Exosome cardiac-targeted delivery system and preparation method and application thereof

Abstract

The present application relates to a delivery system, and discloses an exosome heart-targeting delivery system and a preparation method and application thereof.The exosome heart-targeting delivery system comprises an exosome and a fatty acid translocase ligand modified on the surface of the exosome.The exosome heart-targeting delivery system has good targeting effect, can improve the accumulation of the exosome in the heart after intravenous administration, and is beneficial to improving the drug efficacy.

Description

Technical Field

[0001] This invention relates to a delivery system, specifically an exosome-based cardiac-targeted delivery system. Furthermore, this invention also relates to a method for preparing the exosome-based cardiac-targeted delivery system and its application as a drug delivery system for treating myocardial ischemia-reperfusion injury. Background Technology

[0002] Myocardial ischemia-reperfusion injury can lead to worsening myocardial damage, heart failure, and even death, posing a serious obstacle to the benefits of post-ischemic reperfusion in myocardial infarction patients. However, effective prevention and treatment measures are currently lacking in clinical practice. Stem cell exosomes have been used to treat various diseases due to their ability to inhibit inflammation and apoptosis, and promote tissue repair. However, their poor myocardial targeting, insufficient efficacy, and difficulty in in-situ cardiac administration limit their clinical application in ischemic heart disease. Designing appropriate drug delivery strategies to increase exosome accumulation in the myocardium is a pressing issue that needs to be addressed.

[0003] Most existing studies target damaged cardiomyocytes, which increases the drug's targeting to some extent, but the targeting effect is still limited, resulting in insufficient drug efficacy.

[0004] Based on existing technologies, there is an urgent need to provide an exosome cardiac targeted delivery system that can improve targeting effectiveness. Summary of the Invention

[0005] The purpose of this invention is to overcome the problem of limited drug targeting effect in the prior art, and to provide an exosome cardiac targeted delivery system, its preparation method and application. This exosome cardiac targeted delivery system can improve the accumulation of exosomes in the heart after intravenous administration, has a high targeting effect, and is beneficial to improving drug efficacy.

[0006] To achieve the above objectives, the present invention provides an exosome cardiac targeted delivery system, the system comprising exosomes and fatty acid transloses modified on the surface of the exosomes.

[0007] Preferably, the fatty acid translocase ligand is 1-palmitoyl-2-(5-keto-6-octen-diacylchloro)phosphatidylcholine.

[0008] More preferably, the amount of modification of the fatty acid translocase ligand is 2,500-45,000 relative to one exosome.

[0009] Preferably, the exosomes are plant-derived exosomes and / or animal-derived exosomes.

[0010] More preferably, the animal-derived exosomes are selected from at least one of the following: exosomes from HEK293T cells, exosomes from bone marrow mesenchymal stem cells, exosomes from umbilical cord mesenchymal stem cells, exosomes from adipose-derived mesenchymal stem cells, exosomes from cardiomyocyte-derived cells, exosomes from endothelial progenitor cells, exosomes from hematopoietic stem cells, exosomes from macrophages, exosomes from monocytes, exosomes from plasma, and exosomes from platelets.

[0011] Preferably, the system further includes ischemic myocardial targeting peptides modified on the surface of the exosomes.

[0012] More preferably, the amount of modification of the ischemic myocardial targeting peptide is 2,000-8,000 relative to one exosome.

[0013] Preferably, the amino acid sequence of the ischemic myocardial targeting peptide is shown in SEQ ID NO.1.

[0014] A second aspect of the present invention provides a method for preparing an exosome cardiac targeted delivery system, comprising the following steps:

[0015] The exosomes and / or exosomes coupled with diphenylcyclooctylene are reacted with the substance to be modified and then separated.

[0016] The substance to be modified contains a fatty acid translocase ligand.

[0017] Preferably, the fatty acid translocase ligand is 1-palmitoyl-2-(5-keto-6-octene-diacylchloro)phosphatidylcholine;

[0018] The exosomes are plant-derived exosomes and / or animal-derived exosomes.

[0019] More preferably, the animal-derived exosomes are selected from at least one of the following: exosomes from HEK293T cells, exosomes from bone marrow mesenchymal stem cells, exosomes from umbilical cord mesenchymal stem cells, exosomes from adipose-derived mesenchymal stem cells, exosomes from cardiomyocyte-derived cells, exosomes from endothelial progenitor cells, exosomes from hematopoietic stem cells, exosomes from macrophages, exosomes from monocytes, exosomes from plasma, and exosomes from platelets.

[0020] Preferably, the method includes the following steps: reacting the exosomes of conjugated diphenylcyclooctylene with the substance to be modified in a contact reaction followed by separation; the substance to be modified further includes an ischemic myocardium-targeting peptide with N-terminal modification of azide.

[0021] More preferably, the amino acid sequence of the ischemic myocardial targeting peptide is shown in SEQ ID NO.1.

[0022] Preferably, relative to 1 mg of the exosomes on a protein basis, the amount of fatty acid translocase ligand added is 0.002-0.02 mg, and the amount of the N-terminal modified azide-targeting peptide for ischemic myocardium is 0.001-0.01 mg.

[0023] Preferably, the conditions for the contact reaction include at least: a rotation speed of 50-200 rpm and a time of 1-24 h.

[0024] Preferably, the method for preparing the exosomes coupled with diphenylcyclooctylene includes: reacting the exosomes with diphenylcyclooctylene-sulfonyl-succinimide ester in a contact reaction followed by separation.

[0025] More preferably, the amount of diphenylcyclooctyne-sulfonyl-succinimide ester added is 0.002-0.02 mg relative to 1 mg of the exosomes based on protein.

[0026] Preferably, the conditions for the contact reaction include at least: being carried out in a solvent for 1-6 hours.

[0027] The third aspect of this invention provides an application of the exosome cardiac targeted delivery system described in the first aspect or the exosome cardiac targeted delivery system prepared by the preparation method described in the second aspect as a drug delivery system for treating myocardial ischemia-reperfusion injury.

[0028] Through the above technical solution, the exosome cardiac targeted delivery system provided by the present invention can promote the exosomes to cross the cardiac vascular endothelial barrier and target ischemic myocardial tissue by modifying the surface of exosomes with fatty acid translocase ligands, thereby increasing the accumulation of exosomes in the heart after intravenous administration and effectively improving the targeting effect of the exosome drug delivery system. This effectively improves the bioavailability and efficacy of the drug, providing a more effective way to treat myocardial ischemia-reperfusion injury. Attached Figure Description

[0029] Figure 1 Figure showing the results of flow cytometry characterization of exosome modification efficiency;

[0030] Figure 2 The uptake fluorescence diagram (A) and the results of quantitative fluorescence analysis (B) of targeted modified exosomes in normoxic and hypoxic-induced damaged cardiomyocytes HL-1;

[0031] Figure 3 The uptake of KOdiA-PC modified exosomes in different endothelial cells is shown. Among them, A and C are fluorescence images of KOdiA-PC modified exosomes uptake by different endothelial cells and their fluorescence quantitative analysis results, while B and D are fluorescence images of CMEC cells uptake of exosomes and KOdiA-PC modified exosomes and their fluorescence quantitative analysis results.

[0032] Figure 4 The transendothelial transport of KOdiA-PC modified exosomes loaded with siRNA drugs is shown in Figure 1. A is a schematic diagram of the transendothelial transport model. B and C are fluorescence images and quantitative fluorescence analysis results of the uptake of drugs transported by endothelial cells by HL-1 cells in the lower chamber after different endothelial cells are seeded in the upper chamber. D and E are fluorescence images and quantitative fluorescence analysis results of the uptake of siRNA drugs loaded by exosomes and KOdiA-PC modified exosomes by HL-1 cells in the lower chamber after CMEC cells are seeded in the upper chamber.

[0033] Figure 5 Image (A) showing the accumulation of DiR-labeled exosomes in mouse ischemic-reperfused hearts and the results of their quantitative fluorescence analysis (B). Detailed Implementation

[0034] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0035] As previously described, a first aspect of the present invention provides an exosome cardiac targeted delivery system comprising exosomes and fatty acid translocase ligands modified on the surface of the exosomes.

[0036] When using exosomes as a drug delivery system for cardiac diseases, exosomes must first cross capillaries before entering the myocardial tissue and be taken up by cardiomyocytes. Compared to passively passing through the intercellular spaces of endothelial cells, particle penetration through capillaries relies primarily on active transcytosis by endothelial cells. Solving the problem of exosomes crossing the cardiac endothelial barrier is key to increasing exosome accumulation in the myocardium, and this is precisely the problem that is most easily overlooked.

[0037] During their research, the inventors of this invention unexpectedly discovered that modifying the surface of exosomes with fatty acid translocase ligands enables exosomes to be specifically transported by cardiac microvascular endothelial cells, promoting the crossing of the cardiac vascular endothelial barrier and improving their transendothelial transport efficiency. This, in turn, increases the accumulation of exosomes in the heart after intravenous administration, effectively enhancing the targeting effect of the exosome cardiac targeted delivery system. Consequently, it effectively improves the bioavailability and efficacy of the drug, providing a more effective approach for the treatment of myocardial ischemia-reperfusion injury and improving the therapeutic effect of ischemia-reperfusion injury.

[0038] According to the present invention, preferably, the fatty acid translocase ligand is 1-palmitoyl-2-(5-keto-6-octenyl-diacyl chloride)phosphatidylcholine, which can be inserted between the exosome bilayer membrane, so that the fatty acid translocase and the ligand have a better binding effect, thereby improving the targeting effect of the obtained exosome drug delivery system.

[0039] Preferably, the amount of modification of the fatty acid translocase ligand is 2,500-45,000 relative to one exosome. During their research, the inventors discovered that limiting the amount of modification of the fatty acid translocase ligand within the above range can promote the uptake of exosomes by cardiac microvascular endothelial cells, thereby further increasing the accumulation of exosomes in the heart after intravenous administration, and thus improving the targeting effect of this exosome cardiac targeted delivery system.

[0040] According to the present invention, preferably, the exosomes are plant-derived exosomes and / or animal-derived exosomes. More preferably, the exosomes are animal-derived exosomes; the animal-derived exosomes are selected from at least one of the following: exosomes from HEK293T cells, exosomes from bone marrow mesenchymal stem cells, exosomes from umbilical cord mesenchymal stem cells, exosomes from adipose-derived mesenchymal stem cells, exosomes from cardiomyocyte-derived cells, exosomes from endothelial progenitor cells, exosomes from hematopoietic stem cells, exosomes from macrophages, exosomes from monocytes, exosomes from plasma, and exosomes from platelets.

[0041] Preferably, the system further includes an ischemic myocardial targeting peptide modified on the surface of the exosomes. The ischemic myocardial targeting peptide modified on the exosome surface and the fatty acid translocase ligand can interact to further enhance the accumulation of exosomes in the heart after intravenous administration, thereby improving the targeting effect of the exosome cardiac targeting delivery system. Further preferably, considering further improving the targeting effect of the exosome cardiac targeting delivery system, the amino acid sequence of the ischemic myocardial targeting peptide is as shown in SEQ ID NO.1. Specifically, SEQ ID NO.1 is STSMLKA.

[0042] Preferably, the amount of modification of the ischemic myocardial targeting peptide relative to one exosome is 2000-8000. Studies have found that limiting the amount of modification of the ischemic myocardial targeting peptide within the above range can further improve the targeting effect of the exosome cardiac targeted delivery system.

[0043] Secondly, the present invention provides a method for preparing an exosome cardiac-targeted delivery system, comprising the following steps:

[0044] The exosomes and / or exosomes coupled with diphenylcyclooctylene are reacted with the substance to be modified and then separated.

[0045] The substance to be modified contains a fatty acid translocase ligand.

[0046] During their research, the inventors discovered that the exosome cardiac targeted delivery system prepared by the above method has a better targeting effect.

[0047] According to the present invention, preferably, the fatty acid translocase ligand is 1-palmitoyl-2-(5-keto-6-octen-diacyl)phosphatidylcholine, which can be inserted between the exosome bilayer membrane, so that the fatty acid translocase and the ligand have a better binding effect, thereby improving the targeting effect of the obtained exosome drug delivery system.

[0048] According to the present invention, preferably, the exosomes are plant-derived exosomes and / or animal-derived exosomes. More preferably, the exosomes are animal-derived exosomes; the animal-derived exosomes are selected from at least one of the following: exosomes from HEK293T cells, exosomes from bone marrow mesenchymal stem cells, exosomes from umbilical cord mesenchymal stem cells, exosomes from adipose-derived mesenchymal stem cells, exosomes from cardiomyocyte-derived cells, exosomes from endothelial progenitor cells, exosomes from hematopoietic stem cells, exosomes from macrophages, exosomes from monocytes, exosomes from plasma, and exosomes from platelets.

[0049] Specifically, exosomes can be obtained by isolating at least one of the following: HEK293T cells, bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, adipose-derived mesenchymal stem cells, cardiomyocyte-derived cells, endothelial progenitor cells, hematopoietic stem cells, macrophages, monocytes, plasma, and platelets. The isolation conditions can be determined based on the actual experimental situation.

[0050] Preferably, the method includes the following steps: reacting exosomes coupled with diphenylcyclooctylene with the substance to be modified, followed by separation; the substance to be modified further includes an ischemic myocardial targeting peptide with an N-terminus modified with azide. The ischemic myocardial targeting peptide modified on the exosome surface and the fatty acid translocase ligand can interact to further enhance the accumulation of exosomes in the heart after intravenous administration, thereby improving the targeting effect of the exosome cardiac targeting delivery system. Exosomes coupled with diphenylcyclooctylene can promote the modification of the ischemic myocardial targeting peptide on the exosomes, thereby further improving the targeting effect of the exosome cardiac targeting delivery system. Further preferably, considering further improving the targeting effect of the exosome cardiac targeting delivery system, the amino acid sequence of the ischemic myocardial targeting peptide is as shown in SEQ ID NO.1. Specifically, SEQ ID NO.1 is STSMLKA.

[0051] Preferably, relative to 1 mg of the exosomes (based on protein content), the amount of fatty acid translocase ligand added is 0.002-0.02 mg, and the amount of the N-terminal azide-modified ischemic myocardial targeting peptide is 0.001-0.01 mg. Under these conditions, the fatty acid translocase ligand and the ischemic myocardial targeting peptide can be modified onto the exosomes in a suitable ratio, thereby improving the targeting effect of the prepared exosome drug delivery system.

[0052] Preferably, the contact reaction conditions include: a temperature of 0-8℃, specifically 0℃, 2℃, 4℃, 6℃, 8℃, or any value within the range of any two of the above values; a rotation speed of 50-200 rpm, specifically 50 rpm, 100 rpm, 150 rpm, 200 rpm, or any value within the range of any two of the above values; and a time of 1-24 h, specifically 1 h, 6 h, 12 h, 18 h, 24 h, or any value within the range of any two of the above values. Under these conditions, fatty acid translocase ligands and ischemic myocardial targeting peptides exhibit better modification effects on exosomes while maintaining the structural and functional integrity of the exosomes, thereby improving the targeting effect of the prepared exosome drug delivery system.

[0053] According to the present invention, the separation process may involve placing the liquid obtained after the contact reaction into an ultrafiltration tube to remove excess N-terminal modified azide-dependent ischemic myocardial targeting peptides and fatty acid translocase ligands. Preferably, the ultrafiltration tube has a specification of 100 kDa, which provides better removal of N-terminal modified azide-dependent ischemic myocardial targeting peptides and fatty acid translocase ligands.

[0054] Preferably, the method for preparing the exosomes coupled with diphenylcyclooctylene includes: reacting the exosomes with diphenylcyclooctylene-sulfonyl-succinimide ester and then separating them.

[0055] Preferably, the amount of diphenylcyclooctyne-sulfonyl-succinimide ester added is 0.002-0.02 mg relative to 1 mg of the exosomes (based on protein). Limiting the amount of diphenylcyclooctyne-sulfonyl-succinimide ester within the above-mentioned mass range allows for better coupling between the exosomes and the diphenylcyclooctyne-sulfonyl-succinimide ester, thereby improving the binding effect between the exosomes and the substance to be modified, and consequently enhancing the targeting effect of the exosome cardiac targeted delivery system.

[0056] To further enhance the coupling effect between exosomes and diphenylcyclooctylene-sulfonyl-succinimide ester, thereby improving the targeting effect of the exosome cardiac targeted delivery system, preferably, in the method for preparing exosomes coupled with diphenylcyclooctylene, the contact reaction is carried out under oscillating conditions. The contact reaction conditions further include: carrying out the reaction in a solvent for 1-6 hours, specifically 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, or any value within the range of any two of the above values. Specifically, the solvent is PBS buffer.

[0057] According to the present invention, in the method for preparing the exosomes of the coupled diphenylcyclooctynylene, the separation treatment may involve placing the liquid obtained after coupling in an ultrafiltration tube to remove excess diphenylcyclooctynyl-sulfo-succinimide ester. Preferably, the ultrafiltration tube has a specification of 100 kDa, which has a better removal effect on diphenylcyclooctynyl-sulfo-succinimide ester.

[0058] Thirdly, the present invention provides an application of the exosome cardiac targeted delivery system described in the first aspect or the exosome cardiac targeted delivery system prepared by the preparation method described in the second aspect as a drug delivery system for treating myocardial ischemia-reperfusion injury.

[0059] Specifically, drugs used to treat myocardial ischemia-reperfusion injury can be siRNA drugs, or small molecule drugs such as edaravone and curcumin that can alleviate myocardial injury.

[0060] According to a particularly preferred embodiment of the present invention, a method for preparing an exosome drug delivery system is provided, comprising the following steps:

[0061] S1. Dissolve exosomes (Exo) in PBS to prepare a PBS solution containing exosomes, add diphenylcyclooctylene-sulfo-succinimide ester (DBCO-sulfo-NHS), shake and react at room temperature for 1-6 h, remove excess DBCO-sulfo-NHS with a 100 kDa ultrafiltration tube to obtain DBCO-coupled exosomes (DBCO-Exo);

[0062] S2. 1-Palmitoyl-2-(5-keto-6-octen-diacyl)phosphatidylcholine (KOdiA-PC) and N-terminal azide-modified ischemic myocardial targeting peptide (IMT) were added to DBCO-Exo and reacted at 0-8℃ and 50-200 rpm for 1-24 h. After purification and removal of free KOdiA-PC and IMT using a 100 kDa ultrafiltration tube, the exosome cardiac targeting delivery system was obtained.

[0063] Relative to 1 mg of the exosomes on a protein basis, the amount of the diphenylcyclooctyne-sulfo-succinimide ester added is 0.002-0.02 mg, the amount of the fatty acid translocase ligand added is 0.002-0.02 mg, and the amount of the N-terminal modified azide-targeting peptide for ischemic myocardium added is 0.001-0.01 mg.

[0064] The exosome system constructed in this invention can be specifically and efficiently taken up by cardiac microvascular endothelial cells, and its transendothelial transport efficiency can be improved. At the same time, it can also increase the uptake of exosome system in damaged cardiomyocytes, improve its targeting effect, and accumulate more efficiently in the heart.

[0065] The present invention will be described in detail below through embodiments.

[0066] Example 1

[0067] After culturing S1 and HEK293T cells (purchased from ATCC) for 48 h, the culture supernatant was collected. The supernatant was centrifuged at 2000g for 20 min at 4 °C, then centrifuged at 10000g for 30 min at 4 °C. The supernatant was then filtered through a 0.22 μm filter and centrifuged at 100000g for 70 min. The precipitate was washed with PBS and resuspended to obtain exosomes (Exo). The concentration of exosome protein was quantified using a BCA kit (Solepro, PC0020).

[0068] S2. Dilute the exosomes (Exo) with PBS to prepare an exosome solution of 0.5 mg / mL. Add 2 mg / mL of diphenylcyclooctylene-sulfo-succinimide ester (DBCO-sulfo-NHS, Glen Research, 50-1941) dissolved in dimethyl sulfoxide to the above exosome solution. The final concentration of DBCO-sulfo-NHS is 10 μM. The reaction is carried out at room temperature with shaking for 4 h. Remove excess DBCO-sulfo-NHS with a 100 kDa ultrafiltration tube to obtain DBCO-coupled exosomes (DBCO-Exo).

[0069] S3. 1-Palmitoyl-2-(5-keto-6-octen-diacyl)phosphatidylcholine (KOdiA-PC, APExBio, C4591) and an N-terminal azide-modified ischemic myocardial targeting peptide (IMT, synthesized by Suzhou Qiangyao Biotechnology Co., Ltd.) were added to DBCO-Exo. The mass ratio of KOdiA-PC to exosomes based on protein was 1:100, and the mass ratio of IMT to exosomes based on protein was 1:300. The above reaction solution was reacted at 4°C and 100 rpm for 12 h. After purification and removal of free KOdiA-PC and IMT using a 100 kDa ultrafiltration tube, the exosome cardiac targeting delivery system was obtained.

[0070] The efficiency of dual-target modification was assessed using Cy5-labeled IMT and BodiPY-labeled fatty acid translocase ligands, and characterized by flow cytometry. Figure 1 The results showed that 93.1% of the exosomes were modified by KOdiA-PC and IMT.

[0071] The particle concentration of the dual-targeted modified exosomes was determined using a nanoparticle size analyzer (NS300, Malvern). The fluorescently labeled ligands modified on the exosome surface were quantified using a multi-functional microplate reader (SpectraMax iD3, Molecular Devices). The excitation / emission wavelengths for Cy5 were 640 nm / 680 nm, and for BodiPY, they were 480 nm / 520 nm. The modification density of ligands on the exosomes was calculated, with an average modification amount of 23,020 ligands / exosome for KOdiA-PC and 3,703 ligands / exosome for ischemic myocardial targeting peptides.

[0072] Test Example 1

[0073] Exosomes loaded with edaravone (MCI-186) and siRNA drugs

[0074] First, MCI-186 was loaded into targeted-modified exosomes via co-incubation. MCI-186 (MCE, HY-B0099) and exosomes were mixed at a mass ratio of 2:1 and incubated with shaking at 37°C for 20 min. Then, siRNA (synthesized by Suzhou Gemma Gene Co., Ltd.) was mixed with exosomes at a mass ratio of 1:15, and Exofect reagent (SBI, EXFT20A) was added. The mixture was incubated at 37°C for 10 min, followed by the addition of TC reagent. After precipitation on ice for 30 min, the exosomes were precipitated by centrifugation at 12000g for 3 min at 4°C, removing free MCI-186 and siRNA, resulting in a dual-targeting exosome drug delivery system co-loaded with siRNA and MCI-186. The absorption of MCI-186 at 245 nm was detected by UV-Vis absorption spectroscopy for quantification. The encapsulation efficiency of MCI-186 was calculated using Formula 1: Encapsulation efficiency = (drug dosage - supernatant volume) / drug dosage × 100% (Formula 1). Using TAMRA-labeled siRNA as a model drug, fluorescence in the supernatant after centrifugation was detected, and the encapsulation efficiency of siRNA was calculated according to Formula 1. The calculated encapsulation efficiencies of MCI-186 and siRNA were 8% and 90%, respectively.

[0075] Test Example 2

[0076] Uptake of targeted modified exosomes in HL-1 cardiomyocytes

[0077] HL-1 cells (ATCC) were divided into groups of 5 × 10⁻⁶. 4 HL-1 cells were seeded at a density of 1 cell / well in pre-coated 24-well plates and cultured overnight in DMEM (Gibco) medium supplemented with 10% (v / v) FBS, 1% (v / v) penicillin-streptomycin, and 2 mM L-glutamine. HL-1 cells were treated with a mixture of 10 mM sodium thiosulfate and 2 mM 2-deoxy-D-glucose for 30 min to simulate ischemic injury. Exosomes labeled with CM-DiI (Invitrogen) at 20 μg / mL (Exo), IMT-modified exosomes (I-Exo), and double-modified exosomes (KI-Exo) were added and co-incubated with the cells for 2 h. After washing three times with PBS to remove untaken drugs, cells were fixed with 4% (w / v) paraformaldehyde at room temperature for 10 min, followed by nucleus staining with 5 μg / mL DAPI at room temperature for 5 min. Images were taken under a laser confocal microscope and quantitative fluorescence analysis was performed. Results are as follows: Figure 2 As shown, 2 hours after uptake, under normoxic and simulated hypoxia conditions, only weak red fluorescence was observed in the Exo group. However, under simulated hypoxia conditions, compared to the Exo group, the red fluorescence in the I-Exo and KI-Exo groups was significantly increased, indicating that the modification of the ischemic myocardial targeting peptide increased the uptake of exosomes in damaged cells.

[0078] Test Example 3

[0079] Uptake of fatty acid translocase ligand-modified exosomes in different endothelial cells

[0080] Cardiac microvascular endothelial cells (CMECs) highly express the fatty acid translocase CD36, while brain microvascular endothelial cells (BMECs) and human umbilical vein blood endothelial cells (HUVECs) show lower CD36 expression. The uptake of exosomes K-Exo modified with 1-palmitoyl-2-(5-keto-6-octenyl-diacyl)phosphatidylcholine in these different endothelial cells was compared. 4Cells were seeded at a density of 1 cell / well in 24-well plates pre-coated with glass slides and cultured overnight. 20 μg / mL of CM-DiI-labeled Exo or K-Exo was added, and the cells were co-incubated for 4 h. To verify the role of KOdiA-PC modification in endothelial cell uptake, a CD36 receptor blocking group was set up. CMEC cells were pre-treated with 10 μg / mL free KOdiA-PC for 1 h to block the KOdiA-PC binding site, followed by the addition of 20 μg / mL K-Exo, and co-incubated for 4 h. After uptake, cells were washed three times with PBS to remove unuptaken drug, fixed with 4% (w / v) paraformaldehyde at room temperature for 10 min, and then stained with 5 μg / mL DAPI at room temperature for 5 min. Images were taken under a confocal microscope, and quantitative fluorescence analysis was performed. The results showed that under the same uptake conditions, the red fluorescence intensity of K-Exo in CMEC was significantly higher than that in the BMEC and HUVEC groups. Figure 3 (A and C) demonstrate that KOdiA-PC modification has the ability to specifically target CMECs. Pretreatment with KOdiA-PC significantly reduced the uptake of K-Exo by CMEC cells, with no significant difference compared to the Exo group. Figure 3 B and D) indicate that the binding of CD36 to KOdiA-PC promotes the uptake of exosomes by CMEC.

[0081] Test Example 4

[0082] Transendothelial transport effects of exosomes modified with fatty acid translocase ligands

[0083] like Figure 4 As shown in Figure A, CMEC or HUVEC cells were respectively packed at a density of 1.5 × 10⁻⁶. 5 HL-1 cells were seeded at a density of 1 × 10⁻⁶ cells / well in the upper chamber of a Transwell (0.4 μm pore size), with HL-1 cells seeded at a rate of 1 × 10⁻⁶ cells / well. 5Cells were seeded at a density of [number] cells / well in the lower chamber of a Transwell incubator pre-slided with glass slides and cultured overnight at 37°C and 5% CO2 saturated humidity. Exo cells or K-Exo cells carrying siRNA-Cy3 were added to the upper chamber to achieve a final siRNA-Cy3 concentration of 200 nM, and the cells were transported in a cell culture incubator for 12 h. To verify the role of KOdiA-PC in the transendothelial transport of exosomes, CMEC cells were pretreated with 10 μg / mL free KOdiA-PC for 1 h, and then K-Exo cells carrying siRNA-Cy3 were added to the upper chamber and transported in a cell culture incubator for 12 h. After transport, the upper chamber was removed, and the HL-1 cells in the lower chamber were washed three times with PBS to remove free drugs. Cells were fixed with 4% (w / v) paraformaldehyde at room temperature for 10 min, labeled with 10 μM wheat germ lectin WGA (Sigma, L4895) at room temperature for 20 min, and then stained with 5 μg / mL DAPI at room temperature for 5 min. Images were taken under a laser confocal microscope, and quantitative fluorescence analysis was performed. Results are as follows: Figure 4 As shown in B and C, the groups with HUVEC cells in the upper chamber showed lower translocation rates for both Exo and K-Exo, with no significant difference; while the groups with CMEC cells in the upper chamber showed significantly better translocation rates for K-Exo than Exo. Furthermore, CMEC pretreatment with KOdiA-PC significantly reduced the transendothelial translocation efficiency of K-Exo compared to the Exo group, with no significant difference. Figure 4 (D and E) indicates that KOdiA-PC modification improves the efficiency of exosome transport across cardiac endothelial cells.

[0084] Test Example 5

[0085] Accumulation of exosome cardiac-targeted delivery system in mouse ischemia-reperfusion hearts

[0086] Following ischemia-reperfusion, the intercellular space of local capillary endothelial cells increases. Over time, endothelial cell damage gradually subsides, and microvascular permeability tends to return to normal. Therefore, to verify the effectiveness of this targeting strategy under different degrees of damage, this invention investigated the accumulation of exosomes in the heart immediately after ischemia-reperfusion and two weeks after ischemia-reperfusion, as follows:

[0087] A mouse model of myocardial ischemia-reperfusion injury was established by restoring blood flow to the left anterior descending coronary artery ligated for 30 minutes. To facilitate observation of exosome distribution in vivo, exosomes were labeled with the near-infrared dye DiR (Invitrogen). Specifically, DiR solution was added to exosomes (Exo), KOdiA-PC modified exosomes (K-Exo), IMT modified exosomes (I-Exo), and dual-modified exosomes (KI-Exo) to a final concentration of 5 μM. The mixture was incubated at 37°C for 10 minutes, followed by the addition of PBS solution. The mixture was then ultrafiltered three times using a 100 kDa ultrafiltration tube to remove free DiR dye. Protein quantification of the labeled exosomes was performed using a BCA assay kit.

[0088] Mice were randomly divided into 7 groups, with 3 or 4 mice in each group. Five groups received a tail vein injection of PBS, Exo, K-Exo, I-Exo, or KI-Exo immediately after surgery, while the other two groups received a tail vein injection of I-Exo or KI-Exo two weeks after surgery. The dosage was 100 μg Exo per mouse. Twenty-four hours after administration, mouse hearts were harvested and the accumulation of exosomes in the heart was detected using an IVIS Spectrum (PerkinElmer) system (Ex = 745 nm, Em = 800 nm). Fluorescence was quantitatively analyzed using Living Image software. Results are as follows: Figure 5 As shown, compared with the Exo group, the fluorescence intensity in the heart of the K-Exo, I-Exo, and KI-Exo groups was increased, with the KI-Exo group showing significantly better results than the other control groups. Two weeks after surgery, the fluorescence intensity in the heart of the KI-Exo group was significantly higher than that of the I-Exo group, indicating that modification with fatty acid translocase ligands and ischemic myocardial targeting peptides can increase exosome accumulation in the heart. Furthermore, dual-targeting modification can further increase exosome accumulation in the heart, especially with the targeting effect still present two weeks after reperfusion. These results demonstrate, at the in vivo level, the significant advantage of the dual-targeting modification strategy in promoting exosome accumulation in the heart.

[0089] Example 2

[0090] S1. Mouse bone marrow mesenchymal stem cells were isolated in the laboratory. After culturing for 48 h, the culture supernatant was collected. The supernatant was centrifuged at 2000 g for 20 min at 4 °C, then centrifuged at 10000 g for 30 min at 4 °C. The supernatant obtained after centrifugation was then filtered through a 0.22 μm filter membrane and centrifuged at 100000 g for 70 min. The obtained solid PBS was washed and the precipitate was resuspended to obtain exosomes (Exo). The concentration of exosome protein was quantified using a BCA kit (Solepro, PC0020).

[0091] S2. Dilute the exosomes (Exo) with PBS to prepare an exosome solution of 0.5 mg / mL. Add 2 mg / mL of diphenylcyclooctylene-sulfo-succinimide ester (DBCO-sulfo-NHS, Glen Research, 50-1941) dissolved in dimethyl sulfoxide to the above exosome solution. The final concentration of DBCO-sulfo-NHS is 10 μM. The reaction is carried out at room temperature with shaking for 4 h. Remove excess DBCO-sulfo-NHS with a 100 kDa ultrafiltration tube to obtain DBCO-coupled exosomes (DBCO-Exo).

[0092] S3. 1-Palmitoyl-2-(5-keto-6-octen-diacyl)phosphatidylcholine (KOdiA-PC, APExBio, C4591) and an N-terminal azide-modified ischemic myocardial targeting peptide (IMT, synthesized by Suzhou Qiangyao Biotechnology Co., Ltd.) were added to DBCO-Exo. The mass ratio of KOdiA-PC to exosomes based on protein was 1:500, and the mass ratio of IMT to exosomes based on protein was 1:1000. The above reaction solution was reacted at 4°C and 100 rpm for 12 h. After purification and removal of free KOdiA-PC and IMT using a 100 kDa ultrafiltration tube, the exosome cardiac targeting delivery system was obtained.

[0093] The modification density was characterized using Cy5-labeled IMT and BodiPY-labeled fatty acid translocase ligands. The particle concentration of the dual-targeted modified exosomes was detected using a nanoparticle size analyzer (NS300, Malvern). The fluorescently labeled ligands on the exosome surface were quantified using a SpectraMax iD3 (Molecular Devices) multi-mode microplate reader. The excitation / emission wavelengths for Cy5 were 640 nm / 680 nm, and for BodiPY, they were 480 nm / 520 nm. The ligand modification density on the exosomes was calculated, with an average modification density of 2853 KOdiA-PC ligands per exosome and an average modification density of 2439 ischemic myocardial targeting peptides per exosome.

[0094] Example 3

[0095] S1. Human umbilical cord mesenchymal stem cells were isolated in the laboratory. After culturing for 48 h, the culture supernatant was collected. The supernatant was centrifuged at 2000g for 20 min at 4 °C, and then centrifuged at 10000g for 30 min at 4 °C. The supernatant obtained after centrifugation was then filtered through a 0.22 μm filter membrane and centrifuged at 100000g for 70 min. The obtained solid PBS was washed and the precipitate was resuspended to obtain exosomes (Exo). The concentration of exosome proteins was quantified using a BCA kit (Solepro, PC0020).

[0096] S2. Dilute the exosomes (Exo) with PBS to prepare an exosome solution of 0.5 mg / mL. Add 2 mg / mL of diphenylcyclooctylene-sulfo-succinimide ester (DBCO-sulfo-NHS, Glen Research, 50-1941) dissolved in dimethyl sulfoxide to the above exosome solution. The final concentration of DBCO-sulfo-NHS is 10 μM. The reaction is carried out at room temperature with shaking for 4 h. Remove excess DBCO-sulfo-NHS with a 100 kDa ultrafiltration tube to obtain DBCO-coupled exosomes (DBCO-Exo).

[0097] S3. 1-Palmitoyl-2-(5-keto-6-octen-diacyl)phosphatidylcholine (KOdiA-PC, APExBio, C4591) and an N-terminal azide-modified ischemic myocardial targeting peptide (IMT, synthesized by Suzhou Qiangyao Biotechnology Co., Ltd.) were added to DBCO-Exo. The mass ratio of KOdiA-PC to exosomes based on protein was 1:50, and the mass ratio of IMT to exosomes based on protein was 1:100. The above reaction solution was reacted at 4°C and 100 rpm for 12 h. After purification and removal of free KOdiA-PC and IMT using a 100 kDa ultrafiltration tube, the exosome cardiac targeting delivery system was obtained.

[0098] The modification density was characterized using Cy5-labeled IMT and BodiPY-labeled fatty acid translocase ligands. The particle concentration of the dual-targeted modified exosomes was detected using a nanoparticle size analyzer (NS300, Malvern). The fluorescently labeled ligands on the exosome surface were quantified using a SpectraMax iD3 (Molecular Devices) multi-mode microplate reader. The excitation / emission wavelengths for Cy5 were 640 nm / 680 nm, and for BodiPY, they were 480 nm / 520 nm. The ligand modification density on the exosomes was calculated, with an average modification density of 44,021 KOdiA-PC ligands per exosome and an average modification density of 7,890 ischemic myocardial targeting peptides per exosome.

[0099] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. An exosome cardiac-targeted delivery system, characterized in that, The system includes exosomes and fatty acid translocase ligands modified on the surface of the exosomes; The fatty acid translocase ligand is 1-palmitoyl-2-(5-keto-6-octen-diacyl)phosphatidylcholine; the amount of modification of the fatty acid translocase ligand is 2,500-45,000 relative to one exosome; and the exosome is an animal-derived exosome.

2. The exosome cardiac targeted delivery system according to claim 1, characterized in that, The animal-derived exosomes are selected from at least one of the following: exosomes from HEK293T cells, exosomes from bone marrow mesenchymal stem cells, exosomes from umbilical cord mesenchymal stem cells, exosomes from adipose-derived mesenchymal stem cells, exosomes from cardiomyocyte-derived cells, exosomes from endothelial progenitor cells, exosomes from hematopoietic stem cells, exosomes from macrophages, exosomes from monocytes, exosomes from plasma, and exosomes from platelets.

3. The exosome cardiac targeted delivery system according to claim 1, characterized in that, The system also includes ischemic myocardial targeting peptides modified on the surface of the exosomes; The amount of modification of the ischemic myocardium-targeting peptide is 2,000-8,000 relative to one exosome; The amino acid sequence of the ischemic myocardial targeting peptide is shown in SEQ ID NO.

1.

4. A method for preparing an exosome-based cardiac-targeted delivery system, characterized in that, Includes the following steps: The exosomes and / or exosomes coupled with diphenylcyclooctylene are reacted with the substance to be modified and then separated. The substance to be modified contains a fatty acid translocase ligand; The fatty acid translocase ligand is 1-palmitoyl-2-(5-keto-6-octen-diacyl)phosphatidylcholine; the exosomes are animal-derived exosomes; the amount of fatty acid translocase ligand added is 0.002-0.02 mg relative to 1 mg of the exosomes on a protein basis.

5. The preparation method according to claim 4, characterized in that, The animal-derived exosomes are selected from at least one of the following: exosomes from HEK293T cells, exosomes from bone marrow mesenchymal stem cells, exosomes from umbilical cord mesenchymal stem cells, exosomes from adipose-derived mesenchymal stem cells, exosomes from cardiomyocyte-derived cells, exosomes from endothelial progenitor cells, exosomes from hematopoietic stem cells, exosomes from macrophages, exosomes from monocytes, exosomes from plasma, and exosomes from platelets.

6. The preparation method according to claim 4 or 5, characterized in that, The method includes the following steps: reacting the exosomes of the coupled diphenylcyclooctylene with the substance to be modified in a contact reaction followed by separation. The substance to be modified also contains an ischemic myocardial targeting peptide with an N-terminus modified with azide. The amino acid sequence of the ischemic myocardial targeting peptide is shown in SEQ ID NO.

1.

7. The preparation method according to claim 6, characterized in that, The amount of the N-terminal modified azide-targeting peptide for ischemic myocardium added is 0.001-0.01 mg relative to 1 mg of the exosomes on a protein basis. The conditions for the contact reaction include at least: a rotation speed of 50-200 rpm and a time of 1-24 h.

8. The preparation method according to claim 4 or 5, characterized in that, The method for preparing the exosomes coupled with diphenylcyclooctylene includes: reacting the exosomes with diphenylcyclooctylene-sulfonyl-succinimide ester in a contact reaction followed by separation. The amount of diphenylcyclooctyne-sulfonyl-succinimide ester added is 0.002-0.02 mg relative to 1 mg of the exosomes based on protein. The conditions for the contact reaction include at least the following: carried out in a solvent for 1-6 hours.

9. Use of the exosome cardiac targeted delivery system according to any one of claims 1-3 or the exosome cardiac targeted delivery system prepared by the preparation method according to any one of claims 4-8 in the preparation of formulations for the treatment of myocardial ischemia-reperfusion injury drug delivery systems.

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