Application of desoxyribonucleic acid-poly-L-lysine composite hydrogel in redistribution of endogenous exosomes and treatment of myocardial infarction

Abstract

The invention discloses application of desoxyribonucleic acid-poly-L-lysine composite hydrogel in redistribution of endogenous exosomes and treatment of myocardial infarction, and the desoxyribonucleic acid-poly-L-lysine composite hydrogel comprises a DNA (deoxyribonucleic acid) skeleton and poly-L-lysine wrapping the DNA skeleton, the DNA skeleton contains a nucleic acid aptamer targeting exosome surface protein, and the desoxyribonucleic acid-poly-L-lysine composite hydrogel is used for capturing and releasing endogenous exosomes. According to the invention, the injectable desoxyribonucleic acid-poly-L-lysine composite hydrogel is used for treating myocardial infarction, and targeted enrichment and controllable release of endogenous exosomes are realized through the unique molecular design of the desoxyribonucleic acid-poly-L-lysine composite hydrogel; important breakthrough is achieved in the aspects of targeting, controllability, safety, clinical transformation and the like, and a brand new solution is provided for myocardial infarction treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering technology, specifically relating to the use of a deoxyribonucleic acid-poly-L-lysine composite hydrogel in the redistribution of endogenous exosomes and the treatment of myocardial infarction. Background Technology

[0002] Following myocardial infarction, irreversible necrosis of cardiomyocytes leads to a decline in cardiac function, and current therapies struggle to promote myocardial regeneration. Exosomes possess repair potential, but exogenous exosomes face challenges such as high preparation costs and immune rejection. While endogenous exosomes offer high safety, targeted enrichment methods are lacking, and they are easily and rapidly eliminated. Existing hydrogel carriers exhibit low sensitivity to nucleases, hindering the controlled release of exosomes.

[0003] Currently, exosome repair therapy after myocardial infarction mainly relies on the following treatment options: 1. Intravenous exosome therapy. Exosomes are extracted from mesenchymal stem cells (MSCs) or cardiac progenitor cells and delivered systemically via intravenous injection. This method primarily relies on the natural homing effect of exosomes to accumulate in damaged myocardium, resulting in poor targeting and requiring multiple administrations. Research by Xu Biao's research group has shown that intravenously injected exosomes suffer from off-target effects and short in vivo retention time. Although modifying MSC-derived exosomes with targeted peptides can alleviate myocardial ischemia-reperfusion injury, repeated injections (3 times) per week are still necessary to maintain the therapeutic effect.

[0004] 2. Biodegradable biomaterial scaffolds (such as collagen / hyaluronic acid hydrogels). Exosomes or stem cells are loaded onto biomaterials (such as collagen, fibrin, or polylactic-co-glycolic acid copolymers) and implanted into the infarct area. The therapeutic components are slowly released through material degradation. However, this method has drawbacks such as requiring open-chest or minimally invasive surgery for implantation and uncontrollable degradation. Tan Yin's team used fibrin hydrogel to load exosomes, requiring open-chest surgery for implantation in a rat model. Jie Huiqi's team discovered that polylactic-co-glycolic acid copolymer (PLGA) microspheres require 2-4 weeks to degrade.

[0005] 3. Genetically Modified Exosomes (Targeted Enhancement). This involves genetically engineering exosome membrane proteins (such as Lamp2b fusion targeting peptides) to improve myocardial homing efficiency, or modifying exosomes with myocardial-specific peptides (CSTSMLKAC). However, gene editing may induce immune responses and has high production costs. Shen Zhenya's research group used Lamp2b-CSTSMLKAC to modify exosomes, improving cardiac targeting efficiency, but this requires lentiviral transfection of MSCs.

[0006] Therefore, there is a need to develop a treatment strategy that is highly effective in targeting, has long-lasting sustained-release capability, and is minimally invasive and convenient, in order to overcome the limitations of existing methods in terms of targeting accuracy, dosing frequency, and invasiveness. Summary of the Invention

[0007] The purpose of this invention is to provide a deoxyribonucleic acid-poly-L-lysine composite hydrogel for the redistribution of endogenous exosomes and the treatment of myocardial infarction. It organically combines three major technologies: aptamer active targeting technology, nuclease-responsive sustained-release system, and endogenous exosome utilization, forming a comprehensive treatment plan with significant technical advantages. It has shown outstanding beneficial effects in terms of targeting, controllability, safety, and clinical translation.

[0008] To achieve the above objectives, a specific embodiment of the present invention provides the following technical solution:

[0009] The use of a deoxyribonucleic acid-poly-L-lysine composite hydrogel in the redistribution of endogenous exosomes, the deoxyribonucleic acid-poly-L-lysine composite hydrogel comprising a DNA backbone and poly-L-lysine encapsulating the DNA backbone, the DNA backbone containing nucleic acid aptamers targeting exosome surface proteins.

[0010] In one or more embodiments of the present invention, the deoxyribonucleic acid-poly-L-lysine composite hydrogel is used to capture and release endogenous exosomes.

[0011] In one or more embodiments of the present invention, the deoxyribonucleic acid-poly-L-lysine composite hydrogel captures endogenous exosomes by binding to endogenous exosome surface proteins via nucleic acid aptamers.

[0012] In one or more embodiments of the present invention, the deoxyribonucleic acid-poly-L-lysine composite hydrogel is degraded by nucleases to release endogenous exosomes.

[0013] In one or more embodiments of the present invention, the exosome surface protein is CD63 protein, the nucleic acid aptamer is CD63 DNA aptamer, and the CD63 DNA aptamer sequence is shown in SEQ ID NO.5.

[0014] In one or more embodiments of the present invention, the deoxyribonucleic acid-poly-L-lysine composite hydrogel is obtained by mixing and incubating an RCA product obtained by rolling circle amplification of a circular DNA template containing a CD63 DNA aptamer antisense sequence with a poly-L-lysine aqueous solution.

[0015] The concentration of the poly-L-lysine aqueous solution is 20 μmol / L-160 μmol / L.

[0016] In one or more embodiments of the present invention, the concentration of the poly-L-lysine aqueous solution is 80 μmol / L.

[0017] In one or more embodiments of the present invention, the incubation operation is: shaking at 350 rpm-450 rpm for 4 h-16 h.

[0018] Another specific embodiment of the present invention provides the following technical solution:

[0019] The use of a deoxyribonucleic acid-poly-L-lysine composite hydrogel in the treatment of myocardial infarction, wherein the deoxyribonucleic acid-poly-L-lysine composite hydrogel is used for the redistribution of endogenous exosomes;

[0020] The deoxyribonucleic acid-poly-L-lysine composite hydrogel includes a DNA backbone and poly-L-lysine that encapsulates the DNA backbone. The DNA backbone contains nucleic acid aptamers that target exosome surface proteins.

[0021] In one or more embodiments of the present invention, the deoxyribonucleic acid-poly-L-lysine composite hydrogel is used for the treatment of myocardial infarction via in situ injection.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. Regarding targeting, existing intravenous exosome therapy mainly relies on the passive homing effect of exosomes, with only 1-3% of exosomes reaching the heart tissue, and the vast majority being cleared by mononuclear phagocytic organs such as the liver and spleen. This application, however, utilizes precise modification of the Apt CD63 aptamer to enable the DNA-poly-L-lysine composite hydrogel to actively recognize and capture endogenous exosomes secreted by the cells themselves, thus improving cardiac targeting efficiency. This active targeting mechanism not only significantly improves treatment efficiency but also significantly reduces the required dose of exosomes, avoiding the waste of systemic administration.

[0024] 2. Regarding release controllability, existing biodegradable biomaterials (such as collagen and PLGA) generally suffer from uncontrollable release (initial burst release or requiring 2-4 weeks for complete release). The innovative deoxyribonucleic acid-poly-L-lysine composite hydrogel cross-linked network structure of this application can slow down the biodegradation by nucleases in vivo, achieving linear sustained release for up to 7 days. This precise release characteristic ensures a continuous supply of therapeutic components, avoiding waste caused by sudden initial release and guaranteeing sufficient treatment duration, significantly improving the utilization rate of exosomes.

[0025] 3. Regarding therapeutic safety, existing exogenous exosome therapies require the extraction of exosomes from mesenchymal stem cells or cardiac progenitor cells, which is not only costly to prepare but also carries the potential risk of immune rejection. This application, through the innovative approach of in-situ enrichment of endogenous exosomes, completely avoids the introduction of exogenous substances. Biosafety evaluations have also confirmed the excellent biosafety of this technology.

[0026] 4. In terms of clinical translation, existing biodegradable stents typically require open-chest or minimally invasive surgery for implantation, which is complex and invasive. The injectable hydrogel system developed in this application can be precisely delivered to the infarct area, and its ease of operation is significantly superior to traditional implantable stents. This minimally invasive characteristic makes this technology more suitable for early clinical intervention and has broader application prospects. More importantly, this application can maintain a continuous therapeutic effect for 7 days with a single injection, while traditional methods often require repeated administration. Attached Figure Description

[0027] 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 recorded in the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This is a confocal fluorescence image of a deoxyribonucleic acid-poly-L-lysine composite hydrogel encapsulating endogenous exosomes in one embodiment of the present invention.

[0029] Figure 2 This is a graph showing the evaluation of the capture and release efficiency of D-P80 hydrogel exosomes in one embodiment of the present invention;

[0030] Figure 3 This is an in vitro evaluation of the therapeutic effect of D-P80 hydrogel in one embodiment of the present invention;

[0031] Figure 4 This is an echocardiogram of a rat with myocardial infarction 28 days after injection of D-P80 hydrogel in one embodiment of the present invention.

[0032] Figure 5 This is a representative photograph of a Masson trichrome stained paraffin section taken 28 days after injecting D-P80 hydrogel into the site of a rat with myocardial infarction, according to one embodiment of the present invention.

[0033] Figure 6 This is a biosafety evaluation diagram of D-P80 hydrogel in one embodiment of the present invention. Detailed Implementation

[0034] To enable those skilled in the art to better understand the technical solutions in this disclosure, the technical solutions in the embodiments of this disclosure are described clearly and completely below. Obviously, the described embodiments are only some embodiments of this disclosure, and not all embodiments. Based on the embodiments in this disclosure, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this disclosure.

[0035] One specific embodiment of the present invention provides the use of deoxyribonucleic acid-poly-L-lysine composite hydrogel in the redistribution of endogenous exosomes. The deoxyribonucleic acid-poly-L-lysine composite hydrogel includes a DNA backbone and poly-L-lysine that encapsulates the DNA backbone. The DNA backbone contains nucleic acid aptamers that target proteins on the surface of exosomes.

[0036] Furthermore, deoxyribonucleic acid-poly-L-lysine composite hydrogels are used to capture and release endogenous exosomes.

[0037] Specifically, the deoxyribonucleic acid-poly-L-lysine composite hydrogel achieves targeted enrichment of endogenous exosomes by specifically binding to exosome surface proteins via nucleic acid aptamers. Simultaneously, the deoxyribonucleic acid-poly-L-lysine composite hydrogel can achieve linear release within a certain timeframe through nuclease degradation, thereby releasing endogenous exosomes. This achieves an organic combination of active aptamer targeting, nuclease-responsive release, and endogenous exosome utilization, resulting in significant therapeutic advantages.

[0038] Furthermore, the exosome surface protein is CD63 protein, and the nucleic acid aptamer is CD63 DNA aptamer. The CD63 DNA aptamer sequence is shown in SEQ ID NO.5, specifically CACCCCACCTCGCTCCCGTGACACTAATGCTA.

[0039] Furthermore, the deoxyribonucleic acid-poly-L-lysine composite hydrogel was obtained by mixing and incubating the RCA product obtained by rolling circle amplification of a circular DNA template containing the antisense sequence of CD63 DNA aptamer with an aqueous solution of poly-L-lysine.

[0040] Specifically, the concentration of the poly-L-lysine aqueous solution is 20 μmol / L-160 μmol / L, preferably 80 μmol / L. The incubation procedure is as follows: shaking at 350 rpm-450 rpm for 4 h-16 h.

[0041] Furthermore, the preparation method of the deoxyribonucleic acid-poly-L-lysine composite hydrogel in this invention specifically includes the following steps:

[0042] Step 1: Design the antisense sequence of the CD63 DNA aptamer onto the circular DNA template.

[0043] Specifically, 5' phosphorylated modified ssDNA-1 was designed and synthesized, and primer one for ssDNA-1 was designed. The 5' and 3' ends of ssDNA-1 were complementary to the 3' and 5' ends of primer one, respectively.

[0044] The 5' phosphorylated ssDNA-1 and primer 1 were mixed in a molar ratio of 1:1 to 1:1.2, and NaCl was added to a final concentration of 80 mmol / L. Sterile water was then added to bring the volume to 20 μL. Circular DNA-1 with a notched end and a final concentration of 10 mmol / L was synthesized according to the designed heating annealing program. When ssDNA-1 and primer 1 were mixed in a 1:1 ratio, the concentration of ssDNA-1 in the system was 100 mmol / L, and the concentration of primer 1 was 100 mmol / L.

[0045] Add 1 U of T4 DNA ligase to 100 ng of circular DNA-1, and react at 4℃-16℃ for 6 h-12 h in a final concentration of 1 × T4 buffer to obtain the circular DNA template product.

[0046] Step 2: Use a circular DNA template containing the antisense sequence of the CD63 DNA aptamer for rolling circle amplification to obtain the RCA product.

[0047] Specifically, 5×10 -3 -1×10 -2 A circular DNA template of nmol, phi29 DNA polymerase at a final concentration of 0.2 U / µL-0.5 U / µL, phi29 DNA polymerase buffer at a final concentration of 1×phi29 DNA polymerase, BSA at a final concentration of 0.2 mg / mL, dNTPs at a final concentration of 0.5 mmol / L-1 mmol / L, and NaCl at a final concentration of 80 mmol / L are mixed together and then brought to a final volume of 100 µL with sterile water. The mixture is then shaken at 350 rpm-450 rpm at 35 ℃-37 ℃ for 4 h-16 h to obtain the RCA product.

[0048] Step 3: Inactivate RCA products.

[0049] Specifically, the inactivation conditions are: 65℃-75℃, time 8min-12min.

[0050] Step 4: Mix the inactivated RCA product with the poly-L-lysine solution and incubate to obtain a deoxyribonucleic acid-poly-L-lysine composite hydrogel.

[0051] Specifically, poly-L-lysine (PLL) was dissolved in water to obtain a poly-L-lysine solution with a concentration of 20 μmol / L to 160 μmol / L. The inactivated RCA product and the poly-L-lysine solution were mixed and shaken at 350 rpm to 450 rpm for 4 to 16 hours.

[0052] Another specific embodiment of the present invention provides the use of deoxyribonucleic acid-poly-L-lysine composite hydrogel in the treatment of myocardial infarction. The deoxyribonucleic acid-poly-L-lysine composite hydrogel is used to redistribute endogenous exosomes. The deoxyribonucleic acid-poly-L-lysine composite hydrogel includes a DNA backbone and poly-L-lysine that encapsulates the DNA backbone. The DNA backbone contains nucleic acid aptamers that target exosome surface proteins.

[0053] Specifically, deoxyribonucleic acid-poly-L-lysine composite hydrogel is used for the treatment of myocardial infarction via in situ injection. Through in situ injection, the deoxyribonucleic acid-poly-L-lysine composite hydrogel is directly injected into the infarct area. By actively recognizing and capturing endogenous exosomes secreted by the cells themselves, the deoxyribonucleic acid-poly-L-lysine composite hydrogel improves the cardiac targeting efficiency, significantly improves the treatment efficiency, and also reduces the required dose of exosomes, avoiding the waste of systemic administration.

[0054] Further, an exemplary in vitro treatment involves culturing H9C2 cells in exosome-free, complete DMEM-H medium under hypoxic conditions (37 °C, 5% CO2, 2% O2) for 24 hours. Then, D-P80 hydrogels enriched with different concentrations of H9C2 cell-derived exosomes (5 mg / mL, 10 mg / mL, 15 mg / mL, and 20 mg / mL protein concentration) are added, and the cells are incubated for 24 hours.

[0055] An example of in vivo treatment is as follows: After the establishment of a rat myocardial infarction model, 100 µL of different treatment agents (PBS / D-P80 hydrogel without aptamer / D-P80 hydrogel containing AptCD63 aptamer) are uniformly injected into the infarct area immediately after surgery, and the treatment is observed for 28 days.

[0056] The present invention will be further described in detail below with reference to specific embodiments.

[0057] In this invention, RCA is short for rolling circle amplification, BSA is short for bovine serum albumin, and ssDNA is short for linear single-stranded DNA.

[0058] Example 1

[0059] DNA template preparation method:

[0060] The sequences of ssDNA-1, primer 1, and aptamer AptCD63 modified with phosphorylation at the 5' end are shown in Table 1:

[0061] Table 1: Deoxynucleotide sequences

[0062]

[0063] Preparation of circular DNA template 1:

[0064] a. Design and synthesize ssDNA-1 with phosphorylation modification at the 5' end. The number of bases in ssDNA-1 is 128. Design primer 1 for ssDNA-1 with 20 bases. The 3' and 5' ends of primer 1 are complementary to the 5' and 3' ends of ssDNA-1, respectively.

[0065] ssDNA-1 contains a complementary sequence to the aptamer AptCD63.

[0066] The nucleotide sequence of ssDNA-1 is shown in SEQ ID NO.1, as shown in Table 1.

[0067] The nucleotide sequence of primer 1 is shown in Table 1 as SEQ ID NO.3.

[0068] The nucleotide sequence of aptamer AptCD63 is shown in SEQ ID NO.5, as shown in Table 1.

[0069] b. Mix 5' phosphorylated ssDNA-1 and primer 1 in a 1:1 molar ratio, add NaCl to a final concentration of 80 mmol / L, and dilute with sterile water to a total volume of 20 µL. The concentrations of ssDNA-1 and primer 1 are both 100 mmol / L. Synthesize nicked circular DNA using the following heating annealing procedure.

[0070] Annealing procedure: Steps 3 and 4, the temperature decreases by 0.5 ℃ every 30 seconds, for 80 cycles.

[0071] Step 1: 95 ℃ for 2 min;

[0072] Step 2: 65 ℃ for 2 min;

[0073] Step 3: 60 ℃ for 5 min 30 s;

[0074] Step 4: 20 ℃ for 30 s;

[0075] Step 5: 4 ℃ for 10 min.

[0076] c. Add 2 U of T4 DNA ligase to 200 ng of the product from step b, to a final concentration of 1 × T4 buffer, and react at 22 °C for 8 h to obtain circular DNA template 1, which is then stored at 4 °C.

[0077] Example 2

[0078] DNA template preparation method:

[0079] The sequences of ssDNA-1 modified with phosphorylation at the 5' end and primer 1 are shown in Table 2:

[0080] Table 2: Deoxynucleotide sequences

[0081]

[0082] Preparation of circular DNA template 1:

[0083] a. Design and synthesize ssDNA-1 with phosphorylation modification at the 5' end. The number of bases in ssDNA-1 is 92. Design primer 1 for ssDNA-1 with 24 bases. The 3' and 5' ends of primer 1 are complementary to the 5' and 3' ends of ssDNA-1, respectively.

[0084] ssDNA-1 contains a complementary sequence to the aptamer AptCD63.

[0085] The nucleotide sequence of ssDNA-1 is shown in SEQ ID NO.6, as shown in Table 2.

[0086] The nucleotide sequence of primer 1 is shown in Table 2 as SEQ ID NO. 8.

[0087] b. Mix 5' phosphorylated ssDNA-1 and primer 1 in a 1:1 molar ratio, add NaCl to a final concentration of 80 mmol / L, and dilute with sterile water to a total volume of 20 µL. The concentrations of ssDNA-1 and primer 1 are both 100 mmol / L. Synthesize nicked circular DNA using the following heating annealing procedure.

[0088] Annealing procedure: Steps 3 and 4, the temperature decreases by 0.5 ℃ every 30 seconds, for 80 cycles.

[0089] Step 1: 95℃ for 2 min;

[0090] Step 2: 65℃ for 2 min;

[0091] Step 3: 60℃ for 5 min 30 s;

[0092] Step 4: 20℃ for 30 seconds;

[0093] Step 5: 4℃ for 10 min.

[0094] c. Add 2 U of T4 DNA ligase to 200 ng of the product from step b, to a final concentration of 1 × T4 buffer, and react at 22 °C for 8 hours to obtain circular DNA template 1, which is then stored at 4 °C.

[0095] Example 3

[0096] DNA template preparation method:

[0097] The sequences of ssDNA-1 modified with phosphorylation at the 5' end and primer 1 are shown in Table 3:

[0098] Table 3: Deoxynucleotide sequences

[0099]

[0100] Preparation of circular DNA template 1:

[0101] a. Design and synthesize ssDNA-1 with phosphorylation modification at the 5' end. The number of bases in ssDNA-1 is 174. Design primer 1 for ssDNA-1 with 27 bases. The 3' and 5' ends of primer 1 are complementary to the 5' and 3' ends of ssDNA-1, respectively.

[0102] ssDNA-1 contains a complementary sequence to the aptamer AptCD63.

[0103] The nucleotide sequence of ssDNA-1 is shown in SEQ ID NO.10, as shown in Table 3.

[0104] The nucleotide sequence of primer 1 is shown in Table 3 as SEQ ID NO.12.

[0105] b. Mix 5' phosphorylated ssDNA-1 and primer 1 in a 1:1 molar ratio, add NaCl to a final concentration of 80 mmol / L, and dilute with sterile water to a total volume of 20 µL. The concentrations of ssDNA-1 and primer 1 are both 100 mmol / L. Synthesize nicked circular DNA using the following heating annealing procedure.

[0106] Annealing procedure: Steps 3 and 4, the temperature decreases by 0.5 ℃ every 30 seconds, for 80 cycles.

[0107] Step 1: 95 ℃ for 2 min;

[0108] Step 2: 65 ℃ for 2 min;

[0109] Step 3: 60 ℃ for 5 min 30 s;

[0110] Step 4: 20 ℃ for 30 s;

[0111] Step 5: 4 ℃ for 10 min.

[0112] c. Add 2 U of T4 DNA ligase to 200 ng of the product from step b, to a final concentration of 1 × T4 buffer, and react at 22 °C for 8 hours to obtain circular DNA template 1, which is then stored at 4 °C.

[0113] Example 4

[0114] Method for preparing DNA template II:

[0115] The circular DNA template 2 was prepared using the following method:

[0116] a. Design and synthesize ssDNA-2 with phosphorylation modification at the 5' end. The number of bases in ssDNA-2 is 128. Design primer 2 for ssDNA-2 with 20 bases. The 5' end and 3' end of ssDNA-2 are complementary to the 3' end and 5' end of primer 2, respectively.

[0117] The nucleotide sequence of ssDNA-2 is shown in SEQ ID NO.2, as shown in Table 1.

[0118] The nucleotide sequence of primer 2 is shown in SEQ ID NO.4, as shown in Table 1.

[0119] Phosphorylated ssDNA-2 at the 5' end and primer 2 were mixed in a 1:1 molar ratio. NaCl was added to a final concentration of 80 mmol / L, and the volume was adjusted to 20 µL with sterile water. The concentrations of both ssDNA-2 and primer 2 were 100 mmol / L. Circular DNA-2 with a notched end was synthesized according to the following heating annealing procedure.

[0120] Annealing process: Between steps 3 and 4, the temperature decreases by 0.5 ℃ every 30 seconds, and the cycle is repeated 80 times.

[0121] Step 1: 95 ℃ for 2 min;

[0122] Step 2: 65 ℃ for 2 min;

[0123] Step 3: 60 ℃ for 5 min 30 s;

[0124] Step 4: 20 ℃ for 30 s;

[0125] Step 5: 4 ℃ for 10 min.

[0126] c. Add 2 U of T4 DNA ligase to 200 ng of the product obtained in step b, to a final concentration of 1 × T4 buffer, and react at 22 °C for 5 hours to obtain circular DNA template II, which is then stored at 4 °C.

[0127] Example 5

[0128] Method for preparing DNA template II:

[0129] The circular DNA template 2 was prepared using the following method:

[0130] a. Design and synthesize ssDNA-2 with phosphorylation modification at the 5' end. The number of bases in ssDNA-2 is 92. Design primer 2 for ssDNA-2 with 24 bases. The 5' end and 3' end of ssDNA-2 are complementary to the 3' end and 5' end of primer 2, respectively.

[0131] The nucleotide sequence of ssDNA-2 is shown in SEQ ID NO.7, as shown in Table 2.

[0132] The nucleotide sequence of primer 2 is shown in SEQ ID NO.9, as shown in Table 2.

[0133] b. Mix 5' phosphorylated ssDNA-2 and primer 2 at a molar ratio of 1:1, add NaCl to a final concentration of 80 mmol / L, and dilute with sterile water to a total volume of 20 µL. The concentrations of both ssDNA-2 and primer 2 are 100 mmol / L. Synthesize terminally nicked circular DNA-2 according to the following heating annealing procedure.

[0134] Annealing process: Between steps 3 and 4, the temperature decreases by 0.5 ℃ every 30 seconds, and the cycle is repeated 80 times.

[0135] Step 1: 95 ℃ for 2 min;

[0136] Step 2: 65 ℃ for 2 min;

[0137] Step 3: 60 ℃ for 5 min 30 s;

[0138] Step 4: 20 ℃ for 30 s;

[0139] Step 5: 4 ℃ for 10 min.

[0140] c. Add 2 U of T4 DNA ligase to 200 ng of the product obtained in step b, to a final concentration of 1 × T4 buffer, and react at 22 °C for 5 hours to obtain circular DNA template II, which is then stored at 4 °C.

[0141] Example 6

[0142] Method for preparing DNA template II:

[0143] The circular DNA template 2 was prepared using the following method:

[0144] a. Design and synthesize ssDNA-2 with phosphorylation modification at the 5' end. The number of bases in ssDNA-2 is 174. Design primer 2 for ssDNA-2 with 27 bases. The 5' end and 3' end of ssDNA-2 are complementary to the 3' end and 5' end of primer 2, respectively.

[0145] The nucleotide sequence of ssDNA-2 is shown in SEQ ID NO.11, as shown in Table 3.

[0146] The nucleotide sequence of primer 2 is shown in SEQ ID NO.13, as shown in Table 3.

[0147] Phosphorylated ssDNA-2 at the 5' end and primer 2 were mixed in a 1:1 molar ratio. NaCl was added to a final concentration of 80 mmol / L, and the volume was adjusted to 20 µL with sterile water. The concentrations of both ssDNA-2 and primer 2 were 100 mmol / L. Circular DNA-2 with a notched end was synthesized according to the following heating annealing procedure.

[0148] Annealing process: Between steps 3 and 4, the temperature decreases by 0.5 ℃ every 30 seconds, and the cycle is repeated 80 times.

[0149] Step 1: 95 ℃ for 2 min;

[0150] Step 2: 65 ℃ for 2 min;

[0151] Step 3: 60 ℃ for 5 min 30 s;

[0152] Step 4: 20 ℃ for 30 s;

[0153] Step 5: 4 ℃ for 10 min.

[0154] c. Add 2 U of T4 DNA ligase to 200 ng of the product obtained in step b, to a final concentration of 1 × T4 buffer, and react at 22 °C for 5 hours to obtain circular DNA template II, which is then stored at 4 °C.

[0155] Example 7

[0156] A method for preparing a deoxyribonucleic acid-poly-L-lysine composite hydrogel includes the following steps:

[0157] a. 50 nmol / L circular DNA template (prepared in Example 2), 0.2 U / µL phi29 DNA polymerase, 1×phi29 DNA polymerase buffer, 0.2 mg / mL BSA, 1 mmol / L dNTPs, and 80 mmol / L NaCl were added to a final volume of 100 µL with sterile water. The reaction mixture was incubated at 37 °C and 450 rpm for 4 hours. The product was then transferred to 65 °C and incubated for 10 minutes to inactivate the phi29 DNA polymerase, yielding the RCA product.

[0158] b. Take 100 µL of the RCA product and then add 2 µL of PLL solution (concentration of 20 µmol / L).

[0159] c. After mixing the two solutions, place them in a 37 ℃ constant temperature mixer and incubate at 500 rpm for 15 minutes to finally prepare a DNA-PLL hydrogel sample labeled as D-P20, which is used to separate exosomes.

[0160] Implementing Column 8

[0161] A method for preparing a deoxyribonucleic acid-poly-L-lysine composite hydrogel includes the following steps:

[0162] a. 50 nmol / L circular DNA template II (prepared in Example 5), 0.2 U / µL phi29 DNA polymerase, 1×phi29 DNA polymerase buffer, 0.2 mg / mL BSA, 1 mmol / L dNTPs, and 80 mmol / L NaCl were added to a final volume of 100 µL with sterile water. The reaction mixture was incubated at 37 °C and 450 rpm for 4 hours. The product was then transferred to 65 °C and incubated for 10 minutes to inactivate the phi29 DNA polymerase, yielding the RCA product.

[0163] b. Add 2 µL of PLL solution (concentration of 20 µmol / L) to the above RCA product mixture system.

[0164] c. After mixing the two solutions, place them in a 37 ℃ constant temperature mixer and incubate at 500 rpm for 15 minutes to finally prepare a DNA-PLL hydrogel sample labeled as nD-P20 (without AptCD63 aptamer), thus obtaining the nD-P20 hydrogel for capturing exosomes.

[0165] Example 9

[0166] A method for preparing a deoxyribonucleic acid-poly-L-lysine composite hydrogel includes the following steps:

[0167] a. 50 nmol / L circular DNA template (prepared in Example 2), 0.2 U / µL phi29 DNA polymerase, 1×phi29 DNA polymerase buffer, 0.2 mg / mL BSA, 1 mmol / L dNTPs, and 80 mmol / L NaCl were added to a final volume of 100 µL with sterile water. The reaction mixture was incubated at 37 °C and 450 rpm for 4 hours. The product was then transferred to 65 °C and incubated for 10 minutes to inactivate the phi29 DNA polymerase, yielding the RCA product.

[0168] b. Take 100 µL of the RCA product and then add 2 µL of PLL solution (concentration of 40 µmol / L).

[0169] c. After mixing the two solutions, place them in a 37 ℃ constant temperature mixer and incubate at 500 rpm for 15 minutes to finally prepare a DNA-PLL hydrogel sample labeled as D-P40, which is used to separate exosomes.

[0170] Implementation of Column 10

[0171] A method for preparing a deoxyribonucleic acid-poly-L-lysine composite hydrogel:

[0172] a. 50 nmol / L circular DNA template II (prepared in Example 5), 0.2 U / µL phi29 DNA polymerase, 1×phi29 DNA polymerase buffer, 0.2 mg / mL BSA, 1 mmol / L dNTPs, and 80 mmol / L NaCl were added to a final volume of 100 µL with sterile water. The reaction mixture was incubated at 37 °C and 450 rpm for 4 hours. The product was then transferred to 65 °C and incubated for 10 minutes to inactivate the phi29 DNA polymerase, yielding the RCA product.

[0173] b. Add 2 µL of PLL solution (concentration of 40 µmol / L) to the above RCA product mixture system.

[0174] c. After mixing the two solutions, place them in a 37 ℃ constant temperature mixer and incubate at 500 rpm for 15 minutes to finally prepare a DNA-PLL hydrogel sample labeled as nD-P40 (without AptCD63 aptamer), thus obtaining an nD-P40 hydrogel for capturing exosomes.

[0175] Example 11

[0176] A method for preparing a deoxyribonucleic acid-poly-L-lysine composite hydrogel includes the following steps:

[0177] a. 50 nmol / L circular DNA template (prepared in Example 2), 0.2 U / µL phi29 DNA polymerase, 1×phi29 DNA polymerase buffer, 0.2 mg / mL BSA, 1 mmol / L dNTPs, and 80 mmol / L NaCl were added to a final volume of 100 µL with sterile water. The reaction mixture was incubated at 37 °C and 450 rpm for 4 hours. The product was then transferred to 65 °C and incubated for 10 minutes to inactivate the phi29 DNA polymerase, yielding the RCA product.

[0178] b. Take 100 µL of the RCA product and then add 2 µL of PLL solution (concentration of 80 µmol / L).

[0179] c. After mixing the two solutions, place them in a 37 ℃ constant temperature mixer and incubate at 500 rpm for 15 minutes to finally prepare a DNA-PLL hydrogel sample labeled as D-P80, thus obtaining the D-P80 hydrogel for separating exosomes.

[0180] Implementation of Column 12

[0181] A method for preparing a deoxyribonucleic acid-poly-L-lysine composite hydrogel includes the following steps:

[0182] a. 50 nmol / L circular DNA template II (prepared in Example 5), 0.2 U / µL phi29 DNA polymerase, 1×phi29 DNA polymerase buffer, 0.2 mg / mL BSA, 1 mmol / L dNTPs, and 80 mmol / L NaCl were added to a final volume of 100 µL with sterile water. The reaction mixture was incubated at 37 °C and 450 rpm for 4 hours. The product was then transferred to 65 °C and incubated for 10 minutes to inactivate the phi29 DNA polymerase, yielding the RCA product.

[0183] b. Add 2 µL of PLL solution (concentration of 80 µmol / L) to the above RCA product mixture.

[0184] c. After mixing the two solutions, place them in a 37 ℃ constant temperature mixer and incubate at 500 rpm for 15 minutes to finally prepare a DNA-PLL hydrogel sample labeled as nD-P80 (without AptCD63 aptamer), thus obtaining the nD-P80 hydrogel for capturing exosomes.

[0185] Example 13

[0186] A method for preparing a deoxyribonucleic acid-poly-L-lysine composite hydrogel:

[0187] a. 50 nmol / L circular DNA template (prepared in Example 2), 0.2 U / µL phi29 DNA polymerase, 1×phi29 DNA polymerase buffer, 0.2 mg / mL BSA, 1 mmol / L dNTPs, and 80 mmol / L NaCl were added to a final volume of 100 µL with sterile water. The reaction mixture was incubated at 37 °C and 450 rpm for 4 hours. The product was then transferred to 65 °C and incubated for 10 minutes to inactivate the phi29 DNA polymerase, yielding the RCA product.

[0188] b. Take 100 µL of the RCA product and then add 2 µL of PLL solution (concentration of 160 µmol / L).

[0189] c. After mixing the two solutions, place them in a 37 ℃ constant temperature mixer and incubate at 500 rpm for 15 minutes to finally prepare a DNA-PLL hydrogel sample labeled as D-P160, thus obtaining the D-P160 hydrogel for separating exosomes.

[0190] Implementation List 14

[0191] A method for preparing a deoxyribonucleic acid-poly-L-lysine composite hydrogel includes the following steps:

[0192] a. 50 nmol / L circular DNA template II (prepared in Example 5), 0.2 U / µL phi29 DNA polymerase, 1×phi29 DNA polymerase buffer, 0.2 mg / mL BSA, 1 mmol / L dNTPs, and 80 mmol / L NaCl were added to a final volume of 100 µL with sterile water. The reaction mixture was incubated at 37 °C and 450 rpm for 4 hours. The product was then transferred to 65 °C and incubated for 10 minutes to inactivate the phi29 DNA polymerase, yielding the RCA product.

[0193] b. Add 2 µL of PLL solution (concentration of 160 µmol / L) to the above RCA product mixture.

[0194] c. After mixing the two solutions, place them in a 37 ℃ constant temperature mixer and incubate at 500 rpm for 15 minutes to finally prepare a DNA-PLL hydrogel sample labeled as nD-P160 (without AptCD63 aptamer), thus obtaining the nD-P160 hydrogel for capturing exosomes.

[0195] Example 15

[0196] Validation of endogenous exosome capture using a DNA-PLL hydrogel for capturing endogenous exosomes includes the following steps:

[0197] a. Five-week-old healthy SD rats (weighing 180-220 g) were injected with 100 µL of different hydrogels into the anterior wall of the left ventricle using an insulin injector, and samples were collected 24 hours later.

[0198]

[0199] b. Immediately remove the gel and place it in 4% paraformaldehyde at 4°C for 24 hours.

[0200] c. Dehydration and embedding: Tissue samples were dehydrated using a gradient of ethanol (70%, 80%, 90%, 100%), cleared with xylene, embedded in paraffin, and sectioned (4 µm thick). Immunofluorescence staining was then performed: sections were dewaxed using xylene (2 × 5 minutes) and a gradient of ethanol (100%, 95%, 80%, 70%).

[0201] d. Antigen retrieval: Sodium citrate buffer (pH 6.0, 95℃ for 15 minutes); blocking: 5% BSA, incubation at 37℃ for 1 hour; primary antibody incubation: rat anti-CD63 monoclonal antibody (1:200 dilution); overnight washing at 4℃: PBS (3 × 5 minutes); secondary antibody incubation: red fluorescently labeled goat anti-rat IgG (1:500), incubation at room temperature in the dark for 1 hour.

[0202] e. Observe and photograph using an inverted fluorescence microscope; results are shown below. Figure 1 .

[0203] The results showed that the exosome-specific fluorescence signal of the D-P80 group was stronger than that of the nD-P80 group, confirming the key role of AptCD63 in the specific recognition and enrichment of exosomes in the mediator.

[0204] Example 16

[0205] A method for evaluating the in vitro capture and release performance of D-P80 hydrogel for capturing endogenous exosomes, comprising the following steps:

[0206] a. Resuspend CM-DiI-stained H9C2 cells in serum-free medium and adjust the cell concentration to 1-5 × 10⁻⁵. 5 Cells / mL.

[0207] b. Place the Transwell chambers in a 24-well plate and add 100-200 µL of the above cell suspension (containing 1-5 × 10⁶ cells / well) to each chamber. 4 (each cell), be careful to avoid forming bubbles.

[0208] c. Add serum-free medium containing SYBR Green II stained D-P80 hydrogel to the lower chamber of the 24-well plate to maintain osmotic pressure and ensure that the bottom of the insert is completely immersed in the medium containing hydrogel.

[0209] d. The 24-well plate was then incubated in a 37 °C, 5% CO2 incubator, and samples were collected periodically for observation using an inverted fluorescence microscope.

[0210] See results Figure 2 a. Experimental results show that the capture efficiency of D-P80 hydrogel for exosomes is time-dependent, with the capture efficiency increasing linearly in the first 24 hours and then reaching saturation. Figure 2 Results showed that the hydrogel network began to collapse on day 7, coinciding with the almost complete release of captured exosomes. These results demonstrate the efficient exosome capture and controlled release capabilities of the D-P80 hydrogel.

[0211] Example 17

[0212] An in vitro therapeutic validation method for D-P80 hydrogel used to capture endogenous exosomes includes the following steps:

[0213] a. Live / dead cell assay method: H9C2 cells were inoculated at 1×10⁻⁶ cells / cells. 5 Cells / well were seeded into 6-well plates and cultured for 24 hours in complete DMEM-H medium without exosomes under hypoxic conditions (37 °C, 5% CO2, 2% O2).

[0214] b. After incubating with D-P80 hydrogels containing different concentrations of H9C2 cell-derived exosomes (5 mg / mL, 10 mg / mL, 15 mg / mL and 20 mg / mL protein concentration) for 24 hours, the cells were stained using a calcein-AM / propidium iodide (PI) double staining kit, in which live cells were labeled with calcein-AM and dead cells were labeled with PI.

[0215] c. Observe the stained cells under a fluorescence microscope.

[0216] See results Figure 3 The results showed that as the amount of exosomes loaded with D-P80 increased, the fluorescence intensity of proliferating cells gradually increased, indicating that DNA-PLL hydrogel, as an effective exosome capture and delivery system, exerts a concentration-dependent cardioprotective effect on hypoxia-induced damage.

[0217] Example 18

[0218] An in vitro therapeutic validation method for D-P80 hydrogel used to capture endogenous exosomes includes the following steps:

[0219] a. The protein concentration of cell lysates was determined using the BCA protein quantification kit.

[0220] b. Samples containing 30 µg of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% SDS-PAGE) and transferred to a polyvinylidene fluoride membrane. The membrane was blocked with 5% skim milk containing 0.1% Tween-20 for 1 hour, washed again with TBST, and then incubated overnight at 4 °C with anti-ICAM1 primary antibody. After washing with TBST, the membrane was incubated with horseradish peroxidase-labeled secondary antibody (1:5000 dilution) at 37 °C for 1 hour.

[0221] c. After washing TBST three more times, protein bands were detected using an enhanced chemiluminescence reagent and visualized using a gel imaging system.

[0222] See results Figure 3In Figure b, the results showed that hypoxic H9C2 cells treated with 20 mg / mL D-P80 exhibited a significant reduction in the expression level of intercellular adhesion molecule-1 (ICAM-1), indicating that exosomes delivered by DNA-PLL hydrogel can effectively alleviate hypoxia-induced inflammatory responses in cardiomyocytes by inhibiting the expression of ICAM-1.

[0223] Example 19

[0224] Establishment of a rat model of myocardial infarction and administration methods:

[0225] a. Establishment of a rat model of myocardial infarction: Five-week-old healthy SD rats (weighing 180-220 g) were used. A myocardial infarction (MI) model was established by irradiating the local heart area of ​​the rats with a cobalt-60 radiation source at a dose of 20 Gy. Successful modeling was defined as a decrease in left ventricular ejection fraction (LVEF) ≥ 40% confirmed by small animal echocardiography within 24 hours of model establishment.

[0226] b. Administration method: After anesthetizing rats with isoflurane, different therapeutic agents were uniformly injected into the infarct border area using a transthoracic minimally invasive injection technique with an insulin injector. Vital signs were closely monitored after injection to ensure accurate administration.

[0227]

[0228] Example 20

[0229] A method for evaluating the efficacy of D-P80 hydrogel for capturing endogenous exosomes in the treatment of myocardial infarction includes the following steps:

[0230] a. Cardiac function testing: 28 days after intervention, all rats were anesthetized with isoflurane and tested using an ultrasound imaging system. The rats were fixed in a supine position on a 37 ℃ constant-temperature platform, and cardiac images were acquired using an MS250 probe (frequency 13-24 MHz). Left ventricular end-diastolic and end-systolic diameters were measured in the parasternal long-axis section. Results are shown in […]. Figure 4 a; The system automatically calculates the left ventricular ejection fraction (LVEF) and left ventricular shortening fraction (LVFS), and the results are shown in [the table below]. Figure 4 b, Figure 4 a and Figure 4 In b, nD-P refers to nD-P80 hydrogel, and DP hydrogel refers to D-P80 hydrogel.

[0231] The results showed that cardiac function was significantly restored in the D-P80 treatment group, with the left ventricular ejection fraction (EF) increasing to 56.3%, a significant improvement compared to the PBS group (33.6%). Similarly, the fractional shortening (FS) of MI rats increased to 30.5% after D-P80 treatment, significantly higher than that in the PBS group (16.9%). This indicates that D-P80 hydrogel significantly improves the preservation effect on cardiac function.

[0232] b. Histological analysis: All rats were euthanized after being anesthetized with isoflurane. Heart tissue blocks were immediately removed via thoracotomy and fixed in 4% paraformaldehyde solution at 4°C for 24-48 hours. The tissues were dehydrated with a gradient of ethanol (70%-100%), cleared with xylene, and embedded in paraffin (60°C) to prepare 4 µm serial sections. Masson's trichrome staining was then performed as follows: After dewaxing the sections according to the method in Example 15, staining was performed as follows:

[0233] Weigert iron hematoxylin staining for 5 minutes;

[0234] Stain with Ponceau S and Acidic Fuchsin solution for 5 minutes;

[0235] Treat with phosphomolybdic acid for 5 minutes;

[0236] Aniline blue staining for 5 minutes;

[0237] Differentiation with 1% glacial acetic acid for 1 minute;

[0238] Standard dehydration and clearing, then sealing with neutral resin.

[0239] c. Images were acquired using an optical microscope under a 40x objective lens. Five fields of view were randomly selected from each slice. The results are shown in [Figure number missing]. Figure 5 , Figure 5 In this study, nD-P refers to nD-P80 hydrogel, and DP hydrogel refers to D-P80 hydrogel. The results showed that compared with the PBS group, the treatment group had a significantly reduced fibrotic area and the highest proportion of preserved myocardial tissue. D-P80 hydrogel treatment significantly improved cardiac function in rats with myocardial infarction.

[0240] Example 21

[0241] A method for evaluating the biosafety of D-P80 hydrogel for capturing endogenous exosomes, comprising the following steps:

[0242] a. Histological analysis: Five-week-old healthy SD rats (weighing 180-220 g) were used. 100 µL of different therapeutic agents were injected into the anterior wall of the left ventricle using an insulin injector. After 28 days, blood was collected from the retro-orbital vein, and the rats were euthanized. The heart, liver, lungs, spleen, and kidneys were dissected and isolated, and fixed in 4% paraformaldehyde at 4°C for 24 hours. Dehydration and embedding: The rats were dehydrated with a gradient of ethanol (70%, 80%, 90%, 100%), cleared with xylene, and embedded in paraffin (4 µm thick). Sections were then stained with hematoxylin for 10 minutes and eosin for 30 seconds (HE staining), and photographs were captured using an optical microscope. Results are shown below. Figure 6 a.

[0243]

[0244] b. Hematological analysis: 1 mL of rat blood sample was incubated at room temperature for 30 minutes, then gently mixed 8-10 times (avoiding air bubbles). The following counts were analyzed using an automated hematology analyzer: neutrophil count (Neµ); lymphocyte count (Lym); monocyte count (Mon); eosinophil count (Eos); basophil count (Bas). Results are shown below. Figure 6 b.

[0245] The results of the biosafety evaluation experiment showed that after 4 weeks of treatment with D-P80 hydrogel, the major organ tissue structure and the proportion of various leukocyte subsets in the experimental animals were within the normal range, and no inflammation or allergic reaction was caused, which confirmed its biosafety and supported its potential for therapeutic application.

[0246] In summary, this invention utilizes injectable deoxyribonucleic acid-poly-L-lysine composite hydrogels for the treatment of myocardial infarction. Through the unique molecular design of the deoxyribonucleic acid-poly-L-lysine composite hydrogels, it achieves targeted enrichment and controllable release of endogenous exosomes, representing a significant breakthrough in targeting, controllability, safety, and clinical translation, and providing a novel solution for the treatment of myocardial infarction.

[0247] The main differences between this invention and the prior art include:

[0248] a. Active targeted enrichment: Containing the AptCD63 sequence can specifically bind to the CD63 protein on the surface of exosomes, improving targeting efficiency and overcoming the limitations of traditional passive homing enrichment in the liver / lungs.

[0249] b. Nuclease-responsive sustained release: D-P80 hydrogel achieves 7-day linear release through nuclease degradation, which is superior to the uncontrollable degradation of traditional collagen / PLGA materials.

[0250] c. Utilization of endogenous exosomes: Directly enrich endogenous exosomes in the lesion area to avoid the high cost and immune rejection problems of exogenous extraction.

[0251] d. Minimally invasive delivery: Injectable hydrogels can replace surgical implantation, avoiding secondary damage caused by mismatch in mechanical strength.

[0252] It will be apparent to those skilled in the art that this disclosure is not limited to the details of the exemplary embodiments described above, and that this disclosure can be implemented in other specific forms without departing from the spirit or essential characteristics of this disclosure. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of this disclosure is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within this disclosure.

[0253] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. Use of a DNA-poly-L-lysine complex hydrogel in redistributing endogenous exosomes, characterized in that, The deoxyribonucleic acid-poly-L-lysine complex hydrogel comprises a DNA framework containing a nucleic acid aptamer targeting an exosome surface protein and poly-L-lysine wrapping the DNA framework.

2. Use of the DNA-poly-L-lysine complex hydrogel according to claim 1 for redistribution of endogenous exosomes, characterized in that, The deoxyribonucleic acid-poly-L-lysine complex hydrogel is used for capturing and releasing endogenous exosomes.

3. Use of the DNA-poly-L-lysine complex hydrogel according to claim 2 for redistributing endogenous exosomes, characterized in that, The deoxyribonucleic acid-poly-L-lysine complex hydrogel captures endogenous exosomes by binding to endogenous exosome surface proteins through nucleic acid aptamers.

4. Use of the DNA-poly-L-lysine complex hydrogel according to claim 2 in redistributing endogenous exosomes, characterized in that, The deoxyribonucleic acid-poly-L-lysine complex hydrogel releases endogenous exosomes by degradation through nucleases.

5. Use of the DNA-poly-L-lysine complex hydrogel according to claim 1 in redistributing endogenous exosomes, characterized in that, The exosome surface protein is CD63 protein, the nucleic acid aptamer is CD63 DNA aptamer, and the sequence of the CD63 DNA aptamer is shown in SEQ ID NO.

5.

6. Use of the DNA-poly-L-lysine complex hydrogel according to claim 5 for redistributing endogenous exosomes, characterized in that, The deoxyribonucleic acid-poly-L-lysine complex hydrogel is obtained by mixing a RCA product obtained by rolling circle amplification of a circular DNA template containing an antisense sequence of the CD63 DNA aptamer and a poly-L-lysine aqueous solution and incubating them; The concentration of the poly-L-lysine aqueous solution is 20 μmol / L-160 μmol / L.

7. Use of the DNA-poly-L-lysine complex hydrogel according to claim 6 for redistributing endogenous exosomes, characterized in that, The concentration of the poly-L-lysine aqueous solution is 80 μmol / L.

8. Use of the DNA-poly-L-lysine complex hydrogel according to claim 6 for redistributing endogenous exosomes, characterized in that, The incubation operation is oscillation at 350 rpm-450 rpm for 4 h-16 h.

9. Use of a DNA-poly-L-lysine complex hydrogel in the treatment of myocardial infarction, characterized in that, The deoxyribonucleic acid-poly-L-lysine complex hydrogel is used for redistributing endogenous exosomes. The deoxyribonucleic acid-poly-L-lysine complex hydrogel comprises a DNA framework containing a nucleic acid aptamer targeting an exosome surface protein and poly-L-lysine wrapping the DNA framework.

10. Use of the DNA-poly-L-lysine complex hydrogel according to claim 9 for the treatment of myocardial infarction, characterized in that, The deoxyribonucleic acid-poly-L-lysine complex hydrogel is used for treating myocardial infarction by in situ injection.

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