Application of plant-derived nanoparticles in the preparation of drugs for the prevention and treatment of vascular calcification

Abstract

The present invention belongs to the field of biomedicine and specifically relates to the use of plant-derived nanoparticles in the preparation of drugs for the prevention and treatment of vascular calcification. Experimental studies have confirmed that the grapefruit-derived nanoparticles provided by the present invention can effectively alleviate vascular calcification in mouse vascular smooth muscle cells induced by high phosphorus and in mouse vascular calcification models induced by high vitamin D3. Furthermore, the nanoparticles exhibit no significant changes in particle size or membrane potential after prolonged storage, nor do they cause damage to the mouse's major organs (heart, liver, spleen, lungs, and kidneys). These nanoparticles exhibit high stability and good safety, and are expected to be applied in the prevention and treatment of vascular calcification, providing a new approach and improved prevention and treatment strategy for vascular calcification.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and more specifically relates to the use of plant-derived nanoparticles in the preparation of drugs for preventing and treating vascular calcification. Background Art

[0002] Vascular calcification is the abnormal deposition of minerals such as calcium and phosphorus in the form of hydroxyapatite in blood vessel walls. It is a common pathological manifestation in diseases and processes such as atherosclerosis, hypertension, diabetic vascular disease, vascular injury, chronic kidney disease, and aging. It primarily manifests as increased stiffness and decreased compliance of the vessel wall, and can easily lead to myocardial ischemia, left ventricular hypertrophy, and heart failure, triggering symptoms such as thrombosis and plaque rupture. It is a major factor in the high morbidity and mortality of cardiovascular and cerebrovascular diseases and a key marker for the development of atherosclerotic cardiovascular events, stroke, and peripheral vascular disease.

[0003] Currently, vascular calcification is a common and difficult-to-treat disease, and there is still a lack of effective clinical treatments for vascular calcification. A Chinese patent application discloses the use of a growth hormone-releasing hormone agonist in the preparation of an anti-vascular calcification drug. GHRH-A can increase cAMP levels and protein kinase A (PKA) activity in smooth muscle cells, reduce the expression and activity of NAPDH oxidase in smooth muscle cells, reduce the level of ROS in smooth muscle cells, reduce the expression and activity of Runx2 and cellular alkaline phosphatase (ALP), and reduce the level of inorganic phosphorus in the blood, thereby reducing calcium and phosphorus deposition in the aorta and reducing ALP activity in the aorta, achieving the purpose of treating or preventing vascular calcification and arteriosclerosis. However, this drug easily affects the metabolism of normal hormones in the body, posing certain safety risks, and has not yet been put into practical use. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the lack of drugs for preventing and treating vascular calcification and certain safety risks, and to provide a highly safe plant-derived nanoparticle for use in the preparation of drugs for preventing and treating vascular calcification.

[0005] The purpose of the present invention is to provide a method for preparing plant-derived nanoparticles.

[0006] Another object of the present invention is to provide plant-derived nanoparticles prepared by the preparation method.

[0007] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0008] A method for preparing plant-derived nanoparticles comprises the following steps:

[0009] Grapefruit juice is squeezed, and the juice is subjected to differential centrifugation. The supernatant obtained by removing the pomace is subjected to ultrahigh-speed centrifugation. The separated precipitate is resuspended in a buffer solution and purified by sucrose density gradient centrifugation to obtain grapefruit-derived nanoparticles.

[0010] Furthermore, the ultrahigh-speed centrifugation is performed at 100,000 to 160,000 g for 1.5 to 2.5 h. Preferably, the ultrahigh-speed centrifugation is performed at 100,000 g for 2 h.

[0011] Furthermore, the centrifugation is performed at 3-5°C.

[0012] Furthermore, the differential centrifugation is divided into five stages, namely: the first stage is centrifugation at 400-600g for 8-12 min, the second stage is centrifugation at 1800-2200g for 15-25 min, the third stage is centrifugation at 4500-5500g for 30 min, the fourth stage is centrifugation at 8000-12000g for 1-1.5 h, and the fifth stage is centrifugation at 80000-120000g for 1.5-2.5 h.

[0013] Preferably, the differential centrifugation is divided into five stages, namely: the first stage is centrifugation at 500g for 10 min, the second stage is centrifugation at 2000g for 20 min, the third stage is centrifugation at 5000g for 30 min, the fourth stage is centrifugation at 10000g for 1 h, and the fifth stage is centrifugation at 100000g for 2 h.

[0014] Preferably, the buffer is phosphate buffered saline (PBS).

[0015] Furthermore, the sucrose density gradient centrifugation method is as follows: sucrose with densities of 8%, 30%, 45% and 60% are sequentially added to a centrifuge tube to form a sucrose density gradient, the suspension is transferred to the top layer of the sucrose gradient with densities of 8%, 30%, 45% and 60%, and centrifuged at 140,000-160,000 g for 1.5-2.5 hours. The 30% / 45% band is the purified grapefruit-derived nanoparticles.

[0016] In addition, the present invention also provides plant-derived nanoparticles prepared by the preparation method.

[0017] Furthermore, the particle size of the plant-derived nanoparticles is 104.5 to 122.3 nm.

[0018] The plant-derived nanoparticles (GNVs) prepared by a specific method of the present invention have been confirmed through experimental studies to effectively alleviate vascular calcification in mouse vascular smooth muscle cells induced by high phosphorus and mouse vascular calcification models induced by high vitamin D3. Furthermore, the particle size and membrane potential did not change significantly after long-term storage, nor did they cause damage to the major organs of the mice (heart, liver, spleen, lungs, and kidneys). The nanoparticles have high stability and good safety, and are expected to be applied to the prevention and treatment of vascular calcification diseases, providing a new approach and better prevention and treatment plan for the treatment of vascular calcification.

[0019] Additionally, plant-derived nanoparticles have a liposome-like structure that can be further modified to optimize their function.

[0020] Therefore, the present invention also claims to protect the use of the plant-derived nanoparticles in the preparation of drugs for preventing and treating vascular calcification.

[0021] Furthermore, when applied to mouse aortic smooth muscle cells, the concentration of the plant-derived nanoparticles is 5 to 15 μg / ml. For other different targets, the dosage can be converted according to the conversion rules.

[0022] Furthermore, the dosage form of the drug is oral, injection or inhalation.

[0023] The present invention has the following beneficial effects:

[0024] The plant-derived nanoparticles prepared by a specific method of the present invention have been confirmed through experimental studies to be able to effectively alleviate vascular calcification in mouse vascular smooth muscle cells induced by high phosphorus and mouse vascular calcification models induced by high vitamin D3. Moreover, the particle size and membrane potential did not change significantly after long-term storage, nor did they cause damage to the main organs of the mice (heart, liver, spleen, lungs, and kidneys). The nanoparticles have high stability and good safety, and are expected to be applied to the prevention and treatment of vascular calcification diseases, providing a new approach and better prevention and treatment plan for the treatment of vascular calcification. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a picture related to the identification of grapefruit-derived plant nanoparticles in Example 2 of the present invention, wherein: Figure 1 a is a Western blot identification image of grapefruit-derived plant nanoparticle markers. Figure 1 b is the morphological characteristics of grapefruit-derived plant nanoparticles under an electron microscope. Figure 1 c is the particle size distribution of grapefruit-derived plant nanoparticles measured using DLS. Figure 1 d is the Zeta potential diagram of the membrane surface of grapefruit-derived plant nanoparticles.

[0026] Figure 2 This is a picture related to the stability determination of grapefruit-derived plant nanoparticles in Example 3 of the present invention, wherein Figure 2 a, 2b are the particle size and Zeta potential diagrams of grapefruit-derived plant nanoparticles at different time points in PBS buffer containing 50% fetal bovine serum (FBS). Figure 2 c, 2d are the particle size and Zeta potential diagrams of grapefruit-derived plant nanoparticles in PBS buffer at different time points at 4°C.

[0027] Figure 3 This is a statistical graph showing the effect of different concentrations of grapefruit-derived plant nanoparticles on vascular smooth muscle cell viability as determined by CCK8 in Example 4 of the present invention.

[0028] Figure 4 This is a graph showing the anti-calcification effect of grapefruit-derived plant nanoparticles at different concentrations on high phosphorus-induced mouse aortic smooth muscle cells (MVSMCs) in Example 5 of the present invention.

[0029] Figure 5 This is a vascular staining image of the investigation of the anti-vascular calcification effect of grapefruit-derived plant nanoparticles on the high vitamin D3-induced C57BL / 6 mouse vascular calcification model in Example 6 of the present invention.

[0030] Figure 6 This is a statistical chart of the in vitro drug hemolysis rate of grapefruit-derived plant nanoparticles at different concentrations in Example 7 of the present invention.

[0031] Figure 7 This is a HE staining image of the main organs (heart, liver, spleen, lung, and kidney) of C57BL / 6 mice stained with grapefruit-derived plant nanoparticles in Example 8 of the present invention. DETAILED DESCRIPTION

[0032] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0033] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0034] Cell culture:

[0035] Mouse aortic smooth muscle cells (MVSMCs) were isolated from the tunica media of aortic tissue of male C57BL / 6 mice; MVSMCs were cultured in DMEM (4.5Glu) medium containing 10% FBS (Bi), 100 U / ml penicillin, and 100 μg / ml streptomycin, and cells from passages 3 to 8 were used in the experiments.

[0036] Male C57BL / 6 mice aged 6–8 weeks (18–22 g) were purchased from the Guangdong Medical Laboratory Animal Center, and all animal experiments were performed in accordance with the regulations and guidelines of the Institutional Animal Care and Use Committee (IACUC) of Southern Medical University, Guangzhou, China.

[0037] Alizarin red staining of cells: remove the culture medium from the cells, wash three times with PBS, fix with 4% paraformaldehyde solution at room temperature for 15 min, wash three times with distilled water, stain with 2% Alizarin Red S (PH=4.2) for 5 min, wash away the floating color with distilled water, and photograph using an optical microscope.

[0038] Tissue Alizarin Red Staining: The collected aorta tissue was fixed with 4% paraformaldehyde solution at room temperature for 48 h, stained with 0.004% Alizarin Red S overnight, washed with 2% potassium hydroxide for 5 minutes to remove floating color, and scanned and photographed.

[0039] Statistical analysis: All values ​​are expressed as mean ± SD; statistical differences were determined using the unpaired Student-t test with a two-tailed P value when comparing two independent groups, and one-way analysis of variance was performed using the Student-Newman-Keuls (SNK) or Dunnett post hoc test when comparing three or more independent groups, using IBM SPSS 19.0 software; survival analysis was determined using the Kaplan-Meier estimate (log-rank test); differences were considered significant when *P < 0.05, **P < 0.01, or ***P < 0.001.

[0040] Example 1 Extraction and purification of grapefruit-derived plant nanoparticles

[0041] The grapefruit was washed and peeled, and the juice was squeezed at room temperature. After the juice was collected, it was pre-cooled in a 4°C refrigerator and subjected to differential centrifugation at 4°C (500g for 10 minutes, 2000g for 20 minutes, 5000g for 30 minutes, and 10000g for 1 hour). After removing the pomace, the supernatant was centrifuged at ultra-high speed 100000g for 2 hours. The collected precipitate was resuspended in PBS buffer, and the suspension was separated and purified by sucrose density gradient centrifugation to obtain grapefruit-derived plant nanoparticles (GNVs). The suspension was resuspended in PBS to prepare a grapefruit-derived plant nanoparticle suspension for use. The protein concentration measured by BCA was 1 mg / ml. The protein concentration measured by BCA was used to quantify GNVs in subsequent experiments.

[0042] The sucrose density gradient centrifugation method is as follows: sucrose is dissolved in a 20mM Tris.HCl (pH 7.2) solution to prepare sucrose solutions with densities of 8% (4g sucrose, added to 20mM Tris.HCl (pH 7.2) and fixed to 50g), 30% (8g sucrose, added to 20mM Tris.HCl (pH 7.2) and fixed to 50g), 45% (22.5g sucrose, added to 20mM Tris.HCl (pH 7.2) and fixed to 50g), and 60% (30g sucrose, added to 20mM Tris.HCl (pH 7.2) and fixed to 50g). The sucrose solutions are added sequentially to centrifuge tubes to form a sucrose density gradient, and the suspension is transferred to the top layer of the sucrose gradients with densities of 8%, 30%, 45% and 60%, and centrifuged at 150,000g for 2h. The 30% / 45% band is the purified grapefruit-derived nanoparticles.

[0043] Example 2 Identification of Grapefruit-Derived Plant Nanoparticles

[0044] The protein content of the grapefruit-derived plant nanoparticle suspension obtained in Example 1 was determined using a BCA protein quantitative assay kit, and the grapefruit-derived plant nanoparticles were verified by Western blotting. The specific method was as follows:

[0045] The grapefruit-derived plant nanoparticle suspension obtained in Example 1 was centrifuged at 4°C and 100,000 g for 1.5 h. The grapefruit-derived plant nanoparticles were resuspended in RIPA lysis buffer containing 1% protease inhibitor and 1% phosphatase inhibitor (P1006, Beyotime Biotechnology, China) to extract protein. The total protein concentration was determined using a BCA protein quantification kit, and the sample load was determined to be 50 μg. The resulting proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA) by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blocked with TBST (TBS buffer containing 0.1% Tween-20) containing 5% skim milk at room temperature. The membrane was incubated with primary antibodies for 2 h at 4 °C overnight: rabbit anti-Alix antibody (dilution 1:2000), rabbit anti-TSG101 antibody (dilution 1:1000), rabbit anti-CD9 (dilution 1:1000), rabbit anti-CD63 (dilution 1:1000), rabbit anti-calnexin (dilution 1:1000), washed three times with TBST, and incubated with HRP-conjugated anti-rabbit secondary antibody (dilution 1:10000) at room temperature for 2 h. The signals were detected by enhanced chemiluminescence (Millipore, USA) and detected using Bio-Rad ChemiDoc. TM The images were taken using the MP imaging system (Bio-Rad, USA).

[0046] Results see Figure 1 a. As can be seen from the figure, the grapefruit-derived plant nanoparticles obtained in Example 1 can express exosome-related proteins Alix, CD9, and CD63, but do not express Calnexin compared with cells.

[0047] The morphological characteristics of grapefruit-derived plant nanoparticles were evaluated by conventional procedures using electron microscopy, and the results are shown in Figure 1 b. As can be seen from the figure, the grapefruit-derived plant nanoparticles obtained in Example 1 are relatively uniform in size and have a double-layered circular structure.

[0048] DLS was used to measure the particle size and zeta potential of grapefruit-derived plant nanoparticles. Figure 1 c. Figure 1 d. As shown in the figure, the particle size of grapefruit-derived plant nanoparticles ranges from 104.5 to 122.3 nm, and the zeta potential ranges from -6.4 to -4.6 mV.

[0049] Example 3 Stability Determination of Grapefruit-Derived Plant Nanoparticles

[0050] The grapefruit-derived plant nanoparticles obtained in Example 1 were placed in a PBS buffer solution containing 50% fetal bovine serum (FBS) and incubated at 37°C. At selected time points (0 to 168 h, every 24 h), the particle size and zeta potential of the grapefruit-derived plant nanoparticles were measured using DLS.

[0051] The grapefruit-derived plant nanoparticle suspension obtained in Example 1 was placed at 4° C.; at selected time points (0 to 30 days, every 5 days), the particle size and zeta potential of the grapefruit-derived plant nanoparticles were measured using DLS.

[0052] Results see Figure 2 As can be seen from the figure, no significant changes in the particle size distribution and membrane potential of grapefruit-derived plant nanoparticles were observed in PBS buffer containing 50% fetal bovine serum within 168 hours; similarly, no significant changes in the particle size distribution and membrane potential of grapefruit-derived plant nanoparticles were observed in PBS within 30 days.

[0053] Example 4 Cell viability assay

[0054] Evaluation of the cytotoxicity of grapefruit-derived plant nanoparticles using CCK8 assay: MVSMCs (4 × 10 cells / well in 100 μL culture medium) were cultured in a 4% spherical microporous membrane (100 μL) flask. 3Cells (n = 5) were seeded in 96-well plates and cultured at 37 ° C for 24 h; the culture medium in each well was then replaced with 200 μL DMEM medium or 200 μL DMEM medium containing different concentrations of grapefruit-derived plant nanoparticles (0, 1, 5, 10, 15, 20, 30, 40 μg / mL). Cells cultured with DMEM medium served as the control group. After 48 h of culture, the culture medium was replaced with 200 μL DMEM medium containing 10% CCK8 and cultured for another 0.5 h, 1 h, and 2 h. The absorbance was measured at 450 nm using a microplate reader (Synergy2, Bio-Tek, USA). The cell viability in each group was expressed as the percentage of (absorbance of the experimental group - absorbance of the blank group) / (absorbance of the control group - absorbance of the blank group). The experiment was performed in triplicate, and the results show the representative of three independent experiments.

[0055] Results see Figure 3 As can be seen from the figure, grapefruit-derived plant nanoparticles did not affect cell activity at a concentration of 0 to 15 μg / mL; when the concentration of grapefruit-derived plant nanoparticles was higher than or equal to 20 μg / mL, cell activity decreased with the increase of the concentration of grapefruit-derived plant nanoparticles.

[0056] Example 5 Anti-calcification effect of grapefruit-derived plant nanoparticles on mouse aortic smooth muscle cells (MVSMCs)

[0057] MVSMCs cells were seeded into six-well plates (1×10 6 cells) and cultured for 24 hours to allow the cells to adhere. Fresh DMEM medium was replaced, and when the cell density reached approximately 60-70%, high-phosphate calcification solution with a final phosphorus concentration of 3 mmol / L was added to the calcification group and the treatment group, which was recorded as day 0. The medium was changed every other day, and fresh DMEM medium was replaced on the 6th day of modeling. The calcification group continued to receive 3 mmol / L high-phosphate calcification solution, while the treatment group received high-phosphate calcification solution with a final phosphorus concentration of 3 mmol / L and grapefruit-derived plant nanoparticles at final concentrations of 5, 10, and 20 μg / ml, respectively. Grapefruit-derived plant nanoparticles were added to normal mouse vascular smooth muscle cells at a final concentration of 10 μg / ml as the 10 μg / ml GNVs group. In addition, an equal amount of PBS was added to normal mouse vascular smooth muscle cells as the normal control group. After 48 hours of intervention, the degree of calcification of mouse vascular smooth muscle cells was assessed by Alizarin Red staining and optical microscopy.

[0058] Results Figure 4The degree of calcification in mouse vascular smooth muscle cells was assessed by visual observation of alizarin red-stained tissue and microscopic observation of the alizarin red staining. A greater area stained with alizarin red indicates more severe calcification. As shown in the figure, treatment with 10 μg / ml of grapefruit-derived plant nanoparticles for 48 hours in normal mouse vascular smooth muscle cells did not result in any changes in cell morphology or a decrease in cell number. Treatment with 5 μg / ml of grapefruit-derived plant nanoparticles alleviated calcification in mouse vascular smooth muscle cells, with 10 μg / ml having a more pronounced effect. While the effect of 20 μg / ml of grapefruit-derived plant nanoparticles on vascular calcification was largely similar to that of 10 μg / ml, microscopic observation revealed that this concentration caused morphological changes and a decrease in cell number in vascular smooth muscle cells, consistent with the aforementioned cell viability results. Therefore, 10 μg / ml of grapefruit-derived plant nanoparticles is an optimal concentration for intervention in vascular calcification.

[0059] Example 6: Anti-vascular calcification effect of grapefruit-derived plant nanoparticles on high vitamin D3-induced vascular calcification in C57BL / 6 mice

[0060] Male C57BL / 6 mice aged 6 to 8 weeks (18 to 22 g) were subcutaneously injected with vitamin D3 (dose: 0.2 mg / g) to establish the model, and the aorta of the mice was taken for alizarin red staining after sacrifice to confirm the successful establishment of the model.

[0061] C57BL / 6 mice were randomly divided into three different groups without any deviation in body weight (n=6), namely: normal group, calcification group, and treatment group. The model was established according to the above method, and the day was marked as day 0. At the same time, the mice in the treatment group were treated with PBS and grapefruit-derived plant nanoparticles (6.5 mg / kg), respectively, and administered once every other day by intraperitoneal injection; on the 7th day, the mice were killed, and the aorta was collected and fixed with 4% paraformaldehyde solution at room temperature for 48 hours, stained with 0.004% Alizarin Red S overnight, washed with 2% potassium hydroxide for 5 minutes to remove floating color, and scanned and photographed.

[0062] Results see Figure 5 As can be seen from the figure, compared with the calcification group, the degree of aortic calcification in the treatment group mice was significantly reduced, confirming that grapefruit-derived plant nanoparticles can reduce vitamin D3-induced vascular calcification in C57B / L6 mice.

[0063] Example 7 In vitro drug hemolysis experiment of grapefruit-derived plant nanoparticles

[0064] 1 ml of blood was collected from the eyeball of male 6-8 week old C57BL / 6 mice and placed in an EDTA K3 blood collection tube. After inversion and mixing, 500 μl was taken and placed in a 15 ml centrifuge tube containing 5 ml of PBS. The tube was centrifuged at 3000 r / min for 5 min, the supernatant was discarded, 10 ml of PBS was added and mixed, and the tube was centrifuged at 3000 r / min for 5 min. The supernatant was discarded, and this process was repeated 3-4 times until the supernatant was clear. The red blood cells were resuspended in 10 ml of PBS to prepare a 5% red blood cell suspension. 200 μl of the red blood cell suspension was taken and 800 μl of grapefruit-derived plant nanoparticles at different concentrations (1, 5, 10, 25, 50 μg / ml) were added. 800 μl of distilled water was added to the positive control group, and 800 μl was added to the negative control group. PBS was incubated at 37°C for 2 hours, centrifuged at 3000 rpm for 5 minutes, and 200 μl of the supernatant was transferred to a 96-well plate. The absorbance was measured at 576 nm using a microplate reader to calculate the hemolysis rate. The formula is: Hemolysis rate = (absorbance of the test sample - absorbance of the negative control) / (absorbance of the positive control - absorbance of the negative control) as a percentage. Experiments were performed in quintuplicate, and the results shown are representative of five independent experiments.

[0065] Results see Figure 6 As can be seen from the figure, grapefruit-derived plant nanoparticles will not cause hemolysis to the body at a concentration of 50μg / ml. This concentration is higher than the drug concentration in animals and has better safety.

[0066] Example 8 Organ Safety Experiment of Grapefruit-Derived Plant Nanoparticles

[0067] Male C57BL / 6 mice aged 6 to 8 weeks (18 to 22 g) were subcutaneously injected with vitamin D3 (dose: 0.2 mg / g) to establish the model. After model establishment, the mice were treated with PBS or grapefruit-derived plant nanoparticles (6.5 mg / kg) via intraperitoneal injection every other day. Body weight changes of six mice in each group were measured daily until sacrifice. On day 8 after model establishment, the mice were sacrificed, and the aorta and major organs (heart, liver, spleen, lung, and kidney) were harvested. Organs collected from the different treatment groups (heart, liver, spleen, lung, and kidney) were stained with hematoxylin and eosin, and all tissue sections were observed under a light microscope (Nikon, Japan).

[0068] The results are as follows Figure 7 As shown, the use of grapefruit-derived plant nanoparticles to treat vascular calcification did not cause damage to the main organs of mice (heart, liver, spleen, lungs, and kidneys), and was highly safe.

[0069] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. Application of plant-derived nanoparticles in the preparation of drugs for preventing and treating vascular calcification, characterized in that: The preparation method of the plant-derived nanoparticles specifically comprises the following steps: Grapefruit juice is squeezed, and the juice is subjected to differential centrifugation. The supernatant obtained by removing the pomace is subjected to ultracentrifugation. The separated precipitate is resuspended in a buffer and purified by sucrose density gradient centrifugation to obtain grapefruit-derived nanoparticles. The concentration of the plant-derived nanoparticles is 5-15 μg / ml.

2. The application according to claim 1, characterized in that The ultra-high speed centrifugation is performed at 100,000 to 160,000 g for 1.5 to 2.5 hours.

3. The application according to claim 1, characterized in that The centrifugation is carried out at 3-5°C.

4. The application according to claim 1, characterized in that The differential centrifugation is divided into five stages, namely: the first stage is centrifugation at 400-600g for 8-12 minutes, the second stage is centrifugation at 1800-2200g for 15-25 minutes, the third stage is centrifugation at 4500-5500g for 30 minutes, the fourth stage is centrifugation at 8000-12000g for 1-1.5 hours, and the fifth stage is centrifugation at 80000-120000g for 1.5-2.5 hours.

5. The application according to claim 1, characterized in that: The sucrose density gradient centrifugation method comprises the following steps: sucrose with densities of 8%, 30%, 45% and 60% are sequentially added to a centrifuge tube to form a sucrose density gradient; the suspension is transferred to the top layer of the sucrose gradient with densities of 8%, 30%, 45% and 60%; and the suspension is centrifuged at 140,000 to 160,000 g for 1.5 to 2.5 hours. The 30% / 45% band is the purified grapefruit-derived nanoparticles.

6. The application according to claim 1, characterized in that: The particle size of the plant-derived nanoparticles is 104.5-122.3 nm.

7. The use according to claim 1, characterized in that The dosage form of the drug is oral preparation, injection or inhalation.

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