Bionic self-driven microvesicle, preparation method and application

Abstract

This application provides a biomimetic self-driven microvesicle, its preparation method, and its application, belonging to the field of nanobiodelivery technology. This application utilizes a triple synergistic mechanism of a hybrid biomimetic membrane, a magneto-electro-thermal responsive core, and a pH / H2S dual-response release. Driven by a gradient rotating magnetic field generator, the near-infrared photothermal modulation unit softens the cell membrane through thermal effects, and a focused femtosecond pulsed electric field module forms reversible ultrapores. This allows the biomimetic self-driven microvesicles to precisely pass through the reversible ultrapores, then migrate and accumulate deep within the target cell membrane, significantly improving transmembrane efficiency and reducing non-specific damage. This makes it suitable for high-end applications such as deep tumor treatment, blood-brain barrier penetration, and oral delivery of biological drugs.

Description

Technical Field

[0001] This invention belongs to the field of nanobiodelivery technology, and relates to a biomimetic self-driven microvesicle, its preparation method, and its application. Background Technology

[0002] Cellular microvesicles are formed by budding and shedding directly from the cell membrane. They have good biocompatibility and can be used as a natural nanocarrier, playing a role in immune regulation, inflammatory response, and tissue repair.

[0003] When microvesicles are used as natural nanocarriers, they have the following disadvantages: (1) Passive transmembrane limitation: Microvesicles rely on endocytosis or simple membrane fusion, and have insufficient transmembrane power. When facing dense biological barriers such as the blood-brain barrier and deep tumor stroma, the transmembrane efficiency is less than 15%, and they lack the ability to actively infiltrate deep tissues. (2) Single response defect: Microvesicles rely on pH response or glutathione response technology, which cannot adapt to the complex in vivo microenvironment, resulting in incomplete release and high off-target release rate; (3) Insufficient targeting precision: Traditional ligand / antibody modifications are easily degraded by biological fluids and retained by the liver and spleen, lacking the multi-level targeting capability of "chemotaxis-homing-precise recognition", and the target site enrichment rate is low; (4) Limitations of physical permeation: Microvesicles usually rely on electroporation, magnetic transfection or photothermal technology, which can easily cause damage to non-target cells and cannot form a dynamic combination with the targeting and responsiveness of microvesicles themselves. (5) Bionic membrane has a single function: Traditional single-cell-derived bionic membranes only have a single chemotaxis or targeting ability, and cannot simultaneously achieve biological barrier penetration, immune escape and precise cell recognition. Summary of the Invention

[0004] The purpose of this invention is to provide a biomimetic self-driven microvesicle, its preparation method, and its application, in order to solve the problem of low transmembrane efficiency when existing microvesicles are used as nanocarriers.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, this application provides a biomimetic self-driven microvesicle, the preparation method of which includes: After mixing macrophage cell membranes and mesenchymal stem cell membranes, the mixtures were fused by ultrasound and extruded. The resulting hybrid biomimetic membrane was obtained by anchoring the target cell-specific nucleic acid aptamer through a biotin-avidin system. Superparamagnetic Fe3O4 nanoparticles, polycaprolactone, and conductive pyrrole were ultrasonically mixed and then sequentially added with dithiocarbamate, a support, and (2-ethyl-2-oxazoline)-polylactic acid to obtain a magnetoelectric thermal responsive core. The hybrid biomimetic membrane is mixed with the magnetoelectric-thermal responsive core, and after shearing-electrostatic self-assembly using microfluidic-electrospinning technology, sulfated glycosaminoglycans are covalently linked to obtain biomimetic self-driven microvesicles.

[0006] Secondly, this application provides an application of biomimetic self-driven microvesicles, namely, their application in transmembrane drug delivery.

[0007] The present invention has the following beneficial effects: (1) In this application, through the triple synergistic mechanism of hybrid biomimetic membrane, magnetoelectric thermal response core and pH / H2S dual response release, the gradient rotating magnetic field generator drives the cell membrane to soften the cell membrane through the thermal effect of the near-infrared photothermal regulation unit, and the focused femtosecond pulse electric field module forms a reversible micropore, which enables the biomimetic self-driven microvesicles to accurately pass through the reversible micropore and migrate and accumulate in the target cell membrane, thereby greatly improving the transmembrane efficiency and reducing non-specific damage.

[0008] (2) The hybrid biomimetic membrane and sGAG modification achieve dual immune escape. The femtosecond pulse electric field, low intensity gradient magnetic field and low power near-infrared light are non-cytotoxic. The cell membrane perforation is a reversible ultramicropore, which increases the repair speed by more than 1 times. No inflammatory response, organ damage or nuclear accumulation was detected in in vivo experiments. Biocompatibility and safety are significantly improved.

[0009] (3) According to in vitro testing, the transmembrane efficiency of circRNA-loaded microvesicles reached more than 96%, which is 6-7 times higher than the traditional magnetoelectric coupling technology. The penetration efficiency of blood-brain barrier and blood-retinal barrier is more than 5 times higher than the existing technology. The infiltration depth of deep tumor stroma is increased by 4 times, which shows high transmembrane efficiency and deep penetration.

[0010] (4) The target enrichment rate reached over 85%, the liver and spleen retention rate was reduced to below 8%, and the off-target effect was reduced by 90%, far exceeding the traditional ligand / antibody modification and single biomimetic membrane technology, with significant multi-level targeting accuracy.

[0011] (5) The novel pH / H2S dual-response mechanism is adapted to complex microenvironments such as tumors and the central nervous system. The cascade release efficiency of the load is more than 40% higher than that of the traditional pH / MMP response technology. The off-target release rate in normal tissues is less than 5%, and the microenvironment adaptability and release accuracy are greatly improved.

[0012] (6) This application can achieve full-function integration of "multi-level targeting - multi-field synergistic perforation - novel dual-response release - real-time closed-loop monitoring", which can break through multiple biological barriers such as blood-brain barrier, blood-retinal barrier, deep tumor interstitial barrier, and intestinal mucosal barrier. It is suitable for high-end scenarios such as circRNA / saRNA targeted delivery, treatment of central nervous system diseases, oral peptide / protein drug delivery, and intraocular precision drug delivery. Attached Figure Description

[0013] Figure 1 The images shown are detection images of the biomimetic self-driven microvesicles prepared in Example 1 of this application. Image A is a SEM image of the biomimetic self-driven microvesicles, and image B is a particle size distribution map of the biomimetic self-driven microvesicles. Figure 2 The values ​​represent the blood glucose levels of mice in each group. Detailed Implementation

[0014] This application provides a biomimetic self-driven microvesicle, the preparation method of which includes: S01: Macrophage cell membranes and mesenchymal stem cell membranes are mixed, then fused by ultrasound and extruded. After anchoring the target cell-specific nucleic acid aptamer through the biotin-avidin system, a hybrid biomimetic membrane is obtained.

[0015] Mouse bone marrow-derived macrophage cell membranes and mesenchymal stem cell membranes were mixed homogenously at a mass ratio of 3:2 to 2:1 and fused by sonication at 20–40 kHz, 80–120 W, and an ice bath for 3–5 minutes. The mixture was then passed through a polycarbonate filter with a pore size of 120–180 nm and repeatedly extruded 8–10 times to obtain a homogeneous hybrid membrane. The resulting hybrid membrane was then anchored to a target cell-specific nucleic acid aptamer using a biotin-avidin system to obtain a hybrid biomimetic membrane.

[0016] The hybrid membrane retains the macrophage chemotactic receptor CCR2 and the mesenchymal stem cell tissue homing receptor CXCR4. These two receptors can precisely bind to the target cells of the aptamer, achieving multi-level targeting of "inflammatory chemotaxis-tissue homing-precise cell recognition". At the same time, the low immunogenicity of the mesenchymal stem cell membrane enables efficient immune escape, solving the problem of functional limitations of traditional single biomimetic membranes.

[0017] The target cell-specific nucleic acid aptamers in this application include at least one of tumor cell-specific, nerve cell-specific, and intestinal epithelial cell-specific nucleic acid aptamers.

[0018] S02: Superparamagnetic Fe3O4 nanoparticles, polycaprolactone, and conductive pyrrole were ultrasonically mixed, and then dithiocarbamate, a loading material, and (2-ethyl-2-oxazoline)-polylactic acid were added sequentially to obtain a magnetoelectric thermal response core.

[0019] Superparamagnetic Fe3O4 nanoparticles with a particle size of 8-15 nm, polycaprolactone, and conductive pyrrole were mixed in a mass ratio of 1:4:2 to form a magneto-electro-thermal trifunctional core. Dithiocarbamate and a loading material were added to the magneto-electro-thermal trifunctional core. A dithiocarbamate derivative containing H2S-sensitive disulfide bonds was used as a bridging unit to covalently link the magneto-electro-thermal trifunctional core and the loading material, forming a supported trifunctional core. pH-sensitive (2-ethyl-2-oxazoline)-polylactic acid (PEOz-PLA) was added to the supported trifunctional core to coat the outer layer of the supported trifunctional core with PEOz-PLA, forming a magneto-electro-thermal responsive core.

[0020] In this application, superparamagnetic Fe3O4 nanoparticles can be driven by a magnetic field; conductive pyrrole generates local photothermal activity under near-infrared light and enhances the electric field perforation efficiency; polycaprolactone can improve the biocompatibility and drug loading stability of the magneto-electrothermal responsive core, achieve directional deep migration under an applied gradient rotating magnetic field, and near-infrared photothermal activity can soften the cell membrane and further reduce the transmembrane barrier.

[0021] In the tumor / lesion microenvironment, acidic conditions with a pH of 5.8-6.6 can trigger the degradation of the PEOz-PLA shell, and H2S at a concentration of 50-100 μM can hydrolyze the H2S sensitive bond, causing it to break specifically and achieve stepwise cascade release, avoiding lysosomal degradation and off-target release from normal tissue.

[0022] In this application, the loading material includes one or more of nucleic acid drugs, protein and peptide drugs, and nanocrystalline chemotherapy drugs, such as circRNA, saRNA, GLP-1 analogs, nerve growth factor, doxorubicin nanocrystals, cisplatin nanocrystals, etc.

[0023] The mass ratio of superparamagnetic Fe3O4 nanoparticles, dithiocarbamate, support material, and (2-ethyl-2-oxazoline)-polylactic acid is 1 : (0.1-1) : (0.05-1) : (3-20).

[0024] S03: The hybrid biomimetic membrane is mixed with the magnetoelectric thermal responsive core, and after shearing-electrostatic self-assembly by microfluidic-electrospinning technology, sulfated glycosaminoglycans are covalently linked to obtain biomimetic self-driven microvesicles.

[0025] The hybrid biomimetic membrane and the magneto-electro-thermal responsive core were mixed at a mass ratio of 2.5:1 and sheared-electrostatic self-assembly was performed using a microfluidic-electrospinning technique under conditions of a microfluidic channel width of 30-40 μm and an electrospinning voltage of 8-12 kV. After self-assembly, sulfated glycosaminoglycans (sGAGs) with a molecular weight of 10,000-15,000 Da were covalently linked via mercapto-enyne click chemiluminescence, with a final concentration of 0.1-0.5 mg / mL in the reaction system, resulting in biomimetic self-driven microvesicles with a particle size of 150-200 nm.

[0026] sGAG can specifically bind to adhesion molecules in the interstitial tissue, reduce the non-specific binding of microvesicles to mesenchymal stem cells, enhance deep tissue penetration, and further improve immune escape effects, thus solving the problems of cell internalization inhibition and poor interstitial penetration caused by traditional modifications. The sulfated glycosaminoglycans in this application are selected from chondroitin C sulfate and / or dermatan sulfate.

[0027] In addition, this application also provides an application of biomimetic self-driven microvesicles, namely, their application in transmembrane drug delivery. This transmembrane drug delivery includes one or more of the following: targeted delivery for central nervous system diseases, deep infiltration delivery of solid tumors, oral delivery of peptide drugs, and intraocular blood-retinal barrier penetration delivery.

[0028] The technical solution of the present invention will be further explained and described below through specific embodiments.

[0029] Example 1 This application provides a biomimetic self-driven microvesicle, the preparation method of which includes: S101: Mouse bone marrow-derived macrophage cell membranes and mesenchymal stem cell membranes were mixed at a mass ratio of 2:1 and sonicated for 4 minutes under ultrasonic conditions of 30 kHz, 100 W, and ice bath to achieve membrane fusion. The mixture was then extruded repeatedly 9 times through a 160 nm pore size polycarbonate filter membrane to obtain a homogeneous hybrid membrane. The hybrid membrane was then anchored to a U87 cell-specific nucleic acid aptamer using a biotin-avidin system to obtain a hybrid biomimetic membrane.

[0030] S102: Superparamagnetic Fe3O4 nanoparticles with a particle size of 12 nm, polycaprolactone, and conductive pyrrole were mixed at a mass ratio of 1:4:2 and ultrasonically dispersed for 40 min to form a magneto-electro-thermal trifunctional core. Dithiocarbamate and circRNA were added to the magneto-electro-thermal trifunctional core, and the magneto-electro-thermal trifunctional core and circRNA were linked through a dithiocarbamate derivative containing H2S-sensitive disulfide bonds to form a loaded trifunctional core. pH-sensitive PEOz-PLA was added to the loaded trifunctional core to form a magneto-electro-thermal responsive core.

[0031] S103: A hybrid biomimetic membrane and a magnetoelectric-thermal responsive core were mixed at a mass ratio of 2.5:1 and subjected to shear-electrostatic self-assembly using a microfluidic-electrospinning technique under conditions of a microfluidic channel width of 35 μm and an electrospinning voltage of 10 kV. After self-assembly, chondroitin sulfate C with a molecular weight of 12000 Da was covalently linked via a mercapto-enyne click chemiluminescence to obtain biomimetic self-driven microvesicles.

[0032] Example 2 This application provides a biomimetic self-driven microvesicle, the preparation method of which includes: S201: Mouse bone marrow-derived macrophage cell membranes and intestinal mucosal mesenchymal stem cell membranes were mixed at a mass ratio of 3:2 and fused by sonication at 20 kHz, 80 W, and an ice bath for 3 minutes. The mixture was then passed through a 120 nm polycarbonate filter membrane and extruded repeatedly eight times to obtain a homogeneous hybrid membrane. The hybrid membrane was then anchored to a Caco-2 cell-specific nucleic acid aptamer using a biotin-avidin system to obtain a hybrid biomimetic membrane.

[0033] S202: Superparamagnetic Fe3O4 nanoparticles with a particle size of 8 nm, polycaprolactone, and conductive pyrrole are mixed in a mass ratio of 1:4:2 to form a magneto-electro-thermal trifunctional core. Dithiocarbamate and a GLP-1 analog are added to the magneto-electro-thermal trifunctional core. The magneto-electro-thermal trifunctional core and the GLP-1 analog are connected through a dithiocarbamate derivative containing H2S-sensitive disulfide bonds to form a supported trifunctional core. pH-sensitive PEOz-PLA is added to the supported trifunctional core to coat the outer layer of the supported trifunctional core with PEOz-PLA, forming a magneto-electro-thermal responsive core.

[0034] S203: A hybrid biomimetic membrane and a magneto-electro-thermal responsive core were mixed at a mass ratio of 2.5:1 and subjected to shear-electrostatic self-assembly using a microfluidic-electrospinning technique under conditions of a microfluidic channel width of 30 μm and an electrospinning voltage of 8 kV. After self-assembly, dermatin sulfate with a molecular weight of 10000 Da was covalently linked via a mercapto-enyne click chemo-covalent bond to obtain biomimetic self-driven microvesicles.

[0035] Example 3 This application provides a biomimetic self-driven microvesicle, the preparation method of which includes: S301: Mouse bone marrow-derived macrophage cell membranes and mesenchymal stem cell membranes were mixed at a mass ratio of 3:2 and sonicated for 5 minutes under ultrasonic conditions at a frequency of 40 kHz, a power of 120 W, and an ice bath to achieve membrane fusion. The mixture was then extruded repeatedly 10 times through a polycarbonate filter membrane with a pore size of 180 nm to obtain a homogeneous hybrid membrane. The hybrid membrane was then anchored to a nerve cell-specific nucleic acid aptamer using a biotin-avidin system to obtain a hybrid biomimetic membrane.

[0036] S302: Superparamagnetic Fe3O4 nanoparticles with a particle size of 15 nm, polycaprolactone, and conductive pyrrole are mixed in a mass ratio of 1:4:2 to form a magneto-electro-thermal trifunctional core. Dithiocarbamate and doxorubicin nanocrystals are added to the magneto-electro-thermal trifunctional core, and the magneto-electro-thermal trifunctional core and doxorubicin nanocrystals are connected by a dithiocarbamate derivative containing H2S-sensitive disulfide bonds to form a supported trifunctional core. pH-sensitive PEOz-PLA is added to the supported trifunctional core to coat the outer layer of the supported trifunctional core with PEOz-PLA, forming a magneto-electro-thermal responsive core.

[0037] S303: A hybrid biomimetic membrane and a magneto-electro-thermal responsive core were mixed at a mass ratio of 2.5:1 and subjected to shear-electrostatic self-assembly using a microfluidic-electrospinning technique under conditions of a microfluidic channel width of 40 μm and an electrospinning voltage of 12 kV. After self-assembly, dermatan sulfate with a molecular weight of 15000 Da was covalently linked via a mercapto-enyne click chemiluminescence to obtain biomimetic self-driven microvesicles.

[0038] The biomimetic self-driven microvesicles prepared in Example 1 of this application were subjected to SEM and particle size analysis to obtain the attached... Figure 1 From the appendix Figure 1 As can be seen, the biomimetic self-driven microvesicles prepared in Example 1 are spherical, with particle sizes mainly distributed between 75-120 nm.

[0039] The biomimetic self-driven microvesicles in this embodiment achieve drug transmembrane delivery through a magneto-electro-thermal coupling synergistic system. This magneto-electro-thermal coupling synergistic system includes a gradient rotating magnetic field generator, a focused femtosecond pulse electric field module, a near-infrared photothermal modulation unit, a real-time impedance-fluorescence dual monitoring unit, a temperature feedback control module, and an intelligent main control terminal.

[0040] The process by which biomimetic self-driven microvesicles achieve drug transmembrane delivery is as follows: S01: Bionic self-driven microvesicles are chemotactic to the site of inflammation / lesion via the CCR2 receptor in the hybrid bionic membrane, achieve deep tissue homing via the CXCR4 receptor of the mesenchymal stem cell membrane, and then precisely bind to target cells through surface-anchored nucleic acid aptamers.

[0041] S02: The magneto-electro-thermal coupling synergistic system is activated. The gradient rotating magnetic field generator dynamically adjusts the gradient difference based on the target tissue depth and fluorescence signal feedback, driving biomimetic self-driven microvesicles to penetrate dense biological barriers such as tumor stroma and blood-brain barrier and migrate directionally to the target cell membrane surface. The near-infrared photothermal regulation unit softens the cell membrane phospholipid bilayer through photothermal effect, reducing the energy threshold of electric field perforation. The focused femtosecond pulse electric field module forms reversible ultramicropores with a diameter of 30-150 nm on the cell membrane. This forms a multi-field dynamic synergistic effect of active deep migration, photothermal softening, precise perforation, and rotational permeation, enabling biomimetic self-driven microvesicles to precisely pass through the reversible ultramicropores, migrate directionally to the deep part of the target cell membrane, and accumulate, significantly improving transmembrane efficiency and reducing non-specific damage. Among them, the perforation efficiency of biomimetic self-driven microvesicles reaches over 90%, and the non-target cell perforation rate is less than 3%.

[0042] The parameters of the gradient rotating magnetic field generator are as follows: magnetic field strength 3-18 mT, gradient difference 2-5 mT / mm, and rotation frequency 1.5-6 Hz; the parameters of the focused femtosecond pulse electric field module are as follows: femtosecond pulse width 100-500 fs, voltage intensity 60-250 V / cm, 2-6 pulse groups, with an interval of 3-6 s between each group to avoid irreversible cell membrane damage and apoptosis caused by nanosecond / microsecond pulses; and the parameters of the near-infrared photothermal modulation unit are as follows: near-infrared light wavelength 808 nm, and power density 0.5-2 W / cm². 2 .

[0043] In addition, the real-time impedance-fluorescence dual monitoring unit can regulate and provide real-time feedback on the cell membrane perforation status, and track the enrichment and internalization process of microvesicles. When the microvesicle enrichment rate at the target site reaches 70% and the perforation rate reaches 65%, the intelligent main control terminal automatically stops the electric field and photothermal output. The temperature feedback module accurately maintains the temperature of the action area at 36.5-37.5℃ with an error of ±0.3℃.

[0044] S03: After the biomimetic self-driven microvesicles enter the target cell / pathological microenvironment through reversible ultramicropores, the acidic conditions first trigger the degradation of the pH-sensitive PEOz-PLA shell, exposing the core and the load. Subsequently, the high concentration of H2S sensitive bonds enables the precise cascade release of the load, avoiding lysosomal degradation and off-target release from normal tissues. The load release efficiency is over 95%, and the release rate in normal tissues is less than 5%.

[0045] In this application, after the magneto-electro-thermal coupling synergistic system is stopped, the reversible ultrapores of the cell membrane are rapidly and autonomously repaired within 5-10 minutes, which is much faster than the repair speed of traditional nanosecond pulse perforation. Fe3O4 in the magneto-electro-thermal responsive core is metabolized and excreted through the reticuloendothelial system, and conductive pyrrole and polycaprolactone are slowly degraded into small molecules in vivo, with no risk of long-term accumulation. The hybrid biomimetic membrane and sGAG modified layer can be absorbed by cellular enzymes, exhibiting excellent biocompatibility.

[0046] To verify that the biomimetic self-driven microvesicles provided in the embodiments of this application have a high penetration rate, this application provides a specific description using the biomimetic self-driven microvesicles prepared in Examples 1 and 2.

[0047] (1) Targeted transmembrane delivery of gliomas that penetrate the blood-brain barrier In this embodiment, the samples were divided into a blank group, an empty microvesicle group, a free nucleic acid group, a treatment group 1, and a treatment group 2. After being applied to the lesion site, the transmembrane resistance and blood-brain barrier penetration efficiency of each group were measured, as shown in Tables 1 and 2. When calculating the blood-brain barrier penetration efficiency, the initial total fluorescence of the upper chamber was taken as 100%, and the cumulative fluorescence transmittance of the lower chamber was measured. The groups are grouped as follows: Blank group: circRNA was used directly.

[0048] Empty microvesicle group: Empty microvesicles without circRNA were prepared according to the preparation method in Example 1, and the empty microvesicles were co-cultured with U87 cells.

[0049] Cell-free nucleome: Cell-free circRNA was co-cultured with U87 cells.

[0050] Treatment Group 1: The biomimetic self-driven microvesicles prepared in Example 1 were co-cultured with U87 cells.

[0051] Treatment Group 2: The biomimetic self-driven microvesicles prepared in Example 1 were co-cultured with U87 cells, and the magneto-electro-thermal coupling synergistic system was activated. The parameters of the magneto-electro-thermal coupling synergistic system were as follows: gradient rotating magnetic field strength of 12 mT, gradient difference of 3 mT / mm, rotation frequency of 4 Hz, femtosecond pulse width of 300 fs, voltage intensity of 180 V / cm, number of pulse groups of 4, interval of 3 s between each group, near-infrared wavelength of 808 nm, and power density of 1 W / cm². 2 .

[0052] Table 1: Transepithelial / Endothelial Electrical Resistance (TEER) for each group (Ω·cm) 2 ) Table 2: Blood-brain barrier penetration efficiency (%) As shown in Tables 1 and 2, compared with the blank group, empty microvesicle group, free nucleic acid group, and treatment group 1, treatment group 2 has a smaller in vitro transmembrane resistance and a blood-brain barrier penetration efficiency of 80%. This indicates that the magneto-electro-thermal coupling synergistic system significantly improves the biomimetic self-driven microvesicles and can reversibly enhance blood-brain barrier permeability without causing serious damage to the barrier.

[0053] (2) Drug enrichment and distribution experiment in major organs In this embodiment, the drugs were divided into three groups: a free drug group, a traditional single-membrane microvesicle group, and a biomimetic self-driven microvesicle group. U87 nude mice with gliomas were selected, and the reagents for each group were injected via the tail vein. 24 hours after administration, the heart, liver, spleen, lung, kidney, brain, and tumor tissues were separated, and the relative fluorescence intensity of the drug in each tissue was detected, as shown in Table 3.

[0054] Free drug group: The same amount of free fluorescently labeled drug was injected into the tail vein, without the intervention of the magneto-electro-thermal coupling synergistic system, and the animals were kept in a conventional environment.

[0055] Traditional single-membrane microvesicle group: The same amount of traditional single-cell membrane-encapsulated microvesicles were injected into the tail vein, without the intervention of the magneto-electro-thermal coupling synergistic system, and the animals were raised in a conventional environment.

[0056] Bionic self-driven microvesicle group: Bionic self-driven microvesicles prepared in Example 1 were injected via tail vein, and a magneto-electro-thermal coupling synergistic system was applied to the target region of the brain. The parameters of the magneto-electro-thermal coupling synergistic system were as follows: gradient rotating magnetic field strength 12 mT, gradient difference 3 mT / mm, rotation frequency 4 Hz, femtosecond pulse width 300 fs, voltage intensity 180 V / cm, 4 pulse groups with a 3-s interval between each group, near-infrared wavelength 808 nm, and power density 1 W / cm². 2 .

[0057] Table 3: Relative fluorescence intensity of drugs in major organs of mice in each group (Mean±SD) As shown in Table 3, compared with the free drug group and the traditional single-membrane microvesicle group, the biomimetic self-driven microvesicle group showed a significantly increased enrichment amount in tumor tissue, with a target site enrichment rate of over 85%; the amount retained in the liver and spleen was significantly reduced, with the retention rate dropping to below 8%; and the drug distribution in normal organs such as the heart, lungs, and kidneys was not significantly increased. This indicates that the biomimetic self-driven microvesicles prepared in the embodiments of this application have excellent targeted enrichment ability and low off-target characteristics.

[0058] (3) Oral delivery of GLP-1 analogs that penetrate the intestinal mucosal barrier In this embodiment, mice were divided into a control group, a model group, and a treatment group, and each group of mice was administered the reagent by gavage. Twenty-four hours after gavage, the blood glucose levels of the mice in each group were measured to obtain the results. Figure 2 .

[0059] Control group: Healthy mice were selected and administered an equal volume of physiological saline by gavage. No intervention was applied to the magneto-electro-thermal coupling synergistic system. The mice were fed in a routine environment, and the remaining experimental procedures were the same as those for the model mice.

[0060] Model group: Type 2 diabetic mice that have successfully developed the model were selected and administered an equal volume of physiological saline by gavage. No biomimetic self-driven microvesicles were given, and no intervention of the magneto-electro-thermal coupling synergistic system was applied.

[0061] Treatment group: Type 2 diabetic mice were administered the biomimetic self-driven microvesicles prepared in Example 2 by gavage, and a magneto-electro-thermal coupling synergistic system was applied to the target region in the abdomen. The parameters of the magneto-electro-thermal coupling synergistic system were as follows: gradient rotating magnetic field strength of 8 mT, gradient difference of 2 mT / mm, rotation frequency of 2 Hz, femtosecond pulse width of 200 fs, voltage intensity of 100 V / cm, number of pulse groups of 3, interval of 4 s between each group, near-infrared wavelength of 808 nm, and power density of 0.8 W / cm². 2 .

[0062] From the appendix Figure 2 As can be seen, compared with the control group, the blood glucose level of mice in the model group was significantly increased; compared with the model group, the blood glucose level of mice in the treatment group decreased by 75%, and the difference with the control group was small. This indicates that the biomimetic self-driven microvesicles prepared in the embodiments of this application can effectively penetrate the intestinal mucosal barrier to achieve drug delivery.

[0063] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for preparing biomimetic self-driven microvesicles, characterized in that, include: After mixing macrophage cell membranes and mesenchymal stem cell membranes, the mixtures were fused by ultrasound and extruded. The resulting hybrid biomimetic membrane was obtained by anchoring the target cell-specific nucleic acid aptamer through a biotin-avidin system. Superparamagnetic Fe3O4 nanoparticles, polycaprolactone, and conductive pyrrole were ultrasonically mixed and then sequentially added with dithiocarbamate, a support, and (2-ethyl-2-oxazoline)-polylactic acid to obtain a magnetoelectric thermal responsive core. The hybrid biomimetic membrane is mixed with the magnetoelectric-thermal responsive core, and after shearing-electrostatic self-assembly using microfluidic-electrospinning technology, sulfated glycosaminoglycans are covalently linked to obtain biomimetic self-driven microvesicles.

2. The method for preparing biomimetic self-driven microvesicles according to claim 1, characterized in that, The mass ratio of the macrophage cell membrane to the mesenchymal stem cell membrane is 3:2-2:1; the final concentration of the sulfated glycosaminoglycan in the reaction system is 0.1-0.5 mg / mL, and the molecular weight is 10000-15000 Da.

3. The method for preparing biomimetic self-driven microvesicles according to claim 1, characterized in that, The superparamagnetic Fe3O4 nanoparticles have a particle size of 8-15 nm. The loading material includes one or more of nucleic acid drugs, protein peptide drugs, and nanocrystalline chemotherapeutic drugs. The nucleic acid drugs include circRNA and / or saRNA, the protein peptide drugs include GLP-1 analogs and / or nerve growth factors, and the nanocrystalline chemotherapeutic drugs include doxorubicin nanocrystals and / or cisplatin nanocrystals.

4. The method for preparing biomimetic self-driven microvesicles according to claim 1, characterized in that, The mass ratio of the superparamagnetic Fe3O4 nanoparticles, the polycaprolactone, and the conductive pyrrole is 1:4:

2.

5. The method for preparing biomimetic self-driven microvesicles according to claim 1, characterized in that, The mass ratio of the superparamagnetic Fe3O4 nanoparticles, dithiocarbamate, support, and (2-ethyl-2-oxazoline)-polylactic acid is 1:(0.1-1):(0.05-1):(3-20).

6. The method for preparing biomimetic self-driven microvesicles according to claim 1, characterized in that, The mass ratio of the hybrid biomimetic membrane to the magnetoelectric thermal responsive core is 2.5:

1.

7. The method for preparing biomimetic self-driven microvesicles according to claim 1, characterized in that, The parameters of the microfluidic-electrospinning combined technology are: microfluidic channel width of 30-40μm and electrospinning voltage of 8-12kV.

8. The biomimetic self-driven microvesicles prepared by the preparation method according to any one of claims 1-7.

9. The application of the biomimetic self-driven microvesicles prepared by the preparation method according to any one of claims 1-7 in drug transmembrane delivery.

10. The application according to claim 9, characterized in that, The drug transmembrane delivery includes one or more of the following: targeted delivery for central nervous system diseases, deep infiltration delivery for solid tumors, oral delivery of peptide drugs, and intraocular blood-retinal barrier penetration delivery.

Images