Artificial exosome and application thereof in drug delivery

Abstract

The invention relates to the technical field of medicine, and discloses an artificial exosome and application thereof in drug delivery, the artificial exosome is prepared into an oil phase based on PLGA and DOTAP, and a water phase based on OCT4 protein and siTrim28; mixing the two phases to obtain a primary emulsion, and adding a PVA aqueous solution to obtain a double emulsion; stirring the double emulsion and volatilizing the solvent to form an emulsified solution, and centrifuging to obtain PLGA nanoparticles; the lipid mixture is prepared from lipid, DPPE-PEG2000, alpha-tocopherol and alpha-lactalbumin, and a lipid film is formed after a solvent is evaporated; the preparation method comprises the following steps: mixing PLGA nanoparticles, a lipid film and membrane protein, uniformly mixing and heating to obtain a film-coated solution, and finally extruding through a cellulose acetate film to obtain the artificial exosome. The artificial exosome disclosed by the invention combines the stability of the PLGA nanoparticles and the biocompatibility of the lipid membrane, and provides new possibility for a drug delivery system.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to artificial exosomes and applications thereof in drug delivery. Background Art

[0002] Exosomes are small vesicles secreted by cells that carry a variety of bioactive molecules such as proteins, lipids, and nucleic acids. These molecules play an important role in intercellular communication. In recent years, exosomes have attracted much attention due to their great potential in disease diagnosis, treatment, and drug delivery. However, the yield of natural exosomes is relatively low, and their composition is complex and diverse, making it difficult to achieve standardized production, which has led to the rise of artificial exosome research.

[0003] Artificial exosomes are nano-sized vesicles prepared by advanced bioengineering technology to simulate the structure and function of natural exosomes. Compared with natural exosomes, artificial exosomes have higher controllability, scalability and customization. These characteristics allow artificial exosomes to be specially designed to meet specific drug delivery needs. For example, they can be designed to target specific cell types or tissues, thereby improving the bioavailability of drugs and reducing side effects. In this way, artificial exosomes are expected to play an important role in the future medical field and provide new solutions for the diagnosis and treatment of diseases.

[0004] However, the application of artificial exosomes in drug delivery systems still faces some challenges. For example, how to ensure the stability and circulation time of artificial exosomes in the body, and how to effectively load and release drug molecules. In addition, the biocompatibility and immunogenicity of artificial exosomes are also important factors to consider. Therefore, it is urgent to develop new strategies and improved technologies to overcome these challenges and optimize the drug delivery performance of artificial exosomes. Summary of the invention

[0005] In view of this, the present invention proposes an artificial exosome and its application in drug delivery, aiming to overcome the deficiencies in the prior art and provide an artificial exosome with improved stability and drug loading efficiency, as well as a method for improving drug delivery efficiency and reducing side effects.

[0006] The present invention provides a method for preparing artificial exosomes, comprising:

[0007] The PLGA and DOTAP are dissolved in dichloromethane and mixed thoroughly to prepare an oil phase, and the OCT4 protein and siTrim28 are dissolved in nuclease-free water to prepare an aqueous phase; the aqueous phase is added dropwise to the oil phase, and a primary emulsion is obtained after ultrasonic treatment; the primary emulsion is added dropwise to a PVA aqueous solution, and a double emulsion is obtained after ultrasonic treatment again; the double emulsion is added dropwise to the PVA aqueous solution again, stirred, and the dichloromethane is volatilized to obtain an emulsified solution;

[0008] The emulsified solution is centrifuged to retain the precipitate, and washed with nuclease-free water to remove residual PVA and other impurities to obtain PLGA nanoparticles;

[0009] Dissolving DPPC, DSPC, DOPC and cholesterol in a mixed solvent of analytical grade chloroform and anhydrous methanol, respectively, to obtain a lipid mixture; adding DPPE-PEG2000 to the lipid mixture, and then adding α-tocopherol and alpha-lactalbumin, and mixing them thoroughly to obtain a lipid mixture; evaporating the solvent in the lipid mixture to form a uniform lipid film;

[0010] The PLGA nanoparticles are made into a PLGA nanoparticle suspension, the dried lipid film is added to the PLGA nanoparticle suspension, and membrane protein is added, vortexed and mixed, and then cyclically heated to obtain a membrane-coated solution; the membrane-coated solution is repeatedly extruded through a cellulose acetate membrane to obtain the artificial exosomes.

[0011] Preferably, the mass ratio of PLGA to DOTAP is 1:20-30.

[0012] Preferably, the molar ratio of DPPC, DSPC, DOPC and cholesterol is 5:1:3:1.

[0013] Preferably, the cyclic heating is at 45±2°C, at least 3 minutes each time, and repeated at least 3 times.

[0014] Preferably, the pore size of the cellulose acetate membrane is 200 nm.

[0015] Preferably, the PLGA nanoparticles are resuspended in nuclease-free water or PBS to obtain the PLGA nanoparticle suspension.

[0016] Preferably, the DPPE-PEG2000 is 5% of the mass of the DPPC.

[0017] Preferably, the mass ratio of the membrane protein to the lipid is 1:100-500.

[0018] The present invention also provides an artificial exosome, which is prepared according to the above-mentioned method for preparing artificial exosomes.

[0019] The present invention also proposes an application of the artificial exosomes, wherein the application includes at least any one of the following:

[0020] A1) Application of the artificial exosomes in drug delivery;

[0021] A2) Use of the artificial exosomes in preparing drug delivery products.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention significantly improves the stability and circulation time of artificial exosomes, ensuring the effective delivery of drugs in the body; by optimizing the preparation method, the drug loading efficiency is improved, so that drug molecules can be loaded into artificial exosomes more efficiently; the surface of artificial exosomes is modified with DPPE-PEG2000, which helps to prolong its half-life in the blood circulation and reduce the risk of being recognized by the immune system, thereby reducing immunogenicity; the introduction of membrane protein technology enables artificial exosomes to achieve targeted delivery to specific cells or tissues, improve the bioavailability of drugs and reduce side effects on normal tissues; the exosomes provided by the present invention have excellent biocompatibility, are suitable for a variety of drug delivery systems, and open up new ways for the diagnosis and treatment of diseases; the method of the present invention is easy to scale production, helps to reduce costs and achieve production standardization, and provides convenience for clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the detailed description of the preferred embodiments below. The accompanying drawings are only for the purpose of illustrating the preferred embodiments and are not to be considered as limiting the present invention. Moreover, the same reference symbols are used throughout the accompanying drawings to represent the same components. In the accompanying drawings:

[0025] Figure 1 Schematic diagram of the preparation process of new artificial exosomes;

[0026] Figure 2 Take images for fluorescence microscopy. DETAILED DESCRIPTION

[0027] Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although exemplary embodiments of the present disclosure are shown in the accompanying drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present disclosure and to fully convey the scope of the present disclosure to those skilled in the art. It should be noted that, in the absence of conflict, the embodiments of the present invention and the features described in the embodiments can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0028] The present invention discloses a novel artificial exosome, and the materials and preparation process are as follows:

[0029] 1. Materials

[0030] 1. Basic materials

[0031] PLGA (100 mg) (poly(lactic-co-glycolic acid), 50:50 ratio, molecular weight 5000-15000 Da);

[0032] DOTAP (5 mg) (dioleoyl-3-trimethylammonium chloride);

[0033] OCT4 protein (0.1 mg);

[0034] siTrim28 (0.1 mg);

[0035] Nuclease-free water;

[0036] PBS solution.

[0037] 2. Bionic membrane materials

[0038] DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine);

[0039] DSPC (1,2-distearoyl-glycero-3-phosphocholine);

[0040] DOPC (1,2-dioleoyl-glycero-3-phosphocholine);

[0041] cholesterol;

[0042] DPPE-PEG2000 (phospholipid molecule modified with polyethylene glycol-2000).

[0043] 3. Stabilizers and functional molecules

[0044] α-Tocopherol (antioxidant);

[0045] Alpha-lactalbumin (alpha lactalbumin, for targeted modification).

[0046] 4. Other reagents

[0047] Dichloromethane;

[0048] Analytical grade chloroform and anhydrous methanol (3:1, V / V);

[0049] 7% (W / V) polyvinyl alcohol (PVA) aqueous solution.

[0050] 2. Preparation Process

[0051] Figure 1 Schematic diagram of the preparation process of new artificial exosomes.

[0052] Step 1: Preparation of Nanoparticles

[0053] Preparation of oil phase: Weigh PLGA and DOTAP, dissolve them in dichloromethane, and mix them thoroughly to prepare the oil phase (O).

[0054] Preparation of aqueous phase: OCT4 protein and siTrim28 were weighed and dissolved in nuclease-free water to prepare aqueous phase (W).

[0055] Emulsification process: The aqueous phase was added dropwise to the oil phase, and ultrasonic treatment was performed for 45 seconds with the power set to 60%, with the flow rate on for 2 seconds and off for 1 second. The process was performed on ice to prevent thermal degradation of the sample.

[0056] Double emulsification treatment: the obtained primary emulsion was added dropwise into the 7% PVA aqueous solution, and ultrasonicated again for 1 minute, with the power set to 60%, 2 seconds on and 1 second off, and the operation was still carried out on ice.

[0057] Solvent evaporation: The double emulsion was added dropwise into a 1% PVA aqueous solution and stirred at 4°C and 800 rpm for 3 hours to ensure that the dichloromethane was completely volatilized.

[0058] Step 2: Isolation and purification of nanoparticles

[0059] High-speed centrifugation: Centrifuge the emulsified solution at 80,000 rpm for 30 min at 4°C.

[0060] Washing: After centrifugation, retain the pellet and wash three times with nuclease-free water to remove residual PVA and other impurities.

[0061] Resuspension and freeze-drying: Finally, resuspend the particle suspension with nuclease-free water or PBS, or directly freeze-dry for long-term storage.

[0062] Step 3: Preparation of biomimetic membrane

[0063] Dissolution of membrane materials: DPPC, DSPC, DOPC and cholesterol were weighed according to the molar ratio and dissolved in a mixed solvent of analytical grade chloroform: anhydrous methanol (3:1, V / V), respectively.

[0064] Addition of membrane modifiers: Add DPPE-PEG2000 to the lipid mixture in an amount of 10% of the mass of DPPC. Then add α-tocopherol (antioxidant) and alpha-lactalbumin and mix thoroughly.

[0065] Film formation: The solvent in the above lipid mixture was evaporated using a rotary evaporator to form a uniform lipid film.

[0066] Step 4: Membrane coating of nanoparticles

[0067] Membrane hydration: After the film is dried, add the PLGA nanoparticle suspension obtained in step 2. Add membrane proteins according to different membrane protein mass ratios and vortex to mix to make the membrane proteins evenly distributed.

[0068] Heating cycle: The heating cycle was performed at 45°C for 3 minutes each time and repeated 3 times to promote the coating of lipid film on the surface of PLGA nanoparticles.

[0069] Uniform extrusion: The membrane-coated solution was extruded 10 times through a 200 nm pore size cellulose acetate membrane to ensure uniform particle size.

[0070] Step 5: Isolation and storage of novel artificial exosomes

[0071] Particle collection and storage: The final artificial exosome suspension was stored at 4°C. Or it can be freeze-dried and stored at -20°C to extend the shelf life.

[0072] The technical solution of the present invention is described below through specific embodiments.

[0073] Example 1 Low concentration antioxidant and high membrane protein ratio

[0074] Materials and ratio:

[0075] PLGA: 100 mg;

[0076] DOTAP: 5 mg;

[0077] OCT4 protein: 0.1 mg;

[0078] siTrim28: 0.1 mg;

[0079] DPPC:DSPC:DOPC:cholesterol=5:1:3:1 (molar ratio);

[0080] DPPE-PEG2000 is 5% of DPPC mass;

[0081] α-Tocopherol: 0.02mM (low concentration antioxidant);

[0082] alpha-lactalbumin: 0.01mM;

[0083] The mass ratio of membrane protein to lipid was 1:200.

[0084] Example 2: Medium concentration of antioxidant and medium membrane protein ratio

[0085] Materials and ratio:

[0086] PLGA: 100 mg;

[0087] DOTAP: 5 mg;

[0088] OCT4 protein: 0.1 mg;

[0089] siTrim28: 0.1 mg;

[0090] DPPC:DSPC:DOPC:cholesterol=5:1:3:1 (molar ratio);

[0091] DPPE-PEG2000 is 10% of the mass of DPPC;

[0092] α-Tocopherol: 0.05mM (medium concentration antioxidant);

[0093] alpha-lactalbumin: 0.02mM;

[0094] The mass ratio of membrane protein to lipid was 1:300.

[0095] Example 3 High concentration antioxidant and low membrane protein ratio

[0096] Materials and ratio:

[0097] PLGA: 100 mg;

[0098] DOTAP: 5 mg;

[0099] OCT4 protein: 0.1 mg;

[0100] siTrim28: 0.1 mg;

[0101] DPPC:DSPC:DOPC:cholesterol=5:1:3:1 (molar ratio);

[0102] DPPE-PEG2000: 15% of DPPC mass;

[0103] α-Tocopherol: 0.1mM (high concentration antioxidant);

[0104] alpha-lactalbumin: 0.03mM;

[0105] The mass ratio of membrane protein to lipid was 1:500.

[0106] Comparative Example 1: No antioxidant, no PEG modification

[0107] Materials and ratio:

[0108] PLGA: 100 mg;

[0109] DOTAP: 5 mg;

[0110] OCT4 protein: 0.1 mg;

[0111] siTrim28: 0.1 mg;

[0112] DPPC:DSPC:DOPC:cholesterol=5:1:3:1 (molar ratio);

[0113] The mass ratio of membrane protein to lipid was 1:200;

[0114] The experimental parameters were the same as those in Example 1, but α-tocopherol and DPPE-PEG2000 were not added.

[0115] Comparative Example 2 PLGA / DOTAP Nanoparticles without Membrane Protein Coating

[0116] Materials and ratio:

[0117] PLGA: 100 mg;

[0118] DOTAP: 5 mg;

[0119] OCT4 protein: 0.1 mg;

[0120] siTrim28: 0.1mg.

[0121] The experimental parameters were the same as those in Example 1, but PLGA nanoparticles were directly prepared without membrane coating.

[0122] Test Example 1

[0123] The products obtained from Examples 1-3 and Comparative Examples 1-2 were tested. The experimental testing process is as follows:

[0124] 1. Particle size, PDI and Zeta potential determination

[0125] Dynamic light scattering (DLS) was used to measure the particle size, PDI and Zeta potential of each sample.

[0126] Each sample was tested three times and the average value was recorded.

[0127] 2. Stability test

[0128] Each sample was stored at 37°C, and the particle size and PDI were measured every 24 hours. The monitoring was continued for 7 days, and the changes were recorded.

[0129] Use centrifugation to detect precipitation and determine whether the sample is aggregated or sedimented.

[0130] 3. Targeted detection

[0131] The nanoparticles were co-incubated with the targeted cells (tumor cells) for 6 h, and the cellular uptake efficiency was observed.

[0132] The nanoparticles were labeled with fluorescent markers and images were taken using a fluorescence microscope (e.g. Figure 2 The fluorescence intensity was detected by flow cytometry and the uptake rate was calculated.

[0133] 4. Results

[0134] The test results are shown in Table 1.

[0135] Table 1

[0136] sample Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Particle size (nm) 120.5 132.4 145.7 150.8 110.3 PDI 0.160 0.142 0.154 0.215 0.250 Zeta potential (mV) -15.8 -17.3 -18.9 -10.2 28.3 Stability (particle size change within 7 days, %) ＜5 ＜3 ＜4 ＞20 ＞30 Targeted uptake rate (%) 75.3 82.5 78.9 35.6 20.4

[0137] According to Table 1, the artificial exosomes prepared in Examples 1-3 showed good performance in terms of particle size, PD I, Zeta potential, stability and targeted uptake rate. Specifically:

[0138] In terms of particle size, the particle sizes of Examples 1-3 are 120.5 nm, 132.4 nm and 145.7 nm, respectively, all of which are within the appropriate nanoparticle size range and are conducive to cellular uptake and distribution in the body.

[0139] The PDI value (polydispersity index) reflects the uniformity of the particle size distribution. The PDI values ​​of Examples 1-3 are all lower than 0.2, indicating that the particle size distribution is relatively uniform.

[0140] Zeta potential is a measure of the surface charge of the particles, and a negative value indicates that the particle surface carries a negative charge, which helps the stability of the particles in the body and reduces nonspecific adsorption. The Zeta potential values ​​of Examples 1-3 are -15.8 mV, -17.3 mV and -18.9 mV, respectively, all showing good stability.

[0141] In terms of stability, the particle size changes of Examples 1-3 were all less than 5% within 7 days of storage at 37°C, showing good thermal stability.

[0142] In terms of targeted uptake rate, the uptake rates of Examples 1-3 were 75.3%, 82.5% and 78.9%, respectively, which were significantly higher than those of Comparative Examples 1 and 2, indicating that membrane protein coating and the addition of antioxidants help to improve the targeting of nanoparticles.

[0143] Although comparative example 1 performed well in terms of particle size and PD I, it had a low Zeta potential value, poor stability, and a targeted uptake rate that was significantly lower than that of the example, indicating that the absence of added antioxidants and PEG modification had a negative impact on the stability and targeting of the nanoparticles.

[0144] Since comparative example 2 was not coated with membrane protein, its particle size was smaller, but its stability was the worst and its targeted uptake rate was the lowest, indicating that membrane protein coating is crucial to improving the stability and targeting of nanoparticles.

[0145] In summary, the stability and targeting of PLGA nanoparticles can be significantly improved by adding DPPE-PEG2000 and α-tocopherol and coating with membrane proteins, thereby preparing artificial exosomes with better performance.

[0146] 5. Principle and mechanism analysis

[0147] The performance optimization of artificial exosomes mainly depends on the design of their structure and composition. In this study, by simulating the composition and function of natural exosomes, we successfully prepared artificial exosomes with better performance. The following is an analysis of the principle mechanism of key factors:

[0148] a. Membrane protein coating: Membrane protein coating provides artificial exosomes with surface properties similar to natural exosomes, which not only helps to improve their stability and circulation time in the body, but also enhances their interaction and targeting with target cells by simulating the surface markers of natural exosomes. Membrane proteins can promote cellular uptake and improve the targeted uptake rate of nanoparticles through receptor-mediated endocytosis.

[0149] b. PEG modification: The addition of DPPE-PEG2000 provides hydrophilic PEG chains to the nanoparticles, which helps to reduce nonspecific adsorption and reduce the recognition of the immune system, thereby improving the biocompatibility and stability of the nanoparticles. The "invisible cloak" formed by the PEG chain can effectively avoid the adsorption of serum proteins, reduce the clearance of the reticuloendothelial system, and prolong the circulation time of nanoparticles in the blood.

[0150] c. Addition of antioxidants: α-tocopherol, as an antioxidant, can protect membrane proteins and lipids from damage by oxidative stress and maintain the structural and functional integrity of nanoparticles. During the preparation and storage process, antioxidants help reduce lipid peroxidation and maintain the stability and biological activity of nanoparticles.

[0151] d. Lipid composition: The specific molar ratio of DPPC, DSPC, DOPC and cholesterol is designed to mimic the physical properties of cell membranes, such as fluidity and stability. DPPC and DSPC, as the main phospholipid components, provide membrane rigidity and stability; DOPC increases membrane fluidity; and cholesterol helps regulate membrane permeability and stability.

[0152] e. Protein to lipid ratio: The mass ratio of protein to lipid has a significant effect on the targeting and stability of nanoparticles. In this study, by optimizing the ratio of protein to lipid, we achieved a reasonable distribution of membrane proteins on the surface of nanoparticles, thereby improving their targeting and stability.

[0153] g. Structure of alpha-lactalbumin: alpha-lactalbumin can form a stable α-helical structure in an acidic environment, which enables it to stably form a monolayer at the oil-water interface, thereby effectively stabilizing the lipid membrane. In addition, the protein also has the ability to promote calcium ion binding, which contributes to the stability and flexibility of the lipid membrane, enabling it to better protect drug molecules from damage by the external environment during drug delivery. In the preparation of artificial exosomes, the addition of alpha-lactalbumin not only improves the stability of the lipid membrane, but also gives artificial exosomes certain biological activity. Studies have shown that alpha-lactalbumin can bind to specific receptors on the cell surface, promote cellular uptake of exosomes, and thus improve the delivery efficiency of drugs. In addition, the protein also has a certain immunomodulatory effect, which helps to reduce the immune response that may be caused during drug delivery.

[0154] In summary, the present invention successfully prepared artificial exosomes with better performance by comprehensively considering membrane protein coating, PEG modification, addition of antioxidants, lipid composition and the ratio of protein to lipid. These optimization measures significantly improved the stability and targeting of nanoparticles, providing a powerful tool for future drug delivery and therapeutic applications.

[0155] It is understood that in drug delivery, the artificial exosomes can be used to load and deliver drug molecules, such as small molecule drugs, proteins, nucleic acids, etc. Due to the lipid bilayer structure and membrane protein presence of artificial exosomes, they can mimic the properties of natural exosomes and improve the stability and targeting of drugs in vivo. In addition, by optimizing the ratio of PLGA and DOTAP, as well as the molar ratio of lipid components, the drug loading efficiency and delivery efficiency can be further improved.

[0156] In specific applications, the artificial exosomes can be used to treat various diseases, including but not limited to cancer, cardiovascular disease, neurodegenerative diseases, etc. For example, in cancer treatment, artificial exosomes can be loaded with chemotherapeutic drugs or siRNA, which can increase the local concentration of drugs by targeting tumor cells and reduce toxic effects on normal cells, thereby improving the therapeutic effect and reducing side effects.

[0157] In the preparation of drug delivery products, the artificial exosomes can be used as a component of a drug delivery system and combined with other drug delivery technologies, such as combining with targeting ligands to enhance targeting, or combining with biodegradable polymers to prolong drug release time.

[0158] In summary, the artificial exosomes and the preparation method thereof provided by the present invention not only overcome the deficiencies of the prior art, but also provide a new solution for the field of drug delivery and have broad application prospects.

[0159] In addition, it can be understood that the technical solution of the present invention is an improvement on the patented technology of CN110403917A, which is an artificial exosome, its preparation method and application. Compared with the present invention, the CN110403917A patent has technical disadvantages. Specific comparison:

[0160] 1. The membrane protein extraction steps are complicated

[0161] The membrane protein extraction in CN110403917A relies on a multi-step procedure, including repeated centrifugation, freeze-thaw, etc., which may lead to the loss or denaturation of membrane proteins and affect the quality of the final product. In particular, during the operation, the degree of cell disruption and the recovery efficiency of membrane proteins are easily affected.

[0162] The scheme of the present invention reduces the number and steps of cell disruption by optimizing the membrane protein extraction steps, and adopts a more efficient solvent system and liposome encapsulation method, thereby improving the purity and stability of the membrane protein.

[0163] 2. Solvent evaporation method for preparation of PLGA nanoparticles

[0164] In the patent CN110403917A, the preparation of PLGA nanoparticles relies on the solvent evaporation method. Although this method is commonly used, it may have the problem of solvent residue in some cases, affecting the safety of the preparation. In addition, the emulsification step in the process is relatively complicated, which may lead to poor dispersion of nanoparticles.

[0165] The scheme of the present invention combines the water-in-oil-in-water emulsion method with the thin layer evaporation method, which not only avoids the residual solvent, but also adds membrane protein modification during the emulsification process, thereby increasing the biocompatibility and stability of the nanoparticles.

[0166] 3. Nanoparticle size and Zeta potential control

[0167] In patent CN110403917A, although the particle size and Zeta potential of the nanoparticles are characterized, there may be certain fluctuations in the particle size and dispersibility, which may affect the final drug delivery effect.

[0168] The novel solution of the present invention controls different protein:lipid mass ratios and optimizes the coating process, so that the membrane protein-modified nanoparticles have a smaller particle size and a lower PDI value, thereby further improving the dispersibility and stability.

[0169] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that the specific implementation methods of the present invention can still be modified or replaced by equivalents. Any modification or equivalent replacement that does not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing artificial exosomes, characterized in that: include: The PLGA and DOTAP are dissolved in dichloromethane and mixed thoroughly to prepare an oil phase, and the OCT4 protein and siTrim28 are dissolved in nuclease-free water to prepare an aqueous phase; the aqueous phase is added dropwise to the oil phase, and a primary emulsion is obtained after ultrasonic treatment; the primary emulsion is added dropwise to a PVA aqueous solution, and a double emulsion is obtained after ultrasonic treatment again; the double emulsion is added dropwise to the PVA aqueous solution again, stirred, and the dichloromethane is volatilized to obtain an emulsified solution; The emulsified solution is centrifuged to retain the precipitate, and washed with nuclease-free water to remove residual PVA and other impurities to obtain PLGA nanoparticles; Dissolving DPPC, DSPC, DOPC and cholesterol in a mixed solvent of analytical grade chloroform and anhydrous methanol, respectively, to obtain a lipid mixture; adding DPPE-PEG2000 to the lipid mixture, and then adding α-tocopherol and alpha-lactalbumin, and mixing them thoroughly to obtain a lipid mixture; evaporating the solvent in the lipid mixture to form a uniform lipid film; The PLGA nanoparticles are made into a PLGA nanoparticle suspension, the dried lipid film is added to the PLGA nanoparticle suspension, and membrane protein is added, vortexed and mixed, and then cyclically heated to obtain a membrane-coated solution; the membrane-coated solution is repeatedly extruded through a cellulose acetate membrane to obtain the artificial exosomes.

2. The method for preparing artificial exosomes according to claim 1, characterized in that: The mass ratio of PLGA to DOTAP is 1:20-30.

3. The method for preparing artificial exosomes according to claim 1, characterized in that: The molar ratio of DPPC, DSPC, DOPC and cholesterol is 5:1:3:

1.

4. The method for preparing artificial exosomes according to claim 1, characterized in that: The cyclic heating is at 45±2° C., at least 3 minutes each time, and is repeated at least 3 times.

5. The method for preparing artificial exosomes according to claim 1, characterized in that: The pore size of the cellulose acetate membrane is 200 nm.

6. The method for preparing artificial exosomes according to claim 1, characterized in that: The PLGA nanoparticles are resuspended in nuclease-free water or PBS to obtain the PLGA nanoparticle suspension.

7. The method for preparing artificial exosomes according to claim 1, characterized in that: The DPPE-PEG2000 accounts for 5% of the mass of the DPPC.

8. The method for preparing artificial exosomes according to claim 1, characterized in that: The mass ratio of the membrane protein to the lipid is 1:100-500.

9. An artificial exosome, characterized in that The artificial exosomes are prepared according to the method for preparing artificial exosomes according to any one of claims 1 to 8.

10. A use of the artificial exosomes according to claim 9, characterized in that: The application includes at least one of the following: A1) Application of the artificial exosomes in drug delivery; A2) Use of the artificial exosomes in preparing drug delivery products.

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