, PBS, citrate buffer). The results showed that loading equilibrium was achieved after 30 minutes of incubation in HEPES buffer, and extending the incubation time did not significantly improve loading efficiency. Loading efficiency was slightly lower in PBS buffer (approximately 75%), while it significantly decreased to ~45% in citrate buffer (pH 5.0), possibly related to changes in the surface charge of CeO2.

[0049] Example 4: Preparation of PL-EVs composites supported on miR-424-CeO2 (miR-424-CeO2@PL-EVs) This embodiment describes the process of loading the miR-424-CeO2 complex into PL-EVs.

[0050] (1) Preparation of miR-424-CeO2 complex: The miR-424-CeO2 complex was prepared according to the optimal conditions determined in Example 3, at a CeO2:miRNA mass ratio of 50:1. Specifically, an appropriate amount of CeO2NPs was weighed and diluted to 0.5 mg / mL with HEPES buffer. The miR-424 stock solution was diluted to a suitable concentration. After mixing in the proportions, the mixture was vortexed and incubated at room temperature in the dark for 30 minutes for later use.

[0051] (2) Co-incubation of the load: The PL-EVs suspension (protein concentration of about 1 mg / mL) prepared in Example 1 was mixed with the miR-424-CeO2 complex solution prepared above at a PL-EVs protein to CeO2 mass ratio of 1:5 in a centrifuge tube. The mixture was placed on a horizontal shaker and incubated overnight (about 12-16 hours) in the dark at 37°C and 100 rpm. The complex was passively diffused into the vesicles by utilizing the concentration gradient and the permeability of the vesicle membrane.

[0052] (3) Purification: Transfer the incubated mixture to an ultracentrifuge tube and centrifuge at 4°C and 150,000 ×g for 2 hours. Carefully discard the supernatant; the precipitate is the PL-EVs complex loaded with miR-424-CeO2. To remove any remaining free components, resuspend the precipitate in pre-cooled sterile PBS and wash it again under the same conditions (150,000 ×g, 4°C, 2 hours). Finally, resuspend the precipitate in an appropriate amount of PBS to obtain the purified miR-424-CeO2@PL-EVs complex suspension, which can be stored at -80°C or used immediately for subsequent characterization and experiments.

[0053] (4) Morphological characterization: TEM samples were prepared and observed using the same method as in Example 2(1). Figure 4As shown, miR-424-CeO2@PL-EVs (referred to as CeO2@PL-EVs in the figure) retains its spherical vesicle structure. The particle size is slightly larger than that of blank PL-EVs, but the distribution remains uniform. Under high-magnification electron microscopy, dots or small clumps with high electron density can be observed inside the vesicles, similar in morphology to free CeO2NPs. This indicates that CeO2NPs were successfully encapsulated within the vesicles, and the complex exhibits good dispersion without significant aggregation.

[0054] To optimize the loading process, the following comparative example is set: Comparative Example 3 (Electroporation): Take equal amounts of PL-EVs and miR-424-CeO2 complex, place them in an electroporation cup, and electrolyze once at 350 V and 150 μF. After restoring on ice for 10 minutes, dialyze with PBS to remove free components.

[0055] Comparative Example 4 (Freeze-Thaw Cycle Method): The mixture of PL-EVs and miR-424-CeO2 complex was rapidly frozen in liquid nitrogen for 2 minutes, then rapidly thawed in a 37°C water bath. This cycle was repeated 3 times, and finally purified by ultracentrifugation.

[0056] Comparative Example 5 (incubation ratio changed): The mass ratio of PL-EVs protein to CeO2 was changed to 1:1, and other conditions were the same as in Example 4.

[0057] Comparative Example 6 (incubation ratio changed): The mass ratio of PL-EVs protein to CeO2 was changed to 1:10, and other conditions were the same as in Example 4.

[0058] The above samples were characterized, and the drug loading (DL, μg / mg PL-EVs protein) and encapsulation efficiency (EE, %) were calculated by measuring the remaining CeO2 (indirectly determined by cerium elemental ICP-MS analysis) and miR-424 (NanoDrop) content in the supernatant after loading. The results are shown in Table 3. The passive incubation method in Example 4 achieved a high overall loading effect at a 1:5 ratio, and was the simplest to operate with minimal damage to vesicle structure (TEM confirmed). Although electroporation can improve miRNA encapsulation efficiency, it may lead to vesicle aggregation; the freeze-thaw method has a significant impact on vesicle integrity; while changing the loading ratio directly affects the drug loading or encapsulation efficiency.

[0059] Table 3 Comparison of different load methods and conditions Example 5: Cytotoxicity evaluation of PL-EVs and miR-424-CeO2@PL-EVs The in vitro cytotoxicity of blank PL-EVs and their drug-loaded complexes to RAW 264.7 mouse macrophages was evaluated using the MTT assay.

[0060] (1) Cell culture: RAW 264.7 cells were cultured in a high-glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin in a constant temperature incubator at 37°C and 5% CO2.

[0061] (2) Sample preparation: Take cells in the logarithmic growth phase, digest them with trypsin, and count them at a density of 1.0 × 10⁶ cells per well. 4 Cells were seeded at a density of 100 μL of complete culture medium per well in 96-well plates and cultured for 12 hours to allow cell adhesion. A blank control group (containing only culture medium), a negative control group (containing an equal volume of PBS), and an experimental group were set up.

[0062] (3) Drug administration and incubation: Discard the old culture medium, and add PL-EVs diluted to different particle concentrations (concentration gradient: 10) in serum-free DMEM medium to the experimental groups. 2 10 3 10 4 10 5 10 6 10 7 10 8 (particles / mL) or miR-424-CeO2@PL-EVs complex (based on PL-EVs particle concentration, with the same gradient). Six replicates were created for each concentration. Incubation continued for 24 hours.

[0063] (4) MTT assay: After incubation, add 10 μL of MTT solution (5 mg / mL) to each well and continue incubation for 4 hours. Carefully aspirate the liquid from the wells, add 100 μL of dimethyl sulfoxide (DMSO) to each well, and place on a shaker to shake at low speed for 10 minutes to fully dissolve the purple formazan crystals.

[0064] (5) Detection and analysis: The absorbance (OD) of each well was measured at a wavelength of 490 nm using a microplate reader. 490 The formula for calculating cell viability is: Cell viability (%) = (OD experimental group - OD blank wells) / (OD negative control group - OD blank wells) × 100% (6) Results: such as Figure 5 As shown, blank PL-EVs at concentrations not exceeding 10 3 At a concentration of particles / mL, there was no significant effect on the survival rate of RAW cells (survival rate >95%). However, when the concentration increased to 10... 8At particle / mL concentrations, cell viability remained above 85%, indicating that PL-EVs have excellent biocompatibility. The cytotoxicity trend of the miR-424-CeO2@PL-EVs complex at various concentrations was basically consistent with that of the blank PL-EVs, and no additional toxicity was observed within the experimental concentration range, indicating that the loading process and the loaded components did not significantly increase the cytotoxicity of the system.

[0065] Hemolytic assay: To further evaluate biosafety, an in vitro hemolytic assay was performed. Fresh anticoagulated rat blood was collected, centrifuged to obtain red blood cells, washed with PBS, and prepared into a 2% red blood cell suspension. PL-EVs and miR-424-CeO2@PL-EVs were mixed with an equal volume of red blood cell suspension (final concentration was 10-1 PL-EVs). 8 The concentration of particles / mL was calculated, with PBS and deionized water used as negative controls (0% hemolysis) and positive controls (100% hemolysis), respectively. After incubation at 37°C for 2 hours, the samples were centrifuged, and the absorbance of the supernatant was measured at 540 nm. The hemolysis rate was calculated. The results showed that the hemolysis rates of both methods were less than 2%, meeting the safety requirements for biomaterials.

[0066] Example 6: Study on cellular uptake behavior of PL-EVs To understand the cellular uptake characteristics of PL-EVs, the lipophilic fluorescent dye DiI was used for labeling and tracing.

[0067] (1) DiI-labeled PL-EVs: DiI dye was prepared into a 1 mM stock solution using DMSO. The PL-EVs suspension prepared in Example 1 (particle concentration approximately 10) was taken. 9 Add DiI stock solution (particles / mL) to bring the final DiI concentration to 10 μM, and adjust the final PL-EVs concentration to approximately 10 μM. 8 Particles / mL. After vortexing, incubate at 37°C in the dark for 30 minutes. After incubation, transfer the labeled mixture to an ultracentrifuge tube and centrifuge at 4°C and 120,000 ×g for 60 minutes to remove unbound free DiI dye. Gently resuspend the precipitate in PBS to obtain DiI-labeled PL-EVs (DiI-PL-EVs).

[0068] (2) Cell uptake experiment: RAW 264.7 macrophages, dendritic cells (DCs), and T cells were selected for comparison. Cells were seeded in confocal culture dishes and cultured to approximately 70% confluence. The old culture medium was discarded, and freshly prepared DiI-PL-EVs working solution (final DiI concentration 100 nM) was added to the medium without phenol red. The cells were incubated at 37°C and 5% CO2 for 1, 2, 4, and 8 hours, respectively.

[0069] (3) Sample processing and observation: After reaching the predetermined time point, discard the culture medium and gently wash the cells three times with pre-cooled PBS. Fix with 4% paraformaldehyde at room temperature for 15 minutes, and wash three times with PBS. Add anti-fluorescence quenching mounting medium containing DAPI to stain the cell nuclei. Observe using a laser confocal microscope. The excitation / emission wavelength of DiI is 549 / 565 nm (red fluorescence), and that of DAPI is 358 / 461 nm (blue fluorescence).

[0070] (4) Results analysis: such as Figure 6 As shown, under the same incubation conditions, RAW 264.7 cells exhibited the most significant uptake of DiI-PL-EVs, with numerous bright red fluorescent spots visible within the cells, primarily distributed in the cytoplasm. Fluorescence was also observed in DCs and T cells, but at a significantly weaker intensity. Quantitative analysis (using ImageJ software to analyze average fluorescence intensity) further confirmed that at 4 hours, the DiI fluorescence intensity of RAW cells was approximately 3.5 times and 2.8 times that of HUVEC and A549 cells, respectively (p<0.01). This indicates that the pueraria lobata PL-EVs extracted in this invention have a relatively higher uptake tendency in RAW 264.7 macrophages, potentially suggesting a certain degree of cell selectivity or targeting potential.

[0071] Example 7: In vivo efficacy verification (improvement of acute lung injury pathology in septic mice) By constructing a mouse model of sepsis with cecal ligation and perforation (CLP), we preliminarily explored the in vivo protective effect of miR-424-CeO2@PL-EVs.

[0072] (1) Animals and grouping: C57BL / 6 male mice were randomly divided into 5 groups (n=6): ① normal control group; ② CLP model group; ③ CLP+blank PL-EVs group; ④ CLP+CeO2@PL-EVs group; ⑤ CLP+miR-424-CeO2@PL-EVs group.

[0073] (2) Modeling and drug administration: Except for the normal control group which was injected intraperitoneally with an equal volume of physiological saline, the other groups were CLP-based to establish a mouse model of sepsis. Half an hour after the model was established, the treatment groups were injected with the corresponding preparations via the tail vein (dosage: 5 mg / kg per mouse based on PL-EVs, calculated according to vesicle protein content).

[0074] (3) Sample collection and analysis: 24 hours after administration, mice were sacrificed and serum and tissues from multiple organs were collected.

[0075] Serum inflammatory factor marker detection: ELISA was used to detect the levels of inflammatory factors (IL-6, TNF-α, IFN-γ, IL-10) in the serum of different groups of mice to assess the strength of the inflammatory response.

[0076] Histopathology: After fixation with 4% paraformaldehyde, paraffin embedding, and sectioning, the tissue was stained with hematoxylin and eosin (H&E) and the pathological changes, such as cell necrosis and inflammatory cell infiltration, were observed under an optical microscope.

[0077] (4) Results: Inflammatory factor markers ( Figure 7 The levels of serum pro-inflammatory factors (IL-6, TNF-α, IFN-γ) in the CLP model group were sharply elevated compared to the normal control group. All treatment groups showed varying degrees of reduction, with the miR-424-CeO2@PL-EVs group showing the most significant reduction (p<0.01 vs. model group and other treatment groups). Meanwhile, the level of the anti-inflammatory factor IL-10 in the CLP model group was significantly lower than that in the normal control group, while all treatment groups showed varying degrees of elevation, with the miR-424-CeO2@PL-EVs group showing a significant elevation (p<0.01 vs. model group and other treatment groups).

[0078] Pathological sections: such as Figure 8 and Figure 9 As shown, the normal control group exhibited clear lung tissue structure, thin and intact alveolar walls, and no exudate in the alveolar cavities. The CLP model group showed extensive necrosis of the lung parenchyma (indicated by arrows), alveolar structural destruction, and extensive inflammatory cell infiltration filling the alveolar cavities, with significant hemorrhage and edema. The necrotic areas were reduced in the blank PL-EVs and CeO2@PL-EVs treatment groups, with mild thickening of the alveolar septa. The miR-424-CeO2@PL-EVs treatment group showed the most significant improvement in lung tissue structure, with only a few punctate alveolar epithelial cell necrosis, significantly reduced inflammatory infiltration in the alveolar cavities, marked absorption of hemorrhage and edema, and near-normal restoration of alveolar structure. This indicates that the miR-424-CeO2@PL-EVs complex has a significant protective effect against CLP-induced acute lung injury in vivo, with superior efficacy compared to single functional components. Furthermore, given the multi-organ involvement characteristic of sepsis, we further evaluated the pathological changes in the liver, kidneys, heart, intestines, and brain tissues.

[0079] Liver tissue: In the normal control group, the liver lobule structure was clear, and the hepatocyte cords were neatly arranged. In the CLP model group, large areas of hepatocyte necrosis were observed, accompanied by a large amount of inflammatory cell infiltration and significant congestion. The necrotic area and inflammatory infiltration were reduced in the blank PL-EVs and CeO2@PL-EVs treatment groups. The miR-424-CeO2@PL-EVs treatment group showed the most significant improvement, with only a few punctate necrosis, the hepatocyte arrangement was basically normal, and the inflammatory infiltration was significantly reduced.

[0080] Kidney tissue: In the normal control group, the glomerular structure was intact, the renal tubular epithelial cells were of normal morphology, and no casts were formed. In the CLP model group, extensive necrosis and shedding of renal tubular epithelial cells were observed, the lumen was filled with protein casts, and the interstitium was congested and edematous with inflammatory cell infiltration. The degree of renal tubular damage and the number of casts were reduced in the blank PL-EVs and CeO2@PL-EVs treatment groups. In the miR-424-CeO2@PL-EVs treatment group, the glomerular capsule was clear, with only slight swelling of scattered renal tubular epithelial cells, and the interstitial inflammation was significantly reduced, approaching the morphology of normal kidney tissue.

[0081] Cardiac tissue: In the normal control group, myocardial fibers were neatly arranged with clear striations and no interstitial edema. In the CLP model group, disordered and broken myocardial fibers were observed, with some cardiomyocytes showing vacuolar degeneration, and significant interstitial edema accompanied by inflammatory cell infiltration. In the blank PL-EVs and CeO2@PL-EVs treatment groups, the degree of myocardial cell degeneration was reduced, and interstitial edema was alleviated. In the miR-424-CeO2@PL-EVs treatment group, the myocardial fiber structure was basically restored to normal, with occasional punctate cardiomyocyte atrophy, and interstitial edema and inflammatory infiltration were significantly reduced.

[0082] Intestinal tissue: In the normal control group, the intestinal villi structure was intact, the epithelial cells were tightly arranged, and the crypt structure was clear. In the CLP model group, extensive necrosis and shedding of epithelial cells at the villus tips were observed, the villi were shortened and atrophied, and there was extensive inflammatory cell infiltration in the lamina propria, accompanied by significant edema. In the blank PL-EVs and CeO2@PL-EVs treatment groups, the degree of villus damage was reduced, and the continuity of the epithelium was partially restored. In the miR-424-CeO2@PL-EVs treatment group, the intestinal villus structure was basically intact, the epithelial cell morphology was close to normal, only slight crypt dilation was observed, and the inflammation of the lamina propria was significantly reduced.

[0083] Brain tissue: In the normal control group, neurons in the cerebral cortex and hippocampus showed normal morphology, abundant Nissl bodies, no glial cell proliferation, and no interstitial edema. In the CLP model group, some neurons showed cell body shrinkage, nuclear pyknosis, reduced or absent Nissl bodies, significant glial cell proliferation, and widened perivascular spaces, indicating cerebral edema. The degree of neuronal damage was reduced and glial cell proliferation was alleviated in the blank PL-EVs and CeO2@PL-EVs treatment groups. In the miR-424-CeO2@PL-EVs treatment group, neuronal morphology was basically restored to normal, Nissl bodies were abundant, glial cell proliferation was significantly inhibited, and perivascular spaces were restored.

[0084] In summary, the miR-424-CeO2@PL-EVs complex showed significant protective effects against CLP-induced multi-organ damage (liver, kidney, heart, lung, intestine, and brain), and its efficacy was superior to that of single functional components, suggesting that this composite nanosystem effectively alleviated the systemic pathological damage of sepsis through synergistic effects.

[0085] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A plant exosomal vesicle complex loaded with miR-424 and cerium oxide, characterized in that, Extracellular vesicles of plant origin and a complex composed of cerium oxide nanoparticles and miR-424 loaded inside them; wherein the mass ratio of cerium oxide nanoparticles to miR-424 is (1:1) to (100:1), preferably (30:1) to (70:1), more preferably 50:1; and the mass ratio of the plant extracellular vesicles to the cerium oxide nanoparticles is (1:1) to (1:20), preferably (1:3) to (1:10), more preferably 1:

5. 424; wherein the mass ratio of cerium oxide nanoparticles to miR-424 is (1:1) to (100:1), preferably (30:1) to (70:1), more preferably 50:1; and the mass ratio of the plant extracellular vesicles to the cerium oxide nanoparticles is (1:1) to (1:20), preferably (1:3) to (1:10), more preferably 1:

5.

2. The plant exosomal complex of claim 1, wherein the miR-424 is loaded with cerium oxide. The plant-derived extracellular vesicles have a double-membrane structure, and the average particle size is 80 nm to 180 nm, preferably 100 nm to 150 nm.

3. The plant exosomal complex of claim 1 or 2, wherein the miR-424 is loaded with cerium oxide. The plant-derived extracellular vesicles are extracted from Pueraria lobata.

4. A method for the preparation of the plant exosomal complex of miR-424 and cerium oxide according to any one of claims 1-3, characterized by, The method comprises the following steps: S1. providing a plant extracellular vesicle suspension; S2. mixing and incubating cerium oxide nanoparticles with miR-424 in a buffer to form a miR-424-cerium oxide complex; S3. mixing and incubating the plant extracellular vesicle suspension with the miR-424-cerium oxide complex, so that the complex is loaded inside the plant extracellular vesicles; S4. purifying to obtain a plant extracellular vesicle complex loaded with miR-424 and cerium oxide.

5. The preparation method according to claim 4, characterized in that, In step S1, the extraction method of the plant extracellular vesicles comprises: taking plant material, juicing and filtering, then sequentially performing low-speed centrifugation, medium-speed centrifugation and high-speed centrifugation on the obtained juice, collecting the precipitate, and then purifying by ultracentrifugation to obtain plant extracellular vesicles; wherein the ultracentrifugation is performed at 100000xg to 200000xg for not less than 1 hour.

6. The preparation method according to claim 4, characterized in that, In step S2, the mixing and incubation is performed in a HEPES buffer, the incubation temperature is 15-37°C, and the incubation time is 15-60 minutes.

7. The preparation method according to claim 4, characterized in that, In step S3, the incubation is performed at 37°C for 8-24 hours; in step S4, the purification comprises removing the unloaded free components by ultracentrifugation.

8. Use of the plant extracellular vesicle complex loaded with miR-424 and cerium oxide according to any one of claims 1-3 in the preparation of a medicament.

9. Use according to claim 8, characterized in that, The medicament is used for preventing and / or treating inflammation-related diseases or oxidative stress-related diseases.

10. Use according to claim 9, characterized in that, The inflammation-related diseases or oxidative stress-related diseases include sepsis or acute lung injury.

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