Oral delivery of plant exosome-lipid nanoparticle hybrid vesicles and preparation thereof

Abstract

The application discloses oral delivery of plant exosome-lipid nanoparticle hybrid vesicles and a preparation method thereof, and the preparation method comprises the following steps: mixing DOTAP methyl sulfate, DOPE-PEG2000, DOPE-PEG2000-galactose and cholesterol, dissolving by adding a solvent, removing the solvent to obtain a lipid film, hydrating the lipid film, and freeze-drying to obtain lipid nanoparticles after hydration is completed; the lipid nanoparticles are uniformly mixed with edible plant exosomes in a buffer solution, ultrasonic treatment is performed, finally, a lipid extruder is used in cooperation with a polycarbonate film, and the hybrid vesicles are obtained through cyclic extrusion. The application deeply hybridizes and fuses the high-efficiency nucleic acid delivery capacity of the lipid nanoparticles and the anti-inflammatory and intestinal microenvironment regulation activity of the plant exosomes, through the dual-function synergistic design of "delivery carrier + natural medicinal substances", so that the hybrid vesicles can precisely silence target genes by delivering siRNA, and can also remodel the lesion microenvironment by using the intestinal repair function of the plant exosomes.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an orally delivered plant exosome-lipid nanoparticle hybrid vesicle and its preparation. Background Technology

[0002] Inflammatory bowel disease (IBD) is a group of diseases characterized by chronic inflammation of the intestinal mucosa. Its pathogenesis is complex, the course of the disease is prolonged and the recurrence rate is high. Current drug treatments mostly achieve symptom relief through systemic immunosuppression, but the side effects are significant and the efficacy is limited, making it difficult to achieve fundamental mucosal repair.

[0003] Small interfering RNA (siRNA) therapy, as an RNA interference technology, can regulate the expression of susceptibility genes associated with IBD and the secretion of pro-inflammatory cytokines, thereby guiding mucosal repair and immune modulation at the disease site. It holds promise for long-term inflammation suppression with a single dose, demonstrating good efficacy in acute and chronic colitis models, and providing a promising new treatment strategy for IBD clinical practice. However, siRNA molecules are easily degraded by nucleases in the gastrointestinal environment and have difficulty crossing the intestinal epithelial barrier, making oral delivery one of the biggest bottlenecks in the translation of RNA therapy. Although studies have confirmed that RNA interference therapy can effectively alleviate symptoms in animal models of inflammatory bowel disease by silencing inflammation-related genes, its translation and application are limited by problems such as poor patient compliance, low structural stability of free RNA, poor targeting, low transfection efficiency, and potential off-target toxicity associated with traditional administration methods.

[0004] Nucleic acid drugs are extremely difficult to absorb orally due to their high hydrophilicity, negative charge, and large molecular weight. Existing research indicates that oral administration of nucleic acids must overcome a series of stringent gastrointestinal (GI) barriers. Extracellular barriers include: (i) a highly acidic and enzymatic environment: the gastric pH (1.0-1.8) and abundant digestive enzymes lead to the depurination and degradation of nucleic acids; (ii) the mucus barrier: the main component of intestinal mucus, mucin fibers, form a viscoelastic hydrogel that traps nanocarriers through electrostatic and hydrophobic interactions, hindering their contact with intestinal epithelial cells. Intracellular barriers include cell membrane permeability barriers, endosome / lysosomal traps (pH 4.5-5.5 and high concentrations of hydrolytic enzymes), and difficulties in the localization of specific subcellular components within the cytoplasm.

[0005] Lipid nanoparticles are currently recognized as the most mature non-viral nucleic acid delivery carriers. Their mechanism of action relies on the protonation of ionizable lipids in an acidic endosome environment to achieve high drug loading efficiency and excellent lysosomal escape ability.

[0006] Exosomes (30-200 nm) have attracted much attention for oral nucleic acid delivery due to their low immunogenicity, natural lipid bilayer protection, and surface-modifiable receptors. Plant-derived exosomes from edible plants exhibit good biocompatibility and low immunogenicity. In addition to possessing the advantages of physical delivery carriers, plant exosomes are themselves bioactive natural nanocomponents. Rich in various bioactive lipids, specific proteins, and plant-derived miRNAs, plant exosomes possess biological functions such as regulating gut microbiota homeostasis, mitigating oxidative stress damage, and inhibiting the secretion of pro-inflammatory factors, actively participating in the repair of damaged intestinal mucosa and regulating the intestinal immune microenvironment. However, a fatal flaw of plant exosomes as a single carrier is their extremely low nucleic acid encapsulation efficiency and lack of an effective active loading mechanism, making it difficult to achieve the effective doses required for clinical treatment.

[0007] A previously published patent (CN120189501A) discloses a tumor mRNA vaccine that combines ginseng nanovesicles with lipid nanoparticles. This method primarily employs microfluidic or extrusion techniques to hybridize ginseng exosomes with mRNA-loaded lipid nanoparticles for subcutaneous or intramuscular injection to stimulate an anti-tumor immune response. However, this approach, mainly for injection, cannot overcome the complex obstacles encountered in oral delivery, such as high-intensity gastric acid, enzymatic hydrolysis, bile salts, and the mucus barrier. Furthermore, its mRNA payload differs fundamentally from siRNA, which targets intracellular gene silencing, in terms of delivery requirements.

[0008] Based on the above analysis, existing research lacks a functional hybrid system specifically designed to address the oral gastrointestinal barrier, capable of simultaneously achieving both "efficient encapsulation of nucleic acid drugs by lipid nanoparticles" and "the anti-inflammatory activity and oral tolerability of plant exosomes." The plant exosome-lipid nanoparticle hybrid vesicles of this invention address these technological gaps and limitations in oral delivery, proposing an innovative solution for precise intestinal-targeted therapy. Summary of the Invention

[0009] To address the challenge of oral delivery limitations of existing hybrid vesicles, this invention provides an orally deliverable plant exosome-lipid nanoparticle hybrid vesicle and its preparation method. This invention innovatively integrates the highly efficient nucleic acid delivery capability of lipid nanoparticles with the anti-inflammatory and gut microenvironment-regulating activities inherent in plant exosomes. Through a dual-functional synergistic design of "delivery carrier + natural pharmacological substance," the hybrid vesicles can deliver siRNA to precisely silence target genes while simultaneously utilizing the gut repair function of plant exosomes to reshape the lesion microenvironment, achieving a dual synergistic effect of delivery and treatment, resulting in a comprehensive therapeutic efficacy of "1+1>2".

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0011] The first aspect of this invention provides a method for preparing orally delivered plant exosome-lipid nanoparticle hybrid vesicles, comprising the following steps:

[0012] (1) Mix DOTAP methyl sulfate, PEG lipid and cholesterol, add solvent to dissolve, remove solvent to obtain lipid film, hydrate lipid film, freeze dry after hydration to obtain lipid nanoparticles, and freeze-store.

[0013] (2) The lipid nanoparticles and edible plant exosomes were mixed in a buffer solution, ultrasonicated, and finally extruded in a cyclic manner using a lipid extruder with a polycarbonate membrane to obtain the hybrid vesicles.

[0014] The PEG lipids are DOPE-PEG2000 and DOPE-PEG2000-Galactose.

[0015] This invention uses the special lipid DOPE-PEG2000-Galactose to achieve targeted enrichment of intestinal lesions.

[0016] In step (1), nucleic acid drugs can be added during the hydration of the lipid membrane to achieve nucleic acid drug loading.

[0017] Preferably, in step (1), the solvent removal is performed using a rotary evaporator, and the gradient pressure reduction program of the rotary evaporator can be adjusted appropriately according to the type of solvent to ensure that the organic solvent is completely evaporated and forms a uniform film.

[0018] Preferably, the molar ratio of DOTAP methyl sulfate, PEG lipid and cholesterol is (0.5~1.5):1:(1.5~3); the amount of DOPE-PEG2000-Galactose is 0.5%~5% of the PEG lipid, in molar mass.

[0019] Preferably, the edible plant exosomes are derived from at least one of wolfberry, ginger, lemon, cherry, apple, carrot, and grapefruit; more preferably, the edible plant exosomes are derived from wolfberry.

[0020] Preferably, the edible plant exosomes are prepared by a method comprising the following steps: taking dried or fresh edible plant fruit, removing surface impurities, juicing by cell wall breaking, primary filtration, removing impurities by differential centrifugation, and finally extracting by ultracentrifugation to obtain crude extract of edible plant exosomes, which is then resuspended and freeze-dried to obtain freeze-dried powder of the edible plant exosomes; more preferably, the centrifugal force of the ultracentrifugation is 100,000 g to 150,000 g, and the centrifugation time is 60 to 90 minutes.

[0021] Employing a gentle extraction and hybridization process, this method maximizes the preservation of glycoprotein and lipid distribution on the exosome membrane surface, ensuring that the hybrid vesicles possess the ability to cross the intestinal mucus layer and resist the physicochemical barriers of gastric acid, enzymatic degradation, and bile salt solubilization. This system is specifically molecularly adapted for oral intestinal targeting.

[0022] Preferably, in step (2), the lipid nanoparticles and edible plant exosomes are dissolved together in a buffer solution in equal particle numbers.

[0023] Preferably, the DOPE-PEG2000-Galactose is obtained by reacting NHS-PEG2000-COOH and DOPE to obtain the intermediate product DOPE-PEG2000-COOH, which is then reacted with galactosamine to obtain the final product.

[0024] Preferably, in step (2), the power of the ultrasonic treatment is 100~300 W.

[0025] Preferably, in step (2), the pore size of the polycarbonate film is 100~400 nm, and the number of cyclic extrusions is 5~15.

[0026] A second aspect of the present invention provides an orally delivered plant exosome-lipid nanoparticle hybrid vesicle, which is prepared by the method for preparing the orally delivered plant exosome-lipid nanoparticle hybrid vesicle.

[0027] The third aspect of this invention provides the application of the method for preparing the oral delivery plant exosome-lipid nanoparticle hybrid vesicles in the preparation of an oral delivery nucleic acid drug for the treatment of inflammatory bowel disease.

[0028] Compared with the prior art, the beneficial effects of the present invention are:

[0029] This invention provides a technical solution for the physical hybrid assembly of plant exosomes and lipid nanoparticles. Through the dual mechanical forces of "ultrasonic cavitation" and "microporous membrane extrusion," membrane rearrangement and hybrid fusion of two lipid bilayers originating from different systems are induced. This hybridization is not a simple physical mixing, but rather the formation of a hybrid lipid system with novel membrane physical properties. Specifically, the introduction of trace amounts of DOPE-PEG2000-Galactose (galactosyllipid) into the lipid core formulation actively recognizes and binds to specific receptors on the surface of intestinal epithelial cells and macrophages in the lamina propria, endowing the carrier with an "active intestinal lesion targeting" function that distinguishes it from ordinary injectable lipid nanoparticles. Overcoming the inherent defects of two single carriers: This invention utilizes hybrid membrane recombination technology to successfully combine "high drug loading capacity of lipid nanoparticles" and "high gastrointestinal tolerance of plant exosomes" at the preparation process level, avoiding the extremely low encapsulation rate of single plant exosomes and the inherent shortcomings of single lipid nanoparticles being easily destabilized by bile salts in the gastrointestinal tract and retaining in large quantities in the stomach. While leveraging the gene-silencing function of siRNA mediated by lipid nanoparticles, the inherent activity of wolfberry exosomes is utilized to reshape the intestinal microenvironment, achieving a leap from "physical delivery protection" to "delivery synergistic natural drug efficacy".

[0030] The preparation process of hybrid vesicles is integrated: The preparation process of this invention (from differential centrifugation to extract plant exosomes, to thin film hydration to prepare lipid nanoparticles, and then to the fusion process of ultrasound and extrusion) is mild in overall conditions, does not involve complex chemical reactions or large doses of organic solvents, can effectively preserve the natural biological activity (anti-inflammatory activity) of plant exosomes, and has good potential for industrial scale-up.

[0031] In vitro stability experiments simulating gastric, intestinal, and colonic fluids demonstrated that the hybrid vesicles prepared in this invention can effectively resist the oral digestive tract environment, protecting the payload from premature leakage. This invention, through the barrier protection of the plant exosome shell and the active targeting modification of galactose in the lipid nanoparticle core, pioneered the precise oral delivery of siRNA to colonic lesions. Small animal fluorescence imaging experiments confirmed that after oral administration of the hybrid vesicles of this invention, the drug achieved significant fluorescence enrichment in the colonic lesion area within 12 hours, while the fluorescence signal of the single lipid nanoparticle group mainly remained in the stomach. This specific oral targeted delivery capability to the colonic mucosa is completely unattainable by existing technologies.

[0032] In summary, this invention constructs a plant exosome-lipid nanoparticle hybrid vesicle. This design retains the efficient siRNA loading and controllable release capabilities of lipid nanoparticles while utilizing the natural gastrointestinal stability and anti-inflammatory activity of *Lycium barbarum* exosomes. Furthermore, it endows these exosomes with active targeting function through intestinal epithelial targeting ligand modification. This carrier achieves a deep integration of "engineered delivery" and "natural efficacy," combining protective, targeting, and therapeutic functions, providing a novel, non-injection-dependent strategy for the precise intestinal treatment of oral nucleic acid drugs. Attached Figure Description

[0033] Figure 1 The following are characterization diagrams of plant exosomes (GB), lipid nanoparticles (LNP), and hybrid vesicles (HYBRID) in the embodiments; wherein, Figure A is a particle size diagram, Figure B is a particle zeta potential diagram, Figure C is a typical particle size distribution diagram of plant exosomes, Figure D is a typical particle size distribution diagram of lipid nanoparticles, Figure E is a transmission electron microscope image of plant exosomes, Figure F is a transmission electron microscope image of lipid nanoparticles, and Figure G is a transmission electron microscope image of hybrid vesicles.

[0034] Figure 2 The following are membrane stability test results for plant exosomes (GB), lipid nanoparticles (LNP), and hybrid vesicles in the examples; wherein, Figure A shows the comparison of GP values ​​of the plant exosome group, Figure B shows the comparison of GP values ​​of the lipid nanoparticle group, Figure C shows the comparison of GP values ​​of the hybrid vesicle group, and Figure D shows the comparison of the relative deviation of GP values ​​of each group from their respective control (pH=7.4 group).

[0035] Figure 3 The following are stability test results for plant exosomes (GB), lipid nanoparticles (LNP), and hybrid vesicles (HYBRID) in the examples; wherein, Figure A shows the comparison of acridine orange loading, Figure B shows the relative retention rate of acridine orange in the carrier, Figure C shows the comparison of riboflavin loading, and Figure D shows the relative retention rate of riboflavin in the carrier.

[0036] Figure 4 The following are transfection assay results for plant exosomes (GB), lipid nanoparticles (LNP), and hybrid vesicles (HYBRID) in the examples. Figure A compares the transfection capacity of each vector in the three cell types with commercial transfection reagents; Figure B compares the transfection amount in simulated digestive tract solution (Raw 264.7); Figure C compares the transfection amount in simulated digestive tract solution (Caco 2); Figure D compares the transfection amount in simulated digestive tract solution (MDCK); Figure E compares the transfection efficiency (Raw 264.7); Figure F compares the transfection efficiency (Caco 2); and Figure G compares the transfection efficiency (MDCK).

[0037] Figure 5The following are examples of animal fluorescence imaging tests of plant exosomes (GB), lipid nanoparticles (LNP), and hybrid vesicles (HYBRID); Figure A is a flowchart of small animal fluorescence imaging, Figure B is a time comparison of colon fluorescence intensity in each group, Figure C is a quantitative comparison of fluorescence intensity in the colon of each group after 12 hours, and Figure D is a fluorescence imaging image. Detailed Implementation

[0038] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0039] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0040] The preparation method of DOPE-PEG2000-Galactose used in the embodiments of the present invention is as follows:

[0041] I. Experimental Materials

[0042] NHS-PEG2000-COOH (Chongqing Bodaoyun Biotechnology Co., Ltd., CAS: 2414299-79-7), DOPE (Biode Pharmaceutical, CAS: 4004-05-1), Galactosamine Hydrochloride (Biode Pharmaceutical, CAS: 1772-03-8).

[0043] II. Experimental Procedure

[0044] 1. Synthesis of DOPE-PEG2000-COOH:

[0045] Weigh 1 g of NHS-PEG2000-COOH and dissolve it in 10 ml of chloroform. Add DOPE (348 mg, 1.0 eq) and triethylamine (2.0 eq). React at 40 °C for 2 h. Remove the solvent by rotary evaporation under reduced pressure. Purify with acetone (dissolve the sample obtained after reduced pressure distillation in a small amount of chloroform, and slowly add it dropwise to anhydrous acetone pre-cooled to 4 °C or an ice bath under vigorous stirring. The solution becomes turbid and white or pale yellow flocculent powder appears. Continue stirring for 15 minutes after the addition is complete. After stirring, centrifuge at 4000 rpm for 5 minutes to collect the bottom solid. The purification can be repeated 2-3 times as needed). Solidify and dry with ice-cold ether (dissolve the purified product in a small amount of chloroform, and slowly add it dropwise to anhydrous ether pre-cooled to 4 °C or an ice bath under vigorous stirring. The precipitated product is filtered to remove the ether. Finally, place the solid in a vacuum drying oven to dry thoroughly).

[0046] 2. Synthesis of DOPE-PEG2000-Galactose:

[0047] Weigh 500 mg of DOPE-PEG2000-COOH and dissolve it in 5 mL of DMSO. Add galactosamine (152 mg, 1.05 eq), EDC (2.0 eq), HOBT (2.0 eq), and triethylamine (2.0 eq). React at 40 °C for 5 h. Transfer the solution to a dialysis bag (molecular weight cutoff 1000 Da) and dialyze it in pure water for 24 h. Freeze-dry to obtain the product.

[0048] Example 1

[0049] This embodiment provides a method for preparing plant exosome-lipid nanoparticle hybrid vesicles for oral delivery, specifically including the following steps:

[0050] Step 1: Extraction of plant exosomes:

[0051] (1) Pretreatment: Take clean dried wolfberries and wash them repeatedly with distilled water or deionized water more than 3 times to remove surface impurities. Mix the washed wolfberries with phosphate buffer (PBS, pH = 7.4) pre-cooled to 4°C at a mass-volume ratio of 1:1 and place them in a high-speed blender for intermittent blending and juicing. Set the working program as follows: 15 seconds of operation followed by 15 seconds of cooling is one cycle, and a total of 6 cycles are performed.

[0052] (2) Primary filtration: The suspension obtained by juicing is coarsely filtered through multiple layers of gauze or coarse filter screen, and the filtrate is collected and placed into a 50mL centrifuge tube.

[0053] (3) Differential centrifugation to remove impurities: Take the filtrate from step (2) and centrifuge it at 1000 g for 10 minutes at 4℃ to remove large particles of unbroken tissue and collect the supernatant; then centrifuge the supernatant at 3000 g for 20 minutes at 4℃ to remove large fragments such as fruit residue and broken seeds and collect the supernatant; continue to centrifuge the supernatant at 10000 g for 40 minutes at 4℃ to remove small residues such as organelles and bacteria, collect the supernatant and filter it through a 0.45 μm microporous membrane.

[0054] (4) Ultracentrifugation extraction: The liquid filtered in step (3) is centrifuged at 100,000 g for 90 minutes at 4°C. The precipitate at the bottom of the tube is the crude extract containing plant exosomes.

[0055] (5) Resuspension and storage: Discard the supernatant, resuspend the precipitate in a small amount of pre-cooled PBS at 4°C, and then purify the resuspension through a 0.22 μm sterile microporous membrane. Place the purified plant exosome suspension in an ultra-low temperature freezer at -80°C for at least 48 hours, and then freeze-dry it under vacuum for 24 hours to obtain plant exosome freeze-dried powder, which is then stored in a sealed container at -80°C for long-term use.

[0056] Step 2: Synthesis of lipid nanoparticles:

[0057] (1) Preparation of raw materials: Accurately weigh 1 part of DOTAP methyl sulfate (CAS: 144189-73-1), 0.99 parts of DOPE-PEG2000 (CAS: 474922-84-4, average molecular weight about 2000 Da), 0.01 parts of DOPE-PEG2000-Galactose and 2 parts of cholesterol by molar weight, add them to a clean round bottom flask, add an appropriate amount of chloroform to completely dissolve and mix evenly.

[0058] (2) Rotary evaporation for film formation: The round-bottom flask was placed on a rotary evaporator. At room temperature, the rotation speed was set to 100 rpm, and a gradient pressure reduction program was used for vacuum evaporation to remove the organic solvent. The pressure reduction programs were as follows: 150 hPa for 15 minutes, 50 hPa for 10 minutes, and 13 hPa for 10 minutes. After evaporation, a uniform and transparent lipid film was formed at the bottom of the round-bottom flask.

[0059] (3) Hydration: Preheat the phosphate buffer to 45°C and slowly drop it into the round-bottom flask containing the lipid film. Hydrate at 150 rpm for 2 hours under constant temperature of 45°C to fully disperse the lipid film and allow it to self-assemble into lipid nanoparticles loaded with nucleic acid.

[0060] (4) Preservation and freeze-drying: The lipid nanoparticle suspension obtained by hydration is placed in an ultra-low temperature freezer at -80℃ for at least 48 hours, and then subjected to vacuum freeze-drying for 24 hours to obtain lipid nanoparticle freeze-dried powder, which is then sealed and stored at -80℃ for long-term use.

[0061] Step 3: Construction of plant exosome-lipid nanoparticle hybrid vesicles

[0062] (1) Mixing and resuspension: Take the plant exosome lyophilized powder obtained in step one and the lipid nanoparticle lyophilized powder obtained in step two, respectively, and dissolve them together in 1 mL of phosphate buffered saline (PBS, pH = 7.4) in a ratio of equal particle number (collect the samples in the same sample bottle for long-term storage, take 1 mg from it for NTA test to determine the particle number corresponding to 1 mg / mL, and finally calculate the particle number by weight). Use a vortex mixer to mix and disperse the two thoroughly.

[0063] (2) Ultrasonic-assisted fusion: The mixture from step (1) was placed in an ice bath (temperature maintained at 4°C) and ultrasonically treated using an ultrasonic cell disruptor. The power was set to 160 W, and an intermittent working mode with 10-second ultrasonic on and 10-second intervals was adopted. A total of 30 cycles were run to utilize the cavitation effect of ultrasound to promote the physical rearrangement and fusion of the lipid membranes of the two nanoparticles.

[0064] (3) Extrusion homogenization: The mixture after ultrasonic treatment in step (2) is transferred to a lipid extruder and paired with a polycarbonate membrane with a pore size of 200 nm. It is then cyclically extruded 10 times to make the particle size distribution of the hybrid vesicles more concentrated and uniform by shearing force.

[0065] (4) Standing and storage: Let the extruded liquid stand at room temperature for 15 minutes to stabilize its structure, and then transfer it to a 4°C refrigerator for storage or freeze-drying as needed.

[0066] Figure 1 Characterization images of plant exosomes, lipid nanoparticles, and hybrid vesicles prepared for this example are shown. The sizes of the plant exosomes, lipid nanoparticles, and hybrid vesicles are as follows: Figure 1 As shown in Figure A; the zeta potentials of plant exosomes, lipid nanoparticles, and hybrid vesicles are as follows: Figure 1 As shown in Figure B; the typical particle size distribution of plant exosomes determined by nanoparticle tracking analysis (NTA) is shown in Figure B. Figure 1 As shown in C, the sample was diluted 1:250,000 before measurement to ensure the particle concentration was within the instrument's optimal detection range. Based on the dilution factor, the concentration of the undiluted original stock solution was approximately 1.1 × 10⁻⁶. 11 particles / mL. Typical particle size distribution of lipid nanoparticles determined by nanoparticle tracking analysis (NTA) is shown below. Figure 1 As shown in D, the sample was diluted 1:250,000 before measurement to ensure the particle concentration was within the instrument's optimal detection range. Based on the dilution factor, the concentration of the undiluted original stock solution was approximately 3 × 10⁻⁶. 11 particles / mL. Images of plant exosomes captured by transmission electron microscopy, such as... Figure 1 Image of lipid nanoparticles as shown in E. Figure 1 As shown in F, the hybrid vesicle image is as follows: Figure 1 As shown in G.

[0067] Experiment Example 1: Membrane Stability Experiment

[0068] (1) Experimental materials

[0069] Reagents: Deionized water, DMSO, Laurdan dye;

[0070] Simulated solutions: PBS (pH = 1.2), PBS (pH = 6.8), PBS (pH = 7.0), PBS (pH = 7.4), simulated saliva (SSSF), simulated small intestinal solution (SIF), simulated fasting small intestinal solution (FaSSIF), simulated colonic fluid (SCF), simulated gastric juice (SGF), simulated fasting gastric juice (FaSSGF);

[0071] Carriers: freeze-dried plant exosomes, freeze-dried lipid nanoparticles, plant exosome-lipid nanoparticle hybrid vesicles;

[0072] Instruments: analytical balance, microplate reader, black 96-well plate, 1.5mL centrifuge tubes, shaker, centrifuge.

[0073] (2) Experimental methods

[0074] Preparation of carrier stock solution: Weigh 5.0 mg of carrier into 1 ml of PBS to prepare the stock solution;

[0075] Preparation of fluorescent probe stock solution: Weigh 1.77 mg of Laurdan into 1 ml of DMSO to prepare a 5 mM stock solution. Then, take 2 µL and add it to 98 µL of simulated solution to prepare a 100 µL intermediate solution.

[0076] Experimental Groups:

[0077]

[0078] Experimental procedure: The mixture of each carrier stock solution, fluorescent probe stock solution, and simulation solution was incubated at 37°C, 40 rpm, and in the dark for 1 hour. After incubation, the mixture was centrifuged at 10000g, 4°C for 15 minutes, and the supernatant was finally transferred to a black 96-well plate.

[0079] Fluorescence detection: The membrane was excited at 340 nm using an ELISA reader, and the emission at 435 nm and 490 nm was collected respectively. The GP value (generalized polarization value, used to indicate whether the membrane is ordered, 1 for ordered and -1 for disordered) was calculated based on this.

[0080] Calculation formula: GP = (I 435 –I 490 ) / (I 435 + I 490 )

[0081] (3) Experimental results

[0082] Depend on Figure 2 It can be seen that the plant exosomes exhibit strong membrane stability. Figure 2 A), while lipid nanoparticles exhibit relatively weak membrane stability ( Figure 2 B), hybrid vesicles exhibit superior membrane stability compared to lipid nanoparticles. Figure 2 C). Using PBS (pH=7.4) as a control, the membrane stability of the fused hybrid vesicles was generally improved compared to lipid nanoparticles. Figure 2 D). The above analysis results demonstrate that the hybrid vesicles prepared using the present invention have strong membrane stability.

[0083] Experiment Example 2: Packet Stability Experiment

[0084] (1) Experimental materials

[0085] Reagents: Deionized water, ethanol, riboflavin, acridine orange, phosphate buffer (PBS, pH = 7.4);

[0086] Simulated solutions: PBS (pH = 1.2), PBS (pH = 6.8), PBS pH = 7.0, PBS pH = 7.4, simulated saliva (SSSF), simulated gastric juice (SGF), simulated small intestinal solution (SIF), simulated colonic juice (SCF);

[0087] Carriers: freeze-dried plant exosomes, freeze-dried lipid nanoparticles, plant exosome-lipid nanoparticle hybrid vesicles;

[0088] Instruments: analytical balance, non-contact sonicator, magnetic stirrer, microplate reader, black 96-well plate, 1.5 mL centrifuge tubes, shaker, centrifuge, purification kit (SEC method).

[0089] (2) Experimental methods

[0090] Sample preparation: Weigh 3.75 mg of carrier and measure the number of hybrid vesicles as equal particles.

[0091] Fluorescent encapsulation method:

[0092] Exosomal riboflavin encapsulation: A 1.2 mg / mL supersaturated riboflavin solution was prepared, filtered through a 0.22 μm filter, and added to an exosome concentration of 3.75 mg / mL. The mixture was sonicated (320 W, 4°C, 30 sec on / 30 sec off, 6 cycles). After sonication, the mixture was incubated at room temperature for 2 hours. Free riboflavin was then separated using a purification kit (SEC method) and concentrated by ultrafiltration (100 kDa, 4000 g, 15 min).

[0093] Riboflavin encapsulation in lipid nanoparticles: A 1.2 mg / mL supersaturated riboflavin solution was prepared and filtered through a 0.22 μm filter membrane for later use. Lipid nanoparticles were prepared at a weight of 3.75 mg. Riboflavin stock solution was added during hydration for encapsulation. Free riboflavin was separated using a purification kit (SEC method) and concentrated by ultrafiltration (100 kDa, 4000 g, 15 min).

[0094] Hybrid vesicle riboflavin encapsulation: Using unfiltered lipid nanoparticles encapsulated with riboflavin and blank exosomes as raw materials, 3.75 mg of lyophilized exosomes were added to the liquid phase of the unfiltered lipid nanoparticles. The mixture was sonicated (160 W, 4°C, 10 sec on / 10 sec off, 30 cycles), then extruded 10 times using a lipid extruder with a 200 nm polycarbonate membrane. After standing at room temperature for 15 minutes, the free riboflavin was separated using a purification kit (SEC method) and concentrated by ultrafiltration (100 kDa, 4000 g, 15 min).

[0095] Acridine orange encapsulation on exosomes: 1 mg of acridine orange was dissolved in ethanol. Plant exosomes were incubated at room temperature for 30 minutes at a concentration of 3.75 mg / mL (ethanol:water <10% to prevent vector denaturation). Free acridine orange was then separated using a purification kit (SEC method) and concentrated by ultrafiltration (100 kDa, 4000 g, 15 min).

[0096] Lipid nanoparticle encapsulation of acridine orange: 1 mg of acridine orange was dissolved in ethanol, and the carrier lipid nanoparticles were incubated at room temperature for 30 minutes at a concentration of 3.75 mg / mL (ethanol:water <10% to prevent carrier denaturation). Then, the free acridine orange was separated using a purification kit (SEC method) and concentrated by ultrafiltration (100 kDa, 4000 g, 15 min).

[0097] Hybrid vesicle acridine orange encapsulation: 1 mg of acridine orange was dissolved in ethanol, and blank lipid nanoparticles and blank exosomes were added as raw material carriers at a concentration of 3.75 mg / mL (ethanol:water <10% to prevent carrier denaturation). The mixture was ultrasonically treated (160 W, 4°C, 10 sec on / 10 sec off, 30 cycles). After extrusion 10 times using a lipid extruder with a 200 nm polycarbonate membrane, the mixture was allowed to stand at room temperature for 30 minutes. Free acridine orange was then separated using a purification kit (SEC method) and concentrated by ultrafiltration (100 kDa, 4000 g, 15 min).

[0098] Experimental procedure: The mixture of the carrier containing the fluorescent substance and the simulated solution was incubated at 37°C, 40 rpm, and in the dark for 1 hour. After incubation, the free load was separated using a purification kit (SEC method), centrifuged at 10000g for 1 hour, and the supernatant was transferred to an ultrafiltration tube. The sample was then centrifuged again at 4000g for 30 minutes. The sample was then removed, resuspended in 1 mL of PBS, centrifuged at 10000g for 30 minutes, and finally transferred to an ultrafiltration tube and centrifuged again at 4000g for 15 minutes. The sample was then transferred to a black 96-well plate.

[0099] Fluorescence detection: Acridine orange (490nm excitation, 530nm emission) and riboflavin (450nm excitation, 525nm emission) were detected using an ELISA reader.

[0100] (3) Experimental results

[0101] Depend on Figure 3 It can be seen that the encapsulation capacity of lipid nanoparticles is significantly better than that of plant exosomes, and the encapsulation capacity of fused hybrid vesicles is improved compared with that of single plant exosomes. Figure 3 A, Figure 3 C), while maintaining its carrying capacity, the hybrid vesicles also exhibited the high stability characteristic of plant exosomes, effectively protecting the load in a simulated digestive tract environment. Figure 3 B Figure 3 D). The above analysis results indicate that the hybrid vesicles prepared using the present invention have a good ability to protect drugs from passing through the digestive tract environment.

[0102] Experiment Example 3: Cell Transfection Experiment

[0103] (1) Experimental materials

[0104] Reagents: Deionized water, FAM-siRNA, phosphate buffered saline (PBS, pH = 7.4), DMEM medium, MEM medium, penicillin-streptomycin antibiotics, fetal bovine serum;

[0105] Experimental subjects: Caco-2 (human colorectal adenocarcinoma cells), RAW 264.7 (mouse mononuclear macrophage leukemia cells), MDCK (Madin-Darby canine kidney cells);

[0106] Carriers: freeze-dried plant exosomes, freeze-dried lipid nanoparticles, plant exosome-lipid nanoparticle hybrid vesicles;

[0107] Simulated solutions: PBS (pH = 1.2), PBS (pH = 6.8), PBS (pH = 7.0), PBS (pH = 7.4), simulated saliva (SSSF), simulated gastric juice (SGF), simulated small intestinal solution (SIF), simulated colonic juice (SCF);

[0108] Instruments: analytical balance, magnetic stirrer, non-contact ultrasound, microplate reader, cell culture incubator, black 96-well plate, 1.5mL centrifuge tubes, shaker, centrifuge, ultrafiltration tubes (100kDa).

[0109] (3) Experimental methods

[0110] Sample preparation: Take samples according to the target number of cells, with each cell corresponding to 1×10⁻⁶. 4~6 Order of magnitude of carriers.

[0111] FAM-siRNA loading method:

[0112] Exosome encapsulation: A 2 μM FAM-siRNA solution was prepared using phosphate-buffered saline (PBS, pH = 7.4), and exosomes were encapsulated at a concentration of 1 × 10⁻⁶. 11 Incubate on ice for 30 minutes at a concentration on the order of / mL particles.

[0113] Lipid nanoparticle encapsulation: A 2 μM FAM-siRNA solution was prepared using phosphate-buffered saline (PBS, pH = 7.4). The FAM-siRNA solution was added to the lipid nanoparticles during hydration for encapsulation, and the free FAM-siRNA liquid was separated by ultrafiltration (100 kDa, 4000 g, 15 min).

[0114] Hybrid vesicle encapsulation: Using lipid nanoparticles loaded with FAM-siRNA but not ultrafiltered and blank exosomes as raw materials, lyophilized exosomes were added in equal amounts to the liquid phase of the unfiltered lipid nanoparticles. The mixture was ultrasonicated (160W, 4°C, 10 seconds on / 10 seconds off, 30 cycles), then extruded 10 times with a lipid extruder and a 200nm polycarbonate membrane. After standing at room temperature for 15 minutes, the free FAM-siRNA liquid was separated by ultrafiltration (100 kDa, 4000g, 15 minutes).

[0115] Commercial control group Lipo 2000: Before co-incubation with cells, dilute 0.5 μL of the transfection reagent with 50 μL of serum-free corresponding medium, mix gently by pipetting 3-5 times, and let stand at room temperature for 5 minutes. Then prepare 100 μL / well at a 50 nM gene concentration, mix well by pipetting, and let stand at room temperature for 20 minutes.

[0116] Experimental procedure:

[0117] In vitro incubation: Each vector carrying FAM-siRNA was mixed with a simulated digestive tract solution at a working concentration of 50 nM FAM-siRNA. The mixture was incubated at 37°C, 40 rpm, and in the dark for 1 h. After incubation, the mixture was centrifuged at 10000g for 10 minutes, and the supernatant was collected, concentrated by ultrafiltration to remove the leaked payload, and finally diluted with the corresponding culture medium.

[0118] Cell experiments: Each cell was inoculated at 1-5 × 10⁻⁵ cells / cells. 5 Cells were seeded at a density of 100 cells / well into black 96-well plates. Cells were incubated overnight at 37°C to allow adhesion. After cell adhesion, the old culture medium was removed. 100 μL of fresh culture medium containing the corresponding vector was added to each well, and the plates were returned to the incubator. Cells were co-incubated with Caco-2 and MDCK for 6 hours, and with Raw264.7 for 24 hours.

[0119] Fluorescence detection: Before the test, the cells should be gently rinsed with 200 μL PBS. After washing, 100 μL PBS should be added to each well to cover the cells. Then, the FAM (488nm excitation, 525nm emission) fluorescence intensity should be detected immediately using a microplate reader.

[0120] (4) Experimental results

[0121] Depend on Figure 4 It can be seen that the transfection ability of plant exosomes is relatively weak, while the transfection ability of lipid nanoparticles and hybrid vesicles is stronger than that of commercial transfection reagents. Figure 4 A), and in the transfection experiments of the three cell types, although the transfection ability of hybrid vesicles was slightly inferior to that of lipid nanoparticles, they all showed stronger nucleic acid protection capabilities. Figure 4 (B - G). The above analysis results indicate that the hybrid vesicles prepared using this invention have a good ability to protect nucleic acid drugs from crossing the digestive tract environment and being taken up by cells.

[0122] Experiment Example 4: Fluorescence Imaging Experiment in Small Animals

[0123] (1) Experimental materials

[0124] Reagents: Deionized water, phosphate buffer (PBS, pH = 7.4), DMSO, fluorescent dye DiR, sodium dextran sulfate (DSS);

[0125] Experimental subjects: 16 male C57BL / J mice aged 6-8 weeks; the room temperature for feeding animals was controlled at 20-25 ℃, the relative humidity was 40%-50%, natural day and night light illumination, and the mice had free access to food and water;

[0126] Samples: freeze-dried plant exosomes, freeze-dried lipid nanoparticles, plant exosome-lipid nanoparticle hybrid vesicles, DiR aqueous solution;

[0127] Instruments: analytical balance, magnetic stirrer, ELISA reader, in vivo imaging system, non-contact ultrasound, centrifuge, ultrafiltration tubes (100 kDa), 1.5 mL centrifuge tubes, No. 8 gavage needle, sterile syringe, etc.

[0128] (2) Experimental methods

[0129] Sample preparation: according to 2×10 12 Dosage dose: per kg;

[0130] DiR labeling method: Exosomes, lipid nanoparticles, hybrid vesicles, and DiR in a DMSO solution (DMSO content in the mixture should not exceed 2%) were mixed, sonicated (160W, 10-second on / 10-second off, 15 cycles), and incubated for 60 minutes. After incubation, the mixture was concentrated using an ultrafiltration tube (100 kDa). The concentration of DiR in the control group aqueous solution was equal to the concentration of the hybrid vesicles.

[0131] Experimental model establishment: A 3.5% sodium dextran sulfate solution was prepared and administered to mice via drinking water for 7 consecutive days. Weight changes and fecal characteristics were observed daily. Successful model establishment was indicated by weight loss, decreased appetite, loose stools, and bloody stools. Mice were fasted for 12 hours prior to administration.

[0132] (3) Experimental grouping

[0133] Control group: 4 animals, administered 200 μL of DiR aqueous solution by gavage;

[0134] Exosome group: 4 animals were administered 200 μL of DiR exosome solution by gavage;

[0135] Lipid nanoparticle group: 4 animals were administered 200 μL of DiR lipid nanoparticle solution by gavage;

[0136] Hybrid vesicle group: 4 animals, administered 200 μL of DiR hybrid vesicle solution by gavage;

[0137] Experimental procedure: Mice in each group were dissected and samples were collected 3, 6, 12 and 24 hours after gavage. The digestive tract from the stomach to the colon was freed and subjected to fluorescence imaging.

[0138] (4) Experimental results

[0139] Depend on Figure 5 As can be seen, this curve quantitatively characterizes the changes in average fluorescence intensity in the mouse colon at 3, 6, 12, and 24 hours after oral administration of the free probe group (Control), the wolfberry exosome group (GB), the lipid nanoparticle group (LNP), and the hybrid vesicle group (HYBRID). The free probe group showed a brief intensity peak at 6 hours (approximately 8 × 10⁻⁶). 7 After that, the levels rapidly decreased to extremely low levels at 12 and 24 hours, indicating that the free drug was rapidly metabolized and cleared in the colon. A dramatic signal burst occurred at 6 hours, with a peak value of approximately 2 × 10⁻⁶. 8 However, the fluorescence intensity dropped sharply after 12 hours, and then fell to near baseline levels with the control group after 24 hours. This confirms the phenomenon that "goji berry exosomes can quickly reach the colon, but are difficult to retain for a long time at the lesion site." The fluorescence intensity of the lipid nanoparticle group in the colon remained at a low level at all time points, slowly increasing by only about 5 × 10⁻⁶ after 12 hours.7 Most of the fluorescence signal from lipid nanoparticles remained in the stomach, making it difficult for them to effectively cross the barrier and enter the colon. Compared to other groups, the hybrid vesicle group exhibited a remarkably superior "persistent accumulation" characteristic. Its fluorescence intensity remained consistently high from 6 to 12 hours, reaching the highest peak among all experimental groups at 12 hours (approximately 1.2 × 10⁻⁶). 8 () Figure 5 B). Hybrid vesicles maintained high fluorescence signal expression in the colon for 12 hours after entering the body. Figure 5 C). Comparing the images of each group, the fluorescence in the control group was basically metabolized by 6 hours, and there was almost no signal in the lower digestive tract at 12 and 24 hours; the plant exosome group showed strong signal in the colonic segment at 6 hours, and fluorescence signal could still be detected in the colon at 12 hours, indicating that wolfberry had a good protective effect on the load, but it was rapidly distributed to various parts of the body after entering the body; the lipid nanoparticle group showed that the fluorescence signal was almost concentrated in the stomach, and some signal could reach the small intestine, proving that the lipid nanoparticles had a weak protective ability on the load and could not effectively overcome the acidic environment of the stomach; the hybrid vesicle group showed strong fluorescence in the stomach at the first two time points, and the fluorescence in the stomach weakened at the latter two time points, indicating that most of the drug had successfully passed through the stomach and entered the intestine. Observing the colon, a strong fluorescence signal could still be detected 12 hours after administration, proving that the hybrid vesicles had excellent load protection ability and intestinal lesion enrichment ability. Figure 5 D). The above analysis results demonstrate that the hybrid vesicles prepared using this invention have a good ability to protect the payload as it passes through the digestive tract environment and delivers it to the target region.

[0140] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. A method for preparing orally delivered plant exosome-lipid nanoparticle hybrid vesicles, characterized in that, Includes the following steps: (1) Mix DOTAP methyl sulfate, PEG lipid and cholesterol, add solvent to dissolve, remove solvent to obtain lipid film, hydrate lipid film, freeze dry after hydration to obtain lipid nanoparticles, and freeze-store. (2) The lipid nanoparticles and edible plant exosomes were mixed in a buffer solution, ultrasonicated, and finally extruded in a cyclic manner using a lipid extruder with a polycarbonate membrane to obtain the hybrid vesicles. The PEG lipids are DOPE-PEG2000 and DOPE-PEG2000-Galactose.

2. The method for preparing orally delivered plant exosome-lipid nanoparticle hybrid vesicles according to claim 1, characterized in that, The molar ratio of DOTAP methyl sulfate, PEG lipids and cholesterol is (0.5~1.5):1:(1.5~3); the amount of DOPE-PEG2000-Galactose is 0.5%~5% of the PEG lipids.

3. The method for preparing orally delivered plant exosome-lipid nanoparticle hybrid vesicles according to claim 1, characterized in that, The edible plant exosomes are derived from at least one of the following: goji berries, ginger, lemon, cherry, apple, carrot, and grapefruit.

4. The method for preparing orally delivered plant exosome-lipid nanoparticle hybrid vesicles according to claim 3, characterized in that, The edible plant exosomes are derived from wolfberry.

5. The method for preparing orally delivered plant exosome-lipid nanoparticle hybrid vesicles according to claim 1, characterized in that, The edible plant exosomes are prepared by a method comprising the following steps: Take dried or fresh edible plant fruits, remove surface impurities, and then break the cell walls and extract the juice. After primary filtration, use differential centrifugation to remove impurities, and finally use ultracentrifugation to extract crude edible plant exosomes. After resuspension, freeze-dry to obtain freeze-dried powder of the edible plant exosomes.

6. The method for preparing orally delivered plant exosome-lipid nanoparticle hybrid vesicles according to claim 1, characterized in that, In step (2), the lipid nanoparticles and edible plant exosomes are dissolved together in a buffer solution with an equal number of particles.

7. The method for preparing orally delivered plant exosome-lipid nanoparticle hybrid vesicles according to claim 1, characterized in that, The DOPE-PEG2000-Galactose is obtained by reacting NHS-PEG2000-COOH and DOPE to obtain the intermediate product DOPE-PEG2000-COOH, which is then reacted with galactosamine to obtain the final product.

8. The method for preparing orally delivered plant exosome-lipid nanoparticle hybrid vesicles according to claim 1, characterized in that, In step (2), the power of the ultrasonic treatment is 100~300 W; the pore size of the polycarbonate membrane is 100~400 nm; and the number of cyclic extrusions is 5~15 times.

9. An orally delivered plant exosome-lipid nanoparticle hybrid vesicle, characterized in that, It is prepared by the method for preparing orally delivered plant exosome-lipid nanoparticle hybrid vesicles according to any one of claims 1-8.

10. The method for preparing orally delivered plant exosome-lipid nanoparticle hybrid vesicles according to any one of claims 1-8 is used in the preparation of orally delivered nucleic acid drugs for the treatment of inflammatory bowel disease.

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