Exosome derived from rice and preparation method and application thereof

Abstract

This invention provides exosomes derived from rice, their preparation method, and applications. The exosomes have a bilayered membrane spherical structure with a particle size ranging from 20 to 1000 nm. These exosomes are obtained from rice callus tissue and possess high biocompatibility, stability, and sustainable mass production characteristics. Furthermore, experiments have demonstrated that these exosomes have anti-aging, cell regeneration and tissue repair promotion, and protective functions, and can also significantly improve cell absorption efficiency and the delivery efficiency of active ingredients such as peptides.

Description

Technical Field

[0001] This invention relates to the field of plant exosome technology, specifically to exosomes derived from rice and their preparation methods and applications. Background Technology

[0002] Plant-derived extracellular vesicles (PDEVs), as important mediators for the delivery of bioactive substances, have gradually attracted significant attention in the fields of functional cosmetics and medicinal plants. Plant exosomes are typically tiny vesicles formed through exocytosis via the intracellular membrane system. They range in size from approximately 20 to 1000 nm and possess a typical bilayer phospholipid membrane structure, enabling them to naturally encapsulate various intracellular bioactive molecules, such as proteins, small metabolites, nucleic acids (DNA, mRNA, miRNA), and lipids, facilitating intercellular communication without compromising structural integrity. Compared to traditional synthetic liposomes, plant exosomes exhibit superior transdermal penetration and cellular absorption efficiency. This characteristic is related to their natural lipid-protein hybrid conformation, specific marker proteins on the membrane, and negatively charged surface properties, allowing for more effective penetration of the stratum corneum and fusion with cell membranes. Furthermore, plant exosomes possess high stability and are resistant to degradation, effectively reducing the risk of degradation of active substances during transport and prolonging their retention time in target tissues. Compared to exosomes derived from mammalian cells, PDEVs have lower immunogenicity and biosafety risks, and do not raise concerns about human pathogens or animal-derived components. This effectively avoids immune reactions and ethical controversies, making them more suitable for clinical and aesthetic applications. Recent studies have indicated that PDEVs can effectively encapsulate ingredients such as peptides and antioxidants, thereby enhancing their cellular absorption efficiency and biological function, demonstrating their potential application in the cosmetics field.

[0003] However, regardless of whether the source is plant-based or animal-based, the process of obtaining exosomes generally faces problems such as high extraction costs, strong equipment dependence, difficulty in standardizing raw material sources, and poor stability, which seriously restricts its industrialization process and leads to many obstacles in its practical application. Summary of the Invention

[0004] In view of this, the purpose of this invention is to explore new sources of plant exosomes and their functional applications, in order to solve a series of problems such as high extraction costs, strong equipment dependence, difficulty in standardizing raw material sources, and poor stability of exosomes, and to attempt to transform them into industrialization and clinical applications. Therefore, this invention provides exosomes derived from rice and their preparation methods and applications.

[0005] To achieve the above technical objectives, the technical solution adopted in this application is as follows:

[0006] In a first aspect, the present invention provides an exosome derived from rice callus, the exosome having a double-membrane spherical structure and a particle size ranging from 20 to 1000 nm.

[0007] Preferably, the exosomes have a particle size range of 30~500nm. Exosomes within this range have uniform particle size and extremely low impurity content. The present invention can obtain exosomes within this range.

[0008] Optionally, this invention does not limit the rice variety or source, and includes both transgenic and non-transgenic rice; the transgenic rice is not limited by gene type or recombination method. In some cases of this invention, transgenic rice obtained by transfecting recombinant plasmids of the human recombinant type III collagen gene into Agrobacterium has been used.

[0009] Secondly, the present invention provides a method for preparing exosome stock solution derived from rice callus tissue, comprising the following steps:

[0010] Step 1: Obtain rice callus tissue;

[0011] Step 2: The rice callus tissue obtained in Step 1 is subjected to suspension culture and bioreactor scale-up culture in sequence;

[0012] Step 3: Extract the rice callus culture obtained in Step 2 to obtain cell sap;

[0013] Step 4: The cell fluid obtained in Step 3 is subjected to staged centrifugation. After obtaining the supernatant, it is subjected to ultrafiltration to obtain the exosome stock solution.

[0014] Optionally, step 1 includes: selecting healthy, mature rice seeds without disease spots, removing the shells from the rice seeds to retain the embryos, disinfecting the embryos, and then inducing the culture with 2,4-dichlorophenoxyacetic acid to obtain rice callus tissue.

[0015] The above disinfection process is a routine procedure. Optionally, after surface disinfection with 75% ethanol for 30 seconds to 1 minute, the surface should be treated with 0.1% to 0.5% sodium hypochlorite solution (with a small amount of Tween-20 added) for 10 to 15 minutes, followed by rinsing with sterile distilled water 3 to 5 times to remove residual disinfectant.

[0016] Preferably, the 2,4-dichlorophenoxyacetic acid is added by inoculating the embryos into MS medium containing 1-3 mg / L 2,4-dichlorophenoxyacetic acid and 25-30 g / L sucrose under aseptic conditions; more preferably, the induction culture conditions include: culturing at 25±2°C in the dark for 1-2 weeks.

[0017] Alternatively, the MS medium described above can be replaced with at least one of B5 medium, N6 medium, WPM medium, and DCR medium.

[0018] Preferably, step 2 includes: inoculating the rice callus tissue obtained in step 1 into a liquid culture medium and culturing it in suspension at 110-130 rpm, 25-28°C in the dark, or at a light intensity of 10-100 μmol photons·m⁻²·s⁻¹ for 14-30 days; after the suspension culture is completed, inoculating the callus tissue into a bioreactor and adding liquid culture medium, and culturing it in suspension at an atmospheric air intake rate of 0.25-0.5 VVM, 25-28°C in the dark, or at a light intensity of 10-100 μmol photons·m⁻²·s⁻¹ for 7-14 days;

[0019] More preferably, the inoculum solid-liquid ratio for suspension culture and bioreactor scale-up culture is 1:50~100;

[0020] More preferably, the liquid culture medium comprises, according to the final concentration: 2,4-D, 1.5~2.5 mg / L; sucrose, 25~35 g / L; MS liquid culture medium as the basal medium; and pH adjusted to 5.7~5.8.

[0021] Preferably, step 3 includes: grinding and crushing the rice callus culture obtained in step 2 with water, coarsely filtering it with nylon filter cloth and retaining the filtrate; passing the filtrate through 100-mesh, 200-mesh and 400-mesh sieves in sequence, and retaining the filtrate, which is the cell sap.

[0022] The above grinding and crushing process is a routine procedure, and the grinding and crushing process is based on the premise of not damaging the integrity of the cells.

[0023] Preferably, step 4 includes: centrifuging the obtained cell slurry at 2000g~4000g for 20~40 minutes, then at 5000g~7000g for 20~40 minutes, and finally at 12000g~18000g for 50~70 minutes; filtering the supernatant sequentially through filter membranes with pore sizes of 0.45, 0.22, and 0.1 μm; and then introducing the supernatant sequentially into a hollow fiber membrane column with a specification of 500-1000 kD and a hollow fiber membrane column with a specification of 50-100 kD for ultrafiltration to obtain exosome stock solution.

[0024] Thirdly, the present invention provides an exosome stock solution derived from rice callus tissue obtained by the preparation method described in the second aspect.

[0025] Fourthly, the present invention provides a method for preparing exosome freeze-dried powder derived from rice callus, which involves adding the exosome stock solution obtained by the preparation method described in the second aspect to mannitol and water, and then performing freeze-drying.

[0026] Preferably, the mass percentages of exosome stock solution, mannitol, and water are 60-80%, 3-5%, and 24-28%, respectively; and / or, the control parameters for freeze-drying are: pre-freezing the exosome stock solution at -20±1℃ for 550-650 min; then drying sequentially at -20±1℃ for 150-250 min, -15±1℃ for 250-350 min, and 0±1℃ for 250-350 min; finally drying sequentially at 10±1℃ for 250-350 min, 25±1℃ for 150-250 min, and 36±1℃ for 200-280 min.

[0027] Fifthly, the present invention provides exosome lyophilized powder obtained by the preparation method described in the fourth aspect.

[0028] In a sixth aspect, the present invention provides a delivery carrier for encapsulating an active ingredient, comprising: the exosomes described in the first aspect, or the exosome stock solution described in the third aspect, or the exosome lyophilized powder described in the fifth aspect.

[0029] The active ingredients include, but are not limited to: amino acids, peptides, proteins, polysaccharides, terpenes, sterols, alkaloids, nucleic acids, glycosides, oils, waxes, resins, plant pigments, mineral elements, enzymes, and vitamins.

[0030] In a seventh aspect, the present invention provides a topical skin composition comprising: the exosomes described in the first aspect, or the exosome stock solution described in the third aspect, or the exosome lyophilized powder described in the fifth aspect, or the delivery carrier described in the sixth aspect.

[0031] The exosomes in the topical skin composition contain 0.05-99% by mass, preferably 1-98%; or, the exosome stock solution contains 0.05-100% by mass; or, the exosome lyophilized powder contains 0.05-99% by mass, preferably 1-98%; or, the delivery carrier contains 0.05-99% by mass, preferably 1-98%.

[0032] The topical skin composition includes a pharmaceutical composition or a cosmetic composition, especially a cosmetic composition, such as a cosmetic composition having at least one function such as anti-oxidation, repair, whitening, anti-aging, soothing, spot removal, moisturizing, and promoting penetration and absorption.

[0033] In addition, the topical skin composition may optionally include, but is not limited to, various excipients, such as surfactants, diluents, emulsifiers, thickeners, dispersants, etc. Surfactants include, for example, cetearyl oleate, sorbitan oleate, polysorbate-60, polysorbate-80, methyl glucose sesquistearate, PEG-20 methyl glucose sesquistearate, PEG-40 hydrogenated castor oil, PPG-26-butanol polyether-26, PEG-4 polyglycerol-2 stearate, etc. Diluents include, for example, glycerin, dipropylene glycol, butylene glycol, etc. Emulsifiers include one or more of the following: polyglycerol-3-methylglucose distearate, glyceryl stearate citrate, polyglycerol-10 stearate, polyglycerol-10 myristate, polyglycerol-10 dioleate, polyglycerol-10 laurate, polyglycerol-10 isostearate, polyglycerol-10 oleate, polyglycerol-10 diisostearate, polyglycerol-6 laurate, polyglycerol-6 myristate, sucrose stearate, and sucrose polystearate. Thickeners include one or more of the following: carbomer derivatives, acrylic (ester) derivatives and their derivatives, xanthan gum, gum arabic, polyethylene glycol-14M, polyethylene glycol-90M, succinopolysaccharides, hydroxyethyl cellulose, hydroxypropyl cellulose, and hydroxypropyl methylcellulose. Dispersants include: gelatin, pectin, starch, polyvinyl alcohol, and polyacrylic acid.

[0034] The topical skin composition may optionally contain other active ingredients, such as: tocopherol (vitamin E), retinol, retinyl palmitate, hydrolyzed collagen, hydrolyzed elastin, allantoin, yeast extract, oryzanol, tetrahydrocurcumin, ellagic acid, ubiquinone, whey protein, polypeptides, acetyl hexapeptide-8, palmitoyl pentapeptide-4, salicyl phytosphingosine, concentrated birch sap, silymarin, silk fibroin, sodium tocopheryl phosphate, ribonucleic acid (RNA), dipeptide diaminobutyryl benzylamide diacetate, palmitoyl tripeptide-5, oligopeptide-1, hexapeptide-9, palmitoyl oligopeptide, palmitoyl tetrapeptide-7, grape (VITISVINIFERA) seed extract, rosewood (PTEROCARPUS MARSUPIUM) bark extract, tea (CAMELLIASINENSIS) polyphenols, wine extract, apple seed extract, European beech (FAGUS SYLVATICA) bud extract, hydrolyzed baobab (ADANSONIA) The ingredients include one or more of the following: Digitata extract, Artemisia extract, Iris florigenina root extract, hesperidin, ginsenosides, Salvia miltiorrhiza extract, nicotinamide, ursolic acid, sodium hyaluronate, acetylated sodium hyaluronate, hydrolyzed sodium hyaluronate, lycopene, coffee (Coffea arabica) extract, dipeptide-2, lactic acid, superoxide dismutase (SOD), evening primrose (Oenothera biennes) oil, ceramide, dipalmitoyl hydroxyproline, hydroxystearic acid, salicylic acid, ergothioneine, lysophosphatidylcholine, carnosine, decarboxylated carnosine HCl, lipoic acid, adenosine, glycogen, resveratrol, ferulic acid, Bifida ferment lysate, and lactic acid bacteria ferment lysate.

[0035] The exosomes, exosome stock solution, exosome lyophilized powder, or delivery carrier of the present invention can be mixed with other pharmaceutical or cosmetic ingredients by any method known in the skin topical composition industry to obtain a pharmaceutical or cosmetic composition. The other pharmaceutical or cosmetic ingredients are, as described above, commonly used ingredients in skin topical compositions.

[0036] Furthermore, the topical skin composition can be formulated into various dosage forms, such as solutions, suspensions, ointments, creams, lotions, gels, powders, or sprays, as needed.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] Firstly, given that rice has the advantages of being easy to induce, having stable growth, and being able to be mass-produced through suspension culture in a controlled environment without relying on a large amount of planting space, this invention prepares a rice-derived exosome that simultaneously possesses high biosafety, stability, and sustainable mass production.

[0039] Secondly, experiments have confirmed that the exosomes prepared by this invention possess anti-aging, cell regeneration and tissue repair promotion, and protective functions, and can also significantly improve the efficiency of cell absorption of peptides or other active ingredients. The exosomes of this invention can simultaneously achieve stable peptide delivery and carrier function after recombinant protein expression. While improving peptide stability, cell absorption, and transdermal efficiency, they can also fully utilize the high function of encapsulating recombinant proteins, better meeting the global demand for the manufacture and transdermal delivery of high-performance skincare active ingredients.

[0040] Third, the method for preparing exosomes in this invention has the advantages of simplified process, low equipment requirements, and high industrial adaptability. It can also simultaneously complete recombinant protein expression and exosome enrichment and loading. The entire process does not rely on high-precision equipment, is suitable for GMP or large-scale cosmetic raw material production environments, and achieves cost control and batch consistency.

[0041] Fourth, this invention contains no animal-derived ingredients, no endotoxin risk, and no risk of zoonotic pathogens, which aligns with the trend of green and sustainable biomanufacturing. This exosome can be widely used in functional cosmetics (such as anti-aging, repair, whitening, and transdermal delivery), medical aesthetic raw materials (peptide carriers, postoperative repair exosome therapy), skin repair drugs (wound dressings, burn healing), drug delivery carriers (carrying siRNA, anticancer drugs), and even extends to multiple cutting-edge fields such as oral care, hair regeneration, eye mucosal repair, and biological agent co-carrying systems. Attached Figure Description

[0042] Figure 1 This is a schematic diagram of the exosome preparation process in Example 1 of the present invention.

[0043] Figure 2 The images shown are transmission electron microscopy (TEM) images of the exosome stock solution obtained in Example 1, where A is an image of a few cells focused in the stock solution, and B is an image of multiple cells.

[0044] Figure 3 The graph shows the particle size analysis (NTA) results of the exosome stock solution obtained in Example 1. The horizontal axis represents the particle size (nm), and the vertical axis represents the concentration (particles / mL).

[0045] Figure 4 The graph shows the surface potential analysis results of the exosome stock solution obtained in Example 1.

[0046] Figure 5The particle size characterization (A) and concentration characterization (B) of the exosome stock solution obtained in Example 1 after lyophilization are shown.

[0047] Figure 6 The exosome stock solution obtained in Example 1 is in its freeze-dried and reconstituted form.

[0048] Figure 7 The graph shows the surface potential analysis results of the exosome stock solution obtained in Example 1 after lyophilization.

[0049] Figure 8 The images shown are TEM images of the exosome stock solution obtained in Example 1 after lyophilization. In the images, A is an observation image of multiple cells and B is an observation image of a single cell focused on.

[0050] Figure 9 This is a comparison chart of the clarity of the supernatant after each centrifugation stage in the centrifugation parameter investigation experiment of Example 1.

[0051] Figure 10 The results of the filtration volume of the supernatant through a 0.45 μm filter membrane after each centrifugation in the centrifugation parameter investigation experiment of Example 1 were recorded.

[0052] Figure 11 The antioxidant efficacy of exosomes obtained in Example 1 was evaluated; (a) shows the results of the antioxidant activity detection of DPPH free radical scavenging ability of rice callus exosomes; (b) shows the results of the total phenol content (TPC) analysis of rice callus exosomes.

[0053] Figure 12 The results of the analysis of the effects of exosomes obtained in Example 1 on cell proliferation and wound healing are as follows: (a) In the cell proliferation experiment, HaCaT cells were treated with vitamin C (0.25 mg / mL) and different concentrations of rice callus exosomes for 24 hours; (b) The control group and the cells treated with rice callus exosomes (1 × 10⁻⁶) showed significant differences in cell proliferation and wound healing. 10 (c) Cells treated with rice callus exosomes (particles / mL) for 24 hours were subjected to image analysis for wound healing experiments; 10 Cells treated with particles / mL for 24 hours were used to quantify the healing rate in a wound healing assay. Data are expressed as mean ± standard deviation (mean ± SD), and statistical significance is defined as follows: p<0.001.

[0054] Figure 13 This provides evidence that the exosomes obtained in Example 1 can be endocytosed into cells by HaCaT.

[0055] Figure 14 This is a schematic diagram of the exosome preparation process in Example 2 of the present invention.

[0056] Figure 15 This is evidence that the exosomes obtained in Example 2 contain recombinant type III humanized collagen, where 1 is the original extract of recombinant type III humanized collagen from callus tissue; 2 is the original extract of recombinant type III humanized collagen from rice callus exosomes. The arrow indicates recombinant type III humanized collagen.

[0057] Figure 16 This provides evidence that the recombinant type III humanized collagen rice callus exosomes of Example 2, labeled with DIO dye, can be endocytosed into cells by HaCaT.

[0058] Figure 17 This document describes the preparation process and related characteristics of exosomes coated with pentapeptide-48 obtained in Example 1. A represents the preparation process of exosomes coated with pentapeptide-48 obtained in Example 1; B shows the particle size distribution and particle concentration of rice callus exosomes and rice callus exosomes coated with pentapeptide-48 determined using a nanoparticle size tracer (NTA); and C shows the morphology of rice callus exosomes and rice callus exosomes coated with pentapeptide-48 observed using a transmission electron microscope (TEM), with a scale bar of 100 nanometers.

[0059] Figure 18 The results of the analysis of the cell delivery capacity of pentapeptide-48, exosomes obtained in Example 1, and pentapeptide-48-encapsulated exosomes (OsEVs+Pentapeptide-48) in human skin keratinocytes are presented. (a) Fluorescence images of HaCaT cells show the cell penetration capacity of pentapeptide-48, rice callus exosomes, and pentapeptide-48-encapsulated exosomes. Scale bar: 50 μm; (b) Quantitative analysis of FITC fluorescence intensity in HaCaT cells, reflecting the cell penetration degree of pentapeptide-48, rice callus exosomes, and pentapeptide-48-encapsulated exosomes. Data are expressed as mean ± standard deviation. p<0.001).

[0060] Figure 19 The anti-aging and skin-protective effects of pentapeptide-48 and exosome-encapsulated pentapeptide-48 obtained in Example 1 were investigated. The expression levels of genes such as MMP-3 (A), TIMP-1 (B), Hyal-1 (C), and TGM-1 (D) were analyzed by real-time quantitative polymerase chain reaction (RT-qPCR). Data are expressed as mean ± standard deviation (mean ± SD). This indicates that p < 0.05. This indicates that p < 0.01. This indicates that p < 0.001, which is statistically significant.

[0061] Figure 20 Particle size analysis was performed on the exosomes obtained in Example 1 that encapsulate other active ingredients. Detailed Implementation

[0062] In the description of this invention, it should be noted that unless specific conditions are specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0063] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0064] To facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0065] Example 1:

[0066] This invention provides an embodiment for the preparation of exosomes from rice callus, as detailed below:

[0067] (1) Exosomes were prepared using the Chinese rice variety Zhonghua 11. The preparation process is as follows: Figure 1 As shown, the specific steps include the following:

[0068] Step 1: Select healthy rice seeds, remove the husks, and retain the embryos. Choose healthy, mature rice seeds without disease spots, remove the husks, and retain the intact embryos. After surface disinfection with 75% ethanol for 30 seconds to 1 minute, treat with 0.1%–0.5% sodium hypochlorite solution (with a small amount of Tween-20 added) for 10–15 minutes, followed by rinsing with sterile distilled water 3–5 times to remove residual disinfectant. Under sterile conditions, inoculate the embryos into MS induction medium containing 2 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D) and 30 g / L sucrose, and culture in the dark at 25±2°C for 1–2 weeks to induce callus formation.

[0069] Step 2: Rice callus tissue was cultured in suspension in 0.25L Erlenmeyer flasks for amplification. Primary generation rice callus tissue was inoculated into 0.25L Erlenmeyer flasks at a solid-liquid ratio of 1:50-100, and liquid culture medium (MS liquid medium containing 2 mg / L 2,4-D, with 30 g / L sucrose, pH adjusted to 5.8) was added. The flasks were placed on a shaker and cultured at a constant temperature of 110–130 rpm at 25–28°C in darkness or at light intensities below the light compensation point (10–100 μmol photons·m⁻²·s⁻¹). The culture period was generally 7–14 days, during which the proliferation of cell clumps and the uniformity of the suspension were observed. Cell clumps were visually screened based on size, texture, and morphology, removing undispersed or necrotic clumps. The selected cell clusters were inoculated into new culture flasks at a ratio of 1:50-100, and the above steps were repeated for about one month to obtain stable suspended rice callus.

[0070] Step 3: Take rice callus tissue that has undergone stable suspension culture and inoculate it into a 5L fermenter (bioreactor) at a solid-liquid ratio of 1:50–1:100. Add MS liquid medium containing 2 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D) and 30 g / L sucrose, with the pH adjusted to 5.8. Initially, aeration is performed at an atmospheric gas flow rate of 0.25–0.5 VVM (unadjusted composition). The temperature is controlled at 25–28°C, and the culture is kept in darkness or at a light intensity below the light compensation point (10–100 μmol photons·m⁻²·s⁻¹). The culture period is 7–14 days, during which the aeration ratio is dynamically adjusted by monitoring dissolved oxygen levels. After one week of culture, a 5–10-fold increase in rice callus proliferation can be obtained. After culture, fresh rice callus culture is collected, washed three times with pure water, and used as raw material for exosome extraction.

[0071] Step 4: Add the rice callus culture obtained in Step 3 to purified water at a ratio of 1g:1.5mL and grind to obtain a pulp. Since the grinding equipment generates heat during operation, and high temperatures can damage nanovesicles, the pulp temperature should be controlled below 30℃ during the grinding process. Pour the pulp into a 140-mesh nylon filter cloth and squeeze to filter, removing large fiber residue. Then, pass the filtrate through sieves with pore sizes of 100, 200, and 400 mesh sequentially to remove residue, obtaining the final filtrate.

[0072] Step 5: Centrifuge the obtained filtrate multiple times. Collect the supernatant after each centrifugation and then centrifuge again to obtain the supernatant.

[0073] Step 6: The obtained supernatant is sequentially filtered through a sterile membrane to remove particulate impurities, and then the supernatant is ultrafiltered through a hollow fiber membrane to obtain exosome stock solution.

[0074] Step 7: Mix the exosome stock solution, mannitol, and water in a mass percentage ratio of 70%, 4%, and 26%, respectively, and then freeze-dry the mixture to obtain exosome lyophilized powder. Store the powder in sterile glass bottles.

[0075] In step 5, the obtained filtrate is subjected to staged centrifugation to separate the solid residue: first centrifuged at 3,000 × g for 30 minutes, then at 6,000 × g for 30 minutes, and finally at 12,000 × g for 60 minutes.

[0076] In step 6, the supernatant is sequentially filtered through filter membranes with pore sizes of 0.45, 0.22, and 0.1 μm to obtain a supernatant with particulate impurities removed. The supernatant is then injected into a 500-1000 kD hollow fiber membrane column using a peristaltic pump. Using the principle of tangential flow filtration (TFF), exosomes smaller than approximately 500 nm are separated. These exosomes are then injected into a 50-100 kD hollow fiber membrane column to remove impurities smaller than approximately 30 nm, thus obtaining the exosome stock solution derived from rice tissue culture.

[0077] In step 7, the control parameters for freeze drying are as follows: pre-freeze the exosome stock solution at -20±1℃ for 600 min; then dry it sequentially at -20±1℃ for 200 min, -15±1℃ for 300 min, and 0±1℃ for 300 min; finally, dry it sequentially at 10±1℃ for 300 min, 25±1℃ for 200 min, and 36±1℃ for 240 min.

[0078] (2) Performance identification of exosome stock solution and exosome lyophilized powder

[0079] like Figure 2 As shown, the exosome stock solution observed under a transmission electron microscope (TEM) exhibits a typical double-membrane spherical morphology with clear outlines and complete structure, consistent with the characteristics of cell exosomes.

[0080] like Figure 3 As shown, nanoparticle tracking analysis (NTA) revealed that the exosome stock solution had a particle size range of 30-300 nm, with an average particle size of 94.0 ± 0.5 nm. The particles were uniform in size and had extremely low impurities, meeting the group standard for exosome particle size (20-1000 nm), and the concentration could reach as high as 3 × 10⁻⁶. 12 particles / mL.

[0081] like Figure 4As shown, the average potential of the exosome stock solution measured by the Zeta potentiometer was -17 mV, which is consistent with the characteristic range of cell exosomes (between -25 and -6 mV).

[0082] like Figure 5 As shown, NTA analysis revealed that the exosome particle size distribution after lyophilization ranged from 70 to 140 nm, with an average particle size of approximately 100 nm, showing little difference compared to the original solution. The lyophilized exosomes retained approximately 73–78% of the original nanoparticle concentration, maintaining a high degree of concentration while ensuring stable particle size. The total particle concentration after lyophilization remained as high as 1 × 10¹² particles / mL.

[0083] like Figure 6 As shown, the weight of freeze-dried rice callus exosomes was 0.25 ± 0.05 g / bottle, with a water content of less than 3.5%. They appeared as white, non-collapsed solids. No insoluble solids were observed to form upon reconstitution with 5 mL of pure water, indicating that the freeze-dried rice callus exosome product has excellent water solubility.

[0084] like Figure 7 As shown, the surface potential of rice callus exosomes after freeze-drying was as high as -18.9 mV, and they did not aggregate excessively due to freeze-drying.

[0085] like Figure 8 As shown, under a 100 nm scale field of view, the lyophilized exosomes from rice callus exhibited a stable saucer-like double-membrane spherical structure. The particle size was approximately 120-150 nm in the 100 nm field of view, consistent with NTA results, indicating that the membrane structure remained largely intact after lyophilization.

[0086] (3) Investigation of centrifugation control parameters in step 5 of exosome preparation

[0087] This invention takes into account factors such as process requirements, environmental requirements of equipment, equipment price, and ease of scaling up, and selects a high-speed centrifuge with a centrifugal force of less than 20,000 ×g for process development.

[0088] 1) The filtrate obtained in step 5 is sieved and centrifuged sequentially at 3000×g for 30 min, 6000×g for 30 min, 12000×g for 60 min, and 20000×g for 60 min. The supernatant is collected after each centrifugation, and then the next centrifugation is carried out.

[0089] 2) Compare the clarity of the supernatant after each centrifugation. Results are as follows: Figure 9 As shown, there was no significant difference in clarity between the supernatant after being centrifuged to 12000 ×g (3 centrifugations in total) and after being centrifuged to 20000 ×g (4 centrifugations in total).

[0090] 3) Filter the supernatant of each centrifuged segment through a 0.45 μm pore size filter membrane. Record the volume of solution that can be filtered before the membrane becomes clogged, and calculate the membrane filtration efficiency (filtered volume / original liquid volume × 100%). The results are as follows: Figure 10 As shown, there was no significant difference in the filtration efficiency of the supernatant after sequential centrifugation to 12000 ×g (3 centrifugations) and sequential centrifugation to 20000 ×g (4 centrifugations) via a 0.45 μm membrane.

[0091] After comparative experiments, it was found that centrifugation at 3,000 × g for 30 minutes, 6,000 × g for 30 minutes, and 12,000 × g for 60 minutes in sequence was sufficient for the next step of membrane filtration without significantly affecting the filtration volume. Therefore, these three centrifugation conditions were selected as the optimal treatment steps.

[0092] (4) Evaluation of the antioxidant efficacy of exosomes

[0093] This experiment evaluated the antioxidant activity of rice callus exosomes by using the scavenging rate of 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) and the total phenol content.

[0094] (4-1) DPPH method for detecting the free radical scavenging ability of rice callus exosomes

[0095] Prepare a 0.12 mg / mL DPPH solution (dissolved in 95% ethanol); add 50 μL of 95% ethanol to 950 μL of DPPH solution (blank); take 1×10⁻⁶ exosomes from cucumber. 12 particles / mL), rice callus exosomes (1×10⁻⁶) 12 Add 950 μL of DPPH solution (reaction tube) or RO water (bottom tube) to the particles / mL, mix, and react at room temperature in the dark for about 40 minutes. Finally, measure the absorbance at 517 nm using a microplate reader and calculate the absorbance by passing through ((reaction tube - bottom tube) / blank) × 100%.

[0096] The raw material for preparing cucumber-derived exosome stock solution is cucumber seeds, and the preparation method is the same as steps 1 to 6 above.

[0097] (4-2) Determination of Total Phenolic Content (TPC)

[0098] Rice callus exosomes were mixed with 1 mL of Milli-Q purified water, vortexed for 1 minute, and centrifuged. The supernatant was collected as the sample solution (diluted 10–20 times to ensure the absorbance falls within the standard curve range). The preparation method for fresh plant (fruit or vegetable) extracts included: 1 gram of plant sample was added to 1.5 mL of purified water and ground. The temperature of the slurry should be controlled below 30°C during grinding to avoid component denaturation. After grinding, the resulting slurry was poured into a 140-mesh nylon filter cloth and squeezed to remove larger fibrous impurities. Subsequently, the filtrate was filtered sequentially through sieves with pore sizes of 100 mesh, 200 mesh, and 400 mesh to further remove residues and obtain a relatively pure filtrate. This filtrate was centrifuged at 12000×g in a high-speed centrifuge. The supernatant was collected after centrifugation; this is the plant extract liquid, used for subsequent analysis or applications (diluted 10–20 times to ensure the absorbance falls within the standard curve range). Gallic acid standard solution was prepared at a concentration of 1 mg / mL and diluted to a gradient of 20–200 μg / mL to establish a standard curve. In the reaction system, 200 μL of 90 mg / mL Na₂CO₃ solution, 100 μL of standard or sample solution, and then 100 μL of 50% Folin-Ciocalteu reagent were added sequentially. The mixture was thoroughly mixed (using pipetting), and incubated in a 45°C water bath for 30 minutes. After the reaction, the sample was centrifuged, and 200 μL of the supernatant was added to a 96-well microplate. The absorbance was measured at 750 nm. A linear regression equation (y = ax + b) was established using the standard, and the total phenol content in the sample was calculated using interpolation. The results are expressed as gallic acid equivalents (GAE), in mgGAE / mL.

[0099] The results are as follows Figure 11As shown in Figure (a), the DPPH free radical scavenging capacity of rice callus exosomes was analyzed, and the antioxidant activity test results showed that the scavenging rate of cucumber exosomes was 26%, while that of rice callus exosomes reached 47%, significantly higher than that of the cucumber exosome group (p<0.05). Most existing plant-derived exosomes (EVs / ELNs) exhibit DPPH free radical scavenging capacity between 30% and 70% in DPPH free radical scavenging experiments. The rice callus exosomes of this invention, with a DPPH scavenging rate of approximately 47%, have demonstrated high free radical scavenging capacity and at least possess basic antioxidant function. (b) Analysis of total phenol content (TPC) in rice callus exosomes. The results showed that the total phenol concentration in rice callus exosomes was as high as 14.25 mg GAE / mL, which was significantly higher than that in cucumber exosomes (5.18 mg GAE / mL) and fresh plant extracts in this experiment (approximately 1.5–8.5 mg GAE / mL).

[0100] Further comparison with other plant exosome literature also showed a significant advantage: 1) Ma, L., Ye, Z., Guo, D., Nie, C., & Zhou, Z. (2024). Citri reticulate pericarpium -derivedextracellular vesicles exert antioxidant and anti-inflammatory properties and enhance the bioactivity of nobiletin by forming EVs-nobnanoparticles. Frontiers in Cell and Developmental Biology, 12 Article 1509123 (https: / / doi.org / 10.3389 / fcell.2024.1509123) reported that the total phenolic content of exosomes from citrus peel was 0.173 ± 0.02 mg GAE / mL. (Danh, J., Canup, B., Najjar, R., Meister, M., Laroui, H., & Feresin, R. (2021). Characterization and uptake of strawberry-derived exosome-like nanovesicles by human aortic endothelial cells.) Current Developments in Nutrition, 5The strawberry exosomes reported by nzab0310 (https: / / doi.org / 10.1093 / cdn / nzab0310) were 158.9 ± 22.6 μmol GAE / L (approximately 0.27 mg GAE / mL), while Li, S., Ye, Z., Zhao, L., Yao, Y., & Zhou, Z. (2023). Evaluation of antioxidant activity and drug delivery potential of cell-derived extracellular vesicles from Citrus reticulata blanco cv. 'Dahongpao'. Antioxidants, 12 (9), Article 1706. (https: / / doi.org / 10.3390 / antiox12091706) The content of exosomes from red oranges was measured to be 0.755±0.05 mg GAE / mL. The above values ​​are all significantly lower than the total phenol content of rice exosomes measured in this invention.

[0101] In conclusion, the enhanced capacity of rice callus exosomes to accumulate phenolic metabolites may be one of the key factors contributing to their superior antioxidant properties.

[0102] (5) Effects of exosomes on cell proliferation

[0103] To evaluate the effect of rice callus exosomes on cell proliferation, a dose-response experiment was conducted in human skin keratinocytes (HaCaT) using a cell counting kit (CCK-8). The experimental method is as follows:

[0104] (5-1) Cell Culture Methods

[0105] Human skin keratinocytes (HaCaT) were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% antibiotics, respectively, at 37°C and 5% CO2.

[0106] (5-2) Cell proliferation analysis methods

[0107] In this embodiment, HaCaT human skin keratinocytes were prepared at a density of 5 × 10⁶ cells per well. 3 Cells were seeded at a density of 1000 cells / well in 96-well plates and cultured at 37°C for 24 hours to promote cell attachment. After attachment, cells were seeded at different concentrations (0, 6.25 × 10⁻⁶ cells / well). 8 1.25×10 9 2.5×10 9 5×10 9 1×1010 Cells were treated with rice callus exosomes (OsEVs) in PBS solution and vitamin C (0.25 mg / mL) in PBS solution for 24 hours. After culture, 10 μL of CCK-8 reagent was added to each well and reacted at 37°C for 1 hour. The absorbance was measured at 450 nm using a microplate reader to assess cell proliferation.

[0108] (5-3) Experimental methods for cell wound healing

[0109] HaCaT keratinocytes were seeded in 6-well culture dishes and cultured at 37°C in a 5% CO2 incubator for 24 hours to promote cell attachment. After cell attachment, the insert was gently removed (ensuring no scratching of the culture substrate), forming a standard-width "cell defect area" (i.e., a wound). The cells were then washed twice with phosphate-buffered saline (PBS). Subsequently, the cells were divided into a treatment group and a control group. The treatment group was treated with a 1×10⁻⁶ H⁺ phosphate buffer solution. 10 Rice callus-derived exosomes (OsEVs) in PBS solution were added at a concentration of particles / mL, while the control group did not receive OsEVs. Treatment time was 24 hours for both groups. After treatment, the culture medium was removed, and the cells were washed twice more with PBS. Images of the cell healing area were captured using an optical microscope, and the healing area was quantitatively analyzed using ImageJ software.

[0110] The experimental results in Figure 12 show that (a) rice callus exosomes significantly promoted the proliferation of HaCaT keratinocytes, with a proliferation rate of up to 142.4%, which is 19.0% higher than that of the vitamin C treatment group (approximately 119.6%), demonstrating a significant advantage in in vitro proliferation. In contrast, exosomes from various plant sources have also been reported in the literature to have the potential to promote cell proliferation. For example, the proliferation rate of HaCaT cells treated with ginseng-derived nanoparticles (GDNPs) increased to approximately 120% (Yang, S., Lu, S., Ren, L., Bian, S., Zhao, D., Liu, M., & Wang, J. (2023). Ginseng-derived nanoparticles induce skin cell proliferation and promote wound healing). Journal of Ginseng Research, 47(1), 133–143. (https: / / doi.org / 10.1016 / j.jgr.2022.04.003)); the proliferation rate of exosomes derived from wheatgrass juice is approximately 130% (Şahin, F., Koçak, P., Güneş, MY, Özkan, İ., Yıldırım, E., & Kala, EY (2019). In vitro wound healing activity of wheat-derived nanovesicles. Applied Biochemistry and Biotechnology, 188 , 381–394. (https: / / doi.org / 10.1007 / s12010-018-02944-3)); while grapefruit-derived exosomes increased to approximately 125% (Savcı, Y., Kırbaş, OK, Bozkurt, BT, Abdik, EA, Taşlı, PN, Şahin, F., &Abdik, H. (2021). Grapefruit-derived extracellular vesicles as a promising cell-free therapeutic tool for wound healing. Food & Function, 12 (11), 5144–5156. (https: / / doi.org / 10.1039 / D0FO02953J). Although these exosomes promoted cell activity to some extent, none of them achieved the proliferation rate (142.4%) exhibited by the rice exosomes in this invention.

[0111] Furthermore, the in vitro wound healing experiments shown in Figures (b) and (c) revealed that cells treated with rice callus exosomes exhibited a healing rate of 60.9%, a 38.3% increase compared to the untreated control group. In the literature, exosomes from various plant sources have also demonstrated a promoting effect on HaCaT keratinocyte wound healing: the healing rate in the ginseng-derived nanoparticle (GDNP) treatment group was 60% (Yang, S., Lu, S., Ren, L., Bian, S., Zhao, D., Liu, M., & Wang, J. (2023). Ginseng-derived nanoparticles induce skin cell proliferation and promote wound healing). Journal of Ginseng Research, 47(1), 133–143. (https: / / doi.org / 10.1016 / j.jgr.2022.04.003)); grapefruit-derived exosomes also accounted for 60% (Savcı, Y., Kırbaş, OK, Bozkurt, BT, Abdik, EA, Taşlı, PN, Şahin, F., & Abdik, H. (2021). Grapefruit-derived extracellular vesicles as a promising cell-free therapeutic tool for wound healing. Food & Function, 12 (11), 5144–5156. (https: / / doi.org / 10.1039 / D0FO02953J)); while wheatgrass juice-derived nanovesicles reached 65% (Şahin, F., Koçak, P., Güneş, MY, Özkan, İ., Yıldırım, E., & Kala, EY(2019). In vitro wound wound healing activity of wheat-derived nanovesicles. Applied Biochemistry and Biotechnology, 188 , 381–394. (https: / / doi.org / 10.1007 / s12010-018-02944-3)). The above results indicate that rice callus exosomes, consistent with other plant exosomes reported in existing literature, can significantly promote the migration and regeneration of epidermal cells, demonstrating excellent potential for skin repair and regeneration applications.

[0112] (6) Exosomes can enter human skin keratinocytes via pinocytosis.

[0113] The exosome stock solution was diluted and stained with DIO fluorescent dye, then filtered through a hollow fiber membrane to remove free dye. HaCaT human skin keratinocytes were seeded into 6-well plates, and the seeding density was adjusted after cell counting to achieve a final cell count of 3 × 10⁶ cells per well. 5 Incubate for approximately 24 hours, until cells have fully adhered and grown to cover the bottom of the wells. Wash three times with PBS to remove old culture medium. Add 1×10⁻⁶ cells to each well. 10 Exosomes stained with DIO (particles / mL) were cultured in cells for only 24 hours, followed by three washes with PBS to remove unabsorbed exosomes. The uptake and absorption of vesicles by cells was observed under a fluorescence microscope. Results are as follows: Figure 13As shown, under a fluorescence microscope, the green fluorescence emitted by the dye after excitation with blue light was observed, indicating that the fluorescence signal was concentrated inside the cells, and the exosomes derived from rice tissue culture could be taken up and absorbed by the cells.

[0114] Example 2:

[0115] This invention provides another embodiment for the preparation of exosomes from recombinant type III humanized collagen rice callus, as detailed below:

[0116] (1) The process of exosome preparation is as follows Figure 14 As shown, the specific steps include the following:

[0117] Step 1: Referring to paragraphs

[0036] -

[0047] of CN2023105681380, the recombinant human type III collagen (rhCol3) gene was designed and optimized, synthesized, and inserted into a binary vector that replicates in Escherichia coli and Agrobacterium. The recombinant plasmids containing the rhCol3 gene were transfected into Agrobacterium, and further Agrobacterium infection and transfection of rice callus were performed. The transfected rice callus was inoculated onto a selection medium containing hygromycin B for resistance selection. After successful selection of transgenic callus, it was further amplified and cultured, then transferred to an MS medium suspension culture system. After solid-liquid separation, a suspension culture of recombinant type III humanized collagen rice callus was finally obtained.

[0118] Step 2: The separated recombinant type III humanized collagen rice callus suspension culture is processed according to steps 4 to 7 of Example 1 (1) to obtain exosome stock solution and freeze-dried powder containing recombinant type III humanized collagen.

[0119] (2) Validation of recombinant type III humanized collagen in exosomes

[0120] This experiment used the Western ink dot method with HA-tagged antibodies to detect whether recombinant type III humanized collagen rice callus-derived exosomes contained recombinant type III humanized collagen. The specific procedure was as follows: 1 gram of recombinant type III humanized collagen rice callus suspension culture sample was added to 1.5 ml of purified water and ground. During grinding, the temperature of the slurry should be controlled below 30℃ to avoid component denaturation. After grinding, the filtrate was placed in a high-speed centrifuge and centrifuged at 12000×g for 10 minutes. The supernatant was collected after centrifugation, which is the extract of recombinant type III humanized collagen rice callus suspension culture. Take 20 μL of the extract from the rice callus suspension culture containing recombinant type III humanized collagen and the final obtained exosome stock solution, add 5 μL of 5X sample buffer, mix well, heat at 95℃ for 5 minutes, and then cool. Separate the proteins using 10% SDS-PAGE electrophoresis. Transfer the proteins to a PVDF membrane using conductivity, and perform Western ink dot assay with HA-tagged antibody to detect recombinant type III humanized collagen. The detection results are as follows: Figure 15 As shown, the exosomes obtained in this embodiment contain recombinant type III humanized collagen.

[0121] (3) Recombinant type III humanized collagen derived from rice callus exosomes can enter human skin keratinocytes via pinocytosis.

[0122] 12 μL of DiO fluorescent dye was added to 2000 μL of DiO staining buffer and mixed thoroughly. This mixture was then added to 1000 μL of recombinant type III humanized collagen rice callus-derived exosome sample or rice callus suspension culture extract for fluorescent staining. A blank control group was set up by adding an equal volume of DiO staining solution to 1000 μL of phosphate-buffered saline (PBS) to assess the non-specific background signal of the dye. The solution was incubated at 37°C in the dark for 30 minutes. The solution was filtered through 0.22 μm and 0.1 μm membranes. Subsequent ultrafiltration with 10 kDa was used to remove free DiO dye.

[0123] Take 2% of DiO-labeled exosome stock solution (1×10⁻⁶) 10Rice callus suspension culture extract (protein concentration 0.24 μg / μL) containing the same amount (2%) of recombinant humanized type III collagen was added to the cell culture medium and co-cultured with HaCaT cells for 24 hours. The control group consisted of HaCaT human keratinocytes cultured in 6-well plates with only cell culture medium for approximately 24 hours. The culture medium was replaced before photography. The absorption of DiO2-labeled exosomes by the cells was photographed using a confocal microscope under fluorescence illumination at a wavelength of 480 nm. Results are as follows: Figure 16 As shown, the exosomes exhibited green fluorescence after excitation under a fluorescence microscope, indicating that the fluorescence signal was concentrated within the cells. The recombinant type III humanized collagen rice callus-derived exosomes could be taken up and absorbed by the cells. Collagen plays a vital role in beauty and skincare. It is present in the dermis, accounting for 70% of skin composition, and acts as a natural protective barrier, maintaining skin elasticity and firmness. Collagen also contains a large number of hydrophilic natural moisturizing factors, which can lock in moisture in the stratum corneum, keeping the skin hydrated and supple. Furthermore, the tyrosine residues of collagen can competitively bind to tyrosinase, thereby inhibiting melanin production and promoting brighter, clearer skin. The recombinant type III humanized collagen rice callus-derived exosomes of this invention have multiple effects, including anti-aging, anti-wrinkle, moisturizing, and whitening.

[0124] Example 3:

[0125] Pentapeptides have antioxidant properties, promote collagen synthesis, and can also smooth and firm the skin. Therefore, this invention provides an example of a high-purity OsEVs formulation coated with pentapeptide-48 to investigate the relevant effects of exosomes coated with pentapeptide-48, as follows:

[0126] (1) Pentapeptide-48 was coated with the exosome lyophilized powder obtained in Example 1, and the process is as follows: Figure 17 As shown in (a), the specific steps include the following:

[0127] Pentapeptide-48 or FITC-conjugated pentapeptide-48 was dissolved in ice-cold 15 mM sodium citrate buffer (pH 6.0–6.5) at concentrations of 0.1%–2% (w / v) (0.1%, 0.5%, 1.0%, 1.5%, and 2.0% were tested in this example). After complete dissolution, rice callus exosome lyophilized powder reconstituted at a concentration of 50 mg / mL was added to achieve a final exosome concentration of approximately 1 × 10⁻⁶. 10 -1×1011 The particles / mL were then thoroughly mixed to obtain a uniformly dispersed composite solution. To improve peptide coating efficiency, the mixture was sonicated at 4–8°C for 5 minutes, followed by incubation at 4°C for 30 minutes to promote peptide entry into exosome vesicles. After the coating process, unencapsulated free pentapeptide-48 and pentapeptide-48 encapsulated in extracellular vesicles were separated by ultrafiltration. Since the molecular weight of pentapeptide-48 is less than the 100 kDa ultrafiltration membrane cutoff, while the particle size corresponding to the extracellular vesicle diameter is greater than the 100 kDa ultrafiltration membrane cutoff, no pentapeptide was detected in the flow-through after centrifugation at 4°C, 3000×g for 30 minutes, repeated three times, indicating that free pentapeptide had been effectively removed. Subsequently, pentapeptide-48 was separated and detected by high-performance liquid chromatography (HPLC) at a detection wavelength of 215 nm, utilizing the characteristic absorption of pentapeptide-48 at this wavelength for quantitative analysis. The pentapeptide-48 content in the sample was calculated by comparing it with the peak area corresponding to a known concentration in an established standard curve. The results showed that the number of pentapeptide-48 encapsulated molecules was approximately 7.17 × 10⁻⁶. 6 Molecules / vesicles indicate that a high-purity OsEV formulation coated with pentapeptide-48 has been obtained.

[0128] like Figure 17 As shown in Figure (b), the results for a 2.0% concentration of pentapeptide-48 are presented. Confirmed by a nanoparticle size analyzer (NTA), the reconstituted exosome lyophilized powder from Example 1 exhibits high purity with an average particle size of 112.5 ± 1.2 nm and a Zeta potential of approximately -17 mV. When the pentapeptide was successfully encapsulated in OsEVs, its average particle size increased to 128 ± 7.0 nm, an increase of approximately 14%. This particle size change is presumably related to the peptide molecule's embedding within the exosome structure. The hypothetical results for other pentapeptide-48 concentrations are similar. Figure 17 As shown in (c), the exosomes coated with pentapeptide-48 also exhibit a typical bilayer vesicle structure with a cup-shaped morphology and a particle size between 80 and 130 nm, as observed by transmission electron microscopy (TEM).

[0129] (2) Intracellular delivery efficiency of pentapeptides encapsulated in rice callus exosomes

[0130] To verify the intracellular delivery efficiency of rice callus exosomes coated with pentapeptide, this embodiment uses fluorescein-labeled pentapeptide (Pentapeptide-48-FITC) and encapsulates it in rice callus exosomes to treat HaCaT cells. The preparation method of fluorescein-labeled pentapeptide-48 exosomes is described in (1) of this embodiment.

[0131] HaCaT cells were spaced at 5 × 10⁶ cells per well. 3 Cells were seeded at a density of [number] cells / well in 6-well culture dishes and incubated at 37°C with 5% CO2 for 24 hours. After incubation, the cells underwent the following treatments:

[0132] 1) Fluorescently labeled pentapeptide-48 (FITC), final concentration 20 μg / mL;

[0133] 2) Rice callus exosomes (OsEVs), concentration 1 × 10⁻⁶ 10 particles / mL;

[0134] 3) Rice callus exosomes were coated with pentapeptide-48 (OsEVs+Pentapeptide-48-FITC), and the concentration of rice callus exosomes was 1 × 10⁻⁶. 10 particles / mL, containing 20 μg / mL of fluorescently labeled pentapeptide-48.

[0135] The treatment time for all three cell treatment methods was 24 hours. After treatment, the culture medium was removed, and the cells were washed twice with phosphate-buffered saline (PBS). Fluorescence images were captured using a fluorescence microscope, and the average fluorescence intensity was quantitatively analyzed using ImageJ software. Results are as follows: Figure 18 As shown, compared with the pentapeptide group alone, Pentapeptide-48-FITC encapsulated in rice callus exosomes exhibited the highest fluorescence intensity in cells, indicating that this carrier system effectively improves the intracellular delivery efficiency of peptides. This evidence suggests that rice callus exosomes possess the potential to be natural nanoscale peptide carriers.

[0136] (3) Exosomes of rice callus encapsulated with pentapeptide-48 can effectively enhance the skin's anti-aging and barrier protection functions.

[0137] To further verify the regulatory effect of rice callus exosome-encapsulated pentapeptide-48 on skin-related functional genes, this study used human skin fibroblasts (HSF) to analyze the expression of genes related to aging and skin barrier protection. Three genes related to extracellular matrix (ECM) regeneration were examined: matrix metalloproteinase-3 (MMP-3), tissue inhibitor of matrix metalloproteinases-1 (TIMP-1), and hyaluronidase-1 (Hyal-1), as well as one gene related to skin barrier function, transglutaminase-1 (TGM-1).

[0138] The specific procedure is as follows: Human skin fibroblasts (HSF) were seeded into 6-well cell culture dishes and placed in an incubator containing 5% CO2 at 37°C for 24 hours to promote cell attachment. After attachment, the cells were treated for 24 hours with the following reagents: 1) Phosphate-buffered saline (PBS) as the control group; 2) Pentapeptide-48 at a final concentration of 20 μg / mL; 3) OsEVs-coated pentapeptide-48 combined treatment group, which contained 1 × 10⁶ cells / mL. 10 OsEVs containing particles / mL were coated with 20 μg / mL pentapeptide-48. After treatment, the culture medium was removed, and the cells were washed twice with PBS. Subsequently, total RNA was extracted according to the TRIzol reagent instructions, and the obtained RNA was reverse transcribed using a reverse transcriptase kit to generate complementary DNA (cDNA). Real-time quantitative polymerase chain reaction (RT-qPCR) was used to assess the expression of extracellular matrix (ECM)-related genes, including matrix metalloproteinase-3 (MMP-3), tissue inhibitor of metalloproteinases-1 (TIMP-1), hyaluronidase-1 (Hyaluronidase-1), and transglutaminase-1 (TGM-1). Table 1 lists all primer sequences, and Table 2 shows the PCR reaction system composition. Subsequent gene expression levels were calculated using the ΔCt method, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal reference gene. All experimental data were statistically analyzed using one-way ANOVA, and results are expressed as mean ± standard deviation (mean ± SD). Statistical significance was indicated by p-values, where p < 0.05 was marked with *, p < 0.01 with **, and p < 0.001 with ***. Quantitative image analysis was performed using ImageJ software, while statistical analysis and data presentation were completed using GraphPad Prism 8.0 software.

[0139] Table 1 Primer Sequences

[0140]

[0141] Table 2 Composition of PCR reaction system

[0142]

[0143] Amplification reaction conditions: 95.0℃ for 30s; 95.0℃ for 5s, 60.0℃ for 30s, 40 cycles; 65.0℃ for 5s; 95.0℃ for 5s.

[0144] Amplification results as follows Figure 19 As shown, compared with pentapeptide-48 alone, pentapeptide-48 encapsulated in rice callus exosomes significantly regulated the expression of genes related to extracellular matrix regeneration and skin barrier function. Specifically, MMP-3 and Hyal-1 decreased by approximately 10.4% and 8.6%, respectively, while TIMP-1 and TGM-1 increased by approximately 18.9% and 7.0%, respectively. MMP-3, TIMP-1, and Hyal-1 are mainly involved in the extracellular matrix regeneration process, while TGM-1 is a key enzyme in maintaining skin barrier integrity. These results confirm that pentapeptide-48 delivery via rice callus exosomes can enhance its anti-aging and skin barrier support effects at the molecular level, demonstrating the potential of rice callus exosomes as a natural and highly efficient nanocarrier for active substances. This research indicates that pentapeptide-48 encapsulated in rice callus exosomes has the potential for developing innovative and effective cosmetic applications.

[0145] (4) Particle size analysis of other active ingredients encapsulated in exosomes obtained in Example 1

[0146] In current reports on the engineered encapsulation of exosomes or vesicles (see references [1]-[3]), particle size analysis (NTA) can be used to quickly assess whether active ingredients are encapsulated in exosomes or vesicles, and the average particle size of exosomes or vesicles containing encapsulated ingredients will increase. To evaluate the application potential of engineered encapsulation of exosomes derived from rice tissue culture, this experiment further tested the encapsulation results of common active ingredients: ergothioneine, collagen, and arbutin.

[0147] References:

[0148] [1] A doxorubicin delivery platform using engineered natural membranevesicle exosomes for targeted tumor therapy (Tian, ​​Y. et al. (2014). NTA showed that the average particle size of unencapsulated Exo was 100 nm; after encapsulation with Dox, the average particle size of Exo-Dox increased to 150 nm.

[0149] [2] Mesenchymal Stem Cell Derived Exosomes as Nanodrug Carrier of Doxorubicin for Targeted Osteosarcoma Therapy via SDF1-CXCR4 Axis. (Wei, Y. et al. (2022). NTA showed that the average particle size of uncoated Exosomes was 140 nm; after coating with Dox, the average particle size of Exo-Dox increased to 178 nm.

[0150] [3] Han R. et al., Folic acid-modified ginger-derived extracellular vesicles for targeted treatment of rheumatoid arthritis, J. Nanobiotechnol.23, 41 (2025). The peak size of the original GDEVs was 241 nm, and the peak size of FA-GDEVs increased to 268 nm after FA modification.

[0151] The specific procedure is as follows: Ergothioneine, collagen, and arbutin are dissolved in ice-cold 15 mM sodium citrate buffer (pH 6.0–6.5) at proportions of 1-8% (1%, 2%, 4%, 8%), 0.1-2% (0.1%, 0.5%, 1.0%, 2.0%), and 1-8% (1%, 2%, 4%, 8%) (w / v). After complete dissolution, rice callus exosome lyophilized powder reconstituted at a concentration of 50 mg / mL is added to achieve a final exosome concentration of approximately 1 × 10⁻⁶. 10 -1×10 11 The particles were concentrated at a concentration of [particles / mL], and then thoroughly mixed to obtain a uniformly dispersed composite solution. The solution was then sonicated at 4–8°C for 5 minutes, followed by incubation at 4°C for 30 minutes to promote peptide entry into exosome vesicles. After the coating process, uncoated free peptides were removed by ultrafiltration to obtain a high-purity OsEV formulation coated with ergothioneine, collagen, and arbutin. The nanovesicle particle size was analyzed using NTA, and the results are as follows: Figure 20 As shown in the figure, the results for ergothioneine 2%, collagen 1.0%, and arbutin 2% are presented. The exosomes coated with the active ingredients all show an increased particle size, while the results for other contents are largely similar. Therefore, it can be preliminarily inferred that exosomes derived from rice tissue culture can encapsulate a variety of active ingredients and have the potential for engineered encapsulation applications.

[0152] Furthermore, the present invention has now entered the pilot-scale preparation stage for the preparation of various exosome stock solutions and freeze-dried powders. It is planned to use the existing 250-liter fermenter to carry out large-scale fermentation production, and combine it with an industrial-grade centrifuge system and hollow fiber membrane modules with a filtration area of ​​490–790 square centimeters for the purification and concentration of rice-derived exosomes.

[0153] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for preparing exosome stock solution derived from rice callus, characterized in that, Includes the following steps: Step 1: Obtain rice callus tissue; Step 2 involves sequentially performing suspension culture and bioreactor scale-up culture on the rice callus tissue obtained in Step 1. This process includes: inoculating the rice callus tissue obtained in Step 1 into liquid culture medium and suspending it at 110-130 rpm and 25-28℃ for 14-30 days; after suspension culture, inoculating the callus tissue into a bioreactor and adding liquid culture medium, and suspending it at 0.25-0.5 VVM at 25-28℃ for 7-14 days. Step 3: Add water to the rice callus culture obtained in Step 2, grind and break it up, filter it to obtain cell slurry; Step 4: Perform staged centrifugation on the cell slurry obtained in Step 3: first centrifuge at 2000g~4000g for 20~40 minutes, then centrifuge at 5000g~7000g for 20~40 minutes, and finally centrifuge at 12000g~18000g for 50~70 minutes to obtain supernatant; filter the supernatant sequentially through filter membranes with pore sizes of 0.45, 0.22, and 0.1 μm, and then introduce the supernatant sequentially into hollow fiber membrane columns with specifications of 500-1000 kD and 50-100 kD for ultrafiltration treatment, and obtain the exosome stock solution by using the tangential flow filtration principle.

2. The preparation method according to claim 1, characterized in that, Step 1 includes: removing the shell from rice seeds and retaining the embryo, and then inducing the culture with 2,4-dichlorophenoxyacetic acid to obtain rice callus tissue.

3. The preparation method according to claim 2, characterized in that, The 2,4-dichlorophenoxyacetic acid was added as follows: under aseptic conditions, the embryo was inoculated into MS medium containing 1-3 mg / L 2,4-dichlorophenoxyacetic acid and 25-30 g / L sucrose.

4. The preparation method according to claim 2 or 3, characterized in that, The induction culture conditions include: culture at 25±2°C in the dark for 1 to 2 weeks.

5. The preparation method according to claim 1, characterized in that, In step 2, the rice callus obtained in step 1 is subjected to suspension culture and bioreactor scale-up culture in sequence. This process includes: inoculating the rice callus obtained in step 1 into liquid culture medium and suspending it at 110-130 rpm, 25-28℃ in the dark or at a light intensity of 10-100 μmol photons·m⁻²·s⁻¹ for 14-30 days; after the suspension culture is completed, the callus is inoculated into a bioreactor and liquid culture medium is added, and suspended it at an atmospheric air intake rate of 0.25-0.5 VVM, 25-28℃ in the dark or at a light intensity of 10-100 μmol photons·m⁻²·s⁻¹ for 7-14 days.

6. The preparation method according to claim 1 or 5, characterized in that, The inoculum solid-liquid ratio for suspension culture and bioreactor scale-up culture is 1:50~100.

7. The preparation method according to claim 1 or 5, characterized in that, The liquid culture medium, according to its final concentration, comprises: 1.5–2.5 mg / L 2,4-dichlorophenoxyacetic acid; and 25–35 g / L sucrose; the basal medium is MS liquid medium; and the pH is adjusted to 5.7–5.

8.

8. The preparation method according to claim 1, characterized in that, In step 3, the rice callus culture obtained in step 2 is ground and crushed with water, then coarsely filtered with nylon filter cloth and the filtrate is retained. The filtrate is then passed through 100-mesh, 200-mesh and 400-mesh sieves in sequence and the filtrate is retained, which is the cell sap.

9. The exosome stock solution derived from rice callus obtained by the preparation method according to any one of claims 1 to 8.

10. A method for preparing lyophilized exosome powder derived from rice callus, characterized in that, The exosome stock solution obtained by the preparation method according to any one of claims 1 to 8 is mixed with mannitol and water and then freeze-dried.

11. The preparation method according to claim 10, characterized in that, The mass percentages of exosome stock solution, mannitol, and water were 60-80%, 3-5%, and 24-28%, respectively; and / or the control parameters for freeze-drying were as follows: pre-freezing the exosome stock solution at -20±1℃ for 550-650 min; then drying sequentially at -20±1℃ for 150-250 min, -15±1℃ for 250-350 min, and 0±1℃ for 250-350 min; finally drying sequentially at 10±1℃ for 250-350 min, 25±1℃ for 150-250 min, and 36±1℃ for 200-280 min.

12. Exosome lyophilized powder obtained by the preparation method according to claim 10 or 11.

13. A delivery carrier encapsulating an active ingredient, characterized in that, The delivery carrier includes: the exosome stock solution of claim 9 or the exosome lyophilized powder of claim 12.

14. A topical skin composition, characterized in that, The composition comprises: the exosome stock solution of claim 9, the exosome lyophilized powder of claim 12, or the delivery carrier of claim 13.

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