A combination exosome and a preparation method and application thereof

Abstract

This application relates to the field of targeted drug therapy, specifically to a combination exosome, its preparation method, and its application, comprising the following steps: culture, loading, activation, and modification. Loading: miR-132 or mtDNA is mixed with exosomes, followed by electroporation and purification by incubation to obtain a loaded exosome solution. Activation: Angiopep-2 peptide, RVG peptide, and cRGDfk peptide solution are activated with a sulfonyl-SMCC crosslinking agent to obtain an activated solution. This application loads the active substance miR-132 or mtDNA into exosomes secreted by mesenchymal stem cells via electroporation, and then modifies them with Angiopep-2 peptide, RVG peptide, or cRGDfk peptide. This effectively avoids the electroporation process from damaging the conformation of the targeting peptides, preserving the targeting effect of the combination exosomes. The prepared combination exosomes exhibit good therapeutic effects in nerve repair or skin anti-aging.

Description

Technical Field

[0001] This application relates to the field of targeted drug therapy, specifically to a combination of exosomes, their preparation method, and their applications. Background Technology

[0002] Exosomes are bilayered lipid vesicles with a diameter of 50-100 nm. They have been shown to pose no risk of tumorigenesis and have low immunogenicity. Exosomes can be actively released into the extracellular environment by various mammalian cells. Exosomes derived from mesenchymal stem cells have been shown to carry bioactive molecules such as lipids, proteins, nucleic acids, and non-coding ribonucleic acid. These molecules can be exchanged between different cell types and even across species.

[0003] However, natural exosomes have low loading rates for active substances such as miRNA and mtDNA, making it difficult to precisely reach certain cells, such as brain neurons and dermal fibroblasts. These limitations mean that when treating diseases using natural exosomes carrying active substances such as miRNA and mtDNA, the injection volume needs to be significantly increased to achieve an effective therapeutic level. Summary of the Invention

[0004] To address the issue of low carrying capacity of active substances such as miRNA and mtDNA in natural exosomes, and their difficulty in targeting specific cells, this application provides a method for preparing combinatorial exosomes. This method involves loading the active substance miR-132 or mtDNA onto exosomes secreted during mesenchymal stem cell passage culture via electroporation, and then modifying them with Angiopep-2 peptide, RVG peptide, or cRGDfk peptide. This effectively avoids the electroporation process damaging the conformation of the targeting peptides, preserving the targeting effect of the combinatorial exosomes. The prepared combinatorial exosomes exhibit good therapeutic effects in nerve repair or skin anti-aging.

[0005] In a first aspect, this application provides a method for preparing combined exosomes, employing the following technical solution: A method for preparing combined exosomes includes the following steps: Culture: Mesenchymal stem cells were cultured, and the cell culture supernatant from the second and third passages was collected. After differential centrifugation, the supernatant was filtered to obtain exosomes. Loading: The active substance solution and the exosomes are mixed, then electroporated, and then purified by incubation to obtain a loaded exosome solution; the active substance in the active substance solution includes one of miR-132 and mtDNA; Activation: The labeled protein solution is activated by reacting with a sulfonyl-SMCC crosslinking agent to obtain an activated solution; the labeled protein in the labeled protein solution includes at least one of Angiopep-2 peptide, RVG peptide, and cRGDfk peptide; Activation: The loaded exosome solution is reacted with Traut's Reagent to obtain an activated loaded exosome solution; Modification: The activated loading exosome solution and the activation solution were reacted, and the mixture was ultrafiltered to obtain combined exosomes.

[0006] By employing the above technical solution, the active substance miR-132 or mtDNA is first loaded onto exosomes secreted by mesenchymal stem cells via electroporation. Then, a targeting peptide is chemically coupled to the surface of the exosomes, facilitating the precise delivery of the combined exosomes to specific cells. Using electroporation followed by chemical coupling of the targeting peptide effectively avoids the conformational damage to the targeting peptide during electroporation, preserving the targeting effect of the combined exosomes.

[0007] When miR-132 is used as the active ingredient and angiopep-2 peptide or RVG peptide is used as the labeling protein, the resulting combined exosomes can effectively perform neural repair. When the combined exosomes deliver miR-132 to neurons / microglia, they inhibit PTEN / FOXO1, activate the PI3K / AKT pathway, downregulate inflammatory factors such as NF-κB, and upregulate synaptic proteins such as PSD95, thus improving neuroinflammation and synaptic remodeling. Angiopep-2 peptide or RVG peptide can enhance the ability of the combined exosomes to penetrate the blood-brain barrier, facilitating their delivery to neurons / microglia.

[0008] When mtDNA is used as the active ingredient and cRGDfk peptide is used as the labeling protein, the resulting combined exosomes can effectively combat skin aging. When the combined exosomes deliver mtDNA to dermal fibroblasts, they compensate for mitochondrial genome defects, activate the PGC-1α / NRF2 pathway, promote ATP synthesis and collagen regeneration (COL1A1 / ELN), and inhibit aging markers such as SA-β-gal. The cRGDfk peptide can enhance the enrichment of the combined exosomes in dermal cells, thereby improving its efficacy.

[0009] This application describes a method for loading exosomes secreted during mesenchymal stem cell passage culture with active substances miR-132 or mtDNA via electroporation, followed by modification with Angiopep-2 peptide, RVG peptide, or cRGDfk peptide. This effectively avoids the electroporation process from damaging the conformation of the target peptides, thus preserving the targeting effect of the combined exosomes. The resulting combined exosomes exhibit good therapeutic effects in nerve repair or skin anti-aging.

[0010] Preferably, in the loading step, the active material is miR-132, the electroporation voltage is 700-1200V, and the pulse duration is 5-10ms.

[0011] By employing the above technical solution, miR-132 exhibits high charge density but small molecular size, allowing for the overcoming of the electrostatic barrier of the exosome membrane with a relatively low voltage, thus enabling miR-132 loading. Higher voltages can easily lead to miR-132 degradation; therefore, an electroporation voltage of 700-1200V and a pulse duration of 5-10ms are suitable.

[0012] Preferably, strontium chloride is added to the culture medium during the culturing step.

[0013] By adopting the above technical solution, adding strontium chloride solution during mesenchymal stem cell culture can activate the calcium ion signaling pathway, causing phosphorylation of hnRNPA2B1 protein, which then specifically binds to miR-132, enriching it in exosomes and improving loading efficiency.

[0014] Preferably, in the loading step, the active substance is mtDNA, the electroporation voltage is 1200-1800V, and the pulse duration is 10-20ms.

[0015] By employing the above technical solution, it is found that mtDNA, being a large molecule with extremely strong negative charge repulsion, requires a higher electric field energy to penetrate the exosome membrane, and the exosome membrane pores need to expand sufficiently to accommodate the mtDNA, resulting in a relatively long expansion time. Therefore, when the active substance is mtDNA, an electroporation voltage of 1200-1800V and a pulse duration of 10-20ms are suitable.

[0016] Preferably, in the loading step, the active substance solution, the exosomes, and strontium chloride are mixed.

[0017] By employing the above-mentioned technical solution, the addition of strontium chloride solution allows strontium ions to neutralize the negative charge on the surface of exosomes, reducing the repulsive force between exosomes and negatively charged mtDNA. Strontium ions can also chelate the DNA phosphate backbone, inducing it to contract and coil, facilitating its penetration through membrane pores. The addition of strontium chloride solution further enhances the loading of mtDNA onto exosomes.

[0018] Preferably, in the activation step, the labeled protein solution, CD47 protein solution and sulfonyl-SMCC crosslinking agent are subjected to an activation reaction.

[0019] By employing the above-described technical approach, CD47, a widely expressed signaling protein, is mediated by SIRPα on the surface of macrophages. When CD47 binds to SIRPα, it triggers an inhibitory signal, blocking phagocytosis by macrophages. Modifying this protein onto the surface of exosomes significantly improves its in vivo survival rate and target tissue delivery efficiency.

[0020] Secondly, this application provides a combined exosome, employing the following technical solution: A composite exosome is prepared by the above-described method for preparing composite exosomes.

[0021] Thirdly, this application provides an application of combined exosomes, employing the following technical solution: An application of combined exosomes, including applications in nerve repair or skin anti-aging.

[0022] In summary, this application has the following beneficial effects: 1. Because this application loads the active substance miR-132 or mtDNA onto the exosomes secreted during the passage culture of mesenchymal stem cells via electroporation, and then modifies them with Angiopep-2 peptide, RVG peptide or cRGDfk peptide, it effectively avoids the electroporation from damaging the conformation of the target peptides and retains the targeting effect of the combined exosomes; the prepared combined exosomes can have good therapeutic effects in nerve repair or skin anti-aging. 2. In this application, when the active substance is miR-132, strontium chloride is added to the culture medium in advance, which can activate the calcium ion signaling pathway, phosphorylate the hnRNPA2B1 protein, and thus specifically bind miR-132, enrich it in exosomes, and improve loading efficiency. 3. In this application, when the active substance is mtDNA, the mtDNA, exosomes and strontium chloride need to be mixed first before electroporation. This is mainly because strontium ions can neutralize the negative charge on the surface of exosomes, reducing the repulsive force between exosomes and negatively charged mtDNA; strontium ions can also chelate the DNA phosphate backbone, inducing it to contract and curl up, which facilitates penetration of the membrane pores; with the addition of strontium chloride solution, it is more conducive to the loading of mtDNA onto exosomes. Detailed Implementation

[0023] The present application will be further described in detail below with reference to embodiments and comparative examples.

[0024] Example 1 A method for preparing combined exosomes includes the following steps: Culture: Mesenchymal stem cells were seeded into T75 culture flasks and cultured at 37°C in a constant temperature and humidity incubator with DMEM / F12 medium and 5% CO2. The cell culture supernatant from the second and third passages was collected, centrifuged at differential speed, and filtered to obtain exosomes. Loading: Exosomes were resuspended in sterile PBS to a concentration of 500 μg / mL to obtain an exosome solution; miR-132 was resuspended in sterile PBS to a concentration of 500 μg / mL to obtain a miR-132 solution; the miR-132 solution and the exosome solution were mixed at a volume ratio of 3:2, and then electroporated at a voltage of 1000 V and a pulse duration of 8 ms. After electroporation, the mixture was incubated on ice for 5 min, then incubated at 37 °C for 30 min, and finally purified to obtain the loaded exosome solution; Activation: Angiopep-2 peptide was dissolved in sterile PBS to obtain angiopep-2 peptide solution. The angiopep-2 peptide solution was mixed with sulfonyl-SMCC crosslinking agent at a molar ratio of 1:5. The mixture was then shaken at 25°C in the dark for 30 min to carry out the activation reaction and obtain the activated solution. Activation: Traut's Reagent was added to the exosome loading solution to a final concentration of 50 μM. The reaction was carried out at 25 °C for 1 h to obtain the activated exosome loading solution. Modification: The activation solution was added to the activated loading exosome solution, with a molar ratio of Angiopep-2 peptide to activated loading exosomes of 20:1. The mixture was then shaken at 4°C for 2 hours and ultrafiltered to obtain the combined exosomes.

[0025] Example 2 A method for preparing combined exosomes includes the following steps: Culture: Mesenchymal stem cells were seeded into T75 culture flasks and cultured at 37°C in a constant temperature and humidity incubator with DMEM / F12 medium and 5% CO2. The cell culture supernatant from the second and third passages was collected, centrifuged at differential speed, and filtered to obtain exosomes. Loading: Exosomes were resuspended in sterile PBS to a concentration of 500 μg / mL to obtain an exosome solution; miR-132 was resuspended in sterile PBS to a concentration of 500 μg / mL to obtain a miR-132 solution; the miR-132 solution and the exosome solution were mixed at a volume ratio of 3:2, and then electroporated at a voltage of 1000 V and a pulse duration of 8 ms. After electroporation, the mixture was incubated on ice for 5 min, then incubated at 37 °C for 30 min, and finally purified to obtain the loaded exosome solution; Activation: Dissolve RVG peptide in sterile PBS to obtain RVG peptide solution. Mix RVG peptide solution with sulfonyl-SMCC crosslinking agent at a molar ratio of 1:5. Then, perform activation reaction by shaking at 25°C in the dark for 30 min to obtain activated solution. Activation: Traut's Reagent was added to the exosome loading solution to a final concentration of 50 μM. The reaction was carried out at 25 °C for 1 h to obtain the activated exosome loading solution. Modification: The activation solution was added to the activated loading exosome solution, with a molar ratio of RVG peptide to activated loading exosomes of 20:1. The mixture was then shaken at 4°C for 2 hours and ultrafiltered to obtain the combined exosomes.

[0026] Example 3 A method for preparing combined exosomes includes the following steps: Culture: Mesenchymal stem cells were seeded into T75 culture flasks and cultured at 37°C in a constant temperature and humidity incubator with DMEM / F12 medium and 5% CO2. The cell culture supernatant from the second and third passages was collected, centrifuged at differential speed, and filtered to obtain exosomes. Loading: Exosomes were resuspended in sterile PBS to a concentration of 500 μg / mL to obtain an exosome solution; mtDNA was resuspended in sterile PBS to a concentration of 500 μg / mL to obtain an mtDNA solution; the mtDNA solution and exosome solution were mixed at a volume ratio of 3:2, and then electroporated at a voltage of 1500 V and a pulse duration of 15 ms. After electroporation, the mixture was incubated on ice for 5 min, then incubated at 37 °C for 60 min, and finally purified to obtain the loaded exosome solution; Activation: Dissolve cRGDfk peptide in sterile PBS to obtain cRGDfk peptide solution. Mix cRGDfk peptide solution with sulfonyl-SMCC crosslinking agent at a molar ratio of 1:5. Then, perform activation reaction by shaking at 25°C in the dark for 30 min to obtain activated solution. Activation: Traut's Reagent was added to the exosome loading solution to a final concentration of 100 μM. The reaction was carried out at 25 °C for 1 h to obtain the activated exosome loading solution. Modification: The activation solution was added to the activated loading exosome solution, with a molar ratio of cRGDfk peptide to activated loading exosomes of 10:1. The mixture was then shaken at 4°C for 2 hours and ultrafiltered to obtain the combined exosomes.

[0027] Comparative Examples 1-3 Comparative Example 1, based on the preparation method of Example 1, adjusted the preparation steps as follows: A method for preparing combined exosomes includes the following steps: Culture: Mesenchymal stem cells were seeded into T75 culture flasks and cultured at 37°C in a constant temperature and humidity incubator with DMEM / F12 medium and 5% CO2. The cell culture supernatant from the second and third passages was collected, centrifuged at differential speed, and filtered to obtain exosomes. Loading: Exosomes were resuspended in sterile PBS to a concentration of 500 μg / mL to obtain an exosome solution; miR-132 was resuspended in sterile PBS to a concentration of 500 μg / mL to obtain a miR-132 solution; the miR-132 solution and the exosome solution were mixed at a volume ratio of 3:2, and then electroporated at a voltage of 1000 V and a pulse duration of 8 ms. After electroporation, the mixture was incubated on ice for 5 min and then at 37 °C for 30 min. The loaded exosome solution was then purified; the loaded exosomes were obtained after ultrafiltration.

[0028] Comparative Example 2, based on the preparation method of Example 3, adjusted the steps as follows: A method for preparing combined exosomes includes the following steps: Culture: Mesenchymal stem cells were seeded into T75 culture flasks and cultured at 37°C in a constant temperature and humidity incubator with DMEM / F12 medium and 5% CO2. The cell culture supernatant from the second and third passages was collected, centrifuged at differential speed, and filtered to obtain exosomes. Loading: Exosomes were resuspended in sterile PBS to a concentration of 500 μg / mL to obtain an exosome solution; mtDNA was resuspended in sterile PBS to a concentration of 500 μg / mL to obtain an mtDNA solution; the mtDNA solution and exosome solution were mixed at a volume ratio of 3:2, and then electroporated at a voltage of 1500 V and a pulse duration of 15 ms. After electroporation, the mixture was incubated on ice for 5 min, then incubated at 37 °C for 60 min. The resulting solution was purified to obtain a loaded exosome solution; the loaded exosomes were obtained after ultrafiltration.

[0029] Comparative Example 3, based on the preparation method of Example 1, adjusted the preparation steps as follows: A method for preparing combined exosomes includes the following steps: Culture: Mesenchymal stem cells were seeded into T75 culture flasks and cultured at 37°C in a constant temperature and humidity incubator with DMEM / F12 medium and 5% CO2. The cell culture supernatant from the second and third passages was collected, centrifuged at differential speed, and filtered to obtain exosomes. Activation: Angiopep-2 peptide was dissolved in sterile PBS to obtain angiopep-2 peptide solution. The angiopep-2 peptide solution was mixed with sulfonyl-SMCC crosslinking agent at a molar ratio of 1:5. The mixture was then shaken at 25°C in the dark for 30 min to carry out the activation reaction and obtain the activated solution. Activation: Exosomes were resuspended in sterile PBS to a concentration of 500 μg / mL to obtain an exosome solution; Traut's Reagent was added to the exosome solution to a final concentration of 50 μM, and the mixture was reacted at 25 °C for 1 h to obtain an activated exosome solution; Modification: The activation solution was added to the activated exosome solution, with a molar ratio of Angiopep-2 peptide to activated exosomes of 20:1. The mixture was then shaken at 4°C for 2 hours and ultrafiltered to obtain modified exosomes. The modified exosomes were resuspended in sterile PBS to a concentration of 500 μg / mL to obtain the modified exosome solution. Loading: miR-132 was resuspended in sterile PBS to a concentration of 500 μg / mL to obtain a miR-132 solution; the miR-132 solution and the modified exosome solution were mixed at a volume ratio of 3:2, and then electroporated at a voltage of 1000 V and a pulse duration of 8 ms. After electroporation, the mixture was incubated on ice for 5 min and then at 37 °C for 30 min. The resulting compound exosome solution was then purified and obtained; the compound exosomes were obtained by ultrafiltration.

[0030] Performance testing The combined exosomes of Examples 1-3 and Comparative Example 3, as well as the loaded exosomes of Comparative Examples 1-2, were subjected to the following performance tests, and the test results are shown in Table 1.

[0031] 1. Loading efficiency The content of miR-132 or mtDNA in exosomes after electroporation was determined by qPCR and compared with the content of miR-132 or mtDNA added before electroporation, and the loading efficiency was calculated as (B / A)*100%.

[0032] 2. Level of neural repair Two groups of hTNF-α transgenic mice were randomly divided into an experimental group and a control group. The experimental group was injected daily with 200 μL of a combined exosome solution / loaded exosome solution (the combined exosome / loaded exosome was prepared by adding it to sterile PBS to form a solution, with miR-132 as the active substance), while the control group was injected daily with 200 μL of physiological saline. These injections were repeated for 7 consecutive days. 100 μL of cerebrospinal fluid was collected from each group. The expression level of TNF-α in the cerebrospinal fluid of the experimental group (A) and the expression level of TNF-α in the cerebrospinal fluid of the control group (B) were measured using qPCR. The neural repair level was calculated as (BA / B) * 100%.

[0033] 3. Skin's anti-aging level Two groups of SAMP8 (premature aging) mice were divided into an experimental group and a control group. The experimental group was injected daily with 200 μL of combined exosome solution / loaded exosome solution (combined exosomes / loaded exosomes were prepared by adding them to sterile PBS to form a solution, and the active substance was mtDNA), while the control group was injected daily with 200 μL of physiological saline, for 7 consecutive days. 30 mg of the dermal layer of the midline region of the back of each mouse was taken from the experimental group and the control group. The expression level A of COL1A1 in the dermal layer of the midline region of the back of the experimental group mice and the expression level B of COL1A1 in the dermal layer of the midline region of the back of the control group mice were measured by qPCR. The skin anti-aging level was calculated as (AB / B) * 100%.

[0034] Table 1. Combination exosomes of Examples 1-3 and Comparative Example 3, and the types and performance tests of each component of the loaded exosomes of Comparative Examples 1-2. Referring to Table 1, comparing Examples 1-3 and Comparative Examples 1-3, it can be seen that the combined exosomes prepared using miR-132 as the active substance and angiopep-2 peptide or RVG peptide as the labeling protein can effectively perform nerve repair. Without the labeling of angiopep-2 peptide or RVG peptide, the exosomes' ability to penetrate the blood-brain barrier is reduced, making it difficult to reach neurons / microglia. During preparation, electroporation followed by chemical coupling of the targeting peptide is necessary to effectively avoid the electroporation process damaging the conformation of the targeting peptide and to preserve the targeting effect of the combined exosomes. Furthermore, the combined exosomes prepared using mtDNA as the active substance and cRGDfk peptide as the labeling protein can effectively perform skin anti-aging.

[0035] Examples 4-5 Examples 4-5 are based on the preparation method of Example 1, but the voltage and time of electroporation are adjusted. The specific adjustments are shown in Table 2.

[0036] The combined exosomes from Examples 4-5 were subjected to the performance tests described above, and the test results are shown in Table 2.

[0037] Table 2 shows the voltage, time, and performance test results of electroporation in Examples 1 and 4-5. Referring to Table 2, a comparison of Examples 1 and 4-5 shows that when the voltage is insufficient, even with an extended pulse duration, it is difficult to overcome the electrostatic barrier of the exosome membrane and complete miR-132 loading, resulting in low loading efficiency and reduced neural repair level of the combined secretosomes. When the voltage is high, miR-132 degradation is more likely, which also leads to low loading efficiency and reduced neural repair level of the combined secretosomes.

[0038] Examples 6-7 Examples 6-7 are based on the preparation method of Example 3, but the voltage and time of electroporation are adjusted. The specific adjustments are shown in Table 3.

[0039] The combined exosomes from Examples 6-7 were subjected to the performance tests described above, and the test results are shown in Table 3.

[0040] Table 3. Voltage, time, and performance test results of electroporation in Examples 3 and 6-7. Referring to Table 3, a comparison of Examples 3 and 6-8 shows that when the voltage is insufficient, mtDNA, being a large molecule, exhibits extremely strong negative charge repulsion, making it difficult to penetrate the exosome membrane and complete mtDNA loading. This results in low loading efficiency and reduces the skin's anti-aging level of the combined secretions. Conversely, when the voltage is high, exosomes are easily damaged, similarly leading to low loading efficiency and reducing the skin's anti-aging level of the combined secretions.

[0041] Examples 8-13 Example 8 is based on the preparation method of Example 1, but the preparation method is adjusted as follows: in the culture step, strontium chloride is added to the DMEM / F12 medium at a concentration of 1 mM, while the other conditions remain unchanged.

[0042] Example 9 is based on the preparation method of Example 1, but the preparation method is adjusted as follows: In the loading step, the exosomes are resuspended in sterile PBS to a concentration of 500 μg / mL, and strontium chloride is added at a concentration of 2 mM to obtain an exosome solution.

[0043] Example 10 is based on the preparation method of Example 3, but the preparation method is adjusted as follows: in the culture step, strontium chloride is added to the DMEM / F12 medium at a concentration of 1 mM, while the other conditions remain unchanged.

[0044] Example 11 is based on the preparation method of Example 3, but the preparation method is adjusted as follows: In the loading step, the exosomes are resuspended in sterile PBS to a concentration of 500 μg / mL, and strontium chloride is added at a concentration of 2 mM to obtain an exosome solution.

[0045] Example 12 is based on the preparation method of Example 1, but the preparation method is adjusted as follows: In the activation step, Angiopep-2 peptide and CD47 protein are dissolved in sterile PBS to obtain Angiopep-2 peptide solution and CD47 protein solution, respectively. The Angiopep-2 peptide solution, CD47 protein solution and sulfonyl-SMCC crosslinking agent are mixed with each other in a molar ratio of 1:1:10, and then the mixture is activated by shaking at 25°C in the dark for 30 min to obtain an activated solution.

[0046] Example 13 is based on the preparation method of Example 3, but the preparation method is adjusted as follows: In the activation step, Angiopep-2 peptide and CD47 protein are dissolved in sterile PBS to obtain Angiopep-2 peptide solution and CD47 protein solution, respectively. The Angiopep-2 peptide solution, CD47 protein solution and sulfonyl-SMCC crosslinking agent are mixed with each other in a molar ratio of 1:1:10, and then the mixture is activated by shaking at 25°C in the dark for 30 min to obtain an activated solution.

[0047] The combined exosomes of Examples 8-13 were subjected to the above performance tests, and the test results are shown in Table 4.

[0048] Table 4. Examples 1, 3, and 8-13, and performance test results. Referring to Table 4, a comparison of Examples 1, 3, and 8-13 shows that when the active substance is miR-132, adding strontium chloride to the culture medium is superior to directly mixing strontium chloride with exosomes. This is because, during mesenchymal stem cell culture, the addition of strontium chloride activates the calcium ion signaling pathway, leading to phosphorylation of the hnRNPA2B1 protein, which then specifically binds to miR-132, enriching it in exosomes and improving loading efficiency. However, when the active substance is miR-132, its molecular weight is much smaller than that of mtDNA, resulting in insufficient promoting effect from the addition of strontium chloride.

[0049] When the active substance is mtDNA, mixing strontium chloride directly with exosomes is superior to adding strontium chloride to the culture medium. This is because strontium ions can neutralize the negative charge on the surface of exosomes, reducing the repulsive force between exosomes and negatively charged mtDNA. Strontium ions can also chelate the DNA phosphate backbone, inducing it to contract and coil, facilitating its penetration through membrane pores. The addition of strontium chloride solution further enhances the loading of mtDNA onto exosomes.

[0050] Whether it's miR-132 or mtDNA, the addition of CD47 protein during activation enhances therapeutic efficacy. This is because CD47 is a widely expressed signaling protein whose receptor is SIRPα on the surface of macrophages. When CD47 binds to SIRPα, it triggers an inhibitory signal, blocking macrophage phagocytosis. Modifying this protein onto the surface of exosomes significantly improves its survival rate in vivo and its delivery efficiency to target tissues.

[0051] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method of preparing a combination exosome, characterized by, Includes the following steps: Culture: Mesenchymal stem cells were cultured, and the cell culture supernatant from the second and third passages was collected. After differential centrifugation, the supernatant was filtered to obtain exosomes. Loading: The active substance solution and the exosomes are mixed, then electroporated, and then purified by incubation to obtain a loaded exosome solution; the active substance in the active substance solution includes one of miR-132 and mtDNA; The active material is miR-132, the electroporation voltage is 700-1200V, and the pulse duration is 5-10ms; The active substance is mtDNA, and the electroporation voltage is 1200-1800V with a pulse duration of 10-20ms. Activation: The labeled protein solution and CD47 protein solution are subjected to an activation reaction with a sulfonyl-SMCC crosslinking agent to obtain an activated solution; the labeled protein in the labeled protein solution includes at least one of Angiopep-2 peptide, RVG peptide and cRGDfk peptide; Activation: The loaded exosome solution is reacted with Traut's Reagent to obtain an activated loaded exosome solution; Modification: The activated loading exosome solution and the activation solution were reacted, and the mixture was ultrafiltered to obtain combined exosomes; The active substance is miR-132, and strontium chloride is added to the culture medium during the culturing process; The active substance is mtDNA. In the loading step, the active substance solution, the exosomes, and strontium chloride are mixed.

2. A composite exosome, characterized in that: It is prepared by the method for preparing combined exosomes as described in claim 1.

Images