Maize germ exosome extraction method, exosome preparation and application

Abstract

The invention provides a corn germ exosome extraction method, an exosome preparation and application, and belongs to the technical field of biological medicine. According to the invention, the high-purity corn germ source exosome oral preparation loaded with a medicine for promoting the liver metabolism function is prepared through a pH-triggered in-situ loading and size selective purification technology; the problems of efficiency loss and structural damage caused by drug loading after exosome extraction, purity bottleneck that functionalized exosome and impurities are difficult to separate, product loss caused by a multi-step series process and incapability of realizing large-scale production are solved. The exosome preparation disclosed by the invention is high in purity and uniformity, high in product stability and bioavailability, relatively high in level and good in biocompatibility, and can be applied to the fields of medicines, health care products and high-end functional cosmetics.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for extracting exosomes from corn germ, exosome preparations, and their applications. Background Technology

[0002] Plant exosomes are extracellular vesicles, approximately 30-150 nm in diameter, secreted by plant cells and possessing a phospholipid bilayer structure. They carry mother cell-specific proteins, lipids, mRNA, and miRNA. After release into the extracellular space, exosomes and their carried substances can be received by target cells, participating in intercellular transport and information exchange, thereby regulating physiological and pathological activities and demonstrating great potential in intercellular communication and drug delivery. Compared to traditional artificial nanocarriers, exosomes possess advantages such as natural biocompatibility, low immunogenicity, and high delivery efficiency, thus showing promising prospects in disease treatment.

[0003] Liver diseases are among the most threatening to human health, such as fatty liver, liver fibrosis, and cirrhosis. Among them, metabolic-related fatty liver disease has gradually become the most common chronic liver disease in the world. Gut microbiota dysbiosis or intestinal barrier damage can lead to changes in the levels of microbial metabolites and endotoxins entering the liver via the portal vein, resulting in steatosis and liver inflammation. However, research has found that exosomes from some plants can utilize multi-target therapy to simultaneously repair the intestinal barrier and improve the composition of gut microbiota, thereby comprehensively intervening in the pathological process of metabolic-related fatty liver disease. Li P et al., in *NanoResearch*, Vol. 18, No. 12, 2025, proposed a method to extract exosome nanovesicles from honeysuckle to improve metabolism-related fatty liver disease by regulating the gut microbiota and its metabolites. The study systematically regulates the gut-liver through multiple targets, aiming to optimize liver metabolism by modulating the intestinal barrier and microbiota. However, this extraction method, involving high-speed centrifugation followed by tangential flow filtration, can only extract substances based on size and cannot effectively separate extracellular particles similar in size to exosomes. Furthermore, during tangential flow filtration, the membrane structure may deform, rupture, or fuse due to transmembrane pressure difference and shear force, resulting in impaired vesicle integrity and affecting its bioactivity and stability. While a 750 kDa molecular weight cutoff membrane can remove most soluble macromolecules, small molecule metabolites, free nucleic acids, or lipids may still remain in the final product. This insufficiently purified exosome extract may interfere with subsequent functional experiments.

[0004] To accurately determine experimental results, developing exosomes into practical drug carriers requires the initial collection of purified exosomes. Currently, exosome extraction and collection mainly rely on methods such as ultracentrifugation, size exclusion chromatography, and polymer precipitation. CN114699464B discloses a method for preparing tea exosomes and their application in drugs for treating liver diseases. Since tea exosomes can inhibit the expression of fibrosis marker genes at the gene and protein levels, they can effectively prevent and treat liver diseases including cirrhosis, hepatitis, liver fibrosis, and fatty liver. In this invention, freshly picked tea leaves are cleaned with phosphate buffer and ground into a uniform slurry. The slurry is then processed by multiple differential centrifugations and filtered through a 0.2-0.4 μm filter membrane to obtain tea exosomes. Although this preparation method can specifically prepare products of a certain size, the required centrifugal force ranges from 100g to 200,000g, and the time required is also relatively long. Not only is the process cumbersome, but multi-stage centrifugation can also easily cause a certain degree of inactivation of exosomes, resulting in a reduction in the efficacy of the obtained exosomes and their formulations.

[0005] In summary, plant exosomes can be used to effectively prevent and treat liver diseases. Corn germ is the most nutrient-dense part of corn kernels, and corn germ exosomes contain liver-beneficial components such as glutathione, corn peptides, vitamin E, and unsaturated fatty acids. Therefore, corn germ exosomes can be extracted from corn germ and prepared into exosome preparations to improve liver function. However, current methods for efficient extraction and purification of these plant exosomes are still insufficient. There is an urgent need for an exosome extraction method that can achieve high-yield release, drug loading, and high-purity separation of corn germ exosomes in a one-step process, and prepare them into exosome preparations with defined functions, high purity, high bioavailability, and safety for oral administration to meet the application needs of the pharmaceutical, health care, and cosmetic fields. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a method for extracting exosomes from corn germ, exosome preparations, and their applications. This invention utilizes pH-triggered in-situ loading and size-selective purification technology to prepare high-purity oral formulations of corn germ-derived exosomes loaded with specific active molecules. This solves the problems of efficiency loss and structural damage caused by drug loading after exosome extraction, the purity bottleneck due to the difficulty in separating functionalized exosomes from impurities, and product loss and scalability issues resulting from multi-step cascade processes.

[0007] This invention discloses a method for extracting exosomes from maize germ, such as... Figure 1 As shown, the specific technical solution is as follows: Step 1: Using sodium alginate and carboxymethyl chitosan as raw materials, composite microspheres are prepared by ion crosslinking-gelation method. Salicylic acid is covalently modified on the surface of the microspheres as a plant elicitor. At the same time, a membrane is embedded inside the microspheres to anchor the drug, resulting in functional microspheres with pH-responsive properties.

[0008] Step 2: Add corn germ tissue to the culture medium, then add functional microspheres to the culture container, place the system under constant temperature and gently shake to culture, and continuously monitor the pH change in the system. When the pH reaches a certain acidity, stop the culture to obtain exosomes loaded with specific drugs dispersed in the culture medium.

[0009] Step 3: Centrifuge the culture medium at low speed and collect the supernatant. Then add the glycosylated reversible polymer to the supernatant, adjust the pH of the system to weakly alkaline and stir and incubate until visible flocculent matter appears in the system, thus obtaining exosomes encapsulated by the polymer.

[0010] Step 4: Resuspend the obtained precipitate, then centrifuge the system at low speed, collect the precipitate and add acidic buffer solution, gently shake to dissolve the precipitate, then centrifuge at medium speed, collect the supernatant and concentrate it using an ultrafiltration membrane to obtain pure corn germ exosomes loaded with the specific drug, which are then stored at low temperature for later use.

[0011] This invention also discloses a corn germ exosome preparation, the specific technical solution of which is as follows: The purified corn germ exosomes loaded with specific drugs are resuspended in a solution containing lyophilization protectants such as trehalose, and then pre-frozen and freeze-dried to produce a dry and loose exosome lyophilized powder. The powder is then sieved and prepared into tablets or capsules, which are corn germ exosome preparations.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. By utilizing the metabolic signals of corn germ cells to trigger the entire process, the release, loading, and purification of exosomes can be completed continuously in the same container. This reduces cumbersome operations such as ultra-high-speed centrifugation and transfer, simplifies the centrifugation process, reduces manual intervention and operation time, significantly reduces exosome loss and contamination risk during production, and makes the process more consistent between batches, which can significantly improve the feasibility of large-scale production.

[0013] 2. Specific drugs can be inserted into the membrane in situ upon exosome formation, resulting in extremely high loading efficiency. This allows the vast majority of active molecules to successfully enter the exosome carrier. In contrast, traditional extraction followed by drug loading methods often require long incubation or harsh treatment, leading to low loading rates and damage to the carrier. The loading process of this invention causes zero damage to exosomes and no additional free drugs are present, eliminating the need for subsequent steps to separate free drugs and greatly improving overall efficiency and effective drug loading.

[0014] 3. By using glycosyl reversible polymers to specifically and reversibly crosslink the target exosomes, one-step purification of drug-loaded exosomes is achieved. After the target exosomes are polymerized into large, sedimentable particles, they can be completely separated from small-particle impurities by low-speed centrifugation, resulting in a high-purity exosome formulation free of impurities.

[0015] 4. Since the loading and purification processes are carried out under mild conditions, without the need for organic solvents, ultrasound, or vigorous centrifugation, the bioactivity of exosomes is maintained. At the same time, the active drug molecules loaded are encapsulated in the exosomes or anchored on the membrane, which avoids the adverse effects of the digestive tract environment and improves the bioavailability of the drug delivered to the target tissue via the oral route. Attached Figure Description

[0016] Figure 1 This is a flowchart illustrating the method for extracting corn germ exosomes and the preparation of its formulations according to the present invention. Figure 2 Here is a cryo-electron micrograph of exosomes obtained in Example 1 of this invention; Figure 3 This is a schematic diagram illustrating the cell survival rate of the exosome preparation of the present invention after acting on hepatocytes in vitro; Figure 4 This is a schematic diagram illustrating the ADH enzyme activity of the exosome preparation of the present invention after acting on hepatocytes in vitro; Figure 5 This is a schematic diagram showing the ALT enzyme activity in mice after the exosome preparation of the present invention was fed to them. Figure 6 This is a schematic diagram showing the ADH enzyme activity in mice after the exosome preparation of the present invention was fed to them; Figure 7 This is a schematic diagram showing the acetaldehyde concentration in mouse serum after feeding mice with the exosome preparation of the present invention. Detailed Implementation

[0017] The following embodiments further explain and illustrate the technical solutions of the present invention. It should be specifically noted that each specific embodiment is a concretization and explanation of the technical solution and should not be considered as a limitation on the scope of protection of the present invention. Those skilled in the art still have the right to modify the technical solutions of these embodiments and make equivalent substitutions for some or all of the technical features, and these modifications or substitutions do not change the essence of the corresponding technical solutions, nor do they cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions described in the present invention.

[0018] This invention proposes a method for extracting exosomes from maize germ, such as... Figure 1 As shown, the specific technical solution is as follows: 1. Preparation of pH-responsive microsphere inducers Composite microspheres were prepared using sodium alginate and carboxymethyl chitosan as raw materials via an ion crosslinking-gelation method. Salicylic acid was then modified onto the surface of these microspheres, and a drug-anchoring membrane was embedded within them, resulting in pH-responsive microspheres. Sodium alginate, as an anionic polysaccharide, forms highly stereoselective coordination bonds with guluronic acid structural units in the presence of calcium ions, constructing a three-dimensional network framework centered on ion crosslinking points. Carboxymethyl chitosan, as a cationic modified polysaccharide, has carboxymethyl groups introduced into its molecular chain. These groups not only enhance mechanical strength by forming polyelectrolyte complexes with sodium alginate through electrostatic interactions, but their abundant carboxyl functional groups also serve as the structural basis for pH sensitivity. When the ambient pH is higher than the dissociation constant pKa of chitosan (approximately 6.3–6.5), the carboxyl groups are in a deprotonated COO state. - In the initial state, the electrostatic repulsion between adjacent ionized groups causes the polymer chains to extend, increasing the network porosity and maintaining a swollen state. When the hydrogen ion concentration in the environment increases, causing the pH to drop below pKa, the carboxyl groups ionize to COOH, the electrostatic repulsion weakens sharply, and calcium ions dissociate from the coordination sites of the guluronic acid units. This dual destabilization effect causes the polymer chains to shrink from extended worm-like chains into random coils, ultimately leading to the irreversible disintegration of the entire gel network's macroscopic structure. Salicylic acid, as an exogenous exciter, undergoes an amidation reaction between its carboxyl groups and free amino groups on the chitosan chains on the microsphere surface, catalyzed by carbodiimide and N-hydroxysuccinimide, forming covalent bonds. This ensures that the release of salicylic acid in the physiological environment follows surface erosion kinetics, rather than simple diffusion control, thus achieving long-term regulation of its biological activity. Amphiphilic conjugates formed by hydrophobic drug molecules linked to cholesterol via ester bonds are formed during the sol-gel transition of microspheres. The hydrophobic ends of the conjugates are embedded in the hydrophobic microdomains of the polymer through van der Waals forces. The drug conjugates are embedded in the hydrophobic microdomains of the gel through hydrophobic interactions, thus stably retaining the drug conjugates within the network.

[0019] 2. Inducing maize germ cells to release exosomes and load drugs in situ. The microspheres were co-cultured with a corn germ cell system. The pH drop caused by cell metabolism triggered microsphere disintegration, releasing membrane-anchored drugs that were in situ inserted into the membranes of newly formed exosomes for loading. Salicylic acid, as an exogenous elicitor, activated the salicylic acid signaling pathway within corn germ cells, systematically upregulating the activity of exosome biosynthesis and secretion pathways, leading to increased exosome production. Simultaneously, active cell metabolism, especially enhanced secretory activity, excreted acidic metabolites such as lactic acid, causing continuous and natural acidification of the extracellular microenvironment. This endogenous pH drop signal became the key switch triggering the disintegration of the smart microspheres. However, if membrane-anchored drugs are directly added to the culture system, the drug molecules will randomly diffuse throughout the culture environment. This not only fails to precisely target newly formed exosomes but may also aggregate or insert into the cell membrane at non-target locations, resulting in drug waste and unintended cell disturbances. Therefore, pH-responsive microspheres are needed as intelligent carriers. A pH change triggers the microspheres to disintegrate, releasing drug-cholesterol conjugates that precisely diffuse to the active secretion hotspots of exosomes. The lipid membranes of newly formed exosomes exhibit extremely high fluidity and dynamic instability. Drug-cholesterol conjugates, utilizing the strong hydrophobic interaction and structural compatibility between their hydrophobic cholesterol anchors and phospholipid bilayers, can spontaneously and rapidly insert into the lipid bilayers of these newly formed membranes. Therefore, drug loading can be completed synchronously with the natural formation of exosomes, achieving in-situ integration of secretion-synchronized loading.

[0020] 3. Signal-triggered selective capture and purification of drug-loaded exosomes After secretion, cell debris was removed by centrifugation. A glycosyl recognition polymer containing phenylboronic acid groups was added to the supernatant, allowing it to crosslink with the drug-loaded exosomes under pH recovery conditions to form a complex, which was then captured by low-speed centrifugation. As cell secretion slowed and the culture medium pH naturally recovered to neutral or weakly alkaline, the phenylboronic acid groups transformed from an electrically neutral planar structure to a negatively charged tetrahedral borate form. This activated form can specifically react with sugar structures where adjacent carbon atoms all have hydroxyl groups, forming cyclic borate ester bonds. Glycoproteins and glycolipids enriched on the exosome membrane surface provide numerous cis-diol sites at the ends of their glycan chains. The glycosyl reversible polymer used is a multivalent polymer, meaning that each dextran molecule is densely modified with multiple phenylboronic acid groups. When one end of a polymer molecule binds to a sugar site on the exosome surface, other unbound phenylboronic acid groups on its molecular chain are more likely to bind to other sugar sites on the same exosome surface or to sugar sites on a neighboring exosome surface due to spatial proximity. This multivalent effect generates a powerful synergistic binding force, far stronger than that of individual groups. This allows the long polymer chains to covalently link and bind numerous individual exosomes together, forming a loose, network-like aggregate several micrometers in size. When this aggregate interacts with water molecules, its hydration diameter further increases, thus scaling the target substance from the nanometer scale to the micrometer scale. This means that under the same centrifugal force, its sedimentation rate can be increased by hundreds to thousands of times. This allows nano-exosomes, which previously required long periods of ultra-high-speed centrifugation to barely settle, to now settle efficiently and quantitatively with only short periods of ordinary low-speed centrifugation. Meanwhile, free proteins, small-molecule drugs, and other nanoscale impurities in the solution remain dispersed because they are not cross-linked, allowing for complete separation.

[0021] 4. Separation, collection, and release of exosome products The captured complex was dissociated using an acidic buffer to release pure drug-loaded exosomes. After centrifugation and concentration, the mixture was mixed with a lyophilization protectant and freeze-dried to obtain a solid powder formulation, which was then prepared into tablets or capsules. Utilizing the chemical reversibility of borate ester bonds, when the captured exosome aggregates were placed in a pre-cooled acidic buffer, the low pH environment caused the phenylboronic acid groups to reprotonate, changing from tetrahedral borate groups back to a planar structure. The covalent ester bonds formed with the glycosyl groups subsequently underwent acidic hydrolysis and broke. This chemical reaction was mild and rapid, completely dissociating the entire cross-linked network within minutes, releasing structurally intact, unmodified drug-loaded exosomes. Subsequently, the exosomes were concentrated by medium-speed centrifugation and ultrafiltration. Taking advantage of the significant difference in physical size and mass between the exosomes and the dissociated phenylboronicized polymer fragments in a dissolved or minimally aggregated state, acid-insoluble fragments, incompletely dissociated larger aggregates, and dissolved polymer fragments were removed from the system to obtain a pure exosome suspension. Subsequently, a high concentration of lyophilization protectant was added to the purified exosome suspension. During freezing, the protectant and water together formed a glassy matrix. In the subsequent vacuum sublimation drying stage, the water was removed, while the protectant molecules, through multiple hydroxyl groups on their surface, formed a dense hydrogen bond network with the phospholipid head groups and polar residues of membrane proteins, thus replacing the original role of water molecules. This maintained the natural three-dimensional conformation of the biomolecules and the integrity of the lipid membrane even in the dried state. The resulting porous lyophilized powder firmly locked the exosomes in a rigid glassy matrix, reducing their chemical degradation and physical aggregation rates at room temperature to extremely low levels, achieving long-term stable storage.

[0022] The following are some specific embodiments of the present invention, and Table 1 shows the raw material information used in the embodiments.

[0023] Table 1 Raw Material Information Table

[0024] Example 1 S1: Dissolve 0.97g cholesterol and 0.5g succinic anhydride in anhydrous pyridine and react at 60℃ for 6h under nitrogen protection. Remove the solvent under reduced pressure to obtain cholesterol succinate monoester. Weigh 1.0g cholesterol succinate monoester and 1.29g silymarin and dissolve in 25mL N,N-dimethylformamide. Add 1.55g DCC and 31mg DMAP. Stir at 40℃ in the dark for 18h under nitrogen protection. Filter and collect the filtrate. Concentrate the filtrate under reduced pressure to obtain a white solid. Then weigh 1.5g sodium alginate and dissolve in 100mL deionized water. Weigh 0.75g carboxymethyl chitosan and dissolve in 100mL 1% acetic acid solution. Mix the two solutions and add 0.1g of the above white solid. Sonicate and disperse for 30min. Then add 500mL of 2% acetic acid solution. A w / v calcium chloride solution was stirred for 30 min to allow the ions in the system to cross-link and solidify into spherical gel particles. The gel particles were filtered, washed with deionized water, and redispersed in deionized water. 50 mg of salicylic acid, 100 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 60 mg of N-hydroxysuccinimide were added, and the mixture was stirred at 25 °C for 12 h to allow the coupling reaction to proceed. The microspheres were then filtered out and washed three times with deionized water to obtain pH-responsive microspheres coated with a specific drug.

[0025] S2: Place 10g of activated corn germ tissue in 100mL of B5 medium and supplement it with 2g of sucrose, 0.1mg of 2,4-dichlorophenoxyacetic acid and 0.02mg of 6-benzylaminopurine. Then add 200mg of pH-responsive microspheres coated with specific drugs. Incubate at 90rpm in a constant temperature shaker at 26℃ for 6h. Continuously monitor the pH of the system with a pH meter. Stop shaking when the pH drops to 6.5.

[0026] S3: After culture, the system was centrifuged at 800g for 8 min, and the supernatant was collected. The pH of the supernatant was then adjusted to 7.5 with 1 mol / L Tris buffer. Dextran and 4-hydroxyphenylboronic acid were coupled at pH 6.5 to obtain phenylboronic dextran with a molecular weight of 80 kDa. 20 mL of 10 mg / mL phenylboronic dextran solution was added, and the mixture was stirred and incubated at 25 °C for 30 min. The mixture was then centrifuged at 4000g for 15 min, and the precipitate was collected to obtain the exosome-polymer composite precipitate.

[0027] S4: Resuspend the precipitate in 10 mL of pre-cooled citrate buffer (pH=5.0), let it stand at 4 °C for 30 min, then centrifuge the suspension at 12000 g for 20 min, collect the supernatant and transfer it to an ultrafiltration centrifuge tube with a molecular weight cutoff of 200 kDa. After centrifugation and concentration, pure concentrated exosomes loaded with specific drugs are obtained. Add 2 mL of phosphate buffer containing 5% trehalose to the exosome concentrate, freeze at -80 °C for 4 h, transfer to a refrigerated dryer and freeze-dry at -50 °C and 0.05 mbar for 48 h to obtain dried exosome powder.

[0028] Take 1 mg of exosome powder obtained from S4, resuspend it in 100 mL of deionized water, adjust the protein concentration to 0.5 mg / mL, and drop 3 μL of the reconstituted exosome suspension onto the carbon film surface of the discharge-treated grid for cryogenic sample preparation at 4 °C, 100% humidity, adsorption time 3.5 s, and adsorption force 1. After blotting off excess liquid with filter paper, quickly immerse the grid in liquid alkanes at -183 °C for vitrification. Subsequently, perform low-dose imaging using cryo-transmission electron microscopy at an accelerating voltage of 300 kV and a slit width of 20 eV, with a pixel size of 1.1 Å / pixel, an underfocus of -1.5 to -2.5 Sch, and a cumulative electron dose of -50 eV. - / Å 2 The result is as follows Figure 2 As shown, the obtained exosomes have a clear and complete phospholipid bilayer structure, indicating that the exosome membrane was not damaged after pH change treatment, and there were no large amounts of flocculent or network impurities, indicating that the purification and separation steps S3 and S4 successfully removed free proteins and polymer residues.

[0029] Example 2 The preparation method according to Example 1 differs in that: S1: Sodium alginate addition is 1.0g, carboxymethyl chitosan addition is 0.5g, calcium chloride solution concentration is 1.5%w / v, stirring time for ion crosslinking and curing is 20min, coupling reaction time is 6h, succinic anhydride is replaced with glutaric anhydride, and salicylic acid is replaced with chitosan oligosaccharide. S2: The amount of pH-responsive microspheres added was 100 mg, the shaking incubation time was 4 h, and the shaking speed was 70 rpm. S3: The first centrifugation force is 500g, the phenylboronic acid dextran solution is replaced with phenylboronic acid hyaluronic acid solution, the amount added is 10mL, the pH of the supernatant is adjusted to 7.2, the incubation time is 20min, and the second centrifugation force is 3000g. S4: The centrifugal force during suspension centrifugation is 10000g, the trehalose concentration is 3% w / v, the freeze-drying temperature is -60℃, and the remaining steps are the same.

[0030] Example 3 The preparation method according to Example 1 differs in that: S1: Sodium alginate addition amount is 2.0g, carboxymethyl chitosan addition amount is 1.2g, calcium chloride solution concentration is 3.0%w / v, ion crosslinking curing stirring time is 60min, coupling reaction time is 18h, succinic anhydride is replaced with maleic anhydride, and salicylic acid is replaced with oligosaccharide galacturonic acid. S2: The amount of pH-responsive microspheres added was 300 mg, the shaking incubation time was 8 h, and the shaking speed was 110 rpm; S3: The first centrifugation force is 1200g. The benzyl borate dextran solution is replaced with benzyl borate chitosan solution, and the amount added is 30mL. The pH of the supernatant is adjusted to 7.8, and the incubation time is 40min. The second centrifugation force is 5000g. S4: The centrifugal force during suspension centrifugation is 15000g, the trehalose concentration is 8% w / v, the freeze-drying temperature is -45℃, and the remaining steps are the same.

[0031] Example 4 The preparation method according to Example 1 differs in that: S1: Sodium alginate addition amount is 1.8g, carboxymethyl chitosan addition amount is 0.8g, calcium chloride solution concentration is 2.5%w / v, stirring time for ion crosslinking curing is 40min, and coupling reaction time is 15h; S2: The amount of pH-responsive microspheres added was 250 mg, the shaking incubation time was 5 h, and the shaking speed was 80 rpm. S3: The first centrifugation force is 1000g. The phenylboronic acid dextran solution is replaced with phenylboronic acid branched starch solution, and the amount added is 25mL. The pH of the supernatant is adjusted to 7.4, and the incubation time is 25min. The second centrifugation force is 4500g. S4: The centrifugal force during suspension centrifugation is 14000g, the trehalose concentration is 6% w / v, the freeze-drying temperature is -55℃, and the remaining steps are the same.

[0032] Comparative Example 1 The preparation method according to Example 1 differs in that: S1: Without adding white solid to the mixed solution, only microspheres with salicylic acid modified on the surface were prepared. Then, 100 mg of white solid was dissolved in 10 mL of anhydrous ethanol to prepare a stock solution of 10 mg / mL. S2: After shaking culture, the system was centrifuged at 800g for 8min, and the supernatant was collected. Then, the supernatant was centrifuged at 120000g and 4℃ for 90min, the precipitate was collected and resuspended with PBS to obtain blank maize germ exosomes. S3: Mix blank corn germ exosomes with the stock solution obtained in S1 and incubate in a shaker at 37°C for 1 hour; S4: Centrifuge the incubated mixture at 120000g and 4℃ for 90min to remove the supernatant, and resuspend the precipitate with PBS. The remaining steps are the same.

[0033] This comparative preparation separates the steps of inducing secretion, extracting exosomes, loading drugs, and purifying them to prepare a post-drug-loaded exosome formulation.

[0034] Comparative Example 2 The preparation method according to Example 1 differs in that: S1: Replace carboxymethyl chitosan with regular chitosan; the remaining steps are the same.

[0035] This comparative preparation of exosome formulations resulted in a mismatch between pH response and exosome secretion, leading to a time discrepancy between drug release and exosome secretion.

[0036] Comparative Example 3 The preparation method according to Example 1 differs in that: S3: Without adding the phenylboronic acid dextran solution to the system, directly centrifuge the supernatant at 120000g for 90min. All other steps are the same.

[0037] This comparative preparation of exosome formulations without specific cross-linking and size scaling, obtained by separating from complex mixtures in a single ultracentrifugation step.

[0038] Experimental Example 1 L02 cells were seeded into 96-well plates and cultured at 37°C and 5% CO2 for 24 h to allow them to adhere. The exosome lyophilized powders prepared in Examples 1-4 and Comparative Examples 1-3 were dissolved in sterile phosphate buffer to prepare a stock solution with an exosome protein concentration of 1 mg / mL. Two experimental groups were established: a baseline damage group (fresh medium containing 400 μmol / L H2O2) and a product group (fresh medium containing 400 μmol / L H2O2 and the sample stock solution, resulting in a final exosome protein concentration of 30 μg / mL). Additionally, a blank well was set up on the plate, containing only 100 μL of cell culture medium without seeding any cells, serving as the control group. After culturing for another 24 h, cell viability was assessed using the CCK-8 assay: CCK-8 working solution was added to each well, incubated for 2 h, and the absorbance (OD value) was measured at 450 nm. Cell viability was calculated using the following formula:

[0039] This cell viability indicates the sample's ability to maintain hepatocyte survival under oxidative stress. The results are shown in Table 2 and... Figure 3 As shown.

[0040] Table 2 Cell viability after treatment in the examples and comparative examples

[0041] From Table 2 and Figure 3 As can be seen from the results, the sample in the example can maintain the survival of more hepatocytes under oxidative stress, indicating that a highly bioactive formulation can be obtained through this integrated design and simultaneous in-situ drug delivery. The preparation process of Comparative Example 1 was broken down into multiple discrete steps. Due to the multiple centrifugations and transfers involved, a large amount of exosomes were lost and their structures were damaged. Furthermore, the drug loading efficiency was extremely low. As a result, the exosome activity and effective drug loading in the final product were severely insufficient, the protective effect was greatly reduced, and the cell survival rate was significantly decreased. Comparative Example 2 retained the complete purification steps, but the timing of drug release and cell secretion of exosomes was not synchronized. As a result, most of the drug failed to insert into the membrane of newly formed exosomes and remained free in the system, which was then removed in the purification steps. Therefore, the actual drug loading in the final product was extremely low. Although the exosomes themselves were successfully purified, the drug efficacy was still weak, resulting in a low cell survival rate. Although Comparative Example 3 achieved efficient induced release and in-situ drug loading, the omission of the specific purification step based on glycosylated reversible polymers and reliance solely on ultracentrifugation for separation and purification resulted in a large amount of cell debris, protein aggregates, and other impurities mixed in with the final product. These impurities not only have no therapeutic effect but may also induce cytotoxicity or interfere with the normal function of exosomes, thereby greatly offsetting the protective effect of drug-loaded exosomes and resulting in the lowest final cell survival rate.

[0042] Experimental Example 2 Human liver cancer cells HepG2 were injected at a concentration of 2 × 10⁻⁶ cells per well. 5 Cells were seeded at a density of 100 μL in 24-well plates and cultured until fully adherent. The exosome lyophilized powders prepared in Examples 1-4 and Comparative Examples 1-3 were reconstituted with PBS to a 1 mg / mL stock solution. A baseline damage group and a product group were established. For the baseline damage group, 1 mL of complete culture medium containing 100 mmol / L anhydrous ethanol was added to each well. For the product group, 1 mL of complete culture medium containing 100 mmol / L anhydrous ethanol and 50 μL of the product stock solution was added to each well. After 24 h of treatment, the culture medium was discarded, and the cells were washed once with pre-chilled PBS. 150 μL of commercially available cell lysis buffer was added to each well, and the cells were lysed on ice for 15 min. The plates were centrifuged at 12000 g for 10 min at 4 °C. The supernatant from each well was transferred to a new centrifuge tube. 50 μL of the supernatant was then mixed with 200 μL of the kit-provided ethanol and NAD+. +The yellow reaction solution was mixed in a 96-well plate. The plate was immediately placed in a preheated ELISA reader at 37°C, and absorbance values ​​were read at 340 nm. Data were recorded every 30 seconds for 10 minutes. A curve showing a stable linear increase in absorbance over time was selected, and the rate of change of absorbance ΔOD was calculated within this interval. The ADH enzyme activity was then calculated using the formula:

[0043] Where V is the total reaction volume, i.e., 0.25 mL; ε is the molar extinction coefficient of NADH at 340 nm, which is 6.22 mL / (μmol·cm); 0.6 is the optical path length of the 96-well plate; and V1 is the volume of cell lysate supernatant added to the reaction system, i.e., 0.05 mL.

[0044] The results are shown in Table 3 and Figure 4 As shown.

[0045] Table 3. ADH enzyme activity after treatment in the examples and comparative examples.

[0046] From Table 3 and Figure 4 As can be seen, the sample from the example can significantly enhance the ADH enzyme activity of hepatocytes, indicating that the sample from the example maximizes the delivery of silymarin to hepatocytes and effectively activates the metabolic enzyme system. The multi-step discrete process in Comparative Example 1 resulted in severe exosome loss and structural damage, and the subsequent passive incubation led to low drug loading efficiency. This resulted in a very small number of fully functional drug-loaded exosomes in the final product, and insufficient drug delivery to cells, thus weakening the enhancement effect on metabolic enzymes. This demonstrates that such a disjointed process can impair the product's biological function. In Comparative Example 2, although the exosomes were purified, the misalignment of drug release and secretion timing resulted in empty loads. The purified product mainly consisted of unloaded exosomes, and the exosomes themselves have limited enhancement effects on metabolic enzymes. Silymarin could not be efficiently delivered via exosomes, leading to a loss of efficacy and resulting in the lowest ADH enzyme activity in this group of samples. Although Comparative Example 3 had efficient in-situ drug loading, the lack of specific purification resulted in a large amount of cell debris and other impurities in the product. These impurities may be toxic to cells, inhibiting normal cellular metabolic functions. Simultaneously, these impurities competitively occupy cellular uptake pathways, interfering with the effective utilization of drug-loaded exosomes by cells, leading to low ADH enzyme activity.

[0047] Experimental Example 3 The samples prepared in Examples 1-4 and Comparative Examples 1-3 were divided into two groups and made into tablets and capsules, respectively. The tablets were 2 mm in diameter and each contained 0.5 mg of exosome protein. The capsules were No. 3 capsules encapsulated in enteric-coated capsule shells and each contained 0.5 mg of exosome protein. Twenty healthy male mice aged 8-10 weeks, weighing 20-25 g, were selected and divided into four groups: normal control group, model control group, tablet intervention group, and capsule intervention group. The model control group and intervention group were administered 20% ethanol solution by gavage at 9:00 AM daily, with a dose of 2 mg / g body weight. One hour later, the tablet intervention group received one tablet via a special gavage needle, followed immediately by 0.2 mL of water. The capsule intervention group received one capsule in the same manner. The normal control group and model control group were administered the same volume of physiological saline. After 5 consecutive days, 50 μL of blood was collected via tail vein two hours before and after the last administration. Serum was separated, and the activities of alanine aminotransferase (ALT), alcohol dehydrogenase (ADH), and acetaldehyde concentration were measured using commercially available kits. The degree of hepatocyte damage, the activity of key enzymes in alcohol metabolism, and the levels of toxic intermediates were also detected. The results are shown in Tables 4 and 5. Figures 5-7 As shown.

[0048] Table 4. Serum data of mice 2 hours before the last administration

[0049] Table 5. Serum data of mice 2 hours after the last administration

[0050] From Table 4, Table 5 and Figures 5-7As can be seen, during continuous ethanol administration, the liver load of mice was significantly increased, while feeding the sample of the example could effectively reduce the liver load of mice to a certain extent. Moreover, the effect of the capsule was slightly better than that of the tablet, indicating that the capsule shell protects more drug-loaded exosomes to reach the liver intact, resulting in high bioavailability. Due to the discontinuous multi-step process, the absolute number of effective drug-loaded exosomes in Comparative Example 1 was severely insufficient. After four consecutive days of administration, the concentration of the active ingredient accumulated in the liver was far lower than that of the standard process group. Therefore, the cumulative efficacy was weak before the last administration, and the amount of active ingredient in a single dose was too small to produce a significant acute effect, resulting in poor alcohol metabolism. Most of the exosomes obtained from Comparative Example 2 were blank exosomes, which failed to effectively load the specific drug. Continuous administration of the blank carrier had almost no specific promoting effect on the activity of the liver metabolic enzyme ADH and acetaldehyde clearance. Therefore, the cumulative efficacy was similar to that of the model control group that was never administered the drug. In Comparative Example 3, the drug-loaded exosomes were mixed with impurities such as cell debris. These impurities may have certain side effects or toxicity on the metabolism of hepatocytes. Continuous administration of this drug will not only reduce the therapeutic effect of the drug, but also lead to the continuous accumulation of liver damage caused by impurity toxicity. Therefore, the ADH enzyme activity was the lowest, while the ALT enzyme activity and the concentration of the intermediate product acetaldehyde were the highest.

Claims

1. A method for extracting exosomes from corn germ, comprising extraction via pH-responsive loading and centrifugal purification technology, characterized in that, Includes the following steps: S1: Using sodium alginate solution and carboxymethyl chitosan solution as raw materials, composite microspheres were prepared by ion crosslinking-gelation method. Plant elicitors were modified on the surface of the microspheres by coupling reaction. At the same time, the drug was anchored by embedding a membrane inside the microspheres to obtain functional microspheres with pH response characteristics. S2: Add corn germ tissue to the culture medium, then add functional microspheres to the culture container, place the system under constant temperature and gently shake to culture, and continuously monitor the pH change in the system. When the pH drops to 6.5, stop the culture to obtain exosomes loaded with specific drugs dispersed in the culture medium. S3: Centrifuge the culture medium at low speed and collect the supernatant. Then add the glycosylated reversible polymer to the supernatant, adjust the pH of the system to 7.2~7.8 and stir and incubate until visible flocculent matter appears in the system. Collect the flocculent matter by medium-speed centrifugation to obtain exosomes encapsulated by the polymer. S4: Resuspend the precipitate obtained in S3, then centrifuge the system at low speed, collect the precipitate and add acidic buffer solution, gently shake to dissolve the precipitate, then centrifuge at medium speed, collect the supernatant and concentrate it using a 200kDa ultrafiltration membrane to obtain pure corn germ exosomes loaded with specific drugs, add trehalose solution and freeze-dry into powder for later use.

2. The method for extracting exosomes from maize germ according to claim 1, characterized in that: The plant elicitor in S1 is one or more of salicylic acid, chitosan oligosaccharide, and oligosaccharide galacturonic acid. The membrane-anchored drug is a cholesterol-silymarin conjugate. The cholesterol and the silymarin are connected by a dicarboxylic acid linker, which is one or more of succinic anhydride, glutaric anhydride, and maleic anhydride.

3. The method for extracting exosomes from maize germ according to claim 1, characterized in that: The sodium alginate solution in S1 has a concentration of 1%~2% w / v, the carboxymethyl chitosan solution has a concentration of 0.5%~1.2% w / v, the calcium chloride solution for ionic crosslinking has a concentration of 1.5%~3% w / v, the crosslinking time is 20~60 min, and the coupling reaction time is 6~18 h.

4. The method for extracting exosomes from maize germ according to claim 1, characterized in that: The mass ratio of corn germ tissue to functional microspheres in S2 is (100:1) to (100:3), the oscillation culture time is 4 to 8 hours, and the oscillation rate is 70 to 110 rpm.

5. The method for extracting exosomes from maize germ according to claim 1, characterized in that: The glycosyl reversible polymer described in S3 is one or more of phenylboronic dextran, phenylboronic hyaluronic acid, phenylboronic chitosan, and phenylboronic amylopectin, with a molecular weight of 70-80 kDa.

6. The method for extracting exosomes from maize germ according to claim 1, characterized in that: The centrifugal force during low-speed centrifugation in S3 is 500~1200g, the concentration of the added glycosyl reversible polymer is 0.1%~0.3% w / v, the incubation time is 20~40min, and the centrifugal force during medium-speed centrifugation is 3000~5000g.

7. The method for extracting exosomes from maize germ according to claim 1, characterized in that: The centrifugal force during medium-speed centrifugation in S4 is 10000~15000g, the concentration of the trehalose solution is 3%~8% w / v, and the freeze-drying temperature is -60℃~-45℃.

8. A corn germ exosome preparation, characterized in that: The formulation is prepared by preparing corn germ exosomes loaded with a specific drug into a pharmaceutical preparation, wherein the corn germ exosomes loaded with the specific drug are extracted by the corn germ exosome extraction method according to any one of claims 1 to 7.

9. A corn germ exosome preparation according to claim 8, characterized in that: The medicine is one of the following: powder, tablet, or capsule.

10. A corn germ exosome preparation according to claim 8, characterized in that: The formulation can maintain a hepatocyte survival rate of >75% under oxidative stress, and the activity of alcohol-metabolizing enzymes in hepatocytes after alcohol exposure is >30 U / mg protein.

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