CN115804847B - PH/hydrogen peroxide/MMP 9 time sequence response microsphere, exosome-carrying biological carrier and application - Google Patents

Abstract

The invention discloses a pH/hydrogen peroxide/MMP 9 time sequence response microsphere, a biological carrier carrying exosomes and application thereof, and relates to the technical field of medicine carrying carriers. The invention is based on the protection effect of the regulatory T cell-derived exosome with high SPARC expression on heart function, and pH and H in the infarct area ₂ O ₂ And MMP9 levels, to synthesize conjugated peptides, pH/H ₂ O ₂ MMP9 time-series response microsphere and exosome-carrying biological carrier. First, exosomes and hydrogel materials are released by cleavage of the acylhydrazone bond under acidic conditions. Subsequently, H is used ₂ O ₂ Co is to be ²⁺ Oxidation to Co ³⁺ Gel is formed and the released exosomes are immobilized. Finally, under the hydrolysis of MMP9, the gel gradually degrades and releases exosomes, thereby exerting a continuous and long-term protective effect on the myocardium to improve cardiac function.

Description

PH/hydrogen peroxide/MMP 9 time sequence response microsphere, exosome-carrying biological carrier and application

Technical Field

The invention relates to the technical field of drug-carrying carriers, in particular to a pH/hydrogen peroxide/MMP 9 time sequence response microsphere, a biological carrier carrying exosomes and application thereof.

Background

In Acute Myocardial Infarction (AMI), regulatory T cells (Tregs) can inhibit differentiated CD4 ⁺ T cells, CD8 ⁺ Effector activities of T cells, th17 cells, and functions of natural killer and B cells can also affect the healing process of injury by modulating mononuclear/macrophage differentiation [1 ]]. Tregs infiltrated into the myocardium highly express SPARC (cysteine-rich acidic secreted protein) which can increase collagen content and maturity in myocardial infarction scar to prevent heart rupture after myocardial infarction and increase survival rate [2 ]]. Thus, sparc ^high Tregs play an important protective role in tissue repair following acute myocardial infarction. Although Tregs can infiltrate into the infarcted area after myocardial infarction occurs, it reaches a peak at day 7, suggesting that spark is advanced ^high The peak of Treg in infarct area may contribute to repair of infarct area, decreasing infarct area, but direct in vitro expansion of cells is subject to functional limitations and even loss due to instability of genetic material and cell survival [3 ]]。

The cell-derived exosomes have functions similar to those of cells, which promote angiogenesis, reduce apoptosis, inhibit fibrosis, regulate immune response, etc. [4]. Studies have shown that exosomes comprising dendritic cells secreted after stimulation by necrotic cardiomyocytes can reduce cardiac inflammation by activating Tregs, thereby improving cardiac function and left ventricular remodeling following myocardial infarction [5].

However, the number of exosomes targeted and residing in situ in myocardial tissue limits their effectiveness in treating acute myocardial infarction. Currently genetic engineering has been widely used to improve exosome targeting and stability [6]. Meanwhile, many studies have confirmed that hydrogels are good substrates for fixing exosomes [7]. However, many studies of hydrogel-coated exosomes are currently performed by in situ injection, which can cause secondary damage to myocardial tissue. Therefore, there is an urgent need to develop a hydrogel-exosome system that is compatible with the local microenvironment following acute myocardial infarction to target the infarct site. The hydrogel which can be crosslinked in situ to form gel for fixing the exosome and can be slowly released can provide a potential scheme for treating myocardial infarction.

Analysis of local microenvironment changes after acute myocardial infarction shows that, in the first stage after the activation of aseptic inflammatory response, damaged cardiac muscle releases damage related molecular components and binds to Toll-like receptors to initiate production of chemokines such as CXCL1, CXCL2 and CXCL5, etc., with ligand CXCR2, and the ligand CXCL2 induces neutrophils and Ly6C with high expression of CXCR2 ^high Monocyte recruitment releases a large number of pro-inflammatory factors. At the same time, the continuous decrease of pH in the infarct zone (pH) is caused by the continuous death and rupture of cells accompanied by the continuous accumulation of lactic acid <6.8)[8]Forming an acidic microenvironment [9,10 ]]. In the second stage, inflammatory cells that have chemotactic to the infarcted area exert a pro-inflammatory effect, promoting the production of large amounts of reactive oxygen species, including H ₂ O ₂ Superoxide and hydroxyl radicals, and the like, further damage the damaged myocardium [10 ]]. Third stage, ly6C ^low Monocytes and M2-type macrophages exert anti-inflammatory effects in an environment enriched in IL-10, TGF-beta and VEGF, while releasing matrix metalloproteinase 9 (MMP 9) [11 ]]. Fourth stage, fibroblast is activated and migrated to infarct area, converted into myofibroblast under the action of chemotactic factor and growth factor, gradually accumulated MMP9 enzymolysis reaction is activated, extracellular matrix is degraded, proliferation of myofibroblast is promoted, scar tissue formation is promoted [12 ]]。

In view of this, the present invention has been made.

Disclosure of Invention

The invention aims to provide a pH/hydrogen peroxide/MMP 9 time-series response microsphere, a biological carrier carrying exosomes and application thereof, so as to solve the technical problems.

The invention utilizes the inflammatory reaction process after acute myocardial infarction through the acidic microenvironment (pH<6.8 Release of hydrogel materials (microsphere rupture, exosome release)) With consequent use of H after myocardial infarction ₂ O ₂ The hydrogel is formed in situ in a high-concentration environment to fix the exosome, and then the content of MMP9 is increased continuously to degrade the hydrogel and release the exosome, so that the function of repairing cardiovascular tissues is exerted.

The invention is realized in the following way:

the invention provides a conjugated peptide, which comprises 4A-PEG modified by polypeptide and metallic cobalt ion, wherein the polypeptide comprises histidine and peptide fragments for MMP9 enzymatic hydrolysis, and the peptide fragments for MMP9 enzymatic hydrolysis have the following SEQ ID NO:2, wherein the metal cobalt ion and histidine are combined by a coordination bond; the metal cobalt ion can be H ₂ O ₂ Oxidation thereby promotes gel-like formation of the conjugated peptide.

SEQ ID NO:2 is as follows: GGALGLPG.

Previous studies have shown that amino groups and Co in histidine in aqueous 4A-PEG solutions modified by peptide chemistry ²⁺ Coordination bonds may be formed. At H ₂ O ₂ Under the action of Co ²⁺ Oxidized to Co ³⁺ At the same time, the aqueous 4A-PEG solution was changed from liquid to gel-like [14]. H produced in the second stage of the inflammatory response ₂ O ₂ And MMP9 secreted by the fourth stage is continuously accumulated in the infarcted area [10-12]MMP9 can specifically recognize and hydrolyze the peptide fragment "GALGLP" [15 ]]. Uses the change of local microenvironment at different stages after acute myocardial infarction to ensure that the product has the activity of H ₂ O ₂ The properties of gel formation under the action and hydrolysis under the action of MMP9, the inventors used polypeptides (including histidine and peptide fragments recognized and hydrolyzed by MMP9, i.e., GGALGLPGH) to modify 4A-PEG, conjugated Co based on metal-ligand interactions ²⁺ Finally, 4A-PEG conjugated peptide (4 APPC) was formed. The synthetic structure of 4APPC is shown in fig. 7A.

In addition, it was verified that the conjugated peptide has good fluidity once it is H ₂ O ₂ The triggering, the material exhibits good toughness and strength, which will help it adapt to the beating characteristics of the heart and achieve long-term residence. While once the material is triggered again by MMP9, the toughness and toughness characteristics disappear, the fluidity increases, ensuring thatRelease of the loading agent. The conjugated peptide is based on H ₂ O ₂ Designed in response to, and Co ²⁺ The coordinately linked 4A-PEG can form hydrogel in situ in the infarcted area, fixing exosomes. The conjugated peptide is provided with peptide segments for MMP9 enzyme hydrolysis, so that the formed hydrogel is gradually degraded and slowly released to exosomes under the action of MMP9 enzyme.

The invention also provides a pH/H ₂ O ₂ An MMP9 time responsive microsphere comprising: microspheres formed by distearoyl phosphatidylethanolamine and polyethylene glycol connected by acylhydrazone bonds, and the microspheres are loaded with the conjugated peptide.

The microsphere is made of amphoteric polymer, and can self-assemble in water to form the microsphere. The hydrophobic portion of the polymer forms the core of the encapsulated agent, while the hydrophilic portion forms the outer shell of the microsphere structure [16]. The acylhydrazone bond is pH sensitive and breaks at pH <6.8 [17]. Depending on the local acidic microenvironment of the infarcted area, the lipophilic compound DSPE and the hydrophilic compound PEG may be linked by an acylhydrazone bond (Hyd) to form DSPE-Hyd-PEG (DHP). The inventor verifies that the microsphere is in a uniform spherical structure, is stable in neutral solution and depolymerizes in acidic solution. Microsphere (DHPM) loading 4APPC forms DHPM_ (4 APPC) with a loading capacity of 32.34+ -7.43 wt.%.

In a preferred embodiment of the invention, the microspheres are broken at an acylhydrazone bond in an environment having a pH of less than 6.8. Rupture in the environment of infarct zone pH <6.8 to release the internal load.

The invention provides an exosome-carrying biological carrier, which comprises the pH/H ₂ O ₂ MMP9 time-series response microsphere, CD63 aptamer is connected to the periphery of the time-series response microsphere through acylhydrazone bond, and the CD63 aptamer is specifically bound with the exosome through CD63 marker on the surface of the exosome (first mode).

As one embodiment, regulatory T cells have a positive effect on myocardial infarction, and in other embodiments, other sources of exosomes may be selected to specifically bind to CD63 aptamers via CD63 markers on the exosomes surface, as desired. CD63 was highly expressed on the surface of all sources of exosome membrane.

In an alternative embodiment, the exosomes are derived from stem cells or regulatory T cells.

In an alternative embodiment, the exosomes described above are derived from adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, placental mesenchymal stem cells, urine-derived stem cells, or endothelial progenitor cells.

CD63 acts as the most important marker of exosomes, and is expressed in large amounts on the surface of exosomes [13]. The aptamer is a short oligonucleotide sequence or a short polypeptide obtained by in vitro screening, which can bind to the corresponding ligand with high affinity and strong specificity [18,19]. To modify Tregs-derived exosomes highly expressed by Sparc, the inventors selected CD63 aptamers as linkers. The CD 63-aptamer was screened by exponential enrichment ligand system evolution technique [20], the top 10 aptamer sequences with highest score were synthesized, and the affinity of each sequence for CD63 protein was verified (S table). The highest affinity sequence was selected in this study (FIG. 11A). The results of the binding experiments show that the selected aptamer has strong binding force (kd=4.54±0.27 nM) to CD63 (fig. 11B), and fig. 11B is an affinity curve of CD63 aptamer and CD63 protein with increasing concentration.

The pH-sensitive acylhydrazone linkage is set to facilitate exosome release under acidic conditions.

In an alternative embodiment, the CD 63-aptamer specifically binds to an exosome derived from SPARC-highly expressed regulatory T cells via a CD 63-marker on the surface of the exosome (second mode).

In an alternative embodiment, the CD 63-aptamer specifically binds to a CD 63-marker on the surface of an exosome derived from CXCR 2-high and SPARC-high regulatory T cells (third mode). By utilizing the effect of chemokines in the first stage of the inflammatory response, the exosomes with high CXCR2 expression can be targeted to the infarct area under the effect of chemokines.

The inventors found that the first, second and third modes all inhibit ischemia reperfusion injury, restore cardiac function and prevent fibrosis, while the third mode has a better effect. Namely, the exosomes of the regulatory T cells with high CXCR2 expression and SPARC expression have the effects of better inhibiting ischemia reperfusion injury, recovering heart functions and preventing fibrosis, and can improve cardiac insufficiency after acute myocardial infarction.

The inventor finds that the exosomes derived from the Tregs have a certain protection effect on myocardial infarction, and the exosomes derived from the Tregs with high Sparc expression have a strong protection effect on myocardial infarction. Although Sparc is repeatedly injected in situ ^high Tregs can improve cardiac function, but survival is reduced due to secondary injury to the myocardium. Furthermore, targeted delivery and residence of exosomes in the infarcted area is also a challenge. The inventors have utilized the enrichment of CXCL1/2/5 and other chemokines in the infarct zone after acute myocardial infarction to obtain the chemotactic properties of CXCR2 highly expressed immune cells by a gene editing method. The results indicate that spark, highly expressed from CXCR2 ^high Exosomes extracted from tregs can be targeted rapidly and accurately to infarcted areas. The inventor also develops a system which can form gel fixed exosomes through in-situ crosslinking in an infarct area after acute myocardial infarction and can slowly release exosomes under the action of a local microenvironment.

Specifically, referring to FIG. 16, the inventors successfully modified 4A-PEG with MMP 9-specific hydrolysis and recognition of polypeptides, adding Co to the solution ²⁺ After that, the function of the compound is not affected, and in H ₂ O ₂ Is formed into a gel-like form under the action of (a). Meanwhile, the acylhydrazone bond is utilized to connect DSPE and PEG to form DHPM, the aptamer of CD63 is selected to modify the DHPM, the DHPM is combined with and wraps the gel material, and meanwhile, the DHPM is combined with Tregs ^Sp_hi_ Exos ^CXCR2_hi Anchoring to form a composite structure. The system successfully targets the infarcted area under the action of CXCR2, realizes the rupture of the microsphere under the acidic condition, and utilizes the high H of the infarcted area ₂ O ₂ Content of Co in the gel raw material ²⁺ The transition from the divalent state to the trivalent state forms a gel that entraps exosomes. Then, by utilizing the gradual increase of the later MMP9, the gel is gradually degraded, and the exosomes are promoted to be gradually grownGradual release, achieving protection against acute myocardial infarction by targeted delivery and fixation of exosomes as a non-invasive treatment. The invention provides a new choice for treating acute myocardial infarction.

In an alternative embodiment, the regulatory T cells with high CXCR2 expression and high SPARC expression are such that the regulatory T cells with high SPARC expression achieve high CXCR2 expression by genetic editing means.

The inventors found spark ^high Treg-derived exosomes may provide a new approach for the treatment of acute myocardial infarction. The surface of the exosome membrane is greatly expressed by the gene engineering editing to promote the exosome to rapidly target to reach the infarct zone,

in an alternative embodiment, the sequence of the aptamer is as set forth in SEQ ID NO:1 is shown as follows: UUAGCAGUGUACGAGAAGUCGUUACAAGUUA.

In an alternative embodiment, the aptamer is further modified with a modifier or labeled with a detectable label.

In an alternative embodiment, the modification is biotin.

A detectable label refers to a substance of a type having properties such as luminescence, color development, radioactivity, etc., that can be directly observed by the naked eye or detected by an instrument, by which a qualitative or quantitative detection of the corresponding target can be achieved.

In alternative embodiments, detectable labels include, but are not limited to, fluorescent dyes, enzymes that catalyze the development of substrates, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.

In the actual use process, a person skilled in the art can select a suitable marker according to the detection conditions or actual needs, and no matter what marker is used, the marker belongs to the protection scope of the invention.

In alternative embodiments, the fluorescent dyes include, but are not limited to, fluorescein-based dyes and derivatives thereof (including, but not limited to, fluorescein Isothiocyanate (FITC) hydroxy-light (FAM), tetrachlorolight (TET), etc., or analogs thereof), rhodamine-based dyes and derivatives thereof (including, but not limited to, red Rhodamine (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), etc., or analogs thereof), cy-based dyes and derivatives thereof (including, but not limited to, cy2, cy3B, cy3.5, cy5, cy3, etc., or analogs thereof), alexa-based dyes and derivatives thereof (including, but not limited to, alexa fluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc., or analogs thereof), and protein-based dyes and derivatives thereof (including, but not limited to, for example, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polymethine (cp), etc.).

In alternative embodiments, enzymes that catalyze the development of a substrate include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphoglucose deoxygenase.

In alternative embodiments, the radioisotope includes, but is not limited to ²¹² Bi、 ¹³¹ I、 ¹¹¹ In、 ⁹⁰ Y、 ¹⁸⁶ Re、 ²¹¹ At、 ¹²⁵ I、 ¹⁸⁸ Re、 ¹⁵³ Sm、 ²¹³ Bi、 ³² P、 ⁹⁴ mTc、 ⁹⁹ mTc、 ²⁰³ Pb、 ⁶⁷ Ga、 ⁶⁸ Ga、 ⁴³ Sc、 ⁴⁷ Sc、 ¹¹⁰ mIn、 ⁹⁷ Ru、 ⁶² Cu、 ⁶⁴ Cu、 ⁶⁷ Cu、 ⁶⁸ Cu、 ⁸⁶ Y、 ⁸⁸ Y、 ¹²¹ Sn、 ¹⁶¹ Tb、 ¹⁶⁶ Ho、 ¹⁰⁵ Rh、 ¹⁷⁷ Lu、 ¹⁷² Lu and ¹⁸ F。

in alternative embodiments, chemiluminescent reagents include, but are not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and its derivatives, dioxane and its derivatives, lotensine and its derivatives, and peroxyoxalate and its derivatives.

In alternative embodiments, nanoparticle-based labels include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

In alternative embodiments, colloids include, but are not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.

In alternative embodiments, the colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.

The invention provides a pH/H ₂ O ₂ A method for preparing MMP9 time sequence response microspheres, which comprises the following steps:

microspheres formed by conjugated peptide, distearoyl phosphatidylethanolamine connected by acylhydrazone bond and polyethylene glycol are mixed in a solvent, and then centrifuged and dried in vacuum.

In an alternative embodiment, the ratio of the concentration of conjugated peptide in the solvent to the concentration of the microspheres in the solvent is 2.3-2.7mg/ml to 4.8-5.2mg/ml.

In an alternative embodiment, the solvent is methylene chloride and water is added until the volume of water added is 30% of the total mixed solution volume when the conjugated peptide is mixed with the microspheres; in an alternative embodiment, water is added at a rate of 0.5 to 0.6 ml/h;

in an alternative embodiment, after water is added to 30% of the total mixed liquor volume, water is added at a rate of 2-2.5mL/h to 50% of the total mixed liquor volume.

The invention provides a preparation method of a biological carrier carrying exosomes, which is characterized by comprising the following steps:

combining the time-series response microsphere with CD63 ligand according to 0.23-0.27mM: a mixing reaction was performed at a molar ratio of 0.008-0.012mM to obtain a time-series responsive microsphere having CD 63-aptamer linked thereto via an acylhydrazone bond, and then mixing an exosome derived from regulatory T cells with the time-series responsive microsphere having CD 63-aptamer linked thereto. For example, the time-series response microsphere and CD63 aptamer were in 0.257mM: a mixing reaction was carried out at a molar ratio of 0.01 mM.

In an alternative embodiment, the method further comprises modifying the ethynyl group on the CD63 aptamer prior to the mixing reaction. Modification of the microspheres with azidoacetophenone facilitates subsequent reaction with microspheres.

In an alternative embodiment, the mixing mass ratio of exosomes to time-sequential response microspheres linking CD63 aptamers is 0.5-1.5:1-3. Under the above mixing conditions, a preferable combination ratio is obtained. For example, the mixing mass ratio is 1:2.

In an alternative embodiment, the mixing is incubation at 37 ℃ ± 0.5 ℃ for 3 hours.

Conjugated peptide, pH/H ₂ O ₂ Application of MMP9 time sequence response microsphere or exosome-carried biological carrier in preparing myocardial infarction repairing medicine.

In the above application, the pH triggers the release of exosomes of the biological carrier, active oxygen (H ₂ O ₂ ) The oxidation of the metal cobalt ions is initiated to form a gel to immobilize the released exosomes, which are then triggered to degrade based on the hydrolysis of MMP9 enzymes, releasing the exosomes slowly.

In a preferred embodiment of the present invention, the myocardial infarction is selected from acute myocardial infarction, subacute myocardial infarction, myocardial infarction reperfusion or ventricular tumor.

The invention provides a medicament for treating myocardial infarction, which comprises the conjugated peptide and the pH/H ₂ O ₂ MMP9 time-series response microsphere or the above-mentioned exosome-carrying biological carrier.

In a preferred embodiment of the present invention, the above-mentioned drug for treating myocardial infarction further comprises a pharmaceutically acceptable additive or adjuvant;

in an alternative embodiment, the pharmaceutical dosage form is selected from the group consisting of tablets, pills, powders, suspensions, gels, emulsions, creams, granules, nanoparticles, capsules, suppositories, injections, sprays and injections.

In an alternative embodiment, the above-described medicaments further comprise a combination medicament/therapy, which is a combination of the disclosed pharmaceutical compounds with at least one of the following medicaments/therapies:

chemotherapeutic agents, radiation therapy, photosensitizers, photothermal agents, immunotherapy, androgen receptor antagonists and functional modulators, estrogen receptor antagonists and functional modulators, mineralogical receptor antagonists and functional modulators, FXR agonists, antagonists and functional modulators, GPR30 agonists, antagonists and functional modulators, TGR agonists, antagonists and functional modulators, GLP receptor agonists, antagonists and functional modulators, FGF receptor agonists, antagonists and functional modulators, thyroxine receptor agonists, antagonists and functional modulators, sodium-glucose cotransporter 2 inhibitors, dipeptidyl peptidase-4 inhibitors and TGF receptor agonists, antagonists and functional modulators.

The above-mentioned combination may have the same or different mechanism of action as the compound of the present invention.

The invention has the following beneficial effects:

the invention is based on the protection effect of the regulatory T cell-derived exosome with high SPARC expression on heart function, and pH and H in the infarct area ₂ O ₂ And MMP9 levels, to synthesize conjugated peptides, pH/H ₂ O ₂ MMP9 time-series response microsphere and exosome-carrying biological carrier.

First, conjugated peptides have good fluidity once H-substituted ₂ O ₂ The triggering, the material exhibits good toughness and strength, which will help it adapt to the beating characteristics of the heart and achieve long-term residence. While once the material is triggered again by MMP9, the toughness and toughness characteristics disappear, the flowability increases, ensuring the release of the loading agent. The conjugated peptide is based on H ₂ O ₂ Designed in response to, and Co ²⁺ The coordinately linked 4A-PEG can form hydrogel in situ in the infarcted area, fixing exosomes. The conjugated peptide is provided with peptide segments for MMP9 enzyme hydrolysis, so that the formed hydrogel is gradually degraded and slowly released to exosomes under the action of MMP9 enzyme.

Next, the inventors have verified that the above pH/H ₂ O ₂ MMP9 time-series response microspheres are in uniform spherical structures, remain stable in neutral solution and depolymerize in acidic solution. Such as rupture in the environment of the infarcted area, To release the internal load.

Again, the exosome-carrying biological carrier can release exosomes and hydrogel materials by cleavage of the acylhydrazone bond in response to pH. Subsequently, H is used ₂ O ₂ Co is to be ²⁺ Oxidation to Co ³⁺ Gel is formed and the released exosomes are immobilized. Finally, under the hydrolysis of MMP9, the gel gradually degrades and releases exosomes, thereby exerting a continuous and long-term protective effect on the myocardium to improve cardiac function.

In addition, a preparation method of the time sequence response microsphere and the biological carrier carrying the exosome is also provided, and the preparation method is simple and easy to implement and is easy to produce and apply.

The conjugated peptide provided by the invention, the pH/H ₂ O ₂ MMP9 time sequence response microsphere or the biological carrier carrying exosome can be used for preparing medicines for treating myocardial infarction.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the verification of spark in Experimental example 1 ^high Results of Treg-derived exosomes function graph ((a) acute myocardial infarction model was established using SD rats (female, 180-230 g) according to the described surgical procedure, ischemia for 45 min and reperfusion for 4 weeks, after reperfusion was opened, purified Tregs were administered separately by local in situ injection ^Sp_lo Exos (0.1 mL; containing 0.1X10) ⁸ /mL exosomes) or Tregs ^Sp_hi Exos (0.1 mL; containing 0.1X10) ⁸ Per mL exosomes) once daily for 7 days, then once every 2 days, for 7 days, and then for 2 weeks, the PBS group was injected only 1 time; tregs ^Sp_lo Exos stands for spark ^low Treg-derived exosomes, tregs ^Sp_hi Exos stands for spark ^high Treg-derived exosomes. (B) After 24 hours of reperfusion, the hearts were removed and stained for evans blue-TTC; each group, n=5. (C, D) measuring the areas of AAR, IF and LV based on the results of (B), and calculating and statistically analyzing the results of IF/AAR and AAR/LV; for each group, n=5, one-way analysis of variance; IF: infarct size, AAR: risk area, LV: left ventricle. After 4 weeks of reperfusion, cardiac function was examined using echocardiography, (E) statistics of echocardiography were obtained by pulse doppler and M-mode ultrasound detection, (F) E/a: the ratio of the early and late diastole transventricular flow rates; (G) LVEF: left ventricular ejection fraction; (H) LVFS: left ventricular shortening rate; (I) HR: heart rate; each group, n=5; and (5) single-factor variance analysis. (J, K) immediately after echocardiography examination, the heart was removed and cut into 5 μm sections, subjected to (K) masson staining, the area of fibrosis was measured and (J) statistical analysis was performed; each group, n=5; one-way analysis of variance. (L) in the whole process, monitoring and statistically analyzing death of the rat to obtain a Kaplan-Meier survival curve; each group, n=20; log-rank (Mantel-Cox). * P (P) <0.05)；

FIG. 2 is a graph of 4APPC and DHPM characterization results;

FIG. 3 is a graph of DHPM (4 APPC) _Exo characterization results;

FIG. 4 is a graph showing the results of experiments in Experimental example 5 to verify that DHPM (4 APPC) _Exo improves cardiac insufficiency in patients with acute myocardial infarction;

FIG. 5 separation spark ^high Tregs and modeling acute myocardial infarction for its derived exocrine profile ((A), heart was extracted 3 days later and CD45 was used ⁺ CD4 ⁺ Foxp3 ⁺ Sparc ⁺ Tregs as isolation protocol. Isolating CD45 ⁺ CD4 ⁺ Foxp3 ⁺ Sparc ⁺ Tregs and CD45 ⁺ CD4 ⁺ Foxp3 ⁺ Sparc-Tregs were cultured to obtain exosomes, and CXCR2 overexpressing plasmid was transcribed into CD45 ⁺ CD4 ⁺ Foxp3 ⁺ Sparc ⁺ Tregs and the exosomes from which they were derived. (B) detecting exosome morphology by TEM analysis; (C) Nanoparticle Tracking Analysis (NTA) detects particle size distribution; (D) measuring zeta potential of exosomes; (E) Western blot detection of exosome markers CD63 and CD9Expression, by detecting CXCR2 to confirm the efficiency of gene editing; tregs ^Sp_lo Exos: spark low expression Tregs-derived exosomes, tregs ^Sp_hi Exos: spark high expression Tregs-derived exosomes, tregs ^Sp_hi _Exos ^CR2_hi : the spark high expression Tregs source exosomes with CXCR2 high expression);

FIG. 6 shows the establishment of an acute myocardial infarction model using SD rats (female, 180-230 g), once the blood supply to the ischemic area was restored, the following different protocols ((A) PBS (0.2 ml, n=3), tregs, respectively ^Sp_hi _Exos(0.2mL，1×10 ⁸ /mL，n＝3)、FITC(0.2mL，100μM，n＝3)、Tregs ^Sp_hi _Exos(FITC)(0.2mL，Sparc ^low Tregs-derived exosomes (0.2X10) ⁸ ) FITC (0.02 μmol), n=3) and Tregs were added ^Sp_hi _Exos ^CR2_hi (FITC) (0.2 mL, CXCR2 high-expression spark) ^high Treg-derived exosomes (0.2X10) ⁸ ) FITC (0.02. Mu. Mol)) was added to the ischemic area for recovery from blood supply, and then injected via the tail vein, and after 2 hours, the heart, liver, spleen, lung and kidney were removed for optical bioluminescence imaging analysis. (B) For injection of Tregs ^Sp_hi Exos (FITC) and Tregs ^Sp_hi _Exos ^CR2_hi (FITC) group, after optical bioluminescence imaging analysis, hearts were excised and cut into 20 μm sections and fluorescence intensity was observed by a panoramic tissue slice high resolution fluorescence scanning imaging system. When the ischemic area is restored to blood supply, tregs are injected through tail vein ^Sp_hi _Exos ^CR2_hi (FITC) (0.2 mL, CXCR2 high-expression spark) ^high Treg-derived exosomes (0.2X10) ⁸ ) FITC (0.02 μmol) was added, and after (C) 30 minutes (n=3), 2 hours (n=3), 5 hours (n=3) and 10 hours (n=3), heart, liver, spleen, lung and kidney were extracted for optical bioluminescence imaging analysis, and fluorescence intensity was analyzed. Different days after acute myocardial infarction model establishment, tregs are injected through tail vein ^Sp_hi _Exos ^CR2_hi (FITC) (0.2 mL, CXCR2 high-expression spark) ^high Treg-derived exosomes (0.2X10) ⁸ ) FITC (0.02. Mu. Mol) was added, 2 hours after injection, (E) the major organs were extracted for optical bioluminescence imaging analysis, (F) and fluorescence distribution was analyzed, (G, H) and the heart is removed and sectioned into 20 μm sections, and the fluorescence intensity of the infarct area and distal end is observed and analyzed;

FIG. 7 is a synthetic route pattern of 4APPC and H of 4APP ¹ Nuclear magnetic resonance spectrogram (4 APP: coupling of four-arm polyethylene glycol and MMP9 enzyme conjugated peptide, 4APPC: co based on metal-ligand interaction) ²⁺ Modified 4 APP);

FIG. 8 is Co ²⁺ Quilt H ₂ O ₂ Oxidation to Co ³⁺ Schematic representation of specific peptide fragments of MMP9 enzymatic hydrolysis;

FIG. 9 is a synthetic route diagram for intermediate B;

FIG. 10 is a synthetic route of DSPE-Hyd-PEG;

FIG. 11 is a diagram of the folding structure and affinity detection results of CD 63-aptamer;

FIG. 12 is a DHPM_Apt ^CD63 Is a synthetic roadmap of (2);

FIG. 13 is a DHPM_Apt ^CD63 Is a verification result graph of (1);

fig. 14 (a) based on the results of fig. 4A, areas of AAR and LV were measured, AAR/LV calculated and statistically analyzed, n=5 for each group; single factor analysis of variance; AAR: risk area, LV: a left ventricle; (B) HR was detected and analyzed by echocardiography 4 weeks after injection; HR: heart rate; each group, n=5; single factor analysis of variance;

FIG. 15 is a graph showing the results of toxicity evaluation of DHPM (4 APPC) _Exo in rats;

FIG. 16 is a schematic structural view of an exosome-carrying biological vector and pH/H ₂ O ₂ Schematic of MMP9 time series response.

Detailed Description

Reference now will be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment.

Unless otherwise indicated, practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the ability of a person skilled in the art. This technique is well explained in the literature, as is the case for molecular cloning: laboratory Manual (Molecular Cloning: A Laboratory Manual), second edition (Sambrook et al, 1989); oligonucleotide Synthesis (Oligonucleotide Synthesis) (M.J.Gait et al, 1984); animal cell culture (Animal Cell Culture) (r.i. freshney, 1987); methods of enzymology (Methods in Enzymology) (Academic Press, inc.), experimental immunology handbook (Handbook of Experimental Immunology) (D.M.Weir and C.C.Blackwell, inc.), gene transfer vectors for mammalian cells (Gene Transfer Vectors for Mammalian Cells) (J.M.Miller and M.P.calos, inc., 1987), methods of contemporary molecular biology (Current Protocols in Molecular Biology) (F.M.Ausubel et al, inc., 1987), PCR: polymerase chain reaction (PCR: the Polymerase Chain Reaction, inc., 1994), and methods of contemporary immunology (Current Protocols in Immunology) (J.E.Coligan et al, 1991), each of which is expressly incorporated herein by reference.

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The features and capabilities of the present invention are described in further detail below in connection with the examples.

The materials are as follows:

Boc-H (Trt) GPLGLAGG-OH peptide was purchased from ChinapidesCo.LTD (Shanghai, china); quadrifilar polyethylene glycol amine (#A 163118, MW2000 Da), 1-hydroxybenzotriazole (HOBT; #H242, > 97.0%), N, N-dimethylformamide (DMF; #D112009, > 99.9%)N, N' -diisopropylcarbodiimide (DIC; #D106162, 98%), N, N-diisopropylethylamine (DIPEA; #D109321, 99%), trifluoroacetic acid (TFA; #T103295,>99.5%), triisopropylsilane (#t 107280, 98%), cobalt dichloride (#c 106772, 99.7%), N- (2-hydroxyethyl) -piperazine-N' -ethanesulfonic acid (HEPES; # H109408, > 99.5%), sodium hydroxide (#S 291911,60% (w/v)), NH ₂ PEG-COOH (#A 163238, MW600 Da), 4-acetylbenzoic acid (#A 151401), >98.0%) dichloromethane (#d 116153, > 99.9%), N, N' -dicyclohexylcarbodiimide (DCC; #d106074, 99%), 4-dimethylaminopyridine (DMAP; # D109207, 99%), triethylamine (#t 103284, > 99.5%), T-butyl carbazate (#b 106949, 98%), 1, 2-distearoyl-sn-glycerol-3-phosphate ethanolamine (DSPE; a combination of # D130471,>97%), succinic anhydride (#s104823, 99%), acetonitrile (#a 104439, > 99.9%), copper tetrakis (acetonitrile) hexafluorophosphate (#t 115570, 97%), para-azidoacetone (#p 344177), tetrabutylammonium fluoride solution in tetrahydrofuran (TBAF-THF; # T106821,1.0 MinTHF), 5-ethynyl-2' -deoxyuridine (EdU; #E131265, > 98%), cy5.5 (#C 266425), fluorescein isothiocyanate (#F 272903, > 95%), hydrogen peroxide (#H2 112517,30wt% inH ₂ O) was purchased from Aladin. Methylene chloride (# 288306, > 99%), acetone (# 48358), triethylamine acetate buffer (TEAA; #90358,1.0M), biotin (# B4501, > 99%) were available from Sigma Aldric. 4-amino-DL-phenylalanine (# 235606) was purchased from J&KScientific。

Example 1

The embodiment provides a conjugated peptide and a preparation method thereof.

Reference is made to the method described in:

LiPP,XiaYG,HaoJC,WangX.TransientHealabilityofMetallosupramolecularPolymerNetworksMediatedbyKineticControlofCompetin gChemicalReactions.Macromolecules.2020,53(8):2856-2863

4-ArmPEGANE (5.0 g,2.0mmol NH) ₂ end-group), boc-H (Trt) GPLGLAGG-OH (2.0 g,4.0 mmol) and HOBTHOBT (0.54 g,4.0 mmol) were dissolved in DMF solution (15 mL) and DIPEA (1.07 mL,6.0 mmol) and DIC (0.62 mL,4.0 mmol) were added and the mixture was reacted at room temperature for 24 hours. Washing the crude precipitate with cold diethyl ether for 3 times, vacuum drying the obtained white powder precipitate, and further dissolving in water TFA (95 mL), triisopropylsilane (2.5 mL) and water (2.5 mL) and stirring was continued for 3 hours to remove the protecting groups. The solvent was removed under reduced pressure, the product was further dissolved in DMF in appropriate amount and the product was purified with cold diethyl ether (3X) to give 4APP. For further synthesis of 4APPC, GOX (0.4 mg/mL;. Gtoreq.300U/mg, solaro) was mixed with 4APP (100 mg/mL,10 mM) and CoCl2 (13.3 mM) in HEPES buffer (pH 7.0), the pH of the solution was adjusted to 7.0 with NaOH, and after 2 hours of reaction at room temperature, co was obtained ²⁺ The hydrogel was crosslinked, the product was centrifuged (x 1500 g) for 30 minutes and then dried in vacuo to give the final product as 4APPC.4APPC was performed by nuclear magnetic resonance spectrometer (Bruker Avance400 spectrometer at 400 MHz) ¹ The H-spectrum detection was confirmed.

The synthetic structure of 4APPC is shown in fig. 7A. Successful synthesis of 4APP was confirmed by proton nuclear magnetic resonance spectroscopy (fig. 7B).

Example 2

This example provides a pH/H ₂ O ₂ MMP9 time-series response microsphere (DHPM (4 APPC)) and its preparation method are provided.

DSPE-Hyd-PEG microsphere (DHPM) and DHPM (4 APPC) were prepared as follows:

synthesis of DSPE-Hyd-PEG:

4-Acetylbenzoic acid (0.5 mM) was dissolved in methylene chloride solution (2 mL), followed by addition of DCC (1.0 mM) and DMAP (1.0 mM), and reaction was carried out at room temperature for 6 hours; NH2-PEG-COOH (0.25 mM) was added, along with triethylamine (80. Mu.L). The mixture was withdrawn at room temperature with gentle shaking for 6 hours, the reaction solution was filtered and evaporated, the residue was dissolved in an appropriate amount of ultrapure water and dialyzed (1000 Da; # YA1035, solarbio;500Da, # YA1069, solarbio) at room temperature for 48 hours, and freeze-dried to give product A. Mixing the product A (0.5 mM) with tert-butyl carbamate (0.25 mM), and carrying out condensation reaction on the amino group of the tert-butyl carbamate and the carboxyl group of the product A, and repeating the synthesis step of the product A to finally obtain the product B.

DSPE (0.2 mM) was dissolved in methylene chloride (2 mL), succinic anhydride (0.2 mM) was dissolved in DMSO (0.5 mL), then added dropwise to the DSPE solution, and the mixture was reacted in the dark for 24 hours. After the reaction was completed, acetone (45 mL) was added, the mixture was further reacted at-20℃overnight, centrifuged (15000 rpm) for 15 minutes, and the supernatant was removed to collect a solid as a product C. Product C (0.5 mM), DCC (1.0 mM) and DMAP (1.0 mM) were mixed in methylene chloride solution (2 mL) and reacted at room temperature for 6 hours; tert-butyl carbamate (0.5 mM) and triethylamine (80. Mu.L) were added to the reaction mixture, stirring was continued at room temperature for 6 hours, and finally the reaction solvent was evaporated to dryness and purified by chromatography on a silica gel column (eluent: dichloromethane: methanol=10:1) to give product D. Product D (0.5 mM) was further dissolved in dichloromethane (2 mL), TFA (2 mL) was added, the reaction was stopped by stirring at room temperature for 2 hours, the reaction solvent was distilled off, and the product was purified by chromatography on a silica gel column (eluent: dichloromethane: methanol=10:1) to give product E.

Product B (0.3 mM) and product E (0.3 mM) were mixed in dichloromethane (2 mL), TFA (100. Mu.L) was added, the reaction was stirred at room temperature for 8 hours, the reaction was stopped and the reaction solvent was evaporated, the residue was dissolved in an appropriate amount of ultrapure water, dialyzed at room temperature (1000 Da; # YA1035, solarbio) for 48 hours, and lyophilized to finally give DSPE-Hyd-PEG, nuclear magnetic resonance spectrometer (400 MHz Bruker Avance 400) was used ¹ And H spectrum detection.

DSPE-Hyd-PEG (100 mg) and 4APPC (50 mg) were dissolved in dichloromethane (10 mL) and stirred continuously at low speed while H was slowly added ₂ O (4.3 mL) was added by syringe pump at a rate of 0.5 mL/hr until the water concentration was 30% v/v, stirring was continued for 60 hours, and then H was continuously and slowly added at a rate of 2 mL/hr ₂ O (5.7 mL), stirred for 12 hours. Finally, the mixture was centrifuged (1500 rpm) for 8 minutes, washed 3 times with PBS and the sample was dried in vacuo to give DHPM (4 APPC). If 4APPC is not added, the same procedure is performed, and the final product is DHPM. After DHPM (4 APPC) formation, the supernatant was examined by uv-vis spectrophotometer and the loading of 4APPC was calculated according to beer's law. Loading (wt%) = (4 APPC total weight-4 APPC weight remaining)/(DHPM weight) ×100%.

Example 3

This example provides a method for linking CD63 aptamer via acylhydrazone bond to the periphery of time-series responsive microspheres (DHPM_Apt ^CD63 Preparation).

The selected CD63 Aptamer (shown in SEQ ID NO: 1) was ethynyl modified (Aptamer) ^CD63 -CCH) was synthesized by standard RNA synthesis procedure based on solid phase synthesis, i.e. terminal deoxyuridine was replaced by EdU, nucleotide chain was cut off from solid support, controlled Pore Glass (CPG) support (2 mg) was blow dried with nitrogen, acetonitrile (0.5 mL) and tetrakis (acetonitrile) copper (I) hexafluorophosphate (20 mM) were added, then p-azidoacetophenone (1 μm) was added, shaking was continued at room temperature for 16 hours, centrifugation (3000 rpm) was performed at room temperature for 10 minutes and the supernatant was discarded, and the residual solid phase was washed 3 times with 1mL acetonitrile. Then, a mixture (2 mL) of ethanol and concentrated aqueous ammonia (1:3) was added, the mixture was reacted at 55℃for 18 hours, centrifuged at room temperature (10000 rpm) for 10 minutes, the supernatant was collected, and after discharging the aqueous ammonia/ethanol mixture, a white colloidal solid was obtained, which was further dissolved in a TBAF-THF solution (200. Mu.L), continuously shaken at room temperature for 20 hours, tris-HCl buffer (pH 7.4;0.5 mL) was added, and shaken at room temperature for 15 minutes, and the solution was put into a centrifugal drier to extract 1/2 of the original volume, and THF was removed. Extracting the solution with chloroform (0.5 mL) twice, adding TEAA buffer (1 mL), pouring the mixed solution into a solid phase extraction column, removing excessive salt in the solution according to standard RNA desalting procedure to obtain product F, and determining by MALDI-TOF to confirm theoretical molecular weight [ M+Na+ ]=10092, maldi-TOF detection molecular weight 10094.317.

Similar to the previous step, DHPM (0.5 mM) was dissolved in methylene chloride (2 mL), TFA (2 mL) was added, the reaction was stirred at room temperature for 2 hours, the reaction was stopped and the reaction solvent was distilled off, the residue was washed 3 times with ultrapure water, centrifuged (1500 rpm) at room temperature for 5 minutes, and vacuum-dried to obtain product G.

In addition, as previously described, an acylhydrazone bond may be formed between product F and product G based on the catalytic action of 4-amino-DL-phenylalanine. Briefly, product F (0.257 mM) and product G (0.01 mM) were dissolved in ultrapure water (2 mL), and 4-amino-DL-phenylalanine (0.001 mM) was added to the solution, and NaOH solution (0.6M) was added to adjust the pH to 7.4, and the reaction was carried out with gentle stirring at room temperature for 24 hours, and then centrifuged (3000 rpm) at room temperature for 10 minutes, washed 3 times with ultrapure water (2 mL), and vacuum-dried to obtain DHPM_Apt ^CD63 。

Example 4

This example provides a method for preparing an exosome-carrying biological vector, which comprises preparing the DHPM_Apt prepared in example 3 ^CD63 DHPM (4 APPC) _exo was prepared by mixed incubation with exosomes.

Isolation of cells

The infiltrating cells in the heart were isolated according to methods previously reported (XiaN, luY, guM, liN, liuM, jiaoJ, zhuZ, liJ, liD, tangT, lvB, nieS, zhangM, liaoM, liaoY, yangX, chengX.AUniquePopulationofRegulatoryTCellsinHeartPotentiatesCardiacProtectionFromMyocardialInfarction.Circulation.2020Nov17;142 (20): 1956-1973.). Briefly, an acute myocardial infarction model was established, and after 3 days, heart tissue was taken, minced, and tissue pieces were treated with collagenase II (1 mg/mL) in HEPES buffer (# 17101015, gibco) ^TM ) Digestion, gentle spin at 37℃for 1.5h, cell suspension was filtered with a cell filter (40 μm) (#CL map 831750, corning falcon) ^TM ) The mixture is filtered and the mixture is filtered,PM400 (#F4375, sigma-Aldrich) density centrifugation purified heart mononuclear cells. Cardiac mononuclear cells were collected using CD4 microblades (# 130-090-319, miltenyiBiotec), isolated cells were stained using anti-CD 45 (# ab10558, abcam), anti-CD 4 (# ab6413, abcam), anti-Foxp 3 (ab 215206, abcam) and anti-spark (# ab290647, abcam), and finally CD45 was stained using a flow cytometer FACSAria instrument (BD Bioscience, USA) ⁺ CD4 ⁺ Foxp3 ⁺ Sparc ⁺ Tregs were sorted and analyzed with FlowJo software.

Cell culture and lentiviral transfection

The sorted Tregs were cultured in RPMI-1640 complete medium (10% fbs) containing CD3 epsilon antibody (5 μg/mL; #100340, biolegend) and anti-CD 28 antibody (2.5 μg/mL; #102116, biolegend) at 37 ℃ and 5% co 2. After 24 hours of incubation, CXCR2 high expressing Crispr-cas9 lentiviral-packaged plasmid (GenePharma; sgRNA: GCATAGTCTGAGAGATTCTTGCT) was transfected and incubated for 48 hours.

Exosome purification and characterization

Purification of exosomes by continuous centrifugationA body. Briefly, cell supernatants were collected and centrifuged (300 Xg) at 4℃for 10 min, the supernatants were retained and re-centrifuged (10000 Xg) at 4℃for 30 min, then the supernatants were collected again, centrifugation (140000 Xg) at 4℃was continued for 90min, the supernatants were removed, resuspended and washed with an appropriate volume of PBS, the pellet was collected by centrifugation (140000 Xg) at 4℃for 90min, and frozen at-80℃for further use. Using Pierce ^TM Rapid Gold BCA Protein Assay(#A53226,ThermoScientific ^TM ) The total protein concentration was measured. Particle size distribution and particle concentration were measured using NTA nanosized analyzer nanosigntlm 10 (Malvern Instruments). Furthermore, the morphology of the exosomes was observed using a Transmission Electron Microscope (TEM), the exosome suspension was placed on a carbon grid and stained with uranyl acetate (2%), and the morphology was observed by H-7000 fame at 80-kV. The exosome markers CD9 and CD63 were detected by western blotting.

Sparc with the CXCR2 expressed therein ^high Tregs-derived exosomes and DHPM_Apt prepared in example 3 ^CD63 After mixing for 3 hours at 37 ℃, the mixture was centrifuged (1500 rpm) at room temperature for 10 minutes to obtain dhpm_exo and the water was washed 3 times with ultrapure water. The morphology of dhpm_exo was observed by TEM analysis. Further confirmation of DHPM_Apt by fluorescence observation ^CD63 The fluorescent molecule FITC is loaded on the exosomes, the DHPM is loaded on Cy5.5, the DHPM (Cy5.5) _Exo (FITC) is finally formed, and the fluorescence distribution of the DHPM (Cy5.5) _Exo (FITC) is observed through a fluorescence microscope. DHPM_Apt detection by western blot ^CD63 The ratio of the optimal binding amount to the exosomes was determined by disrupting the exosomes bound in dhpm_exo with RIPA lysate (#p0013b, beyotime) against total exosomes, followed by SDS-PAGE electrophoresis, and detection of the binding rate by detection of CD63. Detection of DHPM_Exo or DHPM (4 APPC) _Exo by western blot (at DHPM_Apt) ^CD63 During the formation, the DHPM-loaded 4APPC forms DHPM (4 APPC) _apt ^CD63 ) For pH value<6.8、H ₂ O ₂ Or MMP9 enzyme (#P 01795, solarbio), the released exosomes were verified by CD63 detection and quantified with NTA.

Experimental example 1

This example examined the spark obtained by purification in example 4 ^high Treg derived outerUrinary body, and verify its function.

The experimental method is as follows:

(1) And constructing an acute myocardial infarction model.

An acute myocardial infarction model was established according to methods reported previously (ChengP, hanH, chenF, chengL, maC, huangH, chenC, liH, caiH, huangH, liG, taoJ.AmeliorationofacutemyocardialinfarctioninjurythroughtargetedferritinnanocagesloadedwithanALKBH5inhibitor.ActaBiomater.2022Mar1; 140:481-491.). Briefly, SPF grade SD rats (female, 180-230 g) were used for modeling, and all animal related procedures were approved by the animal protection and use committee. SD rats were anesthetized with intraperitoneal injection of pentobarbital sodium (50 mg/kg), tracheal cannulated, connected to a small animal ventilator to maintain respiration of the mice, and monitored by Electrocardiogram (ECG) to assess myocardial ischemia. Open chest surgery was performed in the left fourth intercostal area to expose the heart, then the left anterior descending coronary artery was ligated with sterile 6-0 wire, and a slip knot was tied 2-3 mm distal to the LCA origin. When T wave inversion and ST elevation occur in the electrocardiogram, myocardial ischemia is prompted, and after 45min of ischemia, a knot is loosened for reperfusion. Meanwhile, different treatment strategies are adopted, and different detection is carried out at different time points. The sham surgery group was the same as the experimental method except for ligating the coronary artery.

(2) Transmission Electron Microscope (TEM)

The sample was placed on a carbon grid and stained with uranyl acetate (2%), the morphology of the sample was observed and obtained by H-7000 fame, the image was obtained by a digital camera connected to a microscope, and the morphology of the sample was imaged.

(3) Particle size distribution and zeta potential detection

As described above, the zeta potential of the exosomes was measured by nanoparticle size and zeta potential analyzer (Zetasizer NanoZ) using dynamic light scattering method with ultrapure water as dispersant. The particle size distribution and zeta potential of the microspheres were measured by a nanoparticle size and zeta potential analyzer (zetasizer nanoz) using a dynamic light scattering method with ethanol as the dispersant.

(4)Western blot

Exosomes or other samples were lysed with RIPA lysis buffer (#p0013b, beyotidme) on ice for 30 min, then centrifuged (10000×g) at 4 ℃ for 15 min, and western blot analysis was performed using the supernatant. Purified proteins were separated on 10% -15% SDS-PAGE gels and transferred to PVDF membranes, incubated for 1 hour at room temperature in a skim milk solution (5%) at 37 ℃ and then incubated with primary antibodies overnight at 4 ℃, specific antibodies were as follows: anti-CXCR 2 (#ab 65968, abcam), anti-CD 63 (#pa 5-92370, invitrogen), anti-CD 9 (#pa 5-85955, invitrogen) and anti-beta-actin (#ab 8226, abcam). The membranes were then washed 3 times (10 min each) with PBST, incubated with secondary anti-mouse IgG (#ab 6789, abcam) or anti-rabbit IgG (#ab 6721, abcam) for 2 hours at 37℃and then washed 3 times (10 min each) with PBST, chemiluminescent was performed with BeyoECLMon (#P 0018FS, beyotime) and bands were obtained by FluorChemE data system (ProteinSimple, CA, USA).

The test results show that spark ^high Treg plays an important role in tissue repair following acute myocardial infarction [2 ]]. To detect Sparc ^high Influence of Treg-derived exosomes on acute myocardial infarction, tregs were separated into Sparc by flow cell sorter ^high Spark ^low (FIG. 5A) and the exosomes of different cell origins were collected by ultracentrifugation [13 ]]. Transmission electron microscopy showed that the purified exosomes remained well intact (fig. 5B), sparc by nanoparticle tracking analysis and dynamic light scattering detection ^high Spark ^low The source exosomes were not significantly different in particle size distribution and zeta potential (fig. 5C and 5D). Western blot detects specific markers CD9 and CD63 of exosomes, thereby further confirming successful exosomes isolation from spark ^high Tregs and spark ^low Tregs (FIG. 5E).

The inventors determined spark by constructing a Sprague-Dawley (SD) rat acute myocardial infarction model ^high Spark ^low Influence of the derived exosomes on cardiac function. Exosomes were injected in situ 2 times per day in the myocardium of SD rats for the first week and 1 time per day for the second week (fig. 1A). After 24 hours of initial injection, hearts were removed and sectioned, stained with evans blue, and then immersed in triphenyltetrazolium chloride. The results indicate that Tregs sources were injected in situ The exosomes can effectively inhibit ischemia reperfusion injury, while Sparc ^high The effect of Treg-derived exosomes was more pronounced (fig. 1B-1D). Four weeks later, the inventors detected cardiac function by echocardiography, and the results showed Sparc ^high Treg-derived exosomes are effective in promoting restoration of cardiac function, including E/A ratio, left ventricular foreshortening rate, and left ventricular ejection fraction (FIGS. 1E-1I). Furthermore, further detection of the degree of myocardial fibrosis by Masson staining indicated Sparc ^high Treg-derived exosomes effectively inhibited fibrotic scar formation (fig. 1J and 1K). The above results indicate that spark ^high Spark ^low The derived exosomes can improve cardiac function after acute myocardial infarction. But in situ injection also resulted in a sustained decrease in survival rate, eventually approaching 30% (fig. 1L).

Experimental example 2

The experimental example proves that the time-series response microspheres prepared in the examples 2-4 and the biological carrier carrying the exosomes have exosome delivery function and are fixed in the infarcted area, and that the conjugated peptide prepared in the example 1 has good fluidity and is once being H-shaped ₂ O ₂ The triggering, the material exhibits good toughness and strength, which will help it adapt to the beating characteristics of the heart and achieve long-term residence. And once the material is triggered again by MMP9, the toughness and elastic characteristics disappear, the mobility increases, ensuring the release of the loading agent.

In early stages of acute myocardial infarction, a large number of CXCR2 highly expressed immune cells accumulate in the infarct zone under the action of chemokines [9 ]]. To ensure that exosomes can target the infarcted area, the inventors used the CRISPR/Cas9 system to Sparc ^high Tregs undergo gene editing to increase CXCR2 expression at the exosome membrane surface. And this was not significantly different from the characteristics of exosomes obtained in non-edited cells (fig. 5B-5E). The inventors loaded Fluorescein Isothiocyanate (FITC) into exosomes and injected it into veins of SD rats after acute myocardial infarction. Biological luminous imaging shows that CXCR2 high-expression spark ^high Treg-derived exosomes can rapidly target the heart (fig. 6A) and are primarily concentrated in the infarct zone (fig. 6B). Research shows that exosomes are injected singlyAt this time, the number of exosomes in the infarct zone peaked after about 2.5 hours and then gradually decreased (fig. 6C and 6D). If exosomes are injected for 7 consecutive days, the number of exosomes accumulated in the infarcted area will gradually decrease over time (fig. 6E-6H). The results indicate that CXCR2 can effectively guide exosomes to infarct areas, but exosomes do not reside long in infarct areas, and targeting ability decreases with time. This may be due to blood flow washout and gradual decrease of CXCR2 ligand over time, resulting in the exosomes not remaining in the infarct zone for a long time.

To solve this problem, the inventors devised a hydrogel-microsphere-exosome complex system, i.e., DHP microparticles (DHPM) (4 APPC) _exo (see exosome-carrying biovector provided in example 4). Previous studies have shown that in aqueous histidine-modified 4A-PEG solutions, the amino groups in histidine and Co ²⁺ Coordination bonds may be formed. At H ₂ O ₂ Under the action of Co ²⁺ Oxidation to Co ³⁺ At the same time, the aqueous 4A-PEG solution was changed from liquid to gel-like [14]. H produced in the second stage of the inflammatory response ₂ O ₂ And MMP9 formed in the fourth stage is continuously accumulated in the infarcted area [10-12]MMP9 can specifically recognize and hydrolyze the peptide fragment "GALGLP" [15 ]]. Uses the change of local microenvironment at different stages after acute myocardial infarction to ensure that the product has the activity of H ₂ O ₂ The properties of gel formation under the action and hydrolysis under the action of MMP9, the inventors used polypeptides (including histidine and peptide fragments recognized and hydrolyzed by MMP9, i.e., GGALGLPGH) to modify 4A-PEG, conjugated Co based on metal-ligand interactions ²⁺ Finally, 4A-PEG conjugated peptide (4 APPC) was formed. The synthetic structure of 4APPC is shown in fig. 7A, and successful synthesis of 4APPC was confirmed by proton nuclear magnetic resonance spectroscopy (fig. 7B).

The inventors will H ₂ O ₂ Added to the aqueous solution of 4APPC to determine the H-pair of 4APPC ₂ O ₂ And MMP 9. Dissolving 4APPC (40 mM) in water, adding H ₂ O ₂ (1.0 wt%) triggered hydrogel formation and the rheological properties of the conjugated peptide were examined, and degradation was triggered after MMP9 (0.05. Mu.g/mL) was added.

Using HAAKE ^TM MARS 40Rheometer(Thermo Scientific ^TM ) Testing rheological properties, equipped with parallel plate geometry (25mm diameter,0.3mm gap); fixed strain (stress) frequency test: 5% strain, 0.628-100 rad/s,37 ℃, pH7.0; yield stress test: oscillation mode, 37 ℃, ph7.0; modulus test: the mode of oscillation, 6.28rad/s,37℃and pH7.0 was measured for 0-1 hour. H was added to the 4APPC solution (40 mM) ₂ O ₂ (1 wt%) formed a gel and sufficient MMP9 was added to degrade the gel. In the gel formation time test, H ₂ O ₂ After addition of the 4APPC solution, G 'and G' were tested and recorded.

Fig. 2 (a) shows the physical form under different trigger conditions. (B) frequency sweep measurement. (C) dynamic time-scanning measurements. (D) yield stress test in vibration mode.

The results show that by H ₂ O ₂ The liquid became gel-like (fig. 2A) by oxidation (fig. 8A). However, once added to MMP9, the solid again became liquid (fig. 2A), which is apparently caused by hydrolysis of the polypeptide chain (fig. 8B). The inventors evaluated their mechanical properties using a frequency sweep test, which showed H ₂ O ₂ The triggered 4APPC has good elastic performance (G'>G'). Then, once MMP9 triggers again, the elastic properties disappear (fig. 2B). Meanwhile, the inventors detected H using a rheometer ₂ O ₂ Time to trigger gelation. When H is ₂ O ₂ After addition of 4APPC, a crossover point between storage modulus (G') and loss modulus (G ") was observed at 341 seconds, indicating H ₂ O ₂ The triggered 4APPC may form a gel. In addition, the results of the stress strain test show that H ₂ O ₂ The triggered 4APPC has good toughness and strength. However, the addition of MMP9 reduced the tensile strength and elongation, while the toughness and strength of the material disappeared (fig. 2D). Taken together, the results indicate that 4APPC has good flowability once it is H-substituted ₂ O ₂ The triggering, the material exhibits good toughness and strength, which will help it adapt to the beating characteristics of the heart and achieve long-term residence. While once the material is triggered again by MMP9, the toughness and toughness characteristics disappear, the fluidity increases, ensuring the release of the loaded reagentAnd (5) placing.

The following experiments confirm that DHPM remains stable in neutral solution and depolymerizes in acidic solution.

The amphiphilic copolymer can self-assemble in water and form microspheres. The hydrophobic portion of the polymer forms the core of the encapsulated agent, while the hydrophilic portion forms the outer shell of the microsphere structure [16]. The acylhydrazone bond is pH sensitive and breaks at pH <6.8 [17]. According to the local acidic microenvironment of the infarcted area, the lipophilic compound DSPE and the hydrophilic compound PEG can be connected through an acylhydrazone bond (Hyd) to form DSPE-Hyd-PEG (DHP), and the DHP can be self-assembled to form microsphere DHPM. The synthetic structure of DHP is shown in fig. 9A and 11A.

Successful synthesis of intermediate reagent and DHP was confirmed by proton nuclear magnetic resonance spectroscopy (intermediate B is shown in FIG. 9B and DSPE-Hyd-PEG H ¹ Nuclear magnetic resonance spectrum is shown in fig. 11B).

DHPM was dissolved in a solution at pH 7.2 or pH <6.8 (about 6.6, all solutions in the present invention) and its (E) morphology (scale, 500 nm), (F) particle size distribution and (G) zeta potential were detected by TEM, nanoparticle size and zeta potential analyzer.

Each DHPM was shown by transmission electron microscopy to be a uniform spherical structure (fig. 2E). Dynamic light scattering measurements of particle size distribution and zeta potential also confirm uniform distribution of DHPM (fig. 2F and 2G). DHPM was added to solutions of different pH values to test its stability. The results indicate that DHPM remained stable in neutral solution and depolymerized in acidic solution (fig. 2E-2G). Furthermore, loading 4APPC with DHPM forms dhpm_ (4 APPC) with a loading capacity of 32.34±7.43wt%. Dhpm_ (4 APPC) can also be rapidly depolymerized in acidic solutions (pH < 6.8) (fig. 2H).

Experimental example 3

The experimental example verifies the DHPM_Apt in the above embodiment ^CD63 Is a successful synthesis of (a).

CD63 is the most important marker of exosomes and is expressed in large amounts on the surface of exosomes [13]. The aptamer is a short oligonucleotide sequence or short polypeptide obtained by in vitro screening, which binds to the corresponding ligand with high affinity and strong specificity [18,19 ]. To modify the high expression of SparcTregs-derived exosomes, the inventors selected CD63 aptamers as linkers. Ligand system by exponential enrichment [20 ]]The CD63 aptamer was screened, the top 10 aptamer sequences with the highest scores were synthesized again, and each sequence was evaluated for affinity for CD63 protein (table S). The highest affinity sequence was selected in this study (FIG. 11A). The results of the binding experiments showed that the selected aptamer had strong binding to CD63 (kd=4.54±0.27 nM) (fig. 11B). The CD63 aptamer was ethynyl modified (FIG. 12A) and then combined with DHPM to form DHPM_Apt ^CD63 It contains a pH-sensitive acylhydrazone bond (fig. 12B). DHPM_Apt ^CD63 The structure is shown in fig. 12B and 12C.

To verify DHPM_Apt ^CD63 Successful synthesis, the DHPM-loaded fluorescent molecule Cy5.5 forms DHPM (Cy5.5) _Apt by biotin modification of the CD63 aptamer ^CD63 Biotin, then microspheres of different composition are combined with Dynabeads ^TM M-450 strepitavidins (5 uL per 1mL solution; #2850000005,Thermo Fisher Scientific) were incubated with gentle shaking at room temperature for 1 hour, centrifuged (1000 rpm) for 10 minutes at room temperature, observed for color changes and photographed. Another strategy is to modify CD63 aptamer formation by Cy5.5 to form DHPM_Apt ^CD63 -cy5.5 detection of dhpm_apt by flow cytometry ^CD63 Fluorescence intensity of cy 5.5. Both strategies confirm dhpm_apt ^CD63 Is a successful synthesis of (a).

The final structure is shown in fig. 13A. As a result, it was found that the successful modification of the CD63 ligand to the DHPM surface was confirmed by the biotin-CD 63 ligand binding experiment. The inventors loaded cy5.5 with DHPM and incubated with streptavidin magnetic beads capable of specifically binding biotin, followed by centrifugation, liquid fluorescence stratification demonstrated successful modification of CD 63-aptamer on DHPM surface (fig. 13B). While cy5.5 was linked to DHPM by direct binding to CD63 aptamer (fig. 13C), successful binding of CD63 aptamer to DHPM was further confirmed by flow cytometry (fig. 13D).

Table S

Experimental example 4

The experimental example shows that DHPM (4 APPC) _Exo effectively targets the infarcted area through design experiments.

To examine the ability to target to the heart and infarcted areas, the inventors performed optical bioluminescence imaging and fluorescence microscopy. Briefly, once an acute myocardial infarction model was established and different treatment regimens were given, after different times, animals were sacrificed by anesthesia and the extracted hearts, lungs, liver, kidneys and spleens were subjected to optical bioluminescence imaging analysis by the Xenogen biopsy system (IVIS, caliperLifeSciences, USA). Simultaneously, the heart was frozen and cut into 20 μm sections, which were observed by a panoramic tissue slice high resolution fluorescence scanning imaging system (THUNDER Imager Tissue, leica) and the fluorescence intensities were statistically analyzed.

DHPM (4 APPC) _apt ^CD63 With Tregs ^Sp_hi _Exos ^CXCR2_hi (Sparc high-expression Tregs-derived exosomes transformed into CXCR2 over-expressed plasmid) to obtain the composite structure DHPM (4 APPC) _Exo. The structure of DHPM (4 APPC) _exo was obtained by transmission electron microscopy (fig. 3A). The vesicle-like structure was clearly observed to adhere to DHPM (4 APPC) _Apt ^CD63 Thus, successful synthesis of DHPM (4 APPC) _Exo was confirmed. In addition, DHPM_Apt was used before incubation ^CD63 Loading Cy5.5 and Tregs ^Sp_hi _Exos ^CXCR2_hi FITC was loaded. Successful synthesis of dhpm_exo was further confirmed using fluorescence confocal microscopy (fig. 3B).

To determine the optimal binding ratio, tregs were used ^Sp_hi _Exos ^CXCR2_hi And DHPM (4 APPC) _Apt ^CD63 Co-cultures were performed at different ratios. The optimal binding ratio was determined to be 1:2 by Western blot analysis of CD63 (FIG. 3C).

DHPM (4 APPC) _exo was dissolved in ultrapure water, pH was adjusted to <6.8 by adding HCl, centrifuged (1500 rpm) for 5 minutes, and the amount of released exosomes was detected by Western blot to detect the expression of CD63 in the supernatant. It was found that at pH <6.8, CD63 expression of DHPM (4 APPC) _exo was significantly reduced, indicating that DHPM (4 APPC) _exo released Exo from exosomes due to cleavage of the acylhydrazone bond (fig. 3D).

FIG. 3E measures the exosome amount released by DHPM (4 APPC) _Exo by NTA after incubation in solutions with pH <6.8 or 7.2. As shown in fig. 3E, small amounts of exosomes are released at pH 7.2 and large amounts of exosomes are released at pH < 6.8.

To verify H ₂ O ₂ And MMP9 triggered exosome release, DHPM (4 APPC) _Exo was combined with pH prior to MMP9 addition<6.8 and H-containing ₂ O ₂ Is a solution culture of (a). Specifically, in the range of (pH<6.8+1.0wt％H ₂ O ₂ ) Or ((pH)<6.8+1.0wt％H ₂ O ₂ ) After 5 minutes incubation in +0.05 μg/mL MMP 9), the released exosomes were quantified by FIG. 3 (F) western blot and FIG. 3 (G) NTA assay, and the supernatants were collected for exosome detection. CD63 expression in the supernatant was detected by Western blot and found to be at pH<6.8 release of exosomes which are subsequently H-substituted ₂ O ₂ Is immobilized and then released again by the triggering of MMP 9. Quantitative detection of exosomes also confirmed this phenomenon (fig. 3E and 3G).

To further verify the ability of DHPM (4 APPC) _exo to target and reside in the infarcted area following acute myocardial infarction, FITC-loaded Tregs were loaded ^Sp_hi _Exos ^CXCR2_hi DHPM (4 APPC) _exo (FITC) was formed in combination with DHPM (4 APPC) and then injected into the tail vein of SD rats.

In this experimental example, SD rats (female, 180-230 g) were used to establish an acute myocardial infarction model according to the method described above, and DHPM (FITC) (0.2 mL, 0.14X10) was injected via the tail vein once the blood supply in the ischemic region was restored ⁸ /mL; containing 0.02. Mu. Mol of FITC) or DHPM (FITC) _Exo (0.2 mL, 0.2X10) ⁸ /mL; comprising 0.1X10 g ⁸ Per mL exosomes and 0.02 μmol FITC), after 2 hours, hearts, livers, spleens, lungs and kidneys were extracted for optical bioluminescence imaging analysis (n=3 per group) and hearts were removed and sectioned into 20 μm sections for fluorescence distribution observation. FITC forms DHPM (4 APPC) _Exo (FITC) by exosome loading once the ischemic area blood supply is restored in the acute myocardial infarction model, DHPM (4 APPC) _Exo (FITC) is injected by tail vein (0.2 mL, 0.2X10 ⁸ /mL; comprises 2.4mM 4APPC, 0.1X10 ⁸ Excrine/mLAnd 0.02 μmol FITC), 3 (n=3), 5 (n=3) and 7 days (n=3) after injection, the major organs were extracted for optical bioluminescence imaging measurement and analyzed for fluorescence distribution, the heart was sectioned into 20 μm sections, and the fluorescence intensities at the infarct area and distal end were observed and analyzed.

Optical bioluminescence imaging and myocardial slice fluorescence imaging indicated that DHPM (4 APPC) _exo effectively targeted the infarct area (fig. 3H and 3I) and resided for more than 7 days (fig. 3J-3M).

Experimental example 5

The experimental example shows that DHPM (4 APPC) _Exo can improve cardiac insufficiency after acute myocardial infarction through design experiments.

The experimental method is as follows:

evan blue-triphenyltetrazolium chloride (TTC) staining

After establishing the acute myocardial infarction model and administering different treatment regimens, the left anterior descending branch was re-ligated after 24 hours and the area of risk (AAR) was stained by right jugular intravenous injection of erwinz blue dye solution (1%, 0.3 ml). Once the heart turned blue, the heart was rapidly excised and washed 3 times with physiological saline, then frozen at-20 ℃ for 1 hour, cut into 1mm thick sections, incubated with TTC solution (1%) for 15 minutes at 37 ℃, formalin (10%) fixed overnight, and then photographed using a digital camera. Red areas represent AAR, white areas represent infarct, and values of IF/AAR and AAR/LV are calculated.

Echocardiography (UGV)

MyLabSat (Esaote) a phased array probe (SP 3630) is provided for echocardiography. Rats were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg) and placed on a hot plate at a constant temperature of 37 ℃. The medical ultrasonic couplant is adopted to ensure the coupling and the ultrasonic transmission. Detecting the diameter of the back wall, the diameter of the interval between chambers, the length of the left chamber and the diameter of the left chamber in diastole and systole by adopting M-type and pulse wave Doppler echocardiography; heart rate, early left ventricular diastolic filling peak (E peak) and late diastolic filling peak (a peak) are simultaneously detected. An ultrasound image is acquired and various values of different indicators are calculated.

Masson staining

The masson staining was performed using the masson's trichromatic staining kit (#g1340, solarbio) according to the manufacturer's instructions, images were observed and acquired by the AperioCS2 instrument (comes biosystems limited), infarct area was measured by ImageJ software and the infarct ratio was calculated as infarct area/left chamber area x 100%.

Survival monitoring

The mortality of the postoperative rats was monitored daily, and the thrombus around the heart and in the chest, as well as the perforation of the infarcted ventricular wall, were assessed, indicating heart rupture.

To determine the effect of DHPM (4 APPC) _exo on cardiac function following acute myocardial infarction, DHPM (4 APPC) _exo, DHPM (4 APPC) and exosomes (Tregs) were injected intravenously during reperfusion, respectively ^Sp_hi _Exos ^CXCR2_hi ). Evans blue-TTC staining (FIGS. 4A, 4B and 14A), echocardiography (FIGS. 4C-4F and 14B) and Masson staining (FIGS. 4G and 4H) were performed.

Acute myocardial infarction model was established according to the above procedure using SD rats (female, 180-230 g), and once blood supply to the ischemic area was restored, DHPM (4 APPC) (0.2 mL, 0.2X10 ⁸ /mL; comprising 2.4mM 4 APPC), exos (0.2 mL, comprising 0.1X10) ⁸ Per mL exosomes) or DHPM (4 APPC) _Exo (0.2 mL, 0.2X10) ⁸ /mL; comprises 2.4mM 4APPC, 0.1X10 ⁸ /mL exosomes).

In fig. 4, a is a graph of results of heart removal and evans blue-TTC staining 24 hours after injection; each group, n=5. (B) To measure AAR and IF area, calculating the IF/AAR results and performing statistical analysis; each group, n=5; single factor analysis of variance; IF: infarct size, AAR: dangerous areas. 4 weeks after injection, performing echocardiography examination, (C) obtaining pulse wave doppler and M-mode images, obtaining statistics of echocardiography, (D) E/a: ratio of early and late diastolic transventricular flow rate, (E) LVEF: left ventricular ejection fraction, (F) LVFS: left ventricular collapse fraction; each group, n=5; one-way analysis of variance. (G, H) immediately following echocardiography examination, the heart was removed and cut into 5 μm sections, (G) Masson staining was performed, the fibrotic area was measured, (H) statistical analysis was performed; each group, n=5; and (5) single-factor variance analysis. (I) In the whole process, the death of the rat is monitored and statistically analyzed to obtain a Kaplan-Meier survival curve; each group, n=20; log-rank (Mantel-Cox). * P <0.05.

The results indicate that exosomes and DHPM (4 APPC) _exo can inhibit ischemia reperfusion injury, restore cardiac function, and prevent fibrosis. Notably, DHPM (4 APPC) _exo is more effective. Meanwhile, DHPM (4 APPC) _exo injection can improve survival rate to about 95% which is significantly higher than in-situ multiple exosome injections (fig. 4I and 1L).

Next, a blood chemistry analysis was performed to assess systemic toxicity of DHPM (4 APPC). For the acute myocardial infarction model, the control group was SD rats, which were dosed with PBS, DHPM (4 APPC), exo or DHPM (4 APPC) _Exo as shown in FIG. 4, and after 4 weeks, the rat blood was collected and centrifuged at room temperature (1500 rpm) for 10 minutes, and the serum CK, AST, ALT, BUN and Crea were examined by a clinical laboratory.

All data are expressed as mean ± standard deviation. Statistical analysis uses T-test, and the inter-sample differences use non-parametric Mann-Whitney rank sum test. Single factor anova and Bonferroni post hoc test were also used for specific experimental analysis. Differences in animal survival were analyzed using the Kaplan-Meier method. A threshold of P <0.05 was used to determine statistical significance.

Serum creatine kinase (FIG. 15A, CK), aspartate aminotransferase (FIG. 15B, AST), blood urea nitrogen (FIG. 15C, BUN), alanine aminotransferase (FIG. 15D, ALT) and creatinine (FIG. 15E, crea) levels indicated little toxicity or side effects of DHPM (4 APPC). Taken together, DHPM (4 APPC) _exo is a safe option for treating acute myocardial infarction.

In conclusion, acute myocardial infarction triggers an inflammatory response, resulting in the formation of collagen-rich scars, replacing necrotic tissue to prevent heart failure [21 ]]. After acute myocardial infarction, T cells are involved in inflammation and tissue repair, tregs mediate organ-specific regeneration [22 ]]. More importantly, after acute myocardial infarction, sparc ^high Treg infiltration into myocardium can increase collagen content, prevent heart rupture, and improve survival rate [23 ]]. Thus, sparc ^high Tregs can potentially promote repair of cardiovascular tissue following AMI by terminating the pro-inflammatory phase and initiating the anti-inflammatory or repair phase. However, cell expansion cannot be achieved in a short time. In additionThe interaction between the expanded cells and the target tissue microenvironment must be further validated to avoid potential immune rejection risks. Instability or loss of function of cell-derived genetic material and limited cell viability also present additional challenges [24 ]]. Thus, there is an urgent need for a new class of therapeutic regimens that avoid the drawbacks of cell therapies.

Exosomes are small vesicles secreted by cells, having a phospholipid bilayer with a diameter of 50-150nm [25], carrying and transmitting key signal molecules, forming a new intercellular information transmission system, affecting the physiological state of cells and being closely related to the occurrence and development of various diseases [26]. Exosomes can carry a variety of important biomolecules including lipids, proteins, messenger RNAs (mrnas), microRNA (miRNA), and other non-coding RNAs. [27]. Studies have shown that microRNAs in exosomes play a key regulatory role in cardiovascular disease [28], exosomes derived from immune cells can promote immune responses and inflammation in various cardiovascular diseases, and different immune cells have different roles [29]. However, no research has been reported to report the role of exosomes secreted by Tregs in cardiovascular.

To investigate the role of Tregs of a particular group in myocardial infarction, spark was studied on SD rats ^high Treg-derived exosomes have a role in acute myocardial infarction. The result shows that the exosomes derived from the Tregs have a certain protection effect on myocardial infarction, and the exosomes derived from the Tregs with high Sparc expression have a strong protection effect on myocardial infarction. Although Sparc is repeatedly injected in situ ^high Tregs can improve cardiac function, but survival is reduced due to secondary injury to the myocardium. Furthermore, targeted delivery and residence of exosomes in the infarcted area is also a challenge. Thus, a non-invasive treatment regimen is needed.

Scientists have shown to direct their surface to target corresponding tissues and even cells by designing a specifically desired ligand through various modifications of their surface [30]. Currently available modification methods are mainly classified into genetic engineering, covalent modification and non-covalent modification [31 ]]. At present, genetic engineering and covalent bonding are the most important modification methods of exosomes, and genetic engineeringThe program editing is most widely applied because of the advantages of strong editability, low damage, high stability and the like. In the study, CXCL related chemokines in an infarct area after acute myocardial infarction are utilized to be highly expressed, and CXCR2 highly expressed immune cells are obtained through a gene editing method, and have good chemotactic characteristics. The results indicate that spark, highly expressed from CXCR2 ^high Exosomes extracted from tregs can be targeted rapidly and accurately to infarcted areas.

Hydrogels have been used to ameliorate the problem of residence of the infarcted exosomes. Hydrogels are physically or chemically crosslinked three-dimensional hydrophilic polymer networks that can adsorb large amounts of target agents without undergoing a dissolution process. In regenerative medicine, hydrogels can be used as scaffolds, barriers, drug delivery systems, and cell encapsulation matrices [32 ]]. Studies have shown that cells or bioactive molecules incorporating hydrogels can retain their structure and function for longer than without hydrogels [33 ]]. Therefore, development of a system that can form gel-immobilized exosomes by in-situ crosslinking in the infarcted area after acute myocardial infarction and can slowly release exosomes under the action of local microenvironment is urgently needed. There are many current therapies for acute myocardial infarction using 4-Arm-polyethylene glycol (4 Arm-PEG) coupled with histidine (4A-PEG-His) and metallic cobalt ions (Co ²⁺ ) Formation of coordination bond with amino group and nitrogen radical in histidine, at H ₂ O ₂ Under the action of Co ²⁺ Changing from the 2-valent state to the 3-valent state, i.e. Co ³⁺ At the same time 4A-PEG-His also changed from liquid to gel-like. At the same time, MMP9 is taken as a zymolytic protein which can specifically recognize and hydrolyze a special peptide segment, namely 'GALGLP' [34,35 ] ]. On the other hand, after myocardial infarction, the series causes the pH value of the infarcted area to decrease continuously due to the continuous death and rupture of cells accompanied by continuous accumulation of lactic acid (pH<6.8 Local in an acidifying environment [36 ]]. The acylhydrazone bond is a kind of dynamic covalent bond, and is stronger than weak interactions (hydrogen bond, etc.) between molecules, and the acylhydrazone bond is formed under specific pH conditions (pH<6.8 With reversible properties, resulting in an acylhydrazone bond that can be at pH<6.8 environmental fracture [37 ]]. Based on this particular nature of the acylhydrazone bond, a great deal of research has been devoted to acyl groupsHydrazone bond connects related compounds to make them available at pH<6.8 cleavage in an acidic Environment, thereby isolating Compound [38 ]]。

Current research is focused mainly on the microenvironment of the infarct zone after acute myocardial infarction (pH<6.8 High H at early infarct) ₂ O ₂ The content and later micro-environmental MMP9 expression gradually increase. In the design, 4A-PEG is modified successfully by polypeptide specifically hydrolyzed and identified by MMP9, co is added into the solution ²⁺ After that, the function of the compound is not affected, and in H ₂ O ₂ Is formed into a gel-like form under the action of (a). Meanwhile, the acylhydrazone bond is utilized to connect DSPE and PEG to form DHPM, the aptamer of CD63 is selected to modify the DHPM, the DHPM is combined with and wraps the gel material, and meanwhile, the DHPM is combined with Tregs ^Sp_hi _Exos ^CXCR2_hi Anchoring to form a composite structure. The system successfully targets the infarcted area under the action of CXCR2, realizes the rupture of the microsphere under the acidic condition, and utilizes the high H of the infarcted area ₂ O ₂ Content of Co in the gel raw material ²⁺ The transition from the divalent state to the trivalent state forms a gel that immobilizes the exosomes. Then, with the gradual increase of later MMP9, the hydrogel is destroyed, causing the exosomes to gradually release. Protection against acute myocardial infarction is achieved by targeted delivery and immobilization and slow release of exosomes as a non-invasive treatment. The result provides a new choice for the treatment of acute myocardial infarction.

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the above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (20)

1. A conjugated peptide comprising 4A-PEG and a metal cobalt ion modified by a polypeptide comprising histidine and a peptide fragment for MMP9 enzymatic hydrolysis, said peptide fragment for MMP9 enzymatic hydrolysis having the amino acid sequence of SEQ ID NO:2, the metal cobalt ion and histidine are used for preparing the metal cobalt-histidine composite material Coordination bond bonding; the metal cobalt ion energy is based on H ₂ O ₂ In response, making the conjugated peptide gelatinous;

the structural formula of the polypeptide modified 4A-PEG is as follows:

2. PH/H ₂ O ₂ MMP9 time-series responsive microspheres, characterized in that it comprises: microspheres formed by distearoyl phosphatidylethanolamine and polyethylene glycol DSPE-Hyd-PEG connected by acylhydrazone bonds, wherein the microspheres are loaded with the conjugated peptide as claimed in claim 1; the structural formula of DSPE-Hyd-PEG is as follows:

。

3. the pH/H according to claim 2 ₂ O ₂ MMP9 time-series responsive microspheres, wherein the microspheres have an acylhydrazone bond cleavage at a pH of less than 6.8.

4. A pH/H according to claim 3 ₂ O ₂ MMP9 time-series response microsphere, wherein said microsphere has a loading capacity of 32.34+ -7.43 wt% of said conjugated peptide.

5. An exosome-carrying biological vector comprising the pH/H of any one of claims 2 to 4 ₂ O ₂ MMP9 time-series response microsphere, wherein the periphery of the time-series response microsphere is connected with CD63 aptamer through an acylhydrazone bond, and the CD63 aptamer is specifically combined with an exosome of regulatory T cells from CXCR2 high expression and SPARC high expression through a CD63 marker on the surface of the exosome;

The sequence of the aptamer is shown as SEQ ID NO: 1.

6. The carrier of claim 5, wherein the regulatory T cells with high expression of CXCR2 and high expression of SPARC are gene editing means to make the regulatory T cells with high expression of SPARC realize high expression of CXCR2, and finally obtain the exosomes with high expression of CXCR 2.

7. The exosome-carrying biological vector according to claim 5, wherein the aptamer is further modified with a modifier or labeled with a detectable label; the modifier is biotin; the detectable label is selected from the group consisting of fluorescent dyes, enzymes that catalyze the development of substrates, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.

8. A pH/H according to claim 2 to 4 ₂ O ₂ The preparation method of the MMP9 time sequence response microsphere is characterized by comprising the following steps:

microspheres formed by conjugated peptide and DSPE-Hyd-PEG are mixed in a solvent, and then centrifuged and dried in vacuum.

9. The pH/H according to claim 8 ₂ O ₂ A method for producing MMP9 time-series responsive microspheres, characterized in that the ratio of the concentration of the conjugated peptide in the solvent to the concentration of the microspheres in the solvent is 2.3-2.7 mg/ml, 4.8-5.2 mg/ml.

10. The pH/H according to claim 9 ₂ O ₂ The preparation method of the MMP9 time sequence response microsphere is characterized in that the solvent is methylene dichloride, water is added when the conjugated peptide is mixed with the microsphere until the volume of the added water accounts for 30% of the volume of the total mixed solution, and the water is added at a speed of 0.5-0.6 mL/h.

11. The pH/H according to claim 10 ₂ O ₂ MMP9 time sequence response microsphere preparation method is characterized in that water is added to total mixed liquidAfter 30% of the volume, water was added at a rate of 2-2.5mL/h, which is 50% of the total mixed solution volume.

12. A method for preparing the exosome-carrying biological vector according to any one of claims 5 to 7, comprising the steps of:

the time-series response microsphere and CD63 aptamer are mixed according to the following ratio of 0.23-0.27 mM: mixing and reacting at a molar ratio of 0.008-0.012 and mM to obtain a time-series response microsphere with CD63 aptamer connected through an acylhydrazone bond, and then mixing an exosome derived from regulatory T cells with the time-series response microsphere with CD63 aptamer connected;

the CD63 aptamer is subjected to a mixed reaction, and the method further comprises the step of modifying ethynyl on the CD63 aptamer and connecting the ethynyl with the p-azidoacetophenone through spot chemistry.

13. The method for producing a biovector carrying an exosome according to claim 12, wherein a mixing mass ratio of the exosome to the time-series response microsphere to which the CD63 aptamer is attached is 0.5 to 1.5:1 to 3.

14. The method for producing an exosome-carrying biological vector according to claim 13, wherein the mixing is incubation at 37±0.5 ℃ for 3 h.

15. Use of the exosome-carrying biological vector according to any one of claims 5-7 in the preparation of a myocardial infarction repair drug.

16. The use according to claim 15, wherein the myocardial infarction is selected from acute myocardial infarction, subacute myocardial infarction or a ventricular tumor.

17. The use according to claim 15, wherein in said use pH triggers the release of exosomes of said biological carrier, active oxygen H ₂ O ₂ Initiating oxidation of metallic cobalt ions to form a gelThe released exosomes are immobilized, then the degradation of the gel is triggered based on the hydrolysis of MMP9 enzymes, releasing the exosomes slowly.

18. A medicament for treating myocardial infarction, characterized in that it comprises the exosome-carrying biological vector according to any one of claims 5 to 7.

19. The medicament of claim 18, wherein the medicament for treating myocardial infarction further comprises a pharmaceutically acceptable adjuvant.

20. The medicament according to claim 19, wherein the pharmaceutical dosage form is selected from the group consisting of tablets, pills, powders, suspensions, gels, emulsions, creams, granules, nanoparticles, capsules, suppositories, injections and sprays.