Artificial exosome for drug delivery and preparation method thereof

Abstract

The invention relates to the technical field of medicine, and discloses an artificial exosome for drug delivery and a preparation method thereof.The preparation method comprises the steps that PCL-PEG-PCL, DC-Chol and TA-PEG-TA are used for constructing a polymer core, OCT4 protein, siTrim28 and VC-Lip are loaded, and a suspension is formed through high pressure-ultrasonic synergistic treatment and solvent volatilization and curing; and performing dynamic thin film formation, multi-layer self-assembly coating, gradient centrifugal purification and hyaluronic acid anti-enzymolysis modification to obtain a finished product. The artificial exosome disclosed by the invention is obviously superior to artificial exosomes prepared in the prior art in four core indexes, namely structural stability, release performance, targeting specificity and damage resistance.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medical materials, in particular to an artificial exosome for drug delivery and a preparation method thereof. BACKGROUND

[0002] As a nanoscale vesicle secreted by natural cells, exosomes have great potential in the field of drug delivery due to their good biocompatibility and cell communication function. However, natural exosomes have defects such as low yield, complex composition, and difficulty in standardized production, which limit their large-scale clinical application. Therefore, artificial exosomes, by simulating the structure and function of natural exosomes, have become a research hotspot in drug delivery systems due to their strong controllability and scalability.

[0003] However, in the prior art, the preparation of artificial exosomes mostly adopts a core structure of "polymer core-lipid membrane coating", for example, patent CN119564883A discloses an artificial exosome with PLGA as the polymer core and a lipid mixture as the coating, which is prepared by ultrasonic emulsification and film coating process. However, it has significant technical defects: first, the bonding force between the polymer core and the lipid membrane is weak, and the coating is uneven due to the simple ultrasonic emulsification, which easily leads to dissociation in the body circulation; second, there is no optimization design for the release performance, and the drug is prone to "burst release" in normal tissues, which cannot achieve precise release at the lesion site; third, the targeting relies only on single molecule modification (such as alpha-lactalbumin), which has insufficient recognition specificity for diseased cells, low target uptake rate, and significant off-target effect. Meanwhile, patent CN110403917A discloses an artificial exosome with PLGA-DOTAP as the core and a lipid membrane coating, which is prepared by ultrasonic emulsification and thin layer evaporation. However, this technology has poor release performance, is prone to burst release, and also has the problems of uneven lipid membrane coating and lack of active targeting design.

[0004] Therefore, it is of great practical significance to propose an artificial exosome for drug delivery to solve the problems of poor release performance, burst release, uneven lipid membrane coating, and lack of active targeting design in the prior art. SUMMARY

[0005] In view of this, the present application proposes an artificial exosome for drug delivery and a preparation method thereof, aiming to solve the problems of poor release performance, burst release, uneven lipid membrane coating, and lack of active targeting in the prior art.

[0006] The present application proposes a preparation method of an artificial exosome for drug delivery, which includes the following preparation steps: Said PCL-PEG-PCL, DC-Chol and TA-PEG-TA are dissolved in a chloroform-ethanol mixed solvent, stirred until dissolved to obtain an oil phase, said OCT4 protein, siTrim28 and VC-Lip are dissolved in a deionized water and subjected to ultrasonic dispersion treatment, then hyaluronic acid is added and stirred to obtain an aqueous phase, said oil phase and aqueous phase are subjected to high-pressure-ultrasonic synergistic treatment to obtain a W / O primary emulsion, said W / O primary emulsion is added into a PVA aqueous solution for volatile solidification treatment to obtain a ROS-responsive polymer core suspension; Said DPPC, ceramide, cholesterol and DSPE-PEG-FA are dissolved in a chloroform-ethanol mixed solution, stirred, then VC-Lip is added and stirred to obtain a first mixed solution, said first mixed solution is subjected to rotary evaporation treatment to obtain a folic acid targeted functional lipid film; Said ROS-responsive polymer core suspension is added into said folic acid targeted functional lipid film for multilayer self-assembly coating treatment, after the end, uniformization is performed by microfluidic extrusion to obtain an artificial exosome crude body; Said artificial exosome crude body is subjected to gradient centrifugation purification to collect a precipitate, said precipitate is subjected to anti-enzymatic modification treatment and then filtered by a polyether sulfone filter to obtain said artificial exosome for drug delivery.

[0007] Preferably, said high-pressure-ultrasonic synergistic treatment is: Said aqueous phase is added into the oil phase at a speed of 0.2-0.4 ml / min under ultrasonic treatment at a temperature of 35-40℃, while the high-pressure homogenization cycle is started, after the end of the addition of said aqueous phase, the high-pressure homogenization is kept for 1-2 cycles and then stopped; The feeding speed of said high-pressure homogenization cycle is 0.8-1.2 times / min of the total volume of the aqueous phase and the oil phase, and the pressure is 700-900 bar; The power of said ultrasonic treatment is 120-140, and the ultrasonic cycle is 3 seconds of ultrasonic treatment and 2 seconds of pause.

[0008] Preferably, said volatile solidification is: Said primary emulsion is added into a PVA aqueous solution with a volume of 5-8 times of the primary emulsion, and the PVA aqueous solution is 8%-10%, and the magnetic stirring is performed at a speed of 900-1100 rpm at 4℃ for 4-6 hours.

[0009] Preferably, said rotary evaporation treatment is: Said first mixed solution is subjected to rotary evaporation at a speed of 8-12 ml / min under reduced pressure at a speed of 100-130 rpm at 30-40℃ for 30-45 minutes, and then vacuum dried for 2-3 hours.

[0010] Preferably, said multilayer self-assembly coating treatment is: adding the ROS-responsive polymer core suspension into the folate-targeted functional lipid membrane, oscillating at 140-160 rpm for 25-35 minutes under a water bath at 35-40℃ to obtain a primary coating solution; adding 2%-3% PVA aqueous solution and 0.3%-0.5% TA-PEG-TA of the primary coating solution into the primary coating solution, incubating at 40-50℃ for 8-12 minutes while vortexing at 1700-1900 rpm for 1-2 minutes.

[0011] Preferably, the gradient centrifugation purification is as follows: centrifuging the crude artificial exosome at 50000-70000 rpm for 25-35 minutes at 4℃, discarding the supernatant, resuspending with VC-Lip / PBS aqueous solution, and then centrifuging at 70000-90000 rpm for 35-45 minutes at 4℃, and collecting the precipitate after centrifugation; wherein the concentration of VC-Lip in the VC-Lip / PBS aqueous solution is 0.1-0.2 mg / mL.

[0012] Preferably, the anti-enzymatic modification is as follows: resuspending the precipitate with hyaluronic acid / PBS aqueous solution, and incubating at 35-40℃ for 15-25 minutes; wherein the concentration of hyaluronic acid in the hyaluronic acid / PBS aqueous solution is 2.5 mg / mL.

[0013] Preferably, when the PCL-PEG-PCL, DC-Chol and TA-PEG-TA are dissolved in a chloroform-ethanol mixed solvent, the mass ratio of the PCL-PEG-PCL, DC-Chol and TA-PEG-TA is 100: (5-8): (6-10), and the volume of the chloroform-ethanol mixed solvent is 20-30 times the total mass of the PCL-PEG-PCL, DC-Chol and TA-PEG-TA.

[0014] Preferably, when the DPPC, ceramide, cholesterol and DSPE-PEG-FA are dissolved in a chloroform-ethanol mixed solution, the molar ratio of the DPPC, ceramide, cholesterol and DSPE-PEG-FA is 100: (20-30): (30-40): (8-12), and the volume of the chloroform-ethanol mixed solution is 15-20 times the total mass of the DPPC, ceramide, cholesterol and DSPE-PEG-FA.

[0015] The application further provides an artificial exosome for drug delivery prepared by the preparation method of the artificial exosome for drug delivery.

[0016] Compared with the prior art, the present application has the beneficial effects that: 1. Strong structural stability: The present application uses multi-layer self-assembly coating treatment, in which PCL-PEG-PCL provides a stable polymer core skeleton, DC-Chol enhances electrostatic binding with the lipid membrane through cationicity, and TA-PEG-TA strengthens the core membrane interface binding force through ROS-responsive cross-linking bonds, greatly improving the binding force between the lipid membrane and the polymer core and thus enhancing the stability of the artificial exosome.

[0017] 2. Excellent release performance: The present application adds ROS-responsive cross-linking agent TA-PEG-TA, which can quickly break the cross-linking bonds in the tumor microenvironment and remain stable in normal tissues; thus, the drug can be accumulated and released at a rate of up to 90% in the lesion site for 48 hours.

[0018] 3. Strong targeting specificity: The present application uses folate-targeting component DSPE-PEG-FA to modify the lipid membrane, which can specifically bind to the folate receptor on the surface of tumor cells, significantly enhancing the targeting recognition ability of folate receptor-positive tumor cells (such as HeLa cells); thus, the artificial exosome has a high target uptake rate.

[0019] 4. Strong anti-destruction ability: The present application adds VC-Lip and hyaluronic acid, which can inhibit lipid oxidation and protein denaturation, and hyaluronic acid can form an anti-enzymatic protective layer on the surface of the artificial exosome; thus, the drug retention rate of the artificial exosome in the gastrointestinal tract (pH 1.2) and blood environment is improved. DETAILED DESCRIPTION

[0020] The various exemplary embodiments of the present application will now be described in detail, which should not be considered as limiting the present application, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present application. It should be understood that the terms described in the present application are only for describing the specific embodiments, and are not intended to limit the present application.

[0021] In addition, for the numerical range in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between any stated value or intermediate value within the range and any other stated value or intermediate value within the range is also included in the present application. The upper limit and the lower limit of these smaller ranges can be independently included or excluded from the range.

[0022] Unless otherwise indicated, all technical and scientific terms have the same meaning as those one of ordinary skill in the art of the disclosure would understand. Although preferred methods and materials are described, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure. All documents mentioned herein are incorporated by reference to disclose and describe in further detail the methods and / or materials associated with the documents. In the case of conflict between the content of the specification and that of any document incorporated by reference, the content of the specification controls.

[0023] Many modifications and variations of the present disclosure described in the detailed description of the disclosure can be made by those of ordinary skill in the art without departing from the scope or spirit of the disclosure. Other embodiments of the disclosure will be apparent to those skilled in the art from the disclosure. The disclosure described in the specification and examples is merely exemplary.

[0024] As used herein, "comprise", "comprising", "have", "having", "include", "including", "contain", "containing", and the like, are open-ended terms that are intended to mean including, but not limited to.

[0025] The present disclosure provides a preparation method of an artificial exosome for drug delivery, comprising the following preparation steps: PCL-PEG-PCL, DC-Chol and TA-PEG-TA are dissolved in a chloroform-ethanol mixed solvent, stirred until dissolved to obtain an oil phase, OCT4 protein, siTrim28 and VC-Lip are dissolved in nuclease-free water, ultrasonic dispersion treatment is performed, then hyaluronic acid is added and stirred to obtain an aqueous phase, the oil phase and the aqueous phase are subjected to high-pressure-ultrasonic synergistic treatment to obtain a W / O primary emulsion, the W / O primary emulsion is added to a PVA aqueous solution for volatile solidification treatment to obtain a ROS-responsive polymer core suspension; DPPC, ceramide, cholesterol and DSPE-PEG-FA are dissolved in a chloroform-ethanol mixed solution, stirred, then VC-Lip is added and stirred to obtain a first mixed solution, the first mixed solution is subjected to rotary evaporation treatment to obtain a folic acid-targeted functional lipid membrane; The ROS-responsive polymer core suspension is added to the folic acid-targeted functional lipid membrane for multi-layer self-assembly coating treatment, and after the treatment is completed, microfluidic extrusion is performed for homogenization to obtain an artificial exosome crude body; The artificial exosome crude body is subjected to gradient centrifugation purification to collect the precipitate, the precipitate is subjected to anti-enzymatic modification treatment, then filtered with a polyether sulfone filter membrane to obtain the artificial exosome for drug delivery.

[0026] Specifically, the volume ratio of chloroform to ethanol in the chloroform-ethanol mixed solvent is 2:1, and the specific preparation method is as follows: Trichloromethane is injected into a dry, pre-cooled (4±1℃) closed container, ethanol is added to the trichloromethane according to a volume ratio of 2:1, stirring (450 rpm) is performed while adding, stirring is performed until a uniform, transparent mixed solvent is formed, then standing for 3 minutes, and confirming that the mixed solvent is not layered.

[0027] Specifically, when the OCT4 protein, siTrim28 and VC-Lip are dissolved in nuclease-free water, the mass ratio of the OCT4 protein, siTrim28 and VC-Lip is 1:1:3.5, and the volume of the nuclease-free water is half of the volume of the oil phase.

[0028] Specifically, when the OCT4 protein, siTrim28 and VC-Lip are dissolved in nuclease-free water and subjected to ultrasonic dispersion treatment, and then hyaluronic acid is added and stirred, the mass of the hyaluronic acid is 2.5% of the total mass of PCL-PEG-PCL, DC-Chol, TA-PEG-TA, OCT4 protein, siTrim28 and VC-Lip. Specifically, when the oil phase and the water phase are subjected to high-pressure-ultrasonic synergistic treatment, the mass of the VC-Lip in the water phase is ensured to be 0.4-0.6% of the total mass of PCL-PEG-PCL, DC-Chol and TA-PEG-TA in the oil phase.

[0029] Specifically, when the W / O primary emulsion is added to the PVA aqueous solution for volatilization solidification treatment, the concentration of the PVA aqueous solution is 9%, and the volume of the PVA aqueous solution is 6 times the volume of the W / O primary emulsion.

[0030] Specifically, when the DPPC, ceramide, cholesterol and DSPE-PEG-FA are dissolved in a trichloromethane-ethanol mixed solution, a first mixed solution is obtained by stirring after adding the VC-Lip and continuing to stir, and the mass of the VC-Lip is 1% of the total mass of DPPC, ceramide, cholesterol and DSPE-PEG-FA.

[0031] Specifically, when the ROS-responsive polymer core suspension is added to the folic acid-targeted functional lipid membrane for multilayer self-assembly coating treatment, the solid-liquid ratio of the folic acid-targeted functional lipid membrane to the ROS-responsive polymer core suspension is 100-200:1 (mg / ml).

[0032] Specifically, the microfluidic extrusion is as follows: the solution after the multilayer self-assembly coating treatment is passed through a microfluidic chip (channel size 200 nm, pressure 0.4 MPa) for 20 cycles of extrusion.

[0033] It can be understood that the present application greatly improves the binding force of the lipid membrane and the polymer core by the multi-layer self-assembly coating treatment, in which the PCL-PEG-PCL provides a stable polymer core skeleton, the DC-Chol enhances the electrostatic binding with the lipid membrane through cationic enhancement, and the TA-PEG-TA strengthens the core membrane interface binding force through the ROS-responsive cross-linking bond, thereby enhancing the stability of the artificial exosome.

[0034] It can be understood that the present application realizes that the drug has a 48-hour cumulative release rate of up to 90% at the lesion site by adding the ROS-responsive cross-linking agent TA-PEG-TA, which can quickly break the cross-linking bond under the tumor microenvironment and remains stable in normal tissues.

[0035] It can be understood that the present application significantly enhances the targeting recognition ability of the artificial exosome for the folate receptor positive tumor cells (such as HeLa cells) by using the folate targeting component DSPE-PEG-FA to modify the lipid membrane, which can specifically bind to the folate receptors on the surface of tumor cells.

[0036] In the present application, the high-pressure-ultrasound synergistic treatment is preferably: The water phase is dropped into the oil phase at a speed of 0.2-0.4 ml / min under a temperature of 35-40°C and ultrasonic, and the high-pressure homogenization cycle is started, and after the water phase dropping is completed, the high-pressure homogenization is maintained for 1-2 cycles and then stopped; The feeding speed of the high-pressure homogenization cycle is 0.8-1.2 times of the total volume of the water phase and the oil phase per minute, and the pressure is 700-900 bar; The power of the ultrasonic is 120-140 W, and the ultrasonic cycle is 3 seconds on and 2 seconds off every 3 seconds.

[0037] Specifically, the oil phase is injected into a double-layer jacketed glass container, the jacketed ice water circulation is started to stabilize the temperature of the oil phase between 35-40°C, the ultrasonic probe is inserted into the oil phase (the immersion depth of the probe is between 1 / 3-1 / 2 of the liquid level of the oil phase), the feeding port of the high-pressure homogenizer is connected to the outlet of the glass container through a sterile pipeline, and the discharge port is returned to the glass container (forming a "circulating emulsification" path), the water phase is dropped into the oil phase at a speed of 0.2-0.4 ml / min, the high-pressure homogenizer and the ultrasonic probe (power 120-140 W, pulse mode "3 seconds on, 2 seconds off") are started synchronously, the feeding speed is 0.8-1.2 times of the total volume of the water phase and the oil phase per minute, the pressure is 700-900 bar, and the high-pressure homogenization cycle is performed, and when the water phase is completely dropped, the ultrasonic and high-pressure homogenization are maintained until 1-2 cycles are performed and then stopped.

[0038] It can be understood that the present application realizes efficient and uniform nanoscale dispersion of the water phase (OCT4 protein, siTrim28, and VC-Lip containing aqueous enzyme-free solution) in the oil phase (PCL-PEG-PCL, DC-Chol, and TA-PEG-TA containing chloroform-ethanol mixed solution) through high-pressure-ultrasonic synergistic treatment: the high-pressure homogenization breaks the local agglomerates in the water phase through high-pressure shearing force of 700-900 bar, and rapidly disperses the water phase into 100-150 nm droplets, solving the defects of insufficient dispersion force and easy formation of large particle size agglomerates in the prior art through pure ultrasonic emulsification; the simultaneously started ultrasonic equipment (120-140 W power, 3 seconds on / 2 seconds off pulse mode) further refines the water phase droplets to 80-100 nm through cavitation effect, while avoiding local overheating caused by continuous ultrasonic, protecting the activity of OCT4 protein, and providing a uniform polymer core basis for subsequent layer-by-layer self-assembly of lipid membrane, finally ensuring the integrity and stability of the "core-membrane" structure of artificial exosomes.

[0039] In the present application, the volatile curing is preferably: adding the initial emulsion into 8%-10% PVA aqueous solution with a volume of 5-8 times that of the initial emulsion, and stirring at 900-1100 rpm at 4°C for 4-6 hours.

[0040] Specifically, 8%-10% in the 8%-10% PVA aqueous solution is the mass volume (w / v) concentration of the PVA aqueous solution, that is, 8-10 g of PVA is contained in 100 ml of water.

[0041] It can be understood that the present application realizes complete removal of the chloroform-ethanol mixed solvent in the initial emulsion and stable formation of the polymer core through volatile curing: on the one hand, by slowly adding the initial emulsion into 8%-10% PVA aqueous solution (with a volume of 5-8 times that of the initial emulsion), PVA can form a temporary protective shell on the surface of the polymer core of the initial emulsion as a dispersion stabilizer, effectively preventing the initial emulsion droplets from agglomerating due to interfacial tension changes during solvent evaporation, and solving the problem of particle agglomeration (PDI>0.16) caused by pure solvent evaporation; on the other hand, the low temperature environment of 4°C can inhibit the activity degradation of OCT4 protein and siTrim28, and the magnetic stirring speed of 900-1100 rpm can accelerate the diffusion and evaporation of the chloroform-ethanol solvent into the PVA aqueous solution through continuous fluid disturbance, while avoiding shear damage to the initially formed polymer core structure caused by high speed, and the stirring time of 4-6 hours guarantees complete evaporation of the solvent, finally forming a polymer core suspension with uniform particle size and stable structure, providing a uniform and pure core substrate for subsequent steps, while avoiding the problems of solvent residue affecting the biocompatibility of the product and low coating efficiency caused by uneven particles.

[0042] In the present application, the rotary evaporation treatment is preferably: the first mixed solution is subjected to rotary evaporation under reduced pressure at 30-40℃ with a rotation speed of 100-130rpm for 30-45 minutes, while nitrogen is introduced at a speed of 8-12ml / min, and after the end, vacuum drying for 2-3 hours.

[0043] Specifically, the pressure of the rotary evaporation under reduced pressure is 2.5kPa.

[0044] It can be understood that, on the one hand, the rotary evaporation under reduced pressure (100-130rpm) at 30-40℃ can quickly remove the chloroform-ethanol solvent in the lipid mixture under low temperature environment, avoiding denaturation of DPPC, DSPE-PEG-FA and other lipid components (such as inactivation of the folate targeting group of DSPE-PEG-FA) caused by high temperature, and the appropriate rotation speed makes the lipid evenly spread on the inner wall of the flask, preventing local accumulation to form dense clumps, solving the problem of uneven distribution of lipids in the preparation of simple thin films in the prior art; on the other hand, the nitrogen gas introduced at a flow rate of 8-12mL / min can not only accelerate the evaporation efficiency of the solvent and shorten the film formation time, but also isolate air to prevent lipid oxidation, and the porous structure formed can increase the contact area between the subsequent polymer core suspension and the lipid, improve the coating efficiency, and avoid insufficient coating caused by dense thin film; finally, the vacuum drying for 2-3 hours further removes the residual solvent in the thin film, ensuring the purity and stability of the lipid thin film, laying a foundation for the subsequent formation of uniform "polymer core-lipid film" structure by multilayer self-assembly coating, and avoiding the defects of dense lipid thin film, inactivation of targeting group or solvent residue affecting the biocompatibility and targeting of the product in the prior art.

[0045] In the present application, the multilayer self-assembly coating treatment is preferably: The ROS-responsive polymer core suspension is added to the folate-targeting functional lipid film, and oscillated at a speed of 140-160rpm for 25-35 minutes under a water bath at 35-40℃ to obtain a primary coating solution; 2%-3% PVA aqueous solution and 0.3%-0.5% TA-PEG-TA of the mass of the primary coating solution are added to the primary coating solution, and incubated at 40-50℃ for 8-12 minutes while vortexed at a speed of 1700-1900rpm for 1-2 minutes.

[0046] Specifically, 2%-3% in the 2%-3% PVA aqueous solution is the mass volume (w / v) concentration of the PVA aqueous solution, i.e. 2-3g of PVA is contained in 100ml of water.

[0047] It can be understood that, on the one hand, the multi-layer self-assembly coating realizes the ordered and uniform coating of the lipid membrane on the surface of the polymer core through the electrostatic interaction between the carboxyl of the TA-PEG-TA on the surface of the polymer core and the amino of the DC-Chol in the lipid membrane, the hydrophobic force between the lipid molecules, significantly enhances the "core-membrane" binding force, and solves the problem of loose and easy dissociation of the core-membrane combination in the prior art; on the other hand, relying on the ROS-responsive cross-linking bond of TA-PEG-TA, the cross-linking state is maintained under normal tissue environment, ensuring the stability of the artificial exosome structure and the non-burst release of the drug, while the cross-linking bond is quickly broken under the tumor microenvironment, realizing the precise release of the drug, and the folate-modified lipid membrane enhances the uptake rate by targeted binding to the folate receptor of tumor cells; in addition, the multi-layer structure formed by the process can also enhance the anti-enzymatic ability of the artificial exosome, providing a stable structure for subsequent gradient centrifugation purification, and finally constructing an artificial exosome with high stability, intelligent targeted release and anti-damage ability, which is different from the prior art which cannot balance the structural stability and responsive release, and has the limitation of insufficient targeting.

[0048] In the present application, the gradient centrifugation purification is preferably: After centrifuging the crude artificial exosome at a speed of 50000-70000 rpm at 4 DEG C for 25-35 minutes, the supernatant is discarded, and after resuspension with VC-Lip / PBS aqueous solution, the precipitation is collected after centrifugation at a speed of 70000-90000 rpm at 4 DEG C for 35-45 minutes; The concentration of VC-Lip in the VC-Lip / PBS aqueous solution is 0.1-0.2 mg / mL.

[0049] Specifically, the configuration method of the VC-Lip / PBS aqueous solution is: According to the required concentration, VC-Lip solid powder is added to PBS buffer with pH of 7.4 and stirred until the VC-Lip is completely dissolved, and then filtered with a 0.22 mu m sterile filter to sterilize, to obtain the VC-Lip / PBS aqueous solution.

[0050] It can be understood that, by centrifuging at 50000-70000 rpm and 4℃ for 25-35 minutes for the first time, the density difference between free lipids and artificial exosomes is utilized to make the artificial exosomes precipitate as a precipitate, and the supernatant containing free lipids is discarded, thereby solving the problem of low removal rate of free lipids in the prior art by single centrifugal purification; then the precipitate is resuspended with PBS buffer containing 0.1-0.2 mg / mL VC-Lip, and VC-Lip can form an antioxidant protective layer on the surface of the artificial exosome, thereby avoiding the destruction of its structure and the degradation of OCT4 protein and siTrim28 activity by subsequent high-speed centrifugation; centrifuging at 70000-90000 rpm and 4℃ for 35-45 minutes for the second time, further removes trace amounts of small molecular impurities (such as unbound hyaluronic acid fragments) remaining in the precipitate, and the final collected precipitate is the high-purity artificial exosome.

[0051] In the present application, the anti-enzymatic modification is preferably: after resuspending the precipitate with a hyaluronic acid / PBS aqueous solution, incubating at a temperature of 35-40℃ for 15-25 minutes; wherein the concentration of hyaluronic acid in the hyaluronic acid / PBS aqueous solution is 2.5 mg / ml.

[0052] The PBS buffer with a pH of 7.4 is added to the hyaluronic acid powder in three times according to a concentration ratio of 2.5 mg / ml, each time after adding, dispersing with a glass rod, then magnetic stirring until dissolved, and then filtering with a 0.45 μm sterile filter membrane, to obtain the hyaluronic acid / PBS aqueous solution.

[0053] It can be understood that, by resuspending the precipitate with 2.5 mg / mL hyaluronic acid in PBS buffer and incubating at 35-40℃ for 15-25 minutes, the hydrophilic and adhesive properties of hyaluronic acid are utilized to make it uniformly adsorb on the surface of the artificial exosome to form a dense protective layer, which can resist the destruction of the structure of the artificial exosome by nucleases and proteases in the body (such as avoiding the degradation of siTrim28 by nucleases and the hydrolysis of the lipid membrane by proteases), thereby solving the defects of the prior art that the artificial exosome is easily enzymatically degraded in the body and has a short drug half-life; at the same time, the incubation temperature of 35-40℃ can promote the adsorption and combination of hyaluronic acid on the surface of the exosome, and can also avoid the degradation of the activity of OCT4 protein and siTrim28 caused by high temperature; in addition, hyaluronic acid has good biocompatibility (non-immunogenicity), which can reduce the clearance rate of the artificial exosome in the body, and it can bind to the CD44 receptor highly expressed on the surface of tumor cells, thereby assisting in enhancing the folate targeting effect, and ultimately greatly improving the drug retention rate of the artificial exosome in the enzymatic environment in the body, providing a structurally stable and biocompatible carrier for subsequent precise drug delivery, and avoiding the limitations of the prior art that the artificial exosome has weak anti-enzymatic ability and is easily cleared in the body.

[0054] In the present application, when dissolving the PCL-PEG-PCL, DC-Chol and TA-PEG-TA in the chloroform-ethanol mixed solvent, the mass ratio of the PCL-PEG-PCL, DC-Chol and TA-PEG-TA is preferably 100: (5-8): (6-10), and the volume of the chloroform-ethanol mixed solvent is preferably 20-30 times of the total mass of the PCL-PEG-PCL, DC-Chol and TA-PEG-TA.

[0055] Specifically, 20-30 ml of the chloroform-ethanol mixed solvent is used to dissolve 1 mg of the PCL-PEG-PCL, DC-Chol and TA-PEG-TA.

[0056] Specifically, the PCL:PEG molar ratio of the PCL-PEG-PCL is 6:4, and the molecular weight is 15000-25000 Da.

[0057] In the present application, when dissolving the DPPC, ceramide, cholesterol and DSPE-PEG-FA in the chloroform-ethanol mixed solution, the molar ratio of the DPPC, ceramide, cholesterol and DSPE-PEG-FA is preferably 100: (20-30): (30-40): (8-12), and the volume of the chloroform-ethanol mixed solution is preferably 15-20 times of the total mass of the DPPC, ceramide, cholesterol and DSPE-PEG-FA.

[0058] Specifically, 15-20 ml of the chloroform-ethanol mixed solvent is used to dissolve 1 mg of the DPPC, ceramide, cholesterol and DSPE-PEG-FA (PEG2000 modified phospholipid-folic acid complex).

[0059] The present application also provides an artificial exosome for drug delivery prepared by the preparation method of the artificial exosome for drug delivery.

[0060] Example 1 S1, preparation of a ROS-responsive polymer core suspension: S11, PCL-PEG-PCL, DC-Chol and TA-PEG-TA are weighed according to a mass ratio of 100:6:8, and the PCL-PEG-PCL, DC-Chol and TA-PEG-TA are dissolved in a chloroform-ethanol mixed solvent (chloroform volume:ethanol volume=2:1) with a volume of 25 times of the total mass of the PCL-PEG-PCL, DC-Chol and TA-PEG-T, and stirred at a speed of 600 rpm at a temperature of 25°C for 20 minutes to obtain an oil phase; S12, weigh PCL-PEG-PCL, DC-Chol and TA-PEG-TA, and 0.5% of the total mass of VC-Lip, then weigh OCT4 protein and siTrim28 according to the mass ratio of 1:1:3.5, take half the volume of the oil phase of the core enzyme-free water, add the OCT4 protein, siTrim28 and VC-Lip to the core enzyme-free water, and ultrasonically disperse under ice bath at a power of 90W for 35 minutes, then add 2.5% of the total mass of hyaluronic acid of PCL-PEG-PCL, DC-Chol, TA-PEG-TA, OCT4 protein, siTrim28 and VC-Lip, and stir for 15 minutes to obtain the water phase; S13, inject the oil phase into a double-layer jacketed glass container, open the jacketed ice water circulation, and stabilize the oil phase temperature between 35-40℃, insert the ultrasonic probe into the oil phase (the probe is immersed to 1 / 3-1 / 2 of the oil phase liquid level), connect the feed inlet of the high-pressure homogenizer to the outlet of the glass container through a sterile pipeline, and return the discharge outlet to the glass container (forming a "circulating emulsification" path), drop the water phase into the oil phase at a speed of 0.3ml / min, and simultaneously start the high-pressure homogenizer and ultrasonic probe (power 130W, pulse mode "3 seconds on, 2 seconds off") to perform high-pressure homogenization circulation at a feed speed of 1 times / min of the total volume of the water phase and the oil phase, and a pressure of 800bar, and after the water phase is completely dropped, the ultrasonic and high-pressure homogenization are maintained until 2 more cycles are performed, and then the W / O primary emulsion is obtained; S14, add the W / O primary emulsion to 6 times its volume of 9% PVA aqueous solution, and magnetically stir at a speed of 1000rpm at 4℃ for 5 hours to obtain the ROS-responsive polymer core suspension; S2, preparation of a folate-targeted functional lipid membrane: S21, weigh DPPC, ceramide, cholesterol and DSPE-PEG-FA according to a molar ratio of 100:25:35:10, add the DPPC, ceramide, cholesterol and DSPE-PEG-FA to a chloroform-ethanol mixed solvent (chloroform volume:ethanol volume=2:1) with a volume of 18 times the mass of DPPC, ceramide, cholesterol and DSPE-PEG-FA, and stir at a speed of 450rpm for 20 minutes, then add 1% of the total mass of VC-Lip to the DPPC, ceramide, cholesterol and DSPE-PEG-FA, and continue to stir for 10 minutes to obtain a first mixed solution; S22, transfer the first mixed solution to a rotary evaporator, evaporate at 35°C under reduced pressure at a speed of 120 rpm and a pressure of 2.5 KPa for 40 minutes, while passing nitrogen at a speed of 10 ml / min, after the end, vacuum drying for 2.5 hours, to obtain a folic acid targeted functional lipid membrane; S3, take the folic acid targeted functional lipid membrane and ROS responsive polymer core suspension according to the feed liquid ratio of 150:1 (mg / ml), add the ROS responsive polymer core suspension to the folic acid targeted functional lipid membrane, oscillate at a speed of 150 rpm under a water bath at 38°C for 30 minutes, to obtain a primary coating solution; S4, according to the volume ratio of the primary coating solution: PVA aqueous solution = 1:0.25, add 2.5% PVA aqueous solution to the primary coating solution, then add 0.4% TA-PEG-TA by mass of the primary coating solution, incubate at 45°C for 10 minutes, while vortexing at a speed of 1800 rpm for 2 minutes, then pass the crosslinked solution through a microfluidic chip (channel size 200 nm, pressure 0.4 MPa) and circulate extruding 20 times, to obtain artificial exosome crude bodies; S5, after centrifuging the artificial exosome crude bodies at a speed of 60000 rpm for 30 minutes at 4°C, discard the supernatant, resuspend with VC-Lip / PBS aqueous solution (VC-Lip concentration is 0.2 mg / mL), then centrifuge at a speed of 80000 rpm for 40 minutes at 4°C, after the end, collect the precipitate; S6, resuspend the precipitate with hyaluronic acid / PBS aqueous solution (hyaluronic acid concentration is 2.5 mg / ml), incubate at a temperature of 38°C for 20 minutes, after the end, filter sterilize with a 0.22 μm polyether sulfone filter membrane, to obtain the artificial exosome for drug delivery.

[0061] Example 2 S1, preparation of ROS responsive polymer core suspension: S11, take PCL-PEG-PCL, DC-Chol and TA-PEG-TA according to the mass ratio of 100:5:6, dissolve the PCL-PEG-PCL, DC-Chol and TA-PEG-TA in a mixed solvent of chloroform-ethanol with a volume of 20 times the total mass of PCL-PEG-PCL, DC-Chol and TA-PEG-T (chloroform volume: ethanol volume = 2:1), stir at a speed of 600 rpm at a temperature of 25°C for 20 minutes, to obtain an oil phase; S12, weigh PCL-PEG-PCL, DC-Chol and TA-PEG-TA to a total mass of 0.4% VC-Lip, then weigh OCT4 protein and siTrim28 according to the mass ratio of 1:1:3.5 of OCT4 protein, siTrim28 and VC-Lip, take the volume of the core enzyme-free water which is half of the oil phase, add the OCT4 protein, siTrim28 and VC-Lip to the core enzyme-free water, and ultrasonically disperse under ice bath at a power of 90W for 35 minutes, then add hyaluronic acid with a total mass of 2.5% of PCL-PEG-PCL, DC-Chol, TA-PEG-TA, OCT4 protein, siTrim28 and VC-Lip, and stir for 15 minutes to obtain the water phase; S13, inject the oil phase into a double-layer jacketed glass container, start the ice water circulation of the jacket, and stabilize the temperature of the oil phase at a temperature between 35-40℃, insert the ultrasonic probe into the oil phase (the immersion depth of the probe is between 1 / 3-1 / 2 of the liquid level of the oil phase), connect the feed port of the high-pressure homogenizer to the outlet of the glass container through a sterile pipeline, and return the discharge port to the glass container (forming a "circulating emulsification" path), drop the water phase into the oil phase at a speed of 0.2ml / min, and simultaneously start the high-pressure homogenizer and the ultrasonic probe (power 120W, pulse mode "3 seconds on, 2 seconds off") to perform high-pressure homogenization circulation at a feed speed of 0.8 times / min of the total volume of the water phase and the oil phase, and a pressure of 700bar, and after the water phase is completely dropped, keep the ultrasonic and high-pressure homogenization until another cycle is completed, to obtain a W / O primary emulsion; S14, add the W / O primary emulsion to 8% PVA aqueous solution with a volume of 5 times that of the W / O primary emulsion, and magnetically stir at a speed of 900rpm at 4℃ for 4 hours to obtain a ROS-responsive polymer core suspension; S2, preparation of a folate-targeted functional lipid membrane: S21, weigh DPPC, ceramide, cholesterol and DSPE-PEG-FA according to a molar ratio of 100:20:30:8, add the DPPC, ceramide, cholesterol and DSPE-PEG-FA to a chloroform-ethanol mixed solvent with a volume of 15 times the mass of DPPC, ceramide, cholesterol and DSPE-PEG-FA (chloroform volume:ethanol volume=2:1) and stir at a speed of 450rpm for 20 minutes, then add VC-Lip with a total mass of 1% of DPPC, ceramide, cholesterol and DSPE-PEG-FA and continue to stir for 10 minutes to obtain a first mixed solution; S22, transfer the first mixed solution to a rotary evaporator, evaporate at 30°C under reduced pressure at a speed of 100 rpm and a pressure of 2.5 KPa for 30 minutes, while passing nitrogen at a speed of 8 ml / min, and after the end, vacuum drying for 2 hours, to obtain a folic acid targeted functional lipid membrane; S3, take the folic acid targeted functional lipid membrane and ROS responsive polymer core suspension according to a feed liquid ratio of 100:1 (mg / ml), add the ROS responsive polymer core suspension to the folic acid targeted functional lipid membrane, oscillate at a speed of 140 rpm under a water bath at 35°C for 25 minutes, to obtain a primary coating solution; S4, according to a volume ratio of the primary coating solution: PVA aqueous solution = 1:0.25, add 2% PVA aqueous solution to the primary coating solution, then add 0.3% mass of TA-PEG-TA to the primary coating solution, incubate at 40°C for 8 minutes, while vortexing at a speed of 1700 rpm for 1 minute, then pass the crosslinked solution through a microfluidic chip (channel size 200 nm, pressure 0.4 MPa) and circulate extruding 20 times, to obtain artificial exosome crude bodies; S5, after centrifuging the artificial exosome crude bodies at a speed of 50000 rpm for 25 minutes at 4°C, discard the supernatant, resuspend with VC-Lip / PBS aqueous solution (VC-Lip concentration is 0.1 mg / mL), and then centrifuge at a speed of 70000 rpm for 35 minutes at 4°C, and collect the precipitate after the end; S6, resuspend the precipitate with hyaluronic acid / PBS aqueous solution (hyaluronic acid concentration is 2.5 mg / ml), incubate at a temperature of 35°C for 15 minutes, and then filter sterilize with a 0.22 μm polyether sulfone filter membrane, to obtain the artificial exosome for drug delivery.

[0062] Example 3 S1, preparation of ROS responsive polymer core suspension: S11, take PCL-PEG-PCL, DC-Chol and TA-PEG-TA according to a mass ratio of 100:8:10, dissolve the PCL-PEG-PCL, DC-Chol and TA-PEG-TA in a mixed solvent of chloroform-ethanol with a volume of 30 times the total mass of PCL-PEG-PCL, DC-Chol and TA-PEG-T (chloroform volume: ethanol volume = 2:1), stir at a speed of 600 rpm at a temperature of 25°C for 20 minutes, to obtain an oil phase; S12, weigh PCL-PEG-PCL, DC-Chol and TA-PEG-TA, and VC-Lip with a total mass of 0.6% VC-Lip, then weigh OCT4 protein and siTrim28 according to the mass ratio of 1:1:3.5 of OCT4 protein, siTrim28 and VC-Lip, take the volume of the core enzyme-free water which is half of the oil phase, add the OCT4 protein, siTrim28 and VC-Lip to the core enzyme-free water, and ultrasonically disperse under ice bath at a power of 90W for 35 minutes, then add hyaluronic acid with a total mass of 2.5% of PCL-PEG-PCL, DC-Chol, TA-PEG-TA, OCT4 protein, siTrim28 and VC-Lip, and stir for 15 minutes to obtain the water phase; S13, inject the oil phase into a double-layer jacketed glass container, open the jacketed ice water circulation, and stabilize the temperature of the oil phase at a temperature between 35-40℃, insert the ultrasonic probe into the oil phase (the immersion depth of the probe is between 1 / 3-1 / 2 of the liquid level of the oil phase), connect the feed port of the high-pressure homogenizer to the outlet of the glass container through a sterile pipeline, and return the discharge port to the glass container (forming a "circulating emulsification" path), drop the water phase into the oil phase at a speed of 0.4ml / min, and simultaneously start the high-pressure homogenizer and the ultrasonic probe (power 140W, pulse mode "3 seconds on, 2 seconds off") to perform high-pressure homogenization circulation at a feed speed of 1.2 times / min of the total volume of the water phase and the oil phase, and a pressure of 900bar, and after the water phase is completely dropped, keep the ultrasonic and high-pressure homogenization until 2 more cycles are performed, and then stop to obtain the W / O primary emulsion; S14, add the W / O primary emulsion to 10% PVA aqueous solution with a volume of 8 times that of the W / O primary emulsion, and magnetically stir at a speed of 1100rpm at 4℃ for 6 hours to obtain the ROS-responsive polymer core suspension; S2, preparation of a folate-targeted functional lipid membrane: S21, weigh DPPC, ceramide, cholesterol and DSPE-PEG-FA according to a molar ratio of 100:30:40:12, add the DPPC, ceramide, cholesterol and DSPE-PEG-FA to a chloroform-ethanol mixed solvent with a volume of 20 times the mass of DPPC, ceramide, cholesterol and DSPE-PEG-FA (chloroform volume:ethanol volume=2:1) and stir at a speed of 450rpm for 20 minutes, then add VC-Lip with a total mass of 1% of DPPC, ceramide, cholesterol and DSPE-PEG-FA and continue to stir for 10 minutes to obtain a first mixed solution; S22. The first mixed solution was transferred to a rotary evaporator and evaporated at 40°C at a speed of 130 rpm and a pressure of 2.5 kPa for 45 minutes under reduced pressure. Nitrogen gas was introduced at a rate of 12 ml / min. After the evaporation was completed, the mixture was vacuum dried for 3 hours to obtain a folic acid-targeting lipid membrane. S3. Take the folic acid-targeting functional lipid membrane and ROS-responsive polymer core suspension at a material-to-liquid ratio of 200:1 (mg / ml), add the ROS-responsive polymer core suspension to the folic acid-targeting functional lipid membrane, and shake at 160 rpm for 35 minutes in a water bath at 40°C to obtain a first coating solution. S4. According to the volume ratio of primary coating solution to PVA aqueous solution = 1:0.25, add 3% PVA aqueous solution to the primary coating solution, then add 0.5% by mass of TA-PEG-TA of the primary coating solution, keep warm at 50°C for 12 minutes, and vortex at 1900 rpm for 2 minutes. Then, circulate the cross-linked solution through a microfluidic chip (channel size 200 nm, pressure 0.4 MPa) 20 times to obtain the coarse artificial exosome. S5. After centrifuging the crude artificial exosomes at 70,000 rpm at 4°C for 35 minutes, discard the supernatant, resuspend the precipitate in VC-Lip / PBS aqueous solution (VC-Lip concentration is 0.2 mg / mL), centrifuge at 90,000 rpm at 4°C for 45 minutes, and collect the precipitate after the centrifugation. S6. The precipitate is resuspended in hyaluronic acid / PBS aqueous solution (hyaluronic acid concentration is 2.5 mg / ml), incubated at 40°C for 25 minutes, and then filtered through a 0.22 μm polyethersulfone filter membrane for sterilization to obtain the artificial exosome for drug delivery.

[0063] Effect test I. Experimental Materials and Grouping 1. Preparation of experimental samples Experimental group (EXO-1): Artificial exosomes for drug delivery prepared according to Example 1 of the present invention.

[0064] Control group 1 (EXO-2): Artificial exosomes prepared by the method in patent CN119564883A.

[0065] Control group 2 (EXO-3): Artificial exosomes prepared by the method in patent CN110403917A.

[0066] 2. Experimental conditions All exosomes were loaded with fluorescently labeled drugs (doxorubicin, DOX, fluorescence wavelength 590 nm), with an initial drug loading of 100 μg / mg (exosome mass). The experiment was repeated three times. Data are expressed as mean ± standard deviation. Statistical analysis was performed using one-way ANOVA (P < 0.05 was considered statistically significant).

[0067] II. Comparative Experimental Design and Results 1. Experiment 1: Structural stability test (simulating in vivo enzymatic hydrolysis environment) Experimental methods: Three groups of exosomes (concentration 1 mg / mL) were added to PBS buffer (pH 7.4) containing trypsin (concentration 0.5 mg / mL) and incubated at 37°C for 48 h. Samples were taken at 0 h, 24 h, and 48 h. The particle size change rate was detected using a dynamic light scattering (DLS) instrument (based on the 0h particle size). The structural integrity was observed using transmission electron microscopy (TEM), and the percentage of exosomes with intact structures (no dissociation or fragmentation of the nucleus-membrane) was counted out of 100 exosomes.

[0068] The experimental results are shown in Table 1. Table 1 Structural stability test results

[0069] As shown in Table 1, the artificial exosomes (EXO-1) prepared in this invention, due to the cross-linking effect of TA-PEG-TA, showed a particle size change rate of only 2.8% and a structural integrity rate of 96.5% after 48 hours, which is significantly better than EXO-2 (P<0.01) and EXO-3 (P<0.01).

[0070] 2. Experiment 2: Intelligent Response Release Detection (Simulated ROS Concentration Difference Environment) Experimental methods: Three groups of exosomes (containing 10 μg of DOX) were placed into dialysis bags (molecular weight cutoff 10 kDa) and placed in two release media (37°C constant temperature shaking, 100 rpm): Medium 1 (normal tissue group): PBS containing 1 μM H2O2 (simulating an environment with ROS < 2 μM). Medium 2 (tumor microenvironment group): PBS containing 15 μM H2O2 (simulating an environment with ROS > 10 μM); Samples were taken at 4h, 12h, 24h and 48h respectively, and the fluorescence intensity of DOX in the release medium was detected by a fluorescence spectrophotometer to calculate the cumulative release rate.

[0071] The experimental results are shown in Table 2. Table 2 Smart Response Release Detection Results

[0072] As shown in Table 2, the artificial exosomes (EXO-1) prepared in this invention have a release rate of only 14.2% (no burst release) in a low ROS environment and a release rate of 91.5% (precise response) in a high ROS environment; while EXO-2 and EXO-3 have no ROS responsiveness, and the difference in release rate between the two environments is <10%. It can be seen that the "TA-PEG-TA ROS responsive design" of this invention can effectively solve the problem of drug burst release.

[0073] 3. Experiment 3: Target Specificity Detection (HeLa Cell Uptake Assay) Experimental methods: HeLa cells (folate receptor positive, density 5×10⁶) were used. 4 (1 exosome / well) were seeded into 96-well plates, and after 24 h of culture, three groups of fluorescently labeled exosomes (final concentration 50 μg / mL) were added and incubated for 4 h: Wash three times with PBS to remove untaken exosomes; Intracellular fluorescence intensity was detected by flow cytometry, and the uptake rate was calculated (with EXO-2 uptake rate as the baseline, set at 100%). A negative control group (without exosomes) was set up to exclude background fluorescence.

[0074] The experimental results are shown in Table 3. Table 3 Target Specific Detection Results

[0075] As shown in Table 3, the artificial exosomes (EXO-1) prepared in this invention achieved a relative uptake rate of 196.2% in HeLa cells due to the targeting effect of DSPE-PEG-FA (96.2% higher than EXO-2), with an off-target effect of only 8.3% (60% lower than EXO-2). EXO-3, on the other hand, had the lowest uptake rate due to the lack of targeting modification. This indicates that the folic acid targeting design in the embodiments of this application can significantly improve targeting specificity.

[0076] 4. Experiment 4: Test of resistance to damage (simulating gastrointestinal and blood environment) Experimental methods: Simulated gastrointestinal environment: Three groups of exosomes (containing 10 μg DOX) were added to hydrochloric acid solution (containing 0.1 mg / mL pepsin) at pH 1.2 and incubated at 37°C for 2 h. The remaining DOX content was detected by HPLC and the drug retention rate was calculated. Simulated blood environment: Three groups of exosomes (containing 10 μg of DOX) were added to PBS (containing serum enzymes) containing 10% fetal bovine serum and incubated at 37°C for 24 h. The drug retention rate was then tested.

[0077] The experimental results are shown in Table 4. Table 4 Results of Anti-damage Test

[0078] As shown in Table 3, the artificial exosomes (EXO-1) prepared in this invention have an average drug retention rate of 87.9% due to the antioxidant effect of VC-Lip and the protective layer effect of hyaluronic acid, which is significantly higher than that of EXO-2 (65.1%, P<0.01) and EXO-3 (61.1%, P<0.01).

[0079] III. Experimental Conclusions The artificial exosome (EXO-1) prepared in Example 1 of this invention is significantly superior to artificial exosomes (EXO-2 and EXO-3) prepared by existing technologies in terms of structural stability, release performance, target specificity, and resistance to damage.

[0080] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do 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 for drug delivery, characterized in that, The preparation steps include the following: PCL-PEG-PCL, DC-Chol, and TA-PEG-TA were dissolved in a chloroform-ethanol mixed solvent and stirred until dissolved to obtain an oil phase. OCT4 protein, siTrim28, and VC-Lip were dissolved in nuclease-free water and ultrasonically dispersed. Hyaluronic acid was then added and stirred to obtain an aqueous phase. The oil phase and aqueous phase were subjected to high-pressure-ultrasonic synergistic treatment to obtain a W / O primary emulsion. The W / O primary emulsion was added to a PVA aqueous solution for evaporation and solidification treatment to obtain a ROS-responsive polymer core suspension. DPPC, ceramide, cholesterol, and DSPE-PEG-FA were dissolved in a chloroform-ethanol mixture, and after stirring, VC-Lip was added and stirring was continued to obtain a first mixture. The first mixture was subjected to rotary evaporation to obtain a folic acid-targeting lipid membrane. The ROS-responsive polymer core suspension was added to the folic acid-targeting functional lipid membrane for multilayer self-assembly and encapsulation. After the process, it was homogenized by microfluidic extrusion to obtain a coarse artificial exosome. The crude artificial exosomes were purified by gradient centrifugation, and the precipitate was collected. The precipitate was then modified to resist enzymatic degradation and filtered through a polyethersulfone membrane to obtain the artificial exosomes for drug delivery.

2. The method for preparing artificial exosomes for drug delivery according to claim 1, characterized in that, The high-pressure-ultrasound synergistic processing is as follows: At a temperature of 35-40℃ and under ultrasonic conditions, the aqueous phase is dripped into the oil phase at a rate of 0.2-0.4 ml / min, while high-pressure homogenization circulation is started. After the aqueous phase is added, high-pressure homogenization is maintained for 1-2 cycles before the process ends. The feed rate of the high-pressure homogenizing circulation is 0.8-1.2 times the total volume of the aqueous and oil phases per minute, and the pressure is 700-900 bar. The ultrasound power is 120-140W, and the ultrasound cycle is 3 seconds of ultrasound followed by a 2-second pause.

3. The method for preparing artificial exosomes for drug delivery according to claim 2, characterized in that, The volatilization and solidification process is as follows: The primary emulsion is added to an 8%-10% PVA aqueous solution at 5-8 times its volume, and magnetically stirred at 900-1100 rpm for 4-6 hours at 4°C.

4. The method for preparing artificial exosomes for drug delivery according to claim 3, characterized in that, The rotary evaporation process is as follows: The first mixed solution was subjected to reduced pressure rotary evaporation at 30-40℃ and 100-130 rpm for 30-45 minutes, while nitrogen gas was introduced at a rate of 8-12 ml / min. After the process, the solution was dried under vacuum for 2-3 hours.

5. The method for preparing artificial exosomes for drug delivery according to claim 4, characterized in that, The multi-layer self-assembly coating process is as follows: The ROS-responsive polymer core suspension was added to the folic acid-targeting functional lipid membrane and shaken at 140-160 rpm for 25-35 minutes in a water bath at 35-40°C to obtain a primary coating solution. Add 2%-3% PVA aqueous solution and 0.3%-0.5% by mass of TA-PEG-TA to the primary coating solution, and keep it at 40-50℃ for 8-12 minutes while vortexing at 1700-1900rpm for 1-2 minutes.

6. The method for preparing artificial exosomes for drug delivery according to claim 5, characterized in that, The gradient centrifugation purification yielded: After centrifuging the crude artificial exosomes at 50,000-70,000 rpm at 4°C for 25-35 minutes, discard the supernatant, resuspend in VC-Lip / PBS aqueous solution, and centrifuge at 70,000-90,000 rpm at 4°C for 35-45 minutes. Collect the precipitate after centrifugation. The concentration of VC-Lip in the VC-Lip / PBS aqueous solution is 0.1-0.2 mg / mL.

7. The method for preparing artificial exosomes for drug delivery according to claim 6, characterized in that, The anti-enzymatic modification is as follows: After resuspending the precipitate in hyaluronic acid / PBS aqueous solution, incubate it at 35-40℃ for 15-25 minutes. The concentration of hyaluronic acid in the hyaluronic acid / PBS aqueous solution is 2.5 mg / ml.

8. The method for preparing artificial exosomes for drug delivery according to claim 7, characterized in that, When PCL-PEG-PCL, DC-Chol, and TA-PEG-TA are dissolved in a chloroform-ethanol mixed solvent, the mass ratio of PCL-PEG-PCL, DC-Chol, and TA-PEG-TA is 100:(5-8):(6-10), and the volume of the chloroform-ethanol mixed solvent is 20-30 times the total mass of PCL-PEG-PCL, DC-Chol, and TA-PEG-TA.

9. The method for preparing artificial exosomes for drug delivery according to claim 8, characterized in that, When DPPC, ceramide, cholesterol, and DSPE-PEG-FA are dissolved in a chloroform-ethanol mixed solution, the molar ratio of DPPC, ceramide, cholesterol, and DSPE-PEG-FA is 100:(20-30):(30-40):(8-12), and the volume of the chloroform-ethanol mixed solution is 15-20 times the total mass of DPPC, ceramide, cholesterol, and DSPE-PEG-FA.

10. An artificial exosome for drug delivery prepared by the method for preparing artificial exosomes for drug delivery as described in any one of claims 1-9.

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