Bionic nanoparticles for delivering traditional Chinese medicine components in grapefruit vesicles, and preparation method and application thereof

Abstract

The application discloses a grapefruit vesicle delivery traditional Chinese medicine component biomimetic nanoparticle and a preparation method and application thereof. The biomimetic nanoparticle is formed by loading a traditional Chinese medicine component on a grapefruit vesicle carrier, and the grapefruit vesicle carrier is obtained by extraction and separation from grapefruit. The grapefruit vesicle delivery traditional Chinese medicine component biomimetic nanoparticle can be used for treating ischemic stroke and reperfusion injury. The biomimetic nanoparticle prepared by the application has multiple excellent properties, such as high endogenicity, high biological safety, long in-vivo half-life, high blood-brain barrier permeability and the like, and the naringin on the surface of the grapefruit vesicle and the loaded traditional Chinese medicine component can play a synergistic effect. The biomimetic nanoparticle can load hydrophilic and hydrophobic drugs, realize multi-component delivery of traditional Chinese medicine, solve the problems of poor water solubility and low bioavailability of traditional Chinese medicine components, and realize efficient drug delivery and treatment of brain ischemic areas.

Description

Technical Field

[0001] This invention belongs to the field of nanoparticle technology, specifically relating to a biomimetic nanoparticle for delivering traditional Chinese medicine components via grapefruit vesicles, its preparation method, and its application. Background Technology

[0002] Stroke includes two types: ischemic stroke and hemorrhagic stroke, with ischemic stroke accounting for 85% of all stroke cases. Ischemic stroke is an acute cerebrovascular disease caused by blood vessel blockage preventing blood flow to the brain, resulting in brain tissue damage. It is characterized by high morbidity, high mortality, and high disability rates. In 2020, updated data from the World Health Organization showed that stroke had become the second leading cause of death worldwide. Stroke not only seriously threatens the health and lives of patients but also imposes a heavy economic, medical, and social burden on society and patients' families.

[0003] Current treatments for ischemic stroke primarily focus on early recanalization of occluded blood vessels to avoid or limit downstream brain damage and ischemia. These methods mainly include endovascular thrombectomy and rt-PA (recombinant tissue plasminogen activator) thrombolysis. However, due to the short treatment window and low recanalization rate, the disability rate of ischemic stroke remains high. It has been reported that even with standard treatment, only 27% of patients with acute ischemic stroke and large vessel occlusion are disability-free after successful reperfusion, while a significant proportion suffer from varying degrees of limb or cognitive impairment. Therefore, for ischemic stroke patients, regardless of whether they have the opportunity for early recanalization, neuroprotective drug therapy remains a crucial treatment method. However, the efficacy of neuroprotective drugs for ischemic stroke is not ideal. Currently used neuroprotective agents (such as edaravone) are prone to causing serious adverse reactions during use (such as acute renal failure, liver damage, thrombocytopenia, and disseminated intravascular coagulation). Furthermore, most neuroprotective agents have a single target and cannot provide comprehensive protection against the complex pathological processes of ischemic stroke. Therefore, developing neuroprotective drugs with multiple targets, a wide time window, and fewer side effects is of great significance for the treatment of ischemic stroke.

[0004] In recent years, scientists have focused their attention on traditional Chinese medicine. Studies have shown that both single-component compounds and compound preparations of traditional Chinese medicine have played a relatively effective role in the treatment of ischemic stroke, producing significant protective effects with fewer adverse reactions. For example, rhodioloside, an effective component of the traditional Chinese medicine Rhodiola rosea, can improve blood-brain barrier damage, inhibit cell apoptosis, salvage the ischemic penumbra, effectively reduce brain damage caused by ischemic stroke and ischemia-reperfusion, and lower the disability rate after reperfusion. However, the bioavailability of traditional Chinese medicine components still faces problems such as low absorption rate and difficulty in crossing the blood-brain barrier, which to some extent limits their biological activity. These issues can be improved through formulation methods.

[0005] Constructing biomimetic nanomedicine delivery platforms can effectively address the bioavailability challenges of traditional Chinese medicine (TCM) components and enhance their therapeutic effects on ischemic stroke. However, existing nanodelivery systems are costly to prepare and often suffer from poor stability, short in vivo half-life, low encapsulation efficiency, and difficulty in crossing the blood-brain barrier. The introduction of highly toxic organic reagents during preparation also leads to low safety, failing to completely solve the bioavailability problem of TCM components. Therefore, there is an urgent need to develop a low-cost, safe, non-toxic nanobiomimetic drug delivery platform with a long half-life and strong blood-brain barrier penetration capability to load TCM components, thereby overcoming the bioavailability defects of TCM components and enhancing their therapeutic effects on ischemic stroke and reperfusion injury. Furthermore, carrier-based delivery of TCM components typically only delivers single TCM compounds, lacking effective delivery methods for TCM compound components. Summary of the Invention

[0006] Objective of the Invention: To address the aforementioned technical problems, the objective of this invention is to provide biomimetic nanoparticles for delivering traditional Chinese medicine components via grapefruit vesicles, along with their preparation method and applications. This invention utilizes grapefruit vesicles to prolong the half-life of the traditional Chinese medicine components, facilitating their penetration of the blood-brain barrier. Furthermore, the synergistic effect of naringin, a characteristic component on grapefruit vesicles, with the traditional Chinese medicine improves the therapeutic effect on ischemic stroke and reperfusion injury, providing a novel approach for stroke treatment.

[0007] Technical Solution: To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:

[0008] A biomimetic nanoparticle for delivering traditional Chinese medicine components via grapefruit vesicles, wherein the biomimetic nanoparticle is formed by loading the traditional Chinese medicine components onto a grapefruit vesicle carrier, which is extracted and separated from grapefruit.

[0009] As a preferred embodiment, the extraction and separation method of the grapefruit vesicle vector includes the following steps:

[0010] Take fresh grapefruit (Citrus genus of Rutaceae family), peel it, juice it, centrifuge it at differential speed to remove large particles and fibers, collect the supernatant, ultrafilter it, and collect the filtrate to obtain the product.

[0011] Preferably, the differential centrifugation method includes the following steps: 2 × 10⁻⁶ centrifuges in the following order... 3 Centrifuge for 15-25 minutes, 5×10⁻⁵ min. 3 Centrifuge for 25-35 minutes at 1.5 × 10⁻⁶ g. 4 Centrifuge for 55-65 minutes; the ultrafiltration method includes the following: passing the supernatant through 100kDa and 10kDa ultrafiltration membranes in sequence.

[0012] As a preferred embodiment, the traditional Chinese medicine components are selected from single traditional Chinese medicine components or compound traditional Chinese medicine components; more preferably, the single traditional Chinese medicine component is selected from rhodioloside; and the compound traditional Chinese medicine components are selected from extracts of musk, turmeric and gardenia.

[0013] As a further preferred embodiment, the extracts of musk, turmeric, and gardenia are loaded into grapefruit vesicles in the form of a microemulsion, and the preparation method of the microemulsion includes the following steps:

[0014] The volatile oils of musk, turmeric, and gardenia were extracted to obtain the volatile oil fractions. The residues of the musk, turmeric, and gardenia after the volatile oil extraction were taken, and water was added for boiling extraction. The extracts were combined to obtain the water-soluble fractions. An emulsifier was taken and added to the above volatile oil fractions. After thorough mixing, the water-soluble fractions were added and the mixture was sonicated to obtain the microemulsion.

[0015] Preferably, the extraction of volatile oils from musk, turmeric, and gardenia is performed by steam distillation; the boiling extraction with water has a material-to-liquid ratio of 1:(7-9), and is performed 1-3 times, with each extraction lasting 1-2 hours; the emulsifier is selected from soybean lecithin; and the ratio of water-soluble fraction to volatile oil fraction is 1:(3-5).

[0016] Preferably, the biomimetic nanoparticles are further modified with borneol.

[0017] This invention also provides a method for preparing biomimetic nanoparticles for delivering traditional Chinese medicine components via grapefruit vesicles, comprising the following steps:

[0018] (1) Extraction and separation of grapefruit vesicles;

[0019] (2) Mix the traditional Chinese medicine components with the grapefruit vesicles obtained in step (1) evenly, sonicate under ice bath conditions, and incubate after sonication to prepare biomimetic nanoparticles that deliver traditional Chinese medicine components via grapefruit vesicles.

[0020] Preferably, in step (2), the mass ratio of the traditional Chinese medicine component to the grapefruit vesicle is 1:(8-12); the ultrasonic conditions are: ultrasonic disruption time is 2s on / 5s off, 4-6min, ultrasonic power is 120-130W; the incubation conditions are: incubation at 37±2℃ for 55-65min.

[0021] Finally, this invention provides the application of the biomimetic nanoparticles that deliver traditional Chinese medicine components via grapefruit vesicles in the preparation of drugs for treating ischemic stroke and reperfusion injury.

[0022] When applied, it can be dissolved in physiological saline, phosphate buffer, or 5% glucose solution for intravenous injection. The biomimetic nanoparticles that deliver traditional Chinese medicine components via grapefruit vesicles can circulate in the body for a long time and cross the blood-brain barrier to efficiently deliver the traditional Chinese medicine components to the ischemic area of ​​the brain, significantly improving the bioavailability of the loaded traditional Chinese medicine components and having therapeutic effects on ischemic stroke and reperfusion injury.

[0023] This invention prepares grapefruit vesicles by combining ultrafiltration and differential centrifugation. Compared with the traditional ultracentrifugation method, the preparation method is simple and does not require a long ultracentrifugation process, which greatly shortens the preparation time and ensures the integrity of the obtained grapefruit vesicles, allowing them to be stored for a long time.

[0024] This invention improves upon the low encapsulation rate of traditional vesicle drug loading technology by inducing the loading of traditional Chinese medicine components into the hydrophilic core of grapefruit vesicles through ultrasonic disruption.

[0025] The natural naringin component on the surface of grapefruit vesicles provided by this invention can inhibit the neuroinflammatory response caused by cerebral ischemia, reduce ischemic brain injury, and synergistically enhance the therapeutic effect with the loaded traditional Chinese medicine components, further improving the therapeutic effect on ischemic stroke and reperfusion injury.

[0026] This invention selects single compounds or compound formulas of traditional Chinese medicine as drugs, and loads them into grapefruit vesicles derived from natural plants through ultrasonic disruption, constructing a biomimetic nanodrug delivery system that can effectively improve the bioavailability defects of free drugs. It has the following advantages:

[0027] (1) Multi-component delivery of traditional Chinese medicine: Extracellular vesicles have a protein and lipid membrane structure. The vesicle cavity enclosed by the membrane material provides space for loading active molecules and has a wide range of carrying capacity. It can load hydrophilic and hydrophobic drugs to realize the multi-component delivery of traditional Chinese medicine and has good application prospects in combination therapy.

[0028] (2) High endogenous nature: The Chinese medicine components are encapsulated using grapefruit vesicles derived from natural plants, and the natural specific proteins on the surface of grapefruit vesicles are preserved, which has high endogenous nature.

[0029] (3) High biocompatibility: Grapefruit vesicles are derived from natural edible plants. Their main components are acylethanolamine and phosphatidylcholine. They are highly biocompatible, biodegradable, and have no toxic side effects on the human body.

[0030] (4) Long in vivo half-life: Grapefruit vesicles have good stability and immune escape ability as drug carriers. After being injected into the blood via intravenous injection, they can significantly prolong the in vivo circulation time of traditional Chinese medicine components.

[0031] (5) High blood-brain barrier permeability: Grapefruit vesicles have natural specific proteins that can efficiently deliver drugs across the blood-brain barrier, thereby improving the blood-brain barrier permeability of Chinese medicine components.

[0032] (6) Synergistic effect: Grapefruit vesicles contain natural naringin components, which can work synergistically with traditional Chinese medicine components to significantly enhance the protective effect on ischemic neurons and improve the efficacy of treatment for ischemic stroke and reperfusion injury.

[0033] (7) Inexpensive and readily available: The raw materials for preparing grapefruit vesicles are common citrus fruits, which are inexpensive and readily available; the construction methods of the nanodelivery system mainly include ultrafiltration combined with ultracentrifugation and ultrasonic disruption, which are relatively simple and easy to implement.

[0034] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0035] (1) This invention uses ultrafiltration combined with ultracentrifugation and ultrasonic disruption to prepare biomimetic nanoparticles for delivering traditional Chinese medicine components via grapefruit vesicles. This avoids the long ultracentrifugation process, greatly saves time and costs, better ensures the integrity of the obtained vesicles, and improves the encapsulation rate of the drug. The method is simple, low-cost, and easier to industrialize.

[0036] (2) This invention selects plant-derived extracellular vesicles as drug carriers, which offer a wider variety of drug delivery methods compared to liposomes, micelles, supramolecular assemblies, nanoemulsions, etc. They can carry hydrophilic and hydrophobic drugs, with high delivery efficiency and good stability. Utilizing their protein-lipid membrane structure, they can not only deliver single compounds of traditional Chinese medicine, but also achieve the combined delivery of multiple components of traditional Chinese medicine, playing a comprehensive protective role against the complex pathological process of ischemic stroke.

[0037] (3) The present invention retains the natural naringin component on the surface of grapefruit vesicles, which can work synergistically with the loaded Chinese medicine components to enhance the therapeutic effect.

[0038] (4) The present invention retains the specific proteins on the surface of grapefruit vesicles, which can achieve efficient drug delivery across the blood-brain barrier and enhance drug uptake by ischemic neurons.

[0039] The grapefruit vesicle-delivered biomimetic nanoparticles of traditional Chinese medicine components provided by this invention can be used to treat ischemic stroke and reperfusion injury. This drug delivery system possesses excellent properties such as high endogenous activity, high biocompatibility, long in vivo half-life, and strong blood-brain barrier penetration. This invention provides a new approach for the application of biomimetic nanomedicine delivery systems in stroke treatment and has broad prospects for clinical translation. Attached Figure Description

[0040] Figure 1 Transmission electron microscopy images of grapefruit vesicles prepared by differential centrifugation combined with ultrafiltration or density gradient centrifugation;

[0041] Figure 2 Expression of characteristic proteins on the surface of grapefruit vesicles;

[0042] Figure 3 Determination of naringin, a characteristic component of grapefruit vesicle surface;

[0043] Figure 4 The in vitro anti-apoptotic effect of rhodioloside-inspired nanoparticles delivered to grapefruit vesicles was investigated, including the MTT assay (A) and Calcein-AM / PI staining and imaging (B).

[0044] Figure 5 The effects of multi-component biomimetic nanoparticles of traditional Chinese medicine delivered by grapefruit vesicles on mitochondrial function were investigated, including JC-1 staining and imaging (A) and ATP content measurement (B).

[0045] Figure 6 Investigation on the protective effect of biomimetic nanoparticles for delivering rhodioloside to grapefruit vesicles on the blood-brain barrier in vitro;

[0046] Figure 7 Figure showing the particle size determination of grapefruit vesicles, grapefruit vesicles delivering rhodioloside and multi-component biomimetic nanoparticles of traditional Chinese medicine;

[0047] Figure 8 Investigation on the in vitro anti-inflammatory effect of multi-component biomimetic nanoparticles of traditional Chinese medicine delivered via grapefruit vesicles;

[0048] Figure 9 Investigation on the in vitro antioxidant stress effect of multi-component biomimetic nanoparticles of traditional Chinese medicine delivered by grapefruit vesicles. Detailed Implementation

[0049] The invention will be further illustrated by the following embodiments. These embodiments are purely illustrative and are only used to specifically describe the invention, and should not be construed as limiting the invention. The invention will be further described below with reference to the accompanying drawings and embodiments.

[0050] Example 1: Extraction and isolation of vesicles (GEVs) from grapefruit

[0051] GEVs were prepared by differential centrifugation combined with ultrafiltration: Fresh grapefruit was peeled, juiced, and then centrifuged at a differential speed (2×10⁻⁶). 3 Centrifuge for 20 min, 5 × 10⁻⁶ 3 Centrifuge for 30 min at 1.5 × 10⁻⁶ g. 4 Centrifuge for 1 hour to remove large particles and fibers, and collect the supernatant. Ultrafilter the supernatant through 100kDa and 10kDa membranes sequentially, and collect the filtrate.

[0052] GEVs were prepared using differential centrifugation combined with density gradient centrifugation: Fresh grapefruit was peeled, juiced, and centrifuged at a differential speed (3×10⁻⁶). 3 Centrifuge for 20 min, 1×10⁻⁶ 4 Centrifuge for 30 min to remove large particles and fibers, then centrifuge the collected supernatant at 1.0 × 10⁻⁶ g for 30 min. 5 Centrifuge for 1 hour. Discard the supernatant and resuspend the precipitate in PBS to 3 mL. Separate and purify using a sucrose density gradient method, adding 3 mL of 60%, 45%, 30%, and 15% sucrose solution, along with vesicle solution, to the bottom of the centrifuge tube sequentially. (1.0 × 10⁻⁶ g) 5 Centrifuge for 2 hours, and band 3 appears at the 30 / 45% interface. Collect band 3 and dilute it in 20 mL of PBS solution. 5 Centrifuge at g for 2 hours, then resuspend in a trace amount of PBS solution to obtain the product.

[0053] The morphology of the GEVs prepared under Example 1 was observed using a transmission electron microscope.

[0054] Experimental results are as follows Figure 1 As shown in Figure A, GEVs prepared by differential centrifugation combined with ultrafiltration exhibit intact morphology, displaying a typical saucer-like or concave hemispherical shape. Experimental results are as follows... Figure 1 As shown in Figure B, the morphology of GEVs prepared by differential centrifugation combined with density gradient centrifugation was somewhat damaged. Furthermore, ultrafiltration is a simple and quick method; therefore, differential centrifugation combined with ultrafiltration was subsequently used to prepare GEVs.

[0055] GEVs prepared under Example 1 were used to characterize the characteristic membrane proteins on their surface using flow cytometry. GEV samples were added to flow cytometry tubes, with PE-CD81 and APC-CD9 fluorescently labeled antibodies added to each tube, along with corresponding isotype controls. After incubation on ice in the dark for 10 min, protein expression was detected by flow cytometry.

[0056] Experimental results are as follows Figure 2 As shown, CD81 and CD9 were both expressed positively in GEVs, proving that the extracted vesicles are exosomes.

[0057] The GEVs prepared under Example 1 were analyzed by HPLC to determine the characteristic component naringin (NAR). 5 mg of GEVs (based on protein concentration) was suspended in 800 μL of anhydrous ethanol, heated in a water bath at 90 °C for 2 h, and then the extract was cooled to 22 °C and filtered for HPLC analysis.

[0058] Experimental results are as follows Figure 3 As shown, GEVs contain NAR, providing experimental evidence for the synergistic effect of GEVs in the following discussion.

[0059] Example 2: Preparation process of grapefruit vesicles (GEVs / Sal) loaded with rhodioloside (Sal)

[0060] GEVs / Sal were prepared by ultrasonic disruption. 150 μg of GEVs (based on protein content) was added to 15 μg of Sal. The mixture was ultrasonically disrupted for 5 min at 120 W with a 2s on / 5s off setting, allowing Sal to be loaded into the hydrophilic core of the GEVs. The mixture was then incubated at 37 °C for 1 h to allow the membrane structure to close, thus obtaining GEVs / Sal.

[0061] Example 3: Properties of biomimetic nanoparticles for delivering rhodioloside via grapefruit vesicles

[0062] The GEVs / Sal prepared in Example 2 above were subjected to the following investigation:

[0063] 1.1 Investigation of the anti-apoptotic effect of GEVs / Sal on PC12 cells

[0064] Establishment of the OGD / R cell model: PC12 cells in the logarithmic growth phase were seeded into well plates and allowed to adhere overnight. The culture medium was then discarded, and DMEM glucose-free culture medium was added. The cells were placed in a hypoxic chamber and cultured at 37°C for 6 hours under oxygen and glucose deprivation. The glucose-free DMEM culture medium was then replaced with DMEM high-glucose complete culture medium containing 10% FBS. Finally, the cells were cultured in a conventional cell culture incubator for 12 hours under reoxygenation and reglucose conditions.

[0065] To investigate the effective drug concentration of GEVs / Sal for improving the cell viability of OGD / R-induced PC12 cells, different concentrations of GEVs / Sal were co-incubated with OGD / R-induced PC12 cells, and cell viability was detected by the MTT assay. PC12 cells in the logarithmic growth phase were incubated at 1×10⁻⁶ cells / cells. 5 cell·mL -1Cells were seeded at a density of 100 μL per well in 96-well plates and incubated overnight at 37°C with 5% CO2. After overnight cell attachment, OGD / R treatment was performed, followed by discarding the serogroups. Different concentrations of GEVs / Sal (0.00, 6.25, 12.5, 25, 50, 100 μM) were added and incubated for 12 h. After incubation, 100 μL of complete serogroups containing 10% MTT solution (5 mg / mL) was added to each well, and the cells were incubated for another 4 h. All supernatant was discarded, and 100 μL of DMSO was added to each well. The cells were shaken on a microplate shaker for 10 min, and the absorbance was measured at 490 nm using a microplate reader.

[0066] The results are as follows Figure 4 As shown in Figure A, co-incubation of PC12 cells with 50 μM GEVs / Sal significantly improved the viability of PC12 cells induced by OGD / R. Therefore, 50 μM was subsequently selected as the dosage concentration of GEVs / Sal.

[0067] The protective effect of GEVs / Sal on damaged PC12 cells was detected by Calcein-AM / PI live / dead staining. PC12 cells were inoculated at 1×10⁻⁶ cells per cell line. 5 cell·mL -1 Cells were seeded at a density of [insert density here] in 96-well plates. After overnight adhesion, OGD / R treatment was performed and the cells were drug-treated for 12 hours. After culture, the culture medium was discarded, cells were washed with PBS, and Calcein-AM and PI were diluted, mixed, and added to the 96-well plates. The plates were incubated for 30 minutes, observed under an inverted fluorescence microscope, and the data were analyzed using ImageJ software.

[0068] The results are as follows Figure 4 As shown in Figure B, compared with the untreated OGD / R group, the number of apoptotic cells in the GEVs / Sal group was significantly reduced, indicating that GEVs / Sal can effectively protect cells damaged by oxidative stress and reduce their apoptosis.

[0069] 1.2 Investigation on the effect of GEVs / Sal on mitochondrial function in PC12 cells

[0070] The effect of GEVs / Sal on mitochondrial membrane potential in PC12 cells after OGD / R injury was detected using the JC-1 fluorescent probe labeling method. PC12 cells were inoculated at a concentration of 1×10⁻⁶ cells / cells. 5 cell·mL -1Cells were seeded at a density of [insert density here] in 96-well plates. After overnight adhesion, OGD / R treatment was performed, followed by drug administration for 12 hours. After culture, the culture medium was discarded, cells were washed with PBS, and JC-1 working solution was added and thoroughly mixed. The cells were then incubated at 37°C in the dark for 20 minutes. After incubation, the supernatant was discarded, and cells were washed twice with JC-1 staining buffer. Culture medium was added to each well after washing, and the cells were observed under an inverted fluorescence microscope. Data analysis was performed using ImageJ software.

[0071] The results are as follows Figure 5 As shown in Figure A, compared to the untreated OGD / R group, GEVs / Sal significantly reversed the decline in mitochondrial membrane potential, thus rescuing mitochondrial energy production.

[0072] Intracellular adenosine triphosphate (ATP) expression levels were detected using enzyme-linked immunosorbent assay (ELISA). PC12 cells were cultured at a concentration of 2 × 10⁻⁶ cells / year. 5 cell·mL -1 Cells were seeded at a density of [insert density here] into 6-well plates. After overnight adhesion, OGD / R treatment was performed followed by drug administration for 12 hours. Cell supernatant was collected according to the kit instructions, and intracellular ATP levels were measured.

[0073] The results are as follows Figure 5 As shown in Figure B, compared with the untreated OGD / R group, the intracellular ATP content in the GEVs / Sal group was significantly increased, indicating that GEVs / Sal can alleviate mitochondrial structural damage.

[0074] 1.3 Investigation on the protective effect of GEVs / Sal on the blood-brain barrier in vitro

[0075] HCMEC cells in the logarithmic growth phase were harvested at a concentration of 1×10⁻⁶. 5 Cells were seeded at a density of cells / well in 24-well Transwell plates. The cell culture medium was changed every other day, and the transmembrane resistance was measured. The cells were considered healthy when the resistance exceeded 200 Ω·cm. 2 The cells were treated with OGD / R and then drug-treated. After 12 hours of drug treatment, FITC-Dextran was added to the basal medium in the upper chamber of the Transwell. The culture medium in the lower chamber was then collected, and the fluorescence intensity of FITC-Dextran was measured using a microplate reader to calculate the permeability.

[0076] The results are as follows Figure 6 As shown, compared with the untreated OGD / R group, the BBB permeability was significantly reduced after treatment with GEVs / Sal, indicating that GEVs / Sal has a certain protective effect against OGD / R-induced BBB damage.

[0077] Example 4: Preparation process of grapefruit vesicles (Bo-GEVs / XNJM) encapsulating multiple components of traditional Chinese medicine (XNJM)

[0078] The lipid-soluble and water-soluble components of musk, turmeric, and gardenia were used to prepare microemulsions (XNJM). GEVs / XNJM were prepared by ultrasonic disruption, and Bo-GEVs / XNJM was obtained by modification with borneol. The specific procedures are as follows: volatile oils of musk, turmeric, and gardenia were extracted by steam distillation to obtain the volatile oil fractions. The residue after volatile oil extraction was taken, boiled with water 1-3 times, and the extracts were combined to obtain the water-soluble fractions. Soybean lecithin was used as an emulsifier, added to the volatile oil fractions, mixed thoroughly, and then the water-soluble fractions were added and ultrasonicated to obtain the microemulsion (XNJM). The protein concentration of GEVs was determined using a BCA kit. XNJM and GDNPs were mixed at a mass ratio of 10:1, and after thorough mixing, the mixture was processed using an ultrasonic cell disruptor. XNJM was ultrasonically broken up for 5 minutes at 120W power with a 2s on / 5s off condition, allowing it to be loaded onto the hydrophilic core of GEVs. The membrane was then incubated at 37℃ for 1 hour to close the membrane structure, yielding GEVs / XNJM. Furthermore, Bo-GEVs / XNJM was synthesized using chemical reactions such as esterification and amide reactions.

[0079] Example 5: Properties of biomimetic nanoparticles for multi-component traditional Chinese medicine delivery via grapefruit vesicles

[0080] The Bo-GEVs / XNJM prepared in Example 4 above was subjected to the following investigation:

[0081] 1.1 Particle size determination

[0082] The particle size of GEVs prepared in Example 1, GEVs / Sal prepared in Example 2, and Bo-GEVs / XNJM prepared in Example 4 were determined using a Malvern laser particle size analyzer.

[0083] Experimental results are as follows Figure 7 As shown, the prepared GEVs, GEVs / Sal and Bo-GEVs / XNJM all have small particle size and good uniformity.

[0084] 1.2 Investigation on the anti-inflammatory effect of Bo-GEVs / XNJM on PC12 cells

[0085] Intracellular inflammatory cytokine expression levels were detected using enzyme-linked immunosorbent assay (ELISA). PC12 cells were cultured at a concentration of 2 × 10⁶ cells / year. 5 cell·mL -1Cells were seeded at a density of [insert density here] into 6-well plates. After overnight adhesion, OGD / R treatment was performed, followed by drug administration for 12 hours. Cell supernatant was collected according to the kit instructions, and the levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) were measured.

[0086] The results are as follows Figure 8 As shown, compared with the untreated OGD / R group, the expression of intracellular TNF-α, IL-1β and IL-6 was significantly reduced after treatment with Bo-GEVs / XNJM, and the efficacy was comparable to that of the Xingnaojing injection group (XNJ), indicating that Bo-GEVs / XNJM has good anti-inflammatory ability.

[0087] 1.3 Investigation on the antioxidant stress effect of Bo-GEVs / XNJM on PC12 cells

[0088] PC12 cells were loaded at 2×10 5 cell·mL -1 Cells were seeded at a density of [insert density here] into 6-well plates. After overnight cell adhesion, OGD / R treatment was performed, followed by drug administration for 12 hours. Cells were collected according to the kit instructions, the supernatant was removed, lysis buffer was added, proteins were extracted, and the levels of malondialdehyde (MDA), glutathione peroxidase (GSH-PX), and superoxide dismutase (SOD) were measured.

[0089] The results are as follows Figure 9 As shown, compared with the untreated OGD / R group, the expression of MDA in cells was significantly reduced after treatment with Bo-GEVs / XNJM, while the expression of GSH-PX and SOD was significantly increased, indicating that Bo-GEVs / XNJM has a good antioxidant capacity.

Claims

1. A biomimetic nanoparticle for delivering traditional Chinese medicine components via grapefruit vesicles, characterized in that, The biomimetic nanoparticles are formed by loading traditional Chinese medicine components onto grapefruit vesicle carriers, which are extracted and separated from grapefruit. The extraction and separation method of the grapefruit vesicle carriers includes the following steps: Take fresh grapefruit, peel and juice it, centrifuge at differential speed to remove large particles and fibers, collect the supernatant, ultrafilter, and collect the filtrate to obtain the product; the differential centrifugation method includes the following steps: in the following order, 2×10 3 Centrifuge for 15-25 min at 5 × 10⁻⁵ g. 3 Centrifuge for 25-35 min at 1.5 × 10⁻⁶ g. 4 Centrifuge for 55-65 min; the ultrafiltration method includes the following: passing the supernatant sequentially through 100 kDa and 10 kDa ultrafiltration membranes; The Chinese medicine components are selected from rhodioloside, or from extracts of musk, turmeric, and gardenia.

2. The biomimetic nanoparticles for delivering traditional Chinese medicine components via grapefruit vesicles according to claim 1, characterized in that, The extracts of musk, turmeric, and gardenia are loaded in grapefruit vesicles in the form of microemulsions. The preparation method of the microemulsions includes the following steps: The volatile oils of musk, turmeric, and gardenia were extracted to obtain the volatile oil fractions. The residues of the musk, turmeric, and gardenia after the volatile oil extraction were taken, and water was added for boiling extraction. The extracts were combined to obtain the water-soluble fractions. An emulsifier was taken and added to the above volatile oil fractions. After thorough mixing, the water-soluble fractions were added and the mixture was sonicated to obtain the microemulsion.

3. The biomimetic nanoparticles for delivering traditional Chinese medicine components via grapefruit vesicles according to claim 2, characterized in that, The extraction of volatile oils from musk, turmeric, and gardenia is performed using steam distillation. The boiling extraction with water is carried out at a material-to-liquid ratio of 1:7-9, with 1-3 extractions per batch, each extraction lasting 1-2 hours. The emulsifier is selected from soybean lecithin. The ratio of water-soluble fraction to volatile oil fraction is 1:3-5.

4. The biomimetic nanoparticles for delivering traditional Chinese medicine components via grapefruit vesicles according to claim 2, characterized in that, The biomimetic nanoparticles are also modified with borneol.

5. The method for preparing biomimetic nanoparticles for delivering traditional Chinese medicine components via grapefruit vesicles according to any one of claims 1-4, characterized in that, Includes the following steps: (1) Extraction and separation of grapefruit vesicles; (2) Mix the traditional Chinese medicine components with the grapefruit vesicles obtained in step (1) evenly, sonicate under ice bath conditions, and incubate after sonication to prepare biomimetic nanoparticles that deliver traditional Chinese medicine components via grapefruit vesicles.

6. The method for preparing biomimetic nanoparticles for delivering traditional Chinese medicine components via grapefruit vesicles according to claim 5, characterized in that, In step (2), the mass ratio of the Chinese herbal medicine components to grapefruit vesicles is 1:8-12; the ultrasonic conditions are: ultrasonic disruption time is 2 s on / 5 s off, 4-6 min, ultrasonic power is 120-130 W; the incubation conditions are: incubation at 37±2℃ for 55-65 min.

7. The use of the biomimetic nanoparticles of grapefruit vesicles delivering traditional Chinese medicine components as described in any one of claims 1-4 in the preparation of drugs for treating ischemic stroke and reperfusion injury.

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