Treg cell-derived exosome drug delivery system, preparation method and application thereof

Abstract

This invention discloses a Treg cell-derived exosome drug delivery system, its preparation method, and its applications, belonging to the field of biomedical technology. This invention obtains a drug delivery system by ultrasonically loading miR-142-5p onto T cell-derived exosomes. The ultrasonic loading method of this invention has higher loading efficiency, ensuring that each exosome carries a sufficient dose of drug; better membrane integrity, as the membrane pores can self-repair after ultrasound, preserving the natural structure and function of the exosomes to the greatest extent; and relatively simple and controllable operation. The T cell-derived exosomes and the carried drug (miR-142-5p) can produce a synergistic effect, showing good efficacy in the treatment of cardiomyopathy, especially myocardial infarction.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a Tregs cell-derived exosome ultrasound-loaded miR-142-5p drug delivery system, its preparation method, and its application. Background Technology

[0002] Ischemic heart disease, such as myocardial infarction, is a disease with high morbidity and mortality rates worldwide. In recent years, the onset of the disease has shown a trend towards younger ages, seriously endangering human health. Although modern medicine has made significant progress, with early treatments such as percutaneous coronary intervention or thrombolysis significantly reducing the mortality rate of acute myocardial infarction, these methods cannot salvage the irreversible myocardial damage caused by severe ischemia, which can then progress to heart failure.

[0003] Currently, the clinical treatment system for ischemic cardiomyopathy revolves around drug therapy (beta-blockers, RAAS inhibitors, diuretics, etc.) and percutaneous coronary intervention (PCI). While these interventions can improve myocardial perfusion, alleviate symptoms, and reduce short-term mortality (PCI reduces the 30-day mortality rate of acute myocardial infarction patients by 40%-60%), their limitations are becoming increasingly apparent: drug side effects can induce electrolyte disturbances, the therapeutic window narrows, the lack of pathological reversal leads to a high readmission rate of up to 60% for fibrotic patients, and PCI can cause continued progression of fibrosis. Therefore, a revolutionary new treatment model is still needed to meet unmet clinical needs.

[0004] In recent years, novel drugs such as RNA and protein-based drugs have come into focus, but their delivery in vivo faces numerous challenges, including biostability, biodegradation, bioavailability, immune response, and specific delivery. Currently, nanoparticle-based drug delivery systems are being used in many studies to deliver miRNAs, proteins, and other drugs. However, artificial nanoparticle-based drug delivery systems, such as liposomes, also have several problems. For example, they are generally cleared by the reticuloendothelial system (leading to short circulation times and easy accumulation in areas such as the liver and spleen, causing toxic reactions), and small RNAs attached to synthesized nanoparticles are easily cleared by pinocytic vesicles and serum proteins. Therefore, given these limitations, finding a nanoparticle-based drug delivery system that can ensure drug stability, therapeutic targeting, and safe distribution has become an urgent problem to solve. Based on the characteristics of exosomes being endogenously secreted by the human body, they overcome the limitations of artificial nanoparticle-based drug delivery systems and possess advantages such as small particle size, strong in vivo permeability, and low toxicity and immunogenicity risks, making them suitable as drug carriers for local treatment.

[0005] Exosomes are phospholipid bilayer nanovesicles with a diameter of approximately 30-150 nm, carrying important information such as proteins, lipids, DNA, and RNA. They can transfer biologically active miRNAs and, upon entering target cells, regulate mRNA levels, participating in physiological processes such as immune responses, antigen presentation, cell migration, cell differentiation, and cell proliferation. Regulatory T cells (Tregs) play a crucial role in protecting the myocardium and blood vessels from damage and promoting remodeling. In 2013, Tregs were first confirmed to have the function of secreting exosomes. It has been reported that Treg cells of the same cell number secrete significantly more exosomes than other immune cells. Some studies indicate that exosomes secreted by Treg cells have similar functions to those of Treg cells. Currently, most commercially available methods for preparing exosomes are derived from stem cell exosomes, resulting in low nucleic acid drug loading, high treatment costs, and low efficiency, making them unsuitable for clinical trials. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention proposes a Treg cell-derived exosome drug delivery system, its preparation method, and its application.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solution: The first objective of this invention is to provide a method for preparing a Treg cell-derived exosome drug delivery system, comprising the following steps: (1) Preparation of Treg cell-derived exosomes: Primary Treg cells were obtained by immunomagnetic bead sorting, and Treg cell culture supernatant was prepared; Exo-spin buffer was added to the supernatant for incubation, centrifugation was performed, and the precipitate was resuspended in PBS to obtain precipitated exosomes; after purification of the precipitated exosomes, purified Treg cell-derived exosomes were obtained. (2) After centrifugation, FITC-labeled miR-142-5p agomir was added to DEPC water to prepare a suspension; (3) The suspension obtained in step (2) is mixed with the Treg cell-derived exosomes obtained in step (1) to form a mixture. After sonication, the mixture is incubated at 37°C for 60 minutes to restore the exosome membrane. (4) The mixture obtained in step (3) was centrifuged at 120,000 g for 70 min at 4 °C and then resuspended in PBS to obtain Treg cell-derived exosomes sonicated with miR-142-5p, i.e., Treg cell-derived exosome drug delivery system.

[0008] Furthermore, the volume ratio of the supernatant to the buffer solution in step (1) is 2:1.

[0009] Further, the purification of sedimented exosomes in step (1) is as follows: PBS is added to the sedimented exosomes, and centrifuged at 50×g for 60 seconds to obtain Treg cell-derived exosomes.

[0010] Furthermore, in step (3), the mixing ratio of the suspension and T cell-derived exosomes is suspension: Treg cell-derived exosomes = 1 nmol: 300 ug.

[0011] Furthermore, the ultrasound treatment in step (3) is ultrasound on ice, and the ultrasound operation parameters are: 20% amplitude, 30s on / off for 3 minutes, 6 cycles.

[0012] The second objective of this invention is to provide a drug delivery system obtained by a method for preparing a Treg cell-derived exosome drug delivery system.

[0013] The third objective of this invention is to provide an application of a drug delivery system in the preparation of drugs for the prevention and treatment of myocardial infarction.

[0014] The present invention relates to a T-cell-derived exosome drug delivery system, its preparation method, and its application, the beneficial effects of which are as follows: (1) Traditional exosome drug delivery methods are inefficient and may cause irreversible damage to the exosome membrane, leading to drug leakage or destruction of carrier integrity, thus affecting its function. The ultrasonic loading method of this invention has higher loading efficiency, ensuring that each exosome carries a sufficient dose of drug; better membrane integrity, as the membrane pores can repair themselves after ultrasonication, preserving the natural structure and function of the exosome to the greatest extent; and relatively simple and controllable operation.

[0015] (2) The myocardial infarction area is a strong inflammatory response center, which releases a large number of chemokines. Treg cells themselves have the characteristic of homing to the site of inflammation and injury, and their derived exosomes are likely to naturally inherit the ability to target the site of inflammation, achieving "active targeting" without the need for complex artificial modification, and thus more efficient.

[0016] (3) Treg exosomes are not only drug carriers, but their internal proteins, miRNAs and other contents also have inherent anti-inflammatory and immunomodulatory functions, which can produce a synergistic effect with the drug they carry (miR-142-5p). They have good effects in the treatment of cardiomyopathy, especially myocardial infarction. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 These are transmission electron micrographs of Tregs cell-derived exosomes of the present invention, wherein A is an electron micrograph showing the morphological changes of Tregs exosomes; B is an image showing the uptake effect of CFs on Tregs exosomes; C is an electron micrograph showing the morphological changes of Tregs cell-derived exosomes loaded with miR-142-5p by ultrasound; and D is an image showing the expression of miR-142-5p after co-culturing Tregs cell-derived exosomes with CFs.

[0019] Figure 2 The following is a particle size distribution diagram of Tregs cell-derived exosomes ultrasonically loaded with miR-142-5p, which is the nanoparticle tracking analysis of the present invention. A is the particle size distribution diagram of Tregs exosomes (diluted 40 times), and B is the particle size distribution diagram of Tregs cell-derived exosomes ultrasonically loaded with miR-142-5p. Figure 3 To detect the protein expression of miR-142-5p in Tregs exosomes and Tregs cell-derived exosomes under ultrasound loading using Western blot, A shows the expression of surface markers CD9, CD81, and CD63 on the surface of Tregs exosomes, and B shows the protein expression of miR-142-5p in Tregs cell-derived exosomes under ultrasound loading.

[0020] Figure 4 Potential map of Treg cell-derived exosomes loaded with miR-142-5p; Figure 5 To detect the positive rate of miR-142-5p expression in exosomes via nanoflow cytometry; Figure 6 Stability of Treg cell-derived exosomes with negative miR-142-5p by ultrasound; Figure 7 The effect of CFs on the cell uptake capacity of Treg cell-derived exosomes loaded with miR-142-5p; Figure 8 The diagram shows the results of mouse body weight, heart weight, and survival time, where A represents the change in mouse body weight, B represents the mouse heart weight coefficient, and C represents the mouse survival curve. Figure 9 This is a schematic diagram of the imaging changes in isolated mouse tissue. Figure 10This is a schematic diagram of HE staining in mouse tissues, where A shows the HE staining changes in mouse liver, spleen, lung, and kidney tissues; and B shows the HE staining changes in mouse myocardial tissue. Figure 11 This is a schematic diagram of Masson staining changes in mouse tissue; Figure 12 The diagrams show the changes in end-diastolic and end-systolic internal diameters in mice. A represents the end-diastolic internal diameter (LVEDD) of mice, and B represents the change in end-systolic internal diameter (LVESD) of mice.

[0021] Figure 13 The figures represent mouse myocardial parameters, where A represents the left ventricular ejection fraction (LVEF) and B represents the changes in the short-axis shortening fraction (LVFS) of the mouse heart.

[0022] Figure 14 The encapsulation efficiency of the Tregs cell-derived exosome drug delivery system of this invention is given. Detailed Implementation

[0023] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0024] The first objective of this invention is to provide a drug delivery system for Treg cell-derived exosomes to be ultrasonically loaded with miR-142-5p.

[0025] The second objective of this invention is to provide a method for preparing a drug delivery system for Treg cell-derived exosomes loaded with miR-142-5p via ultrasound.

[0026] A third objective of this invention is to provide the application of a drug delivery system for Treg cell-derived exosomes loaded with miR-142-5p via ultrasound in the preparation of drugs for the prevention and treatment of myocardial infarction.

[0027] Example 1

[0028] A method for preparing a drug delivery system for Treg cell-derived exosomes loaded with miR-142-5p via ultrasound includes the following steps: (1) Preparation of T cell-derived exosomes: Primary Treg cells were obtained by immunomagnetic bead sorting, and Treg cell culture supernatant was prepared. Exo-spin buffer was added to the supernatant for incubation, centrifugation was performed, and the precipitate was resuspended in PBS to obtain precipitated exosomes. After purification of the precipitated exosomes, purified Treg cell-derived exosomes (Tregs-exo) were obtained. (2) After centrifuging the FITC-labeled miR-142-5p agomir at 3000g for 1 min, DEPC water was added to prepare a suspension with a concentration of 100 μM / L; (3) Take 10 μL (equivalent to 1 nmol) of suspension and mix it with the Tregs cell-derived exosomes obtained in step (1) (at a ratio of suspension: Tregs cell-derived exosomes = 1 nmol: 300 μg) to form a mixture. Mix the mixture into a tube, place it on ice, and perform sonication on ice. The treatment conditions are: sonication on ice, 20% amplitude, 30s on / off for 3 minutes, 6 cycles. After sonication, the mixture is incubated at 37°C for 60 minutes to restore the exosome membrane. (4) The mixture obtained in step (3) was centrifuged at 4°C and 120,000g for 70 min, and then resuspended in PBS to obtain Tregs cell-derived exosomes with sonicated loading miR-142-5p, i.e., Tregs cell-derived exosome drug delivery system (Tregs-exo_miR-142-5p).

[0029] The specific preparation method of Treg cell-derived exosomes in step (1) is as follows: ① Primary Treg cells were obtained by immunomagnetic bead sorting. The sorted Tregs were seeded into cell culture plates coated with CD3 antibody overnight, and RPMI 1640 medium containing CD28 antibody, penicillin antibody and 10% exosome-free serum was added. The cells were cultured at 37°C and 5% CO2 saturated humidity for 48 hours to obtain Treg cell culture supernatant. ②In a 50mL centrifuge tube, add 40mL of cell culture supernatant and centrifuge at 300×g for 10min; ③ After centrifugation, transfer the supernatant into a new 50mL centrifuge tube and centrifuge at 16000×g for 30 minutes; ④ Take a new 50mL centrifuge tube, put in the supernatant obtained in step ③, and add Exo-spin buffer at a supernatant:buffer volume ratio of 2:1. In this step, the preferred supernatant volume is 40mL, then add 20mL of Exo-spin buffer. ⑤ Mix the liquid from step ④ thoroughly, and centrifuge the mixture several times by inverting the centrifuge tube, centrifuging for a few seconds each time. Incubate overnight at -4℃; the next day, centrifuge the mixture at 4℃, 16000×g for 1 hour; discard the supernatant that is carefully and slowly aspirated, being careful not to aspirate too cleanly (to avoid drying out the sample and damaging the exosomes), and obtain a white precipitate; ⑥ Resuspend the white precipitate containing exosomes in 100 μL of PBS to obtain an exosome resuspension; ⑦ Remove the top and bottom caps of the Exo-spin purification column, and connect the matching waste liquid tube to the bottom of the purification column. Remove the preservative solution from the top of the purification column using a 200 μL pipette, being careful not to let the pipette tip touch the purification membrane at the top of the column to avoid damage. Add 200 μL of PBS to the top of the purification column and centrifuge at 50×g for 30 seconds to equilibrate the column. (If there is PBS residue at the very top, centrifuge again at 50×g for 30 seconds. Note: Do not use high speeds or excessively long centrifugation times to avoid damaging the purification column). ⑧ Add 100 μL of the exocrine body suspension obtained in step ⑥ to the top of the purification column, centrifuge at 50×g for 60 seconds, and discard the eluent. ⑨ Place the purification column into a new sterile 1.5 mL microcentrifuge tube, add 200 μL of PBS to the top of the purification column; centrifuge at 50 × g for 60 seconds. After centrifugation, the purified exosomes will be approximately 200 μL of wash solution, which is the Tregs cell-derived exosomes.

[0030] After obtaining Treg cell-derived exosomes in step (1), the Treg cell-derived exosomes were traced using immunofluorescence detection. The specific tracing method included steps i, ii, and iii, as follows: Step i: PKH26 staining of Treg cell-derived exosomes a. Add 50 μg of Tregs cell-derived exosomes to dilution buffer C to make a final volume of 500 μL, mix well, and obtain the dilution buffer; b. Add 2 μL of PKH26 to 500 μL of diluent C and mix well to obtain a mixed solution; c. Add the mixed solution from step b to the diluent from step a, mix gently, and incubate at room temperature for 5 minutes.

[0031] d. Stop staining with 1 mL of 1% BSA for 1 min; e. Add 1% BSA to 2 ml, centrifuge at 120000 g for 60 min at 4℃, discard the supernatant, and obtain the precipitate (exosomes labeled PKH26); f. Resuspend the precipitate obtained in step e (which is a PKH26-labeled exosome) in 500 μl of DMEM basal medium for later use.

[0032] Step ii: Validation by co-culturing exosomes derived from fibroblasts (CFs) and Tregs cells. The specific method is as follows: a. CFs cells were digested, counted to 1×10^4, and seeded into 24-well plates containing glass slides (for growth on slides) and cultured overnight. They were divided into three groups: Control group (CFs without Tregs exosomes), Tregs-exo group (Tregs exosomes co-cultured with CFs), and Tregs+GW4869)-exo group (Tregs and exosome inhibitor GW4869 co-cultured with exosomes derived from CFs). b. On the second day, wash the well plate three times with PBS, add the stained exosomes from step (1) into the cells in the well plate according to the group, and put it into a carbon dioxide incubator to co-culture with the cells for 24 hours.

[0033] Step iii: Immunofluorescence staining a. After co-culturing cells and exosomes for 24 hours, remove the culture medium and wash three times with PBS; b. Fix with 4% paraformaldehyde at room temperature for 10 min, then wash 3 times with PBS; c. Add Mounting Medium containing DAPI to the slide, seal the slide, and observe and photograph it under a laser confocal scanning microscope.

[0034] Example 2

[0035] Characterization of a drug delivery system for Treg cell-derived exosomes loaded with miR-142-5p via ultrasound 1. Detection of morphology, particle size, potential, and fluorescence information The resuspension of Tregs cell-derived exosomes and exosome drug delivery systems obtained in Example 1 was used as a sample.

[0036] (1) Electron microscopy morphology: Tregs cell-derived exosomes (Tregs-exo) and exosome drug delivery system (Tregs-exo_miR-142-5p) were resuspended in PBS to obtain resuspensions. 20 μL of the resuspension was pipetted onto a copper grid and allowed to adsorb naturally for 5-10 minutes. Excess droplets were then removed with filter paper, and the cells were allowed to dry slightly. 20 μL of 2% phosphotungstic acid solution was then pipetted onto the copper grid and allowed to stand for 3-5 minutes. Excess droplets were removed with filter paper, and the cells were allowed to dry under an incandescent lamp. The cells were then observed and photographed under a transmission electron microscope. The results are shown below. Figure 1 As shown.

[0037] from Figure 1 As shown in Figure A, the exosomes derived from pre-loaded Tregs exhibit a double-membrane, saucer-like structure. The results obtained from the CFs uptake function assay are as follows: Figure 1 As shown in Figure B, DAPI stained the nuclei of CF cells blue, and PHK26 labeled Tregs-exo in red. After merging, a large number of red fluorescent exosomes surrounded the CF cell nuclei. Figure 1 Immunofluorescence assay in sample B showed that CFs had a good uptake capacity for Tregs-exo. Compared with Tregs-derived exosomes, electron microscopy analysis of the morphology of drug-loaded Tregs-exo_miR-142-5p yielded the following results: Figure 1 As shown in Figure C, there was no change, and all exhibited a double-membrane saucer-like structure. PCR detection confirmed miR-142-5p expression in Treg cell-derived exosomes co-cultured with CFs, as shown in the figure. Figure 1 As shown in Figure D, miR-142-5p is expressed at low levels in myocardial fibroblasts.

[0038] (2) Particle size detection: Tregs cell-derived exosomes (Tregs-exo) were diluted with sterile PBS, filtered through a 0.22 μm filter, and measured using a particle size analyzer. The results are shown below. Figure 2 As shown in A, from Figure 2 Electron microscopy analysis in A shows that the diameters of Treg-derived exosomes are concentrated in the range of 30-150 nm.

[0039] Tregs cell-derived exosomes (Tregs-exo) and the exosome drug delivery system (Tregs-exo-miR-142-5p) were analyzed using nanoparticle tracking. The results are as follows: Figure 2 A and Figure 2 As shown in B, from Figure 2 As shown in Figure A, the number of T cell-derived exosomes (Tregs-exo) was 4.0 × 10⁻⁶. 8 The particle size is mainly concentrated in the range of 30-150 nm, with a density of [number] particles per mL. Figure 2 As can be seen from B, the particle size of the exosome drug delivery system (Tregs-exo-miR-142-5p) is mainly concentrated in the range of 30-150 nm.

[0040] (3) Western blot detection of exosome surface protein expression: The specific steps are as follows: Proteins were extracted from Tregs cell-derived exosomes (Tregs-exo) and the Tregs cell exosome drug delivery system (Tregs-exo_miR-142-5p), respectively, to obtain total protein from Tregs cell-derived exosomes and total protein from the exosome drug delivery system. These proteins were then mixed with loading buffer and incubated at 100°C for 10 min. After centrifugation, the mixture was placed on ice for loading. SDS-PAGE separating gels and 5% stacking gels were prepared, loaded, electrophoresed, transferred, blocked with 5% milk, and incubated overnight at 4°C with primary antibodies (CD63, CD9, CD81, and TSG101 antibodies). The mixture was washed with 1X TBST, incubated with secondary antibody, then with primary antibody, and developed. Results are shown below. Figure 3 As shown.

[0041] from Figure 3 As can be seen, CD63, CD9, and CD81 are proteins specific to the exosome membrane, while TSG101 is a protein specific to the exosome cytoplasm. From... Figure 3 In sample A, Tregs-derived exosomes (Tregs-exo) showed detectable expression of exosome surface markers CD63, CD9, and CD81, indicating that the exosomes extracted and purified from the supernatant were Tregs cell exosomes, not cell debris or other substances. Figure 3 As shown in Figure B, both Tregs-exo before and after drug loading can detect the expression of exosome surface markers CD9, CD81, and TSG101, indicating that the surface protein function of the exosome drug loading system remains unchanged.

[0042] (4) Potential detection: The sample pool was washed with 1×PBS buffer; Tregs cell-derived exosomes (Tregs-exo) and the Tregs cell-derived exosome drug delivery system (Tregs-exo_miR-142-5p) were diluted with 1×PBS buffer, injected, and the potential changes were detected. The results are as follows: Figure 4 As shown.

[0043] The changes in Tregs exosome potentials before and after drug loading were detected using a Zeta potential analyzer. Figure 4 The detection results showed no significant difference in potential between the exosome drug loading state before (Tregs-exo) and after (Tregs-exo_miR-142-5p). The potential of Tregs-exo_miR-142-5p after drug loading was (-20.77±1.23mV). According to the colloidal stability assessment, the potential of the exosome drug loading system (Tregs-exo_miR-142-5p) was between -20 and -30mV, indicating that the system has good stability. The exosome drug loading system (Tregs-exo_miR-142-5p) showed a negative shift trend, indicating that miR-142-5p was successfully loaded into the exosomes of Tregs cells.

[0044] (5) Nanoflow cytometry detection of exosome positive expression: Take 10 μL of the Tregs cell-derived exosome drug delivery system (Tregs-exo_miR-142-5p) and dilute it to an appropriate fold. Inject the samples sequentially according to the serial dilution. After the sample detection is completed, the fluorescence information of the exosomes detected by the instrument can be obtained. The results are as follows: Figure 5 As shown.

[0045] Fluorescence changes in exosome samples were detected using nanoflow cytometry. Figure 5The test results showed that the positive rate of the Tregs cell-derived exosome drug delivery system (Tregs-exo_miR-142-5p) was 12%.

[0046] Example 3

[0047] Loading efficiency and stability of miR-142-5p on T cell-derived exosomes in drug delivery systems I. The loading efficiency of T cell-derived exosomes for miR-142-5p was detected using qPCR. The specific steps are as follows: (1) Take each Tregs-exo_miR-142-5p sample and add 1 mL of RNA extraction solution to lyse for 5 min. Add 200 μL of chloroform, shake to mix, and let stand at room temperature for 5 min.

[0048] (2) Centrifuge at 4℃, 12000 g for 15 min, and transfer the upper aqueous phase to another centrifuge tube.

[0049] (3) Add an equal volume of isopropanol and mix by inverting. Let stand at -20℃ for 10 min, then centrifuge at 12000 g for 10 min at 4℃. Discard the supernatant and let the RNA settle at the bottom of the tube.

[0050] (4) Add 1 mL of 75% ethanol, gently invert the centrifuge tube, centrifuge at 7500 g for 5 min at 4℃, and discard the supernatant as much as possible.

[0051] (5) Air dry at room temperature for 10 min, then dissolve the RNA precipitate with 20 μL DEPC H2O.

[0052] (6) Detect RNA purity and concentration, and amplify miRNA by reverse transcription and quantitative real-time PCR according to the kit. Obtain miRNA expression.

[0053] The results are as follows Figure 14 As shown in the figure, the encapsulation efficiency of miR-142-5p by exosomes derived from Treg cells using ultrasound was 5.85%. This value is close to the encapsulation efficiency of similar miRNA drugs loaded using existing technologies with similar strategies, demonstrating the effectiveness of this drug delivery strategy.

[0054] II. Stability of T-cell-derived exosome drug delivery systems 30 μg of the drug-loaded system (drug-loaded exosomes) obtained in Example 1 was taken. The T-cell-derived exosomes and the drug-loaded system were incubated at -80℃ for 0, 7, and 14 days, respectively. After incubation at 4℃ and 120,000g for 70 min, they were resuspended in an appropriate amount of PBS. qPCR was used to detect miR-142-5p expression in the drug-loaded system to determine stability. The results are as follows: Figure 6 As shown.

[0055] From Figure 6 The results showed that there was no significant difference in the expression of miR-142-5p, and the expression of miR-142-5p could not be detected in the supernatant after ultracentrifugation, indicating that the stability of miR-142-5p in the drug delivery system was good.

[0056] III. Uptake ability of CFs to the drug delivery system (Tregs-exo_miR-142-5p) Using the same method as in Example 1, DIPA was used to counterstain the cell nuclei, PKH26 was used to red-label Tregs-exo, and FITC was used to green-label miR-142-5p. For the exosome uptake process: A laser confocal microscope was used to observe the uptake of Tregs-exo_miR-142-5p by CFs, and the results are as Figure 7 shown. DAPI stained the nuclei of CFs blue. After adding the PHK26-labeled exosomes to CFs and performing Merge, a large number of red fluorescent exosomes surrounded the cell nuclei, and the miR-142-5p loaded in the green fluorescent exosomes was also around the cell nuclei, indicating that CFs had good uptake ability for Tregs-exo_miR-142-5p.

[0057] Example 4

[0058] Pharmacodynamic evaluation of the T cell-derived exosome drug delivery system 1. Experimental animals and feeding 50 male C57BL / 6J mice, 6 - 8 weeks old (Vital River Laboratory Animal Technology Co., Ltd., production license number: SCXK(Zhe)2025 - 0001, issued by Zhejiang Science and Technology Commission). SPF-class animal room, temperature 20 - 26 °C, humidity 40 - 70%, 12-hour light-dark cycle; free diet except when fasting is required.

[0059] 2. Establishment of a mouse myocardial infarction model A permanent occlusion myocardial infarction surgery was performed on 2-month-old male C57BL / 6J mice. The anesthetized mice were fixed in the supine position, tracheally intubated, and the ventilator ventilation was controlled. The animals were maintained at 37 °C using a far-infrared heating pad. A left thoracotomy was performed under an anatomical microscope, and the proximal left anterior descending coronary artery was ligated and then the surgical wound was closed.

[0060] 3. Animal husbandry and management were conducted in accordance with the Standard Operating Procedures (SOPs) of the Animal Experiment Center of Xinjiang Medical University, while also referring to the 8th edition of the Guide for the Care and Use of Laboratory Animals (2010) and the Public Law 99-198. The methods of animal use were approved by the Institute of Animal Ethics (IACUC). The IACUC approval number for this experiment is: IACUC-20230508-06.

[0061] 4. Treatment Plan The experiment lasted for 4 weeks. Animals were randomly divided into 4 groups and administered drugs according to Table 1.

[0062] Note: po: oral gavage; IV: intravenous injection; IM: intramyocardial injection; QD: once daily; Biw: twice weekly. 5. Evaluation Indicators ①Weighing: Weigh once before model establishment, and twice a week after drug administration; ② Echocardiography before the end of the experimental drug administration; Four weeks after myocardial infarction, changes in left ventricular ejection fraction (EF), fractional shortening (FS), end-systolic diameter (LVSD), and end-diastolic diameter (LVDD) were measured in surviving mice using echocardiography.

[0063] ③ Weigh the isolated heart and calculate the cardiac index; After weighing the mice in each group, blood was collected from the abdominal aorta, and all mice hearts were harvested, washed with physiological saline, dried with filter paper, weighed, and the cardiac weight index was calculated. The results are as follows: Figure 8 and Figure 9 As shown.

[0064] Heart weight coefficient = Total heart weight (g) / Body weight (100g) ④ Distribution of Tregs carrying drugs in the heart Mice in each group were sacrificed at 4 weeks. Tissue samples were collected via perfusion with physiological saline, taking care to avoid light exposure. A negative control group (Negiative) and tissue samples from the Tregs exosome-loaded group (Tregs-exo_miR-142-5p) were placed on black cardstock and photographed using a small animal imaging device. Results are as follows: Figure 9 As shown.

[0065] ⑤ HE staining Tissue blocks were fixed in 4% paraformaldehyde for at least 24 hours. After tissue trimming, rinsing, dehydration, embedding, sectioning, mounting, baking, HE staining, and mounting, the tissue morphology and pathological changes were observed under an optical microscope at 100x magnification.

[0066] ⑥ Masson staining Heart tissue was fixed in formalin solution, routinely dehydrated, embedded in paraffin, sectioned, stained with Masson's stain, and then observed under a microscope for pathological morphology. The collagen deposition in the myocardial tissue was observed, and the collagen fiber area was measured using ImageJ software.

[0067] 6. Data Analysis All data were analyzed using ANOVA (GraphPad Prism 8.0 software). *P<0.05, **P<0.01. Data are expressed as mean ± standard deviation, and differences between groups were analyzed using t-tests.

[0068] from Figure 8 It can be seen that after 14 days, compared with the model group, the mice in the Treg cell-derived exosome drug delivery system (Tregs-exo_miR-142-5p) group had significantly increased body weight. Figure 8 A). Compared with the sham surgery control group, the model group showed a significant increase in heart weight and heart weight coefficient. P <0.05)( Figure 8 B), while in the group simultaneously administered Treg cell-derived exosome delivery system (Tregs-exo_miR-142-5p) or positive telmisartan, heart weight and heart weight coefficient were significantly reduced (B). P <0.05 or P <0.01). Compared with the model group, the survival time of mice in the drug-treated group was significantly prolonged ( Figure 8 C).

[0069] from Figure 9 It can be seen that in the negative control group (Negiative), after 2 hours of administration of the FITC-labeled exosome-loaded miR-142-5p exosome drug delivery system, fluorescence accumulated in the liver. Compared with the negative control group, the Treg cell-derived exosome drug delivery system (Tregs-exo_miR-142-5p) group showed enrichment of FITC-labeled exosome-loaded miR-142-5p, with enrichment also observed in the liver and kidneys. This indicates that FITC-labeled exosome-loaded miR-142-5p can accumulate in the ischemic area of ​​myocardial infarction and is highly likely to be metabolized by the liver and kidneys.

[0070] Based on the conclusions of HE staining ( Figure 10As can be seen, compared with the sham surgery control group, there were no significant changes in the pathological changes of liver, spleen, lung and kidney tissues in mice in the model group, the positive drug telmisartan group and the Treg cell-derived exosome drug delivery system (Tregs-exo-miR-142-5p) group. In the control group (sham surgery), HE staining revealed intact myocardial tissue with well-organized fibers, clearly visible nuclei and cytoplasm, and intact intercellular junctions. In the model group, the cardiac microstructure was severely abnormal, with damage to most of the left ventricular wall, thinning of the wall, disordered arrangement of myocardial fibers, and a relative reduction in cardiomyocytes. Numerous cardiomyocytes showed shrinkage, degeneration, or necrosis (blue arrows), and myocardial fibers were damaged and degenerated into connective tissue with abundant collagen fiber proliferation (green arrows). Extensive inflammatory cell infiltration (black arrows) and hemorrhage (red arrows) were observed at the site of injury. The positive control group (telmisartan) and the exosome-loaded drug system (Treg-exo_miR-42-5p) showed improved myocardial structure, reduced inflammatory cell infiltration (black arrows), gradual recovery of myocardial fiber rupture (green arrows), and reduced congestion and edema (red and yellow arrows). This indicates that the exosome-loaded drug system can significantly improve ventricular remodeling after myocardial infarction.

[0071] From the conclusions of Masson staining ( Figure 11 As can be seen, no fibrotic lesions were observed in the myocardial tissue of the sham-operated control group, and the myocardial cell structure remained intact. The myocardial tissue of the model group mice showed more collagen staining and fibrotic lesions. After administration of the exosome delivery system or telmisartan, the fibrotic lesions were significantly reduced. Collagen area analysis results showed that in the model group, the collagen area was significantly increased (…). P <0.01), while the positive control group (telmisartan) and the exosome delivery system (Tregs-exo_miR-42-5p) significantly reduced collagen area ( P <0.01). Compared with the sham surgery control group, the model group showed significantly increased end-diastolic diameter (LVEDD) and end-systolic diameter (LVESD). P <0.01)( Figure 12 Compared with the model group, the positive control drug telmisartan group and the exosome delivery system Tregs-exo_miR-42-5p group showed significantly reduced end-diastolic diameter and end-systolic diameter. P <0.01). Compared with the sham surgery group, the model group had a significantly lower LVFS (lower left lateral fallopian tube stenosis) score. P <0.01); Compared with the model group, the telmisartan positive control group and the Tregs-exo_miR-42-5p exosome delivery system group showed significantly increased left ejection fraction (LVEF) and left ejection fraction shortening (LVFS). P <0.01)( Figure 13).

[0072] The technical solutions provided by the embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the embodiments of the present invention. The descriptions of the embodiments above are only for helping to understand the principles of the embodiments of the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the embodiments of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A method for preparing a Treg cell-derived exosome drug delivery system, characterized in that: Includes the following steps: (1) Preparation of Treg cell-derived exosomes: Primary Treg cells were obtained by immunomagnetic bead sorting, and Treg cell culture supernatant was prepared; Exo-spin buffer was added to the supernatant for incubation, centrifugation was performed, and the precipitate was resuspended in PBS to obtain precipitated exosomes; after purification of the precipitated exosomes, purified Treg cell-derived exosomes were obtained. (2) After centrifugation, FITC-labeled miR-142-5p agomir was added to DEPC water to prepare a suspension; (3) The suspension obtained in step (2) is mixed with the Treg cell-derived exosomes obtained in step (1) to form a mixture. After sonication, the mixture is incubated at 37°C for 60 minutes to restore the exosome membrane. (4) The mixture obtained in step (3) is centrifuged at 4°C and 120,000g for 70 min and then resuspended in PBS to obtain Treg cell-derived exosomes sonicated with miR-142-5p, i.e., Treg cell-derived exosome drug delivery system.

2. The method for preparing the Treg cell-derived exosome drug delivery system according to claim 1, characterized in that: The volume ratio of the supernatant to the Exo-spin buffer in step (1) is 2:

1.

3. The method for preparing the Treg cell-derived exosome drug delivery system according to claim 1, characterized in that: The purification of sedimented exosomes in step (1) is as follows: PBS is added to the sedimented exosomes, and the mixture is centrifuged at 50×g for 60 seconds to obtain Treg cell-derived exosomes.

4. The method for preparing the T cell-derived exosome drug delivery system according to claim 1, characterized in that: The mixing ratio of the suspension and Treg cell-derived exosomes in step (3) is suspension: T cell-derived exosomes = 1 nmol: 300 ug.

5. The method for preparing the Treg cell-derived exosome drug delivery system according to claim 4, characterized in that: The ultrasound treatment in step (3) is on-ice ultrasound, and the ultrasound operation parameters are: 20% amplitude, 30s on / off for 3 minutes, 6 cycles.

6. A drug delivery system obtained by the preparation method of the Treg cell-derived exosome drug delivery system according to any one of claims 1-5.

7. The use of the drug delivery system according to claim 6 in the preparation of drugs for the prevention and treatment of myocardial infarction.

Images