A sesame leaf exosome nanoparticle, a preparation method thereof, and application thereof in active ingredient delivery

Abstract

The application belongs to the technical field of nanomaterials and its preparation, and specifically discloses a sesame leaf exosome nanoparticle, a preparation method thereof and application thereof in active ingredient delivery. The preparation method of the exosome nanoparticle comprises the following steps: homogenizing sesame leaves, performing differential centrifugation, and then performing ultrafiltration to obtain supernatant concentrate of sesame leaf exosomes; then, the obtained supernatant concentrate is subjected to exosome purification through an ultra-pure chromatographic column; subsequently, the obtained exosomes are further concentrated through an ultrafiltration tube, and the exosome nanoparticle is obtained. The preparation method has the advantages of fewer extraction steps, high extraction efficiency, mild conditions, high purity of the obtained product and the like. The exosome nanoparticle can realize high loading of natural active ingredients through ultrasonic auxiliary technology, slow down the release speed of the loaded active ingredients, improve the light stability and bioavailability of the active ingredients, enhance the anti-inflammatory capacity of the active ingredients, and has good biocompatibility and low toxicity.

Description

A sesame leaf exosome nanoparticle, its preparation method, and its application in the delivery of active ingredients. Technical Field

[0001] This invention belongs to the field of nanomaterials and their preparation technology, specifically relating to a sesame leaf exosome nanoparticle, its preparation method, and its application in the delivery of active ingredients. Background Technology

[0002] Plant-derived natural active ingredients, such as curcumin, resveratrol, and luteolin, possess antioxidant, anti-inflammatory, and anti-obesity bioactivities. However, their strong hydrophobicity, poor chemical stability, and rapid metabolism result in low bioavailability, limiting their development and utilization. Nanotechnology has been applied to improve the stability and physiological activity of these active ingredients. Plant exosomes are biological nanostructures with a diameter of approximately 30–150 nm and a lipid bilayer. They are naturally rich in bioactive proteins, lipids, RNA, and other pharmacologically active molecules, which can regulate gene and protein expression levels in recipient cells, thereby mediating the physiological and pathological functions of multicellular organisms. They can also deliver various active ingredients, including chemical drugs, proteins and peptides, gene drugs, and natural molecules, exhibiting high biocompatibility, high tolerability, and biodegradability, making them considered an ideal natural delivery carrier for both food and medicine.

[0003] Sesame, belonging to the genus *Sesamum* of the family Pedaliaceae, is an annual herbaceous plant and one of the world's most important oilseed crops. The global sesame cultivation area is estimated at 7 to 8 million hectares. 2 The annual output is approximately 4.5 million tons. The total sesame planting area in my country is about 700,000 hectares. 2 The annual output is between 600,000 and 650,000 tons. Sesame seeds are mainly used for oil extraction, while sesame leaves, as a byproduct of sesame production, are rich in protein, fat, minerals, polysaccharides, polyphenols, flavonoids, and other substances. They possess health benefits such as anti-inflammatory, antioxidant, anti-obesity, diabetes relief, and cardiovascular disease prevention. However, their utilization rate is currently low, with most being discarded, resulting in significant resource waste. This invention prepares sesame leaf exosomes and develops related delivery vectors, which not only enhance the physiological activity of sesame leaves themselves but also improve the physiological activity of the loaded active ingredients. Compared to animal exosomes, plant exosomes have a wider range of applications, including food, medicine, and daily necessities. Furthermore, sesame leaf exosomes are a plant byproduct, making them abundant and readily available.

[0004] Existing methods for preparing plant exosomes mainly include ultracentrifugation, density gradient centrifugation, polymer precipitation, ultrafiltration, size exclusion chromatography, and immunoaffinity chromatography. Ultracentrifugation is generally time-consuming, typically exceeding 5 hours, and requires expensive separation equipment (e.g., CN110964694A). Polymer precipitation and ultrafiltration yield exosomes with lower purity, with polymer precipitation often requiring the addition of extra chemical reagents (e.g., CN112574940A, CN114015640A). Size exclusion chromatography offers advantages such as simple operation, good reproducibility, and high purity, but the resulting exosome concentration is low, limiting its application (e.g., CN114591892A). While immunoaffinity chromatography separates exosomes with high specificity and purity, and intact morphology, the affinity chromatography media are expensive, resulting in high purity costs (e.g., CN105934670B). In view of the shortcomings of the prior art described above, the present invention combines ultrafiltration and size exclusion chromatography to improve the purity of exosomes and increase their content. The method is simple, easy to control, and can obtain sesame leaf exosomes with high quality and high yield. It has good loading capacity and can be used in food, daily chemical products and pharmaceuticals. Summary of the Invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a sesame leaf exosome nanoparticle, a method for preparing it with high purity, and its application.

[0006] The technical problem to be solved by this invention is to provide an exosome nanoparticle loaded with active ingredients, its preparation method and application. Sesame leaf exosome nanoparticles are used as delivery carriers for active ingredients with anti-inflammatory effects, thereby realizing the utilization of by-product resources and improving the physiological activity of active ingredients.

[0007] Technical solution: To achieve the above technical objective, the present invention provides sesame leaf exosome nanoparticles. The sesame leaf exosome nanoparticles are obtained by homogenizing sesame leaves, centrifuging at differential speed, and then ultrafiltration to obtain a supernatant concentrate of sesame leaf exosomes. The obtained supernatant concentrate is then purified by ultrapure chromatography column, and the obtained exosomes are further concentrated by ultrafiltration tube.

[0008] The sesame leaf exosome nanoparticles have a particle size of 50-60 nm.

[0009] The present invention also includes an exosome nanoparticle loaded with an active ingredient, wherein the exosome nanoparticle loaded with the active ingredient is obtained by loading the active ingredient into the sesame leaf exosome nanoparticle.

[0010] The active ingredient is a physiologically active substance.

[0011] Preferably, the active ingredient is the natural flavonoid luteolin (Lu).

[0012] The present invention also includes a method for preparing the sesame leaf exosome nanoparticles, comprising the following steps:

[0013] (1) Take sesame leaves, rinse off surface stains, rinse with ultrapure water, add PBS solution to soak and homogenize, centrifuge, and collect the supernatant;

[0014] (2) Centrifuge the supernatant from step (1) and collect the supernatant;

[0015] (3) Take the supernatant from step (2) and ultrafilter it to obtain a concentrated solution of sesame leaf exosomes;

[0016] (4) The concentrate was purified using an ultrapure chromatography column. The concentrate was added to the ultrapure chromatography column. After all samples entered the column, 1 / 12 of the column bed volume of PBS solution was added for elution each time, and the fraction (i.e., eluent) was collected. When no liquid flowed out of the outlet, the fraction was considered collected. The fraction obtained from the first elution was called the first fraction. Subsequent elutions yielded the second, third, fourth, fifth, and so on fractions. Fractions 2-5 were combined and concentrated using an ultrafiltration tube. Based on particle size and morphology analysis, combined with protein molecular weight and lipid composition, it was determined to be exosomes. Protein quantification was performed using BCA, and the protein content could reach 500 μg / mL-3 mg / mL.

[0017] In step (1), the pH of the PBS solution is 7.0 and the concentration is 0.01 mol / L. The mass-to-volume ratio of the sesame leaves to the PBS solution is 1 g: 2 mL.

[0018] In step (1), the centrifugal force is 1000g, and in step (2), the centrifugal force is 10000g.

[0019] The present invention also includes a method for preparing the aforementioned exosome nanoparticles loaded with active ingredients, comprising the following steps: dissolving the active ingredient in ethanol to obtain an active ingredient solution, mixing sesame leaf exosome nanoparticles with the active ingredient solution, and preparing the exosome nanoparticles loaded with active ingredients by ultrasonication.

[0020] The mass ratio of the active ingredient to the protein in sesame leaf exosomes is 2:1.

[0021] The present invention also includes the application of the sesame leaf exosome nanoparticles and the exosome nanoparticles loaded with active ingredients in the delivery of active ingredients or in the preparation of drugs for treating anti-inflammatory diseases.

[0022] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0023] This invention utilizes exosomes derived from sesame leaves as a carrier, representing a rational use of byproduct resources. The natural active ingredient Lu is loaded into these exosomes with ultrasound assistance, resulting in high-load exosomes. Compared to artificially synthesized nanoparticles such as liposomes, this invention, using exosomes derived from sesame leaves as a carrier for the active ingredient, offers advantages such as readily available materials, low cost, high biocompatibility, high loading rate, wide application range, and ease of operation. Furthermore, this invention determined the loading potential of sesame leaf exosomes, studied the stability, in vitro release characteristics, and bioavailability of luteolin-loaded exosome nanoparticles, as well as their in vitro anti-inflammatory activity, demonstrating the advantages of using exosomes to load active ingredients and providing a foundation for research on sesame leaf exosome delivery systems. Attached Figure Description

[0024] Figure 1 shows the electron microscopy (EM) images and particle size distribution of the prepared sesame leaf exosomes (Exo) and Lu-loaded exosomes (Exo@Lu). In Figure 1, ab represents the transmission electron microscopy (TEM) images of sesame leaf Exo and Exo@Lu; cd represents the scanning electron microscopy (SEM) images of sesame leaf Exo and Exo@Lu; and ef shows the particle size distribution of Exo and Exo@Lu, which are 156.4 nm (PDI = 0.242) and 134.2 nm (PDI = 0.289), respectively.

[0025] Figure 2 shows the basic characteristics of the obtained sesame leaf Exo and Exo@Lu. a) Protein distribution of exosomes observed by Coomassie brilliant blue staining; b) Lipid composition of exosomes; c) Solubility of Lu in H2O and Exo; d) FT-IR spectrum; e) Release of Lu under simulated gastrointestinal digestion conditions.

[0026] Figure 3 shows the stability test results. a represents temperature stability; b represents light stability; and c represents particle size and potential stability. Specifically, I and II represent the particle size stability of Exo and Exo-Lu, respectively, while III and IV represent the potential stability of Exo and Exo-Lu.

[0027] Figure 4 shows the cytotoxicity assay. Ab represents the cytotoxicity assay of Exo and Exo@Lu at concentrations of 0-40 μg / mL; c represents the cytotoxicity assay of Lu at concentrations of 0-10 μg / mL.

[0028] Figure 5 shows the oxidation index test. Among them, a represents the intracellular reactive oxygen species (ROS) level; b represents the intracellular glutathione (GSH) level.

[0029] Figure 6 shows the NO release level and inflammatory factor level test. In the figure, a represents the NO level; b and d represent the levels of tumor necrosis factor (TNF-α), interleukin-1 (IL-1β), and interleukin-6 (IL-6), respectively. Detailed Implementation

[0030] The following specific embodiments further illustrate the evaluation of the bioactivity of the loaded active ingredient exosomes described in this invention. The following examples are only used to further illustrate the invention and should not be construed as limiting the scope of protection of this invention. Those skilled in the art can make some non-essential improvements and adjustments to this invention based on the above-described invention content, and these improvements and adjustments still fall within the scope of protection of this invention.

[0031] In the following embodiments:

[0032] The sesame leaves used are freshly picked sesame leaves;

[0033] All PBS solutions used were pH 7.0 and had a concentration of 0.01 mol / L.

[0034] Example 1: Preparation of sesame leaf exosome nanoparticles (Exo)

[0035] (1) Take sesame leaves, rinse off surface stains, rinse with ultrapure water, add PBS solution (mass-volume ratio of sesame leaves to PBS solution is 1g:2mL) to soak, homogenize, centrifuge at 4℃ for 30min, collect the supernatant, and centrifuge at 1000g.

[0036] (2) Take the supernatant from step (1) and centrifuge at 4°C for 30 min. Collect the supernatant with a centrifugal force of 10000g.

[0037] (3) Take the supernatant from step (2) and ultrafilter it to obtain the filtrate. The filter membrane has a molecular weight cutoff of 100kDa to obtain a concentrated solution of plant-derived exosomes.

[0038] (4) Purify the concentrate using a SuperEV5.0 ultrapure chromatography column with a column bed volume of 60 mL: Add the concentrate obtained in step (3) (injection volume of 5.0 mL) to the ultrapure chromatography column. Elute with PBS solution of 1 / 12 of the column bed volume (i.e., 5.0 mL) each time. Collect the eluent obtained after each elution as the fraction number. Combine the liquids of fractions 2-5 (based on particle size and morphology analysis, combined with protein molecular weight and lipid composition). Then concentrate with a 100 kDa ultrafiltration tube to obtain exosomes Exo. Quantify the protein content by BCA protein quantification. The protein content is 500 μg / mL.

[0039] Example 2: Preparation of exosome nanoparticles loaded with active ingredients (Exo@Lu)

[0040] The exosomes Exo (sesame leaf exosome nanoparticles) prepared in Example 1 were selected for loading the active ingredients.

[0041] (1) Take 100 μL of Lu (2 mg / mL Lu in ethanol) and add it to 200 μL of exosomes (500 μg / mL protein concentration). Use an ultrasonic cell disruptor to sonicate the reaction system, on / off for 2 seconds, for a total of 10 cycles, 9 W.

[0042] (2) Place the ultrasonically treated mixture at 37°C for 1 hour to allow the exosome membrane to return to its original state. Then, centrifuge the mixture at 3500g through an ultrafiltration tube (100kDa) at 4°C for 10 minutes to obtain Lu-loaded exosomes, i.e., exosomes loaded with active ingredients.

[0043] (3) Measure the absorbance at an absorption wavelength of 348 nm and calculate the loading rate (DLE%) according to the following formula.

[0044]

[0045] The amount of Lu loaded into the exosomes was 184 μg, and the total amount of exosome nanoparticles was 900 μg. Based on this formula, the loading rate of the active ingredient can be calculated to be 20.44%.

[0046] Example 3: Characterization and performance testing of sesame leaf exosome nanoparticles (Exo) and exosome nanoparticles loaded with active ingredients (Exo@Lu)

[0047] The sesame leaf exosome nanoparticles (Exo) prepared in Example 1 and the exosomes loaded with active ingredients prepared in Example 2 were characterized and their performance was determined.

[0048] (1) Characterization of sesame leaf exosome nanoparticles (Exo) and exosome nanoparticles loaded with active ingredients (Exo@Lu)

[0049] TEM and SEM images of Exo prepared in Example 1 and Exo@Lu prepared in Example 2 were performed, respectively. As shown in Figure 1, the particle size of Exo and Exo@Lu is 50-60 nm. The hydrated particle size was determined using a Malvern Nano-ZS90 laser particle size analyzer. Figures 1e-f show the particle size distribution of Exo and Exo@Lu, which are 156.4 nm (PDI = 0.242) and 134.2 nm (PDI = 0.289), respectively. SDS-PAGE gel electrophoresis in Figure 2a shows that the molecular weight of Exo is 10-17 kDa. Figure 2b shows the lipid composition of sesame leaf exosomes, which includes EtherDG (46.65%), TG (13.75%), DG (11.04%), PE (7.76%), Cer_AP (6.24%), PC (1.71%), etc. Figure 2c shows that Lu is more soluble in Exo than in water. Figure 2d is an FT-IR spectrum, indicating that Lu was successfully loaded into exosomes. Figure 2e shows the Lu release behavior under simulated gastrointestinal digestion conditions. After 6 hours of gastrointestinal digestion, the Lu release rate was 30 ± 0.06%, and the Lu release rate in Exo@Lu was 12 ± 0.09%, indicating that Exo@Lu has the effect of slowly releasing the active ingredient, while also showing that the bioavailability increased from 70% to 88%.

[0050] (2) Stability test of exosomes loaded with active ingredients

[0051] As shown in Figure 3a, Exo@Lu was placed at 25℃, 50℃, 60℃, 70℃, 80℃, and 90℃ for 1 hour, and the retention rate of Lu was tested. The figure shows that the remaining rates of Lu at 25℃, 50℃, 60℃, 70℃, 80℃, and 90℃ were 100%, 94±5.89%, 92±7.01%, 79±2.48%, 71±5.23%, and 71±7.92%, respectively, indicating that Lu-loaded exosomes have good thermal stability. As shown in Figure 3b, after exposing Lu and Exo@Lu to ultraviolet irradiation for 7 hours, the remaining rate of free Lu was 44.9%, while the remaining rate of Lu in Exo@Lu was 70.7%, indicating that Lu-loaded exosomes have good photostability. As shown in Figure 3c, the particle size and potential of Exo and Exo@Lu prepared in Examples 1-2 were measured over 7 days using a Malvern laser particle size analyzer. The particle size of Exo was approximately 150 nm, and the particle size of Exo@Lu was approximately 130 nm. The potential fluctuated around -7 mV, indicating that Exo and Exo@Lu have good particle size and potential stability.

[0052] (3) Cytotoxicity test

[0053] The sesame leaf exosome nanoparticles (Exo) prepared in Example 1, the exosome nanoparticles loaded with active ingredients prepared in Example 2, and free Lu were evaluated cytologically using mouse mononuclear macrophage Raw264.7 cells as a model. Raw264.7 cells were prepared into 5×10⁶ cells... 4 Cell suspension was seeded at 100 μL per well in 96-well plates and cultured overnight at 37°C with 5% CO2. DMEM medium was then removed, and six concentration gradients of Exo, Exo@Lu, and Lu (prepared in DMEM medium) were added and co-cultured with the cells for 24 h (Exo concentration gradient: 5, 10, 15, 20, 25, 40 μg / mL; Exo@Lu concentration gradient: 5, 10, 15, 20, 30, 40 μg / mL; Lu concentration gradient: 0.3, 0.6, 1.3, 2.5, 5, 10 μg / mL). Following the instructions of the CCK-8 assay kit (Cell Proliferation and Toxicity Assay Kit, Dojindo, CK04-01), 10 μL of the solution was added to each well. CCK-8 reagent was used for incubation at room temperature for 45 min. The absorbance of each well in each group was measured at 450 nm. Cell viability (%) was calculated using the formula: (Experimental group - Blank control group) / (Control group - Blank control group) × 100%, where the blank control group consisted of uninoculated wells, the experimental group consisted of wells inoculated with the drug, and the control group consisted of wells inoculated with the drug. The viability of Exo, Exo@Lu, and Lu on Raw264.7 cells was calculated. The results are shown in Figure 4. When the concentrations of Exo and Exo@Lu were 0-40 μg / mL, and the concentration of Lu was 0-10 μg / mL, the viability of Raw264.7 cells was higher than 90%, indicating that Lu-loaded exosomes exhibited good biocompatibility.

[0054] (4) Evaluation of bioactivity

[0055] 4.1 Antioxidant Activity

[0056] Raw264.7 cells were prepared into 5×10⁻⁶ cells. 5Cell suspensions were seeded at 500 μL per well in 24-well plates and cultured overnight at 37°C with 5% CO2. DMEM medium was removed, and Exo (20 μg / mL), Lu (1.28 μg / mL), and Exo@Lu (equal to free Lu) prepared in DMEM medium were added and co-cultured with the cells for 24 h. ROS levels were measured according to the ROS manufacturer's instructions. The same method was used for seeding, drug administration, and cell detachment to measure intracellular GSH levels. As shown in Figure 5, after lipopolysaccharide (LPS)-induced inflammation, the ROS level was 129 ± 1.68% in the normal group, 118 ± 0.49% in the free Lu group, and 112 ± 2.70% in the Exo@Lu group. Compared with the free Lu group, Exo@Lu reduced ROS levels. After treatment with Exo@Lu, the GSH level was 328.1 μmol / gprot, which was higher than that of the LPS group (275.0 μmol / gprot) and the free Lu group (282.4 μmol / gprot). Therefore, Exo@Lu can significantly improve the GSH level, indicating that exosome nanoparticles loaded with active ingredients have good antioxidant properties.

[0057] 4.2 Anti-inflammatory activity

[0058] Raw264.7 cells were prepared into 5×10⁻⁶ cells. 5 / well cell suspension was seeded in 24-well plates at 500 μL per well. After 24 h, the DMEM medium was removed, and Exo (20 μg / mL), Lu (1.28 μg / mL), and Exo@Lu (equal to free Lu) prepared in DMEM medium were added and co-cultured with the cells for 2 h. Then, the cells were treated with 0.5 μg / mL lipopolysaccharide for 24 h. The cell supernatant was collected, and the levels of NO, tumor necrosis factor (TNF-α), interleukin-1 (IL-1β), and interleukin-6 (IL-6) were measured. As shown in Figure 6, after treatment with Exo@Lu, the NO content was 13.5 μmol / gprot, lower than that of the LPS group (28.3 μmol / gprot) and the Lu group (15.3 μmol / gprot). The TNF-α level of Exo@Lu was 338.8 ng / L, lower than that of the LPS group (564.1 ng / L) and the Lu group (505.6 ng / L). The IL-1β level of Exo@Lu was 2.6 ng / L, lower than that of the LPS group (17.1 ng / L) and the Lu group (6.0 ng / L). The IL-6 level of Exo@Lu was 42.1 ng / L, lower than that of the LPS group (55.3 ng / L) and the Lu group (51.4 ng / L). Therefore, compared with free Lu, Exo@Lu can significantly reduce the levels of NO and pro-inflammatory factors, indicating that exosome nanoparticles loaded with active ingredients have stronger anti-inflammatory activity.

Claims

1. An exosome nanoparticle loaded with an active ingredient, characterized in that, The exosome nanoparticles loaded with the active ingredient are obtained by loading the active ingredient into sesame leaf exosome nanoparticles. The sesame leaf exosome nanoparticles are obtained by homogenizing sesame leaves, centrifuging at differential speed, and then ultrafiltration to obtain a supernatant concentrate of sesame leaf exosomes. The supernatant concentrate is then purified by ultrapure chromatography, followed by further concentration of the exosomes using an ultrafiltration tube. The particle size of the sesame leaf exosome nanoparticles is 50-60 nm, and the protein content is 500 μg / mL-3 mg / mL. The active ingredient is the natural flavonoid luteolin. The preparation method of the exosome nanoparticles loaded with the active ingredient includes the following steps: dissolving the active ingredient in ethanol to obtain an active ingredient solution; mixing the sesame leaf exosome nanoparticles with the active ingredient solution; and preparing the exosome nanoparticles loaded with the active ingredient by ultrasonication. The mass ratio of the natural flavonoid luteolin to the protein in the sesame leaf exosomes is 2:

1.

2. The exosome nanoparticles loaded with active ingredients according to claim 1, characterized in that, The preparation method of the sesame leaf exosome nanoparticles includes the following steps: (1) Take sesame leaves, rinse off surface stains, rinse with ultrapure water, soak in PBS solution, homogenize, centrifuge, and collect the supernatant; (2) Centrifuge the supernatant from step (1) and collect the supernatant; (3) Ultrafilter the supernatant from step (2) to obtain a concentrated solution of sesame leaf exosomes; (4) Purify the concentrated solution from step (3) using an ultrapure chromatography column, add the concentrated solution to the ultrapure chromatography column, elute with PBS solution at 1 / 12 of the column bed volume each time, collect and combine the eluents obtained from the 2nd to 5th elutions, and then concentrate with an ultrafiltration tube to obtain sesame leaf exosome nanoparticles with a protein content of 500 μg / mL-3 mg / mL.

3. The exosome nanoparticles loaded with active ingredients according to claim 2, characterized in that, The pH of the PBS solution in step (1) is 7.0, the concentration is 0.01 mol / L, the mass-to-volume ratio of the sesame leaves to the PBS solution is 1 g: 2 mL, the centrifugal force is 1000 g, and the centrifugal force in step (2) is 10000 g.

4. The method for preparing exosome nanoparticles loaded with active ingredients according to any one of claims 1 to 3, characterized in that, The process includes the following steps: dissolving the active ingredient in ethanol to obtain an active ingredient solution, mixing sesame leaf exosome nanoparticles with the active ingredient solution, and preparing exosome nanoparticles loaded with the active ingredient by ultrasonication, wherein the mass ratio of the natural flavonoid luteolin to the protein of sesame leaf exosomes is 2:

1.

5. The use of the exosome nanoparticles loaded with active ingredients as described in any one of claims 1 to 3 in the preparation of anti-inflammatory drugs.

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